CLOSPATH 7 - Conference Planning and Management - Iowa State ...
CLOSPATH 7 - Conference Planning and Management - Iowa State ...
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7 TH INTERNATIONAL CONFERENCE ON THE<br />
MOLECULAR BIOLOGY AND PATHOGENESIS<br />
OF THE CLOSTRIDIA<br />
<strong>CLOSPATH</strong> 7<br />
PROGRAM & ABSTRACT BOOK<br />
October 25 to 29, 2011<br />
<strong>Iowa</strong> <strong>State</strong> Center<br />
Ames, <strong>Iowa</strong> USA
7 th International <strong>Conference</strong> on the Molecular Biology <strong>and</strong> Pathogenesis of the Clostridia<br />
Abstract Book<br />
Organizing Committee:<br />
Jimmy Ballard (USA)<br />
Neil Fairweather (UK)<br />
Miia Lindström (Finl<strong>and</strong>)<br />
Paola Mastrantonio (Italy)<br />
Bruce A. McClane (USA)<br />
Nigel Minton (UK)<br />
Tom Riley (Australia)<br />
Julian Rood (Australia)<br />
Maja Rupnik (Slovenia)<br />
Bo-Moon Shin (South Korea)<br />
Linc Sonenshein (USA)<br />
J.Glenn Songer (USA)<br />
Rick Titball (UK)<br />
Rod Tweten (USA)<br />
Gayatri Vedantam (USA)<br />
Vince Young (USA)<br />
Scientific Advisory Board:<br />
Klaus Aktories (Germany)<br />
Frederic Barbut (France)<br />
Marietta Flores Diaz (Costa Rica)<br />
Bruno Dupy (France)<br />
Dale Gerding (USA)<br />
Gerhard Gottschalk (Germany)<br />
Eric Johnson (USA)<br />
Haru Kato (Japan)<br />
Cesare Montecucco (Italy)<br />
Peter Mullany (UK)<br />
Shinichi Nakamura (Japan)<br />
Michael R. Popoff (France)<br />
Ian Poxton (UK)<br />
Tohru Shimizu (Japan)<br />
Francisco A. Uzal (USA)
The 7 th International <strong>Conference</strong> on the Molecular Biology of the<br />
Clostridia would like to acknowledge the contributions of<br />
Funding for this conference was made possible [in part] by R13 AI098104-01 from<br />
NIH. The views expressed in written conference materials or publications <strong>and</strong> by<br />
speakers <strong>and</strong> moderators do not necessarily reflect the official policies of the<br />
Department of Health <strong>and</strong> Human Services, nor does mention of trade names,<br />
commercial practices, or organizations imply endorsement by the U.S.<br />
Government.
IOWA STATE UNIVERSITY<br />
OF SCIENCE AND TECHNOLOGY<br />
Dear Colleague <strong>and</strong> Fellow Delegate,<br />
Many of you may have thought you‘d never have a good reason to come to <strong>Iowa</strong>, but here you are!<br />
<strong>Iowa</strong> is not, on the whole, great for nightlife, although I suspect that many of you would be surprised by<br />
the variety <strong>and</strong> extent of after hours entertainment. I‘ve lived in or been in some way connected to<br />
<strong>Iowa</strong> for more than 50 years, <strong>and</strong> have yet to observe real, live cow-tipping.<br />
<strong>Iowa</strong> IS a great agricultural state, leading the US most years in production of corn <strong>and</strong> hogs, <strong>and</strong><br />
ranking high in cattle production, as well. Disease caused by members of the genus Clostridium in<br />
humans is as common here as anywhere, <strong>and</strong> the plethora of domestic animals have their share of<br />
infections, as well. So, maybe <strong>Iowa</strong> is a good place for the 7 th International <strong>Conference</strong> on the<br />
Molecular Biology <strong>and</strong> Pathogenesis of the Clostridia. In any case, it‘s here.<br />
And all of you are absolutely welcome! Members of the Organizing Committee have done our best to<br />
assemble a program that will be stimulating to everyone. We hope that each of you will have the<br />
opportunity to forge new relationships, make friends, find new collaborators, <strong>and</strong> above all to learn a<br />
lot about the current state of clostridial research – from basic to translational to applied. In addition to<br />
the science, I think you‘ll be pleased with the meals <strong>and</strong> breaks – we should have great volumes of<br />
food <strong>and</strong> drink, with enough variety to satisfy everyone‘s tastes.<br />
I owe a great debt of gratitude to the Organizing Committee, <strong>and</strong> especially to the ―executive<br />
committee,‖ consisting of Julian Rood, Bruce McClane, <strong>and</strong> Linc Sonenshein. Finally, my ―right h<strong>and</strong><br />
woman‖ has been Patti Thrasher. She has dealt with matters of registration, housing, <strong>and</strong> all local<br />
arrangements at the venue single-h<strong>and</strong>edly. I cannot possibly thank her enough for all she‘s done.<br />
Without her, I would have been, long sense, a jabbering mass of coffee-stained blue jeans.<br />
If you have trouble while here, seek help at the registration desk, where Patti <strong>and</strong> a plethora of helpers<br />
from my laboratory will be around to assist you. As a last resort, you may come to me, but I‘m likely to<br />
be of less value than the others. Enjoy yourselves, <strong>and</strong> let us know if there‘s anything we can do to<br />
make your stay more fulfilling.<br />
All the best,<br />
Dr. J. Glenn Songer, Fellow AAM, Diplomate ACVP<br />
Boehringer Ingelheim Vetmedica Chair in Food Animal Infectious Disease<br />
Department of Veterinary Microbiology <strong>and</strong> Preventive Medicine<br />
College of Veterinary Medicine<br />
<strong>Iowa</strong> <strong>State</strong> University<br />
Ames, IA 50011
<strong>CLOSPATH</strong> 7 CONFERENCE PROGRAM
Tuesday, October 25, 2011 — Scheman Building (1 st Floor)<br />
5:30 pm — Open for Registration (Lobby Registration Desk )<br />
6:00 pm — Welcome: Wine <strong>and</strong> Cheese Reception (Lobby)<br />
7:00 pm — Keynote Address (Benton Auditorium): Rodney K. Tweten, University of<br />
Oklahoma, USA. ―Cholesterol-Dependent Cytolysins.‖<br />
Wednesday, October 26, 2011 — Fisher Theater<br />
7:00 am — Open for Registration (Lobby)<br />
— Continental Breakfast (Lobby)<br />
Session I (Fisher Theater): Epidemiology <strong>and</strong> Laboratory Diagnosis (Chair: Nigel<br />
Minton):<br />
8:30 – 9:00 am — Maja Rupnik, University of Maribor, Slovenia. ―Epidemiology of<br />
Clostridium difficile – new environments <strong>and</strong> new types.‖<br />
9:00 – 9:20 am — David Aronoff, University of Michigan, USA. ―Lack of association<br />
between ribotype <strong>and</strong> severe Clostridium difficile infection.‖<br />
9:20 – 9:40 am — Cornelis Knetsch, Leiden University, The Netherl<strong>and</strong>s. ―Unique genetic<br />
markers for hypervirulent Clostridium difficile.”<br />
9:40 – 10:00 am — Ying Cheng, Chinese Center for Disease Control <strong>and</strong> Prevention,<br />
China. ―Genomic characteristics of a toxin A-negative, toxin B-positive C. difficile<br />
strain from Beijing, China.‖<br />
10:00 am — Break (Lobby)<br />
Session II (Fisher Theater): Large Clostridial Glycosylating Toxins (Chair: Klaus<br />
Aktories):<br />
10:15 – 10:45 am — Jimmy Ballard, University of Oklahoma, USA. ―Critical differences<br />
between historical <strong>and</strong> hypervirulent TcdB.‖<br />
10:45 – 11:05 am — Borden Lacy, V<strong>and</strong>erbilt University, USA. ―Structural determinants of<br />
the Clostridium difficile toxin A glucosyltranserase activity.‖<br />
11:05 – 11:25 am — Aimee Shen, University of Vermont, USA. ―Chemically interrogating<br />
Clostridium difficile glucosylating toxin activation.‖<br />
11:25 – 11:45 am — Shan Li, Tufts University, USA. ―A neutralizing intrabody to study<br />
autocleavage of Clostridium difficile toxin B.‖<br />
11:45 am – 12:05 pm — Wensheng Wei, Peking University, China. ―Identification of novel<br />
endocytosis pathway for TcdB of Clostridium difficile through shRNAmir library<br />
screening <strong>and</strong> high-throughput sequencing analysis.‖
12:05 pm — Buffet Lunch (Scheman Bldg., Rooms 167-179)<br />
Session III (Fisher Theater): Genetics, including genomes <strong>and</strong> plasmids (Chair: Miia<br />
Lindström):<br />
1:20 – 1:50 pm — Marian Wachtel, Program Officer, NIH. ―NIAD Support of Clostridial<br />
Research <strong>and</strong> Resources for the Research Community‖<br />
1:50 –2:20 pm — Julian Rood, Monash University, Australia. ―The mechanism of<br />
conjugation in C. perfringens.”<br />
2:20 – 2:50 pm — Bruce McClane, University of Pittsburgh, USA. ―Characterization of the<br />
toxin plasmids of Clostridium perfringens.”<br />
2:50 – 3:20 pm — Nigel Minton, University of Nottingham, UK. ―The development of<br />
clostridial gene systems <strong>and</strong> their exploitation.‖<br />
3:20 – 3:40 pm — Anne Collignon, Université Paris-Sud, France. ―Analysis of the<br />
genome-wide temporal expression of a Clostridium difficile 027 hypervirulent strain<br />
in monoxenic mice.‖<br />
3:40 – 4:00 pm — Eric Johnson, University of Wisconsin, USA. ―Phylogenomic analysis of<br />
Clostridium botulinum strains.‖<br />
4:00 – 6:30 pm — POSTERS I (Scheman Bldg., Lounge 182)<br />
4:30 pm — Registration Closes (Fisher Theater, Lobby)<br />
6:30 pm — Buffet Dinner (Scheman Bldg., Rooms 167-179)<br />
Thursday, October 27, 2011—Fisher Theater<br />
7:00 am — Continental Breakfast (Lobby)<br />
Session IV (Fisher Theater): Membrane active toxins, binary toxins <strong>and</strong> enzymes<br />
(Chair: Joe Barbieri):<br />
8:30 – 9:00 am — Klaus Aktories, Alert-Ludwigs Universität, Freiburg, Germany.<br />
―Clostridium difficile CDT <strong>and</strong> Clostridium perfringens Iota Toxin: Identification of<br />
their membrane receptors <strong>and</strong> actions on the cytoskeleton of target cells.‖<br />
9:00 – 9:20 am — Mark McClain, V<strong>and</strong>erbilt University, USA. ―Identification of host factors<br />
contributing to Clostridium perfringens epsilon-toxin-induced cell death.‖<br />
9:20 – 9:40 am — Sérgio Fern<strong>and</strong>es da Costa, University of Exeter, UK. ―Expression <strong>and</strong><br />
functional characterization of Clostridium perfringens NetB Toxin.‖<br />
9:40 – 10:00 am — Ajit Basak, Birkbeck College, UK. ―Structural Relationships in<br />
clostridial beta-pore-forming toxins.‖<br />
10:00 am — Break (Lobby)
Session V (Fisher Theater): Regulation of virulence genes, including transcriptomics<br />
(Chair: Linc Sonenshein):<br />
10:20 – 10:50 am — Bruno Dupuy, Institut Pasteur, Paris, France. ―Role of carbon<br />
catabolite repression (CCR) in the pathogenicity of Clostridium difficile.”<br />
10:50 – 11:10 am — Revathi Govind, Kansas <strong>State</strong> University, USA. ―A new role for C.<br />
difficile glutamate dehydrogenase.‖<br />
11:10 – 11:30 am — Kaori Ohtani, Kanazawa University, Japan. ―Novel regulatory<br />
mechanism for spore formation <strong>and</strong> enterotoxin production in Clostridium<br />
perfringens.”<br />
11:30 – 11:50 am — Wiep Klaas Smits, Leiden University Medical Center, The<br />
Netherl<strong>and</strong>s. ―Clostridium difficile Spo0A regulates sporulation, but not toxin<br />
production, by direct binding to target DNA.‖<br />
11:50 am – 12:10 pm — Kate Mackin, Monash University, Australia. ―Regulator-mediated<br />
modulation of toxin production <strong>and</strong> sporulation in an epidemic clinical isolate of C.<br />
difficile.”<br />
12:10 pm — Buffet Lunch (Scheman Bldg., Rooms 167-179)<br />
Session VI (Fisher Theater): Food animal diseases; animal models (Chair: Neil<br />
Fairweather):<br />
1:30 – 2:00 pm — Francisco Uzal, University of California, Davis, USA. ―Animal models to<br />
study C. perfringens diseases.‖<br />
2:00 – 2:20 pm — Kevin Chen, Tufts University, USA. ―Systemic dissemination of C.<br />
difficile toxins A <strong>and</strong> B is associated with severe fatal disease in the piglet <strong>and</strong><br />
mouse models.‖<br />
2:20 – 2:40 pm — Anthony Buckley, University of Glasgow, UK. ―Aspects of the hamster<br />
model of infection.‖<br />
2:40 – 3:00 pm — Xingmin Sun, Tufts University, USA. ―A mouse relapse model of C.<br />
difficile infection <strong>and</strong> its application in evaluating immunotherapies against<br />
disease.‖<br />
3:00 – 3:20 pm — Horst Posthaus, University of Berne, Switzerl<strong>and</strong>. ―Lessons from<br />
natural disease in pigs: pathogenesis of C. perfringens type C enteritis.‖<br />
3:20 – 3:40 pm — Duncan MacCannell, CDC, USA. ―The molecular epidemiology of<br />
Clostridium difficile in the United <strong>State</strong>s‖<br />
3:40 – 6:30 pm — POSTERS II (Scheman Bldg., Lounge 182)<br />
6:30 pm — Buffet Dinner (Scheman Bldg., Rooms 167-179)
Friday, October 28, 2011—Scheman Building (1 st Floor)<br />
7:00 am — Continental Breakfast (1 st Floor Lobby)<br />
Session VII (Benton Auditorium): Physiology, including proteomics/metabolomics <strong>and</strong><br />
sporulation/germination (Chair: Gayatri Vedantam):<br />
8:30 – 9:00 am — William Self, University of Central Florida, USA. ―Elucidating the role<br />
<strong>and</strong> requirement for selenoenzymes in growth of Clostridium difficile.”<br />
9:00 – 9:30 am — Linc Sonenshein, Tufts University, USA. ―Regulation of C. difficile<br />
metabolism.‖<br />
9:30 – 9:50 am — Robert Fagan, Imperial College, UK. ―Clostridium difficile has two<br />
parallel <strong>and</strong> essential Sec secretion systems.‖<br />
9:50 – 10:10 am — Mahfuzur Sarker, Oregon <strong>State</strong> University, USA. ―The molecular<br />
mechanism of Clostridium perfringens spore germination.‖<br />
10:10 – 10:30 am — Laurel Saujet, Institut Pasteur, France. ―The key sigma factor of<br />
transition phase, SigH, controls sporulation, metabolism, <strong>and</strong> virulence factor<br />
expression in Clostridum difficile.‖<br />
10:30 am — Break (1 st Floor Lobby)<br />
Session VIII (Benton Auditorium): Control <strong>and</strong> treatment (including therapeutics,<br />
vaccines/immune responses <strong>and</strong> exploitation) (Chair: Anne Collignon):<br />
10:50 – 11:20 am — Hanping Feng, Tufts University, USA. ―Novel C. difficile vaccines.‖<br />
11:20 – 11:40 am — Anjana Chakravorty, Monash University, Australia. ―Opioid-based<br />
analgesics block the progression <strong>and</strong> development of C. perfringens mediated<br />
myonecrosis.‖<br />
11:40 – 12:00 am — Louis Charles Fortier, Universite de Sherbrooke, Canada. ―Subinhibitory<br />
concentrations of tigecycline inhibit sporulation of Clostridium difficile in<br />
vitro.‖<br />
12:00 am – 12:20 pm — Simon Cutting, Imperial College, UK. ―Immunization with Bacillus<br />
spores expressing toxin A peptide repeats protects against infection with<br />
Clostridium difficile strains producing toxins A <strong>and</strong> B.‖<br />
12:20 – 12:40 pm — Shonna McBride, Tufts University, USA. ―The C. difficile CPR locus<br />
mediates resistance to antimicrobial peptides through molecular mimicry.‖<br />
12:40 – 1:00 pm — Rebecca McQuade, University of Arizona, USA. ―Differential<br />
sensitivity of Clostridium difficile clinical isolates to mammalian cationic<br />
antimicrobial peptides.‖<br />
1:00 pm — Buffet Lunch (Rooms 167-179)
Session IX (Benton Auditorium): Neurotoxins <strong>and</strong> toxin applications (Chair: Rod<br />
Tweten):<br />
2:15 – 2:45 pm — Joe Barbieri, Medical College of Wisconsin, USA. ―Botulinum toxin<br />
structure/function relationships.‖<br />
2:45 – 3:05 pm — Miia Lindström, University of Helsinki, Finl<strong>and</strong>. ―A two-component<br />
system negatively regulates botulinum neurotoxin expression.‖<br />
3:05 – 3:25 pm — David Kirk, University of Helsinki, Finl<strong>and</strong>. ―Disruption of sigK in C.<br />
botulinum ATCC 3502 prevents sporulation.‖<br />
3:25 – 6:00 pm — POSTERS III (Lounge 182)<br />
6:30 pm — <strong>Conference</strong> Banquet (Rooms 167-179)<br />
Saturday, October 29, 2011—Scheman Building(1 st Floor)<br />
7:00 am — Continental Breakfast (Lobby)<br />
Session X (Benton Auditorium): Host-pathogen interactions involving toxins (Chair:<br />
Bruce McClane):<br />
8:30 – 9:00 am — Dena Lyras, Monash University, Australia. ―Roles of Toxins in C.<br />
difficile pathogenesis.‖<br />
9:00 – 9:20 am — Sarah Kuehne, University of Nottingham, UK. ―C. difficile toxins <strong>and</strong><br />
pathogenesis.‖<br />
9:20 – 9:40 am — Jianming Chen, University of Pittsburgh, USA. ―The AGR quorum<br />
sensing system is a global regulator of Clostridium perfringens toxin production<br />
<strong>and</strong> virulence.‖<br />
9:40 – 10:00 am — Kevin D‘Auria, University of Virginia, USA. ―Global gene expression of<br />
epithelial cells from an in vivo model of C. difficile toxin A <strong>and</strong> B intoxication.‖<br />
10:00 am — Break (Lobby)<br />
Session XI (Benton Auditorium): Host-pathogen interactions involving nontoxic<br />
virulence factors (Chair: Dena Lyras):<br />
10:20 – 10:50 am — Gayatri Vedantam, University of Arizona, USA. ―Sporulation <strong>and</strong><br />
other non-toxin virulence factors.‖<br />
10:50 – 11:20 am — Steve Melville, Virginia Tech, USA. ―Motility <strong>and</strong> adherence to<br />
myoblasts.‖<br />
11:20 – 11:50 am — Neil Fairweather, Imperial College, UK. ―Antigenic <strong>and</strong> phase<br />
variation in cell wall proteins of Clostridium difficile.
11:50 am – 12:10 pm — Jihong Li, University of Pittsburgh, USA. ―Sialidases affect the<br />
host cell adherence <strong>and</strong> epsilon toxin-induced cytotoxicity of C. perfringens type D<br />
strain CN3718.‖<br />
12:10 – 12:30 pm — Craig Ellermeier, ―University of <strong>Iowa</strong>, USA. ―Extra-cytoplasmic<br />
function sigma factor CSFV regulates lysozyme resistance of Clostridium difficile”.<br />
12:30 – 12:45 pm Meeting Closing<br />
12:45 pm — Buffet Lunch (Rooms 167-179)<br />
DEPART
ABSTRACTS OF ORAL PRESENTATIONS<br />
Keynote Address<br />
O1 to O52
Keynote<br />
THE STRUCTURAL BIOLOGY OF CLOSTRIDIUM PERFRINGENS PERFRINGOLYSIN O:<br />
NEW PARADIGMS, VACCINE DEVELOPMENT AND EVOLUTION<br />
Rodney K. Tweten, Department of Microbiology <strong>and</strong> Immunology, University of Oklahoma<br />
Health Sciences Center, Oklahoma City, OK 73104 USA,<br />
The cholesterol-dependent cytolysins (CDCs) are a family of pore forming toxins produced<br />
by many species of Gram-positive pathogenic <strong>and</strong> commensal bacteria. Insights into the<br />
structural biology of pore formation of the CDCs have been largely accomplished by the<br />
study of the archetype CDC, Clostridium perfringens perfringolysin O (PFO). The study of<br />
PFO has revealed many new paradigms of how the CDCs recognize a cell <strong>and</strong> form a pore<br />
in its membrane. These studies, however, have impacted a much broader expanse of<br />
science than could have been predicted. The structure/function studies of PFO were key to<br />
(1) revealing that the eukaryotic membrane attack complex/perforin (MACPF) proteins may<br />
utilize a pore-forming mechanism that exhibits features of the CDC mechanism <strong>and</strong> may be<br />
ancient ancestors of the CDCs, (2) the presence of CDCs in Gram-negative bacteria <strong>and</strong> (3)<br />
the rational design of a pneumolysin toxoid as a component for a protein-based vaccine for<br />
Streptococcus pneumoniae. Furthermore, methods developed to trap PFO at different stages<br />
of its pore forming mechanism have been used to show that formation of the pore is not the<br />
only way that CDCs can contribute to pathogenesis, which suggests that CDCs are<br />
multifunctional proteins. Hence, the investigation of the PFO structure <strong>and</strong> pore forming<br />
mechanism has impacted many fields of study <strong>and</strong> its continued study will likely reveal new<br />
paradigms for how the CDCs function <strong>and</strong> contribute to bacterial survival <strong>and</strong> pathogenesis.
O1<br />
EPIDEMIOLOGY OF CLOSTRIDIUM DIFFICILE – NEW ENVIRONMENTS AND NEW<br />
TYPES<br />
Maja Rupnik. Institute of Public Health Maribor, Centre for Microbiology, Maribor; University<br />
of Maribor, Faculty of Medicine, Maribor; Centre of Excellence CIPKeBIP, Ljubljana,<br />
Slovenia.<br />
Clostridium difficile is currently still regarded as a nosocomial pathogen, but its importance in<br />
the community is also rising. The hospital, as well as the non-hospital, environment can be<br />
the source of infection that occurs either in outbreaks or as isolated cases. Even within<br />
hospitals new modes of exposure, such as airborne dissemination, are taken into account.<br />
Isolation of C. difficile from substantial numbers of normal or diseased food animals or<br />
companion animals was reported relatively recently. Species infected most commonly<br />
include piglets, calves, poultry, cats <strong>and</strong> dogs. Genotypes present in humans are much more<br />
diverse ( > 300 ribotypes) than in animals or food (approx. 30 ribotypes) <strong>and</strong> the prevalent<br />
genotype in human <strong>and</strong> animal reservoirs is still different. But there is also a large overlap of<br />
genotypes from food, food animals, <strong>and</strong> humans, suggesting a contribution of food <strong>and</strong><br />
contact with animals to the infection process. The presence of most common PCR ribotypes<br />
in many different sources (humans, animals, food, soil, <strong>and</strong> water) suggests that the ability to<br />
survive in different environments plays a role in successful distribution <strong>and</strong> high prevalence<br />
of a given genotype. Underst<strong>and</strong>ing of the broader epidemiology (e.g. comparison of<br />
ribotypes in different countries, <strong>and</strong> spread of more virulent genotypes) is still limited by the<br />
use of non-exchangeable typing techniques. Interestingly, there seems to be a large change<br />
in circulating toxin production types. A few decades ago, C. difficile strains were distributed<br />
into two main groups, toxinogenic <strong>and</strong> nontoxinogenic. Toxinogenic strains always produced<br />
both toxins A <strong>and</strong> B, <strong>and</strong> nontoxigenic strains produced neither. Later discovery of A-B+<br />
strains has dramatically changed laboratory diagnostics. In the non-outbreak situation,<br />
approximately 25% of strains will be A-B+ or variant forms of A+B+. So far, existence of A+Bstrains<br />
has not been confirmed. There are some interesting new A-B+ strains that in genetic<br />
background resemble nontoxinogenic strains, but have an entire functional tcdB as well,<br />
suggesting an association with mobile genetic elements.
O2<br />
LACK OF ASSOCIATION BETWEEN RIBOTYPE AND SEVERE CLOSTRIDIUM DIFFICILE<br />
INFECTION<br />
S. T. Walk 1,2 , D. Micic 1 , R. Jain 1,2 , I. Trivedi 1 , E. W. Liu 1 , L. M. Almassalha 1 , S.A. Ewing 1 , D.<br />
W. Newton 3,4 , A. T. Galecki 1,5,6 , C. Ring 1,2 , L. Washer 1 , V. B. Young 1,2,5 , <strong>and</strong> D. M.<br />
Aronoff* 1,2,7 . 1 Department of Internal Medicine; 2 Division of Infectious Diseases; 3 Clinical<br />
Microbiology Laboratories; 4 Department of Pathology; 5 Institute of Gerontology; 6 Department<br />
of Biostatistics, <strong>and</strong> 7 Department of Microbiology, University of Michigan Health System, Ann<br />
Arbor, MI 48109 USA.<br />
Epidemiologic studies suggested that closely related strains of C. difficile caused more<br />
severe clinical disease than other strains. Although new studies have challenged this<br />
hypothesis, they were limited by heterogeneous clinical criteria defining severe disease. We<br />
readdressed this question using recommended clinical definitions of severity <strong>and</strong> isolates<br />
from 150 independent cases of C. difficile infection (CDI) from a single institution. Toxigenic<br />
isolates were selectively cultured from stool samples, characterized using PCR virulence<br />
factor screening, <strong>and</strong> typed using high-throughput, fluorescent PCR ribotyping, yielding 47<br />
distinct ribotypes. Ribotype 014-020 was the most prevalent (21% of all isolates). Sixteen<br />
cases met the definition of severe CDI <strong>and</strong> were caused by 9 ribotypes. CDI caused by<br />
isolates previously implicated with more severe disease (ribotypes 027 <strong>and</strong> 078) was not<br />
more severe than CDI caused by other ribotypes. Similarly, severe CDI cases caused by<br />
isolates of ribotypes commonly associated with outbreaks were not more prevalent than<br />
cases caused by non-outbreak isolates. Six epidemiologic characteristics were significantly<br />
associated with severe CDI using univariate logistic regression but on multivariate analysis<br />
only albumin level remained significantly (<strong>and</strong> inversely) associated with disease severity.<br />
However, ribotype did not predict hypoalbuminemia (
O3<br />
UNIQUE GENETIC MARKERS FOR HYPERVIRULENT CLOSTRIDIUM DIFFICILE<br />
C.W. Knetsch* 1 , M.P.M. Hensgens 1 , C. Harmanus 1 , M.W. van der Bijl 2 , P.H.M. Savelkoul 2 ,<br />
E.J. Kuijper 1 , J. Corver 1 * <strong>and</strong> H.C. van Leeuwen 1 . 1 Department of Medical Microbiology,<br />
Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherl<strong>and</strong>s;<br />
2 Department of Medical Microbiology <strong>and</strong> Infection Control, VU University Medical Center,<br />
Amsterdam, The Netherl<strong>and</strong>s.<br />
Clostridium difficile is an anaerobic bacillus that resides in the gut <strong>and</strong> has rapidly emerged<br />
as a leading cause of antibiotic associated diarrheal disease in humans. Recently, the<br />
incidence, complications <strong>and</strong> mortality of a Clostridium difficile infection have increased<br />
dramatically due to the emergence of hypervirulent strains PCR ribotype 027 (NAP01) <strong>and</strong><br />
PCR ribotype 078 (NAP7/8). In this study we identified unique loci in the PCR ribotypes 027<br />
<strong>and</strong> 078 <strong>and</strong> used these markers to detect specifically hypervirulent C. difficile strains. Whole<br />
genome sequences of C. difficile PCR ribotypes 001, 012, 017, 027 <strong>and</strong> 078 were compared<br />
using a bioinformatics approach. This resulted in a list of c<strong>and</strong>idate marker genes unique for<br />
hypervirulent types 027 <strong>and</strong> 078. Of these we selected the most promising c<strong>and</strong>idates (i.e.<br />
genes that are stably integrated or not abundantly present in genomes). Next, a large<br />
collection of C. difficile strains (N=68) was screened for the presence of the selected<br />
markers. Surprisingly, we found that the markers were also present in a few other strains.<br />
Therefore, we analyzed if strains with the insert were genetically more related to each other<br />
compared to strains without the insert. The strains with the insert were indeed more closely<br />
related as evidenced by AFLP, MLST, similarity in PCR ribotype pattern <strong>and</strong> toxin gene<br />
profile (tcdA, tcdB, tcdC, binary toxin). We have identified six PCR ribotypes that resemble<br />
the hypervirulent type 078 <strong>and</strong> eleven PCR ribotypes that resemble the hypervirulent type<br />
027. These markers enable us to categorize these strains into two clades of genetically<br />
related strains. Analysis of the 078 <strong>and</strong> 027 loci revealed several interesting open reading<br />
frames. The 078-region introduces an enzyme that is involved in the biosynthesis of<br />
antibiotics. The 027-insert disrupts the thymidylate synthetase (thyX) gene (involved in DNA<br />
replication) <strong>and</strong> replaces it by an equivalent, catalytically more efficient, thyA gene. Both<br />
inserts may confer a selective advantage to the pathogen. The identified markers enable<br />
rapid <strong>and</strong> specific recognition of hypervirulent strains <strong>and</strong> other C. difficile ribotypes with<br />
similar characteristic features.
O4<br />
GENOMIC CHARACTERISTICS OF A TOXIN A-NEGATIVE, TOXIN B- POSITIVE C.<br />
DIFFICILE STRAIN FROM BEIJING, CHINA<br />
Y. Cheng* 1 , P. Du 1 , C. Chen 1 , Q. Yan 1 , J. Lu 1 . 1 Hospital Acquired Infection Department,<br />
<strong>State</strong> Key Laboratory for Infectious Disease Prevention <strong>and</strong> Control (SKLID), National<br />
Institute for Communicable Disease Control <strong>and</strong> Prevention, Chinese Center for Disease<br />
Control <strong>and</strong> Prevention, Beijing, 102206 China.<br />
Recently, TcdA-negative, TcdB-positive (A-/B+) C. difficile strains have been reported<br />
frequently in C. difficile infection (CDI) worldwide. However, its published isolation rate<br />
ranged drastically from 3% to 75% in different regions, <strong>and</strong> the Asians <strong>and</strong> Latin Americans<br />
seemed more prone to suffer from infection by A-/B+ strains. To further underst<strong>and</strong> this<br />
distinct variation in isolation rate between different regions, <strong>and</strong> to investigate the genomic<br />
characteristics of A-/B+ C. difficile from Asia, we performed sequence comparison with seven<br />
other published C. difficile sequences <strong>and</strong> bioinformatic analysis. First, we compared widelyused<br />
primers NK1-NK2, NK2- NK3, NK104-NK105, A1C-A2N, <strong>and</strong> others (used to conduct<br />
epidemic surveys in Japan, France, Argentina, <strong>and</strong> other countries). Second, we performed<br />
bioinformatic <strong>and</strong> comparative genome analysis on the primer regions of 7 published C.<br />
difficile sequences, looking for genomic sequence variations. Third, we selected an A-/B+ C.<br />
difficile strain (BJ08) from a Beijing hospital. Then, a shotgun method was used to acquire its<br />
genome sequence. Two DNA libraries, 500bp <strong>and</strong> 2kb DNA fragments for high throughput<br />
genome sequencing with SOLEXA, were constructed. Our results show that the PCR primers<br />
used in different countries for toxin detection do not provide uniform results. The intergroup<br />
of toxin A+B+, <strong>and</strong> the intra group between toxin A+B+ <strong>and</strong> Toxin A-B+, shows more<br />
diversity in the Paloc region. These diversities or single nucleotide polymorphisms (SNPs)<br />
that occur in primers may weaken our signal when we use PCR methods to detect TcdA/B<br />
regions. The genome of BJ08 was sequenced <strong>and</strong> assembled into 19 scaffolds, with N50 at<br />
460,923 bp. Compared with sequenced genome of C. difficile 630, CD196 <strong>and</strong><br />
R20291(ribotype 017), our sequence shows a large degree of conservation in size, number<br />
of open reading frames (ORF), GC content, gene length, <strong>and</strong> density. Thus, the genomic<br />
information of BJ08 will provide us new primer positions for toxin detection for the coming<br />
China CDI survey. Furthermore, our results may shed new light on the distinctly higher A-B+<br />
incidence rate in Asia <strong>and</strong> Latin America.
O5<br />
CRITICAL DIFFERENCES BETWEEN HISTORICAL AND HYPERVIRULENT TcdB.<br />
Jimmy D. Ballard. The University of Oklahoma Health Sciences Center, Department of<br />
Microbiology <strong>and</strong> Immunology, Oklahoma City, OK USA.<br />
Infection with hypervirulent strains of Clostridium difficile is associated with a higher morbidity<br />
<strong>and</strong> mortality compared to infection with historical strains of this pathogen. The underlying<br />
reasons for the increased lethality of hypervirulent C. difficile strain are unknown. A major<br />
virulence factor of C. difficile is the cytotoxin TcdB, which is responsible, in part, for the<br />
pathology of the intestines as well as systemic effects. Unlike other proteins encoded within<br />
the PaLoc of hypervirulent C. difficile, the sequence of TcdB has diverged from that of<br />
historical strains. Our work has focused on determining how these sequence differences<br />
impact the function <strong>and</strong> antigenic make-up of TcdB. Antibodies raised against the antigenic<br />
carboxy-terminus of historical TcdB (TcdB HIST ) were found to not cross-neutralize<br />
hypervirulent TcdB (TcdB HV ), suggesting the neutralizing epitopes differ between the two<br />
forms of TcdB. Fine specificity mapping of epitopes in TcdB HIST <strong>and</strong> TcdB HV revealed clear<br />
differences in the antigenic profiles of these proteins. The two forms of TcdB were found to<br />
differ in autoprocessing, a critical step in cell entry. TcdB HV underwent more efficient<br />
autoprocessing <strong>and</strong> was activated by previously unreported factors, including UDP <strong>and</strong> GDP.<br />
Further studies using an activity based fluorescent probe found that TcdB HV engaged<br />
intramolecular substrate more efficiently than TcdB HIST , providing an underlying mechanistic<br />
explanation for differences in autoprocessing between the two forms of TcdB. These data<br />
provide insight into a critical change in TcdB activity <strong>and</strong> indicate that variations in the<br />
sequence of TcdB have altered the toxin‘s antigenic characteristics <strong>and</strong> efficiency of<br />
autoprocessing.
O6<br />
STRUCTURAL DETERMINANTS OF THE CLOSTRIDIUM DIFFICILE TOXIN A<br />
GLUCOSYLTRANSFERASE ACTIVITY<br />
R. N. Pruitt 1 , N. M. Chumbler 1 , M. A. Farrow 1 , S. A. Seeback 1 , D. B. Friedman 2 , B. W.<br />
Spiller 1,3 , <strong>and</strong> D. B. Lacy* 1,2 . Departments of 1 Pathology, Microbiology <strong>and</strong> Immunology;<br />
2 Biochemistry; Pharmacology; V<strong>and</strong>erbilt University School of Medicine, Nashville TN 37232<br />
USA.<br />
The principle virulence factors in Clostridium difficile pathogenesis are TcdA <strong>and</strong> TcdB,<br />
homologous glucosyltransferases capable of inactivating small GTPases within the host cell.<br />
We present crystal structures of the TcdA glucosyltransferase domain (GTD) in the presence<br />
<strong>and</strong> absence of the co-substrate UDP-glucose. While the enzymatic core is similar to that of<br />
TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is<br />
comparable to TcdB in its modification of Rho-family substrates <strong>and</strong> that, unlike TcdB, TcdA<br />
is also capable of modifying Ras-family GTPases both in vitro <strong>and</strong> in cells. The<br />
glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but<br />
can be restored with autoproteolytic activation <strong>and</strong> GTD release. We propose a model<br />
wherein the receptor-binding domain occludes the binding of GTPase substrates <strong>and</strong> discuss<br />
the importance of cellular activation in determining the array of substrates available to the<br />
toxins once delivered into the cell.
O7<br />
CHEMICALLY INTERROGATING CLOSTRIDIUM DIFFICILE GLUCOSYLATING TOXIN<br />
ACTIVATION<br />
A. Shen* 2 , P.J. Lupardus 3 , A.W. Puri 4 , M.M. Gersch 4,5 , V.E. Albrow 1,3,6 , K.C. Garcia 3,6 , M.<br />
Bogyo 1,2,4 . 1 Department of Pathology, Stanford University, Stanford, CA 94305 USA;<br />
2 Department of Microbiology <strong>and</strong> Molecular Genetics, University of Vermont, Burlington, VT,<br />
05405 USA; 3 Department of Molecular <strong>and</strong> Cellular Physiology, Stanford University,<br />
Stanford, CA, 94305 USA; 4 Department of Chemical <strong>and</strong> Systems Biology, Stanford<br />
University, Stanford, CA, 94305 USA; 5 Department of Chemistry, Center for Integrated<br />
Protein Science Munich (CIPSM), Technische Universitat Munchen, Garching, Germany;<br />
6 Howard Hughes Institute, Stanford School of Medicine, Stanford, California, 94305 USA.<br />
Multidomain toxins TcdA <strong>and</strong> TcdB are the primary mediators of Clostridium difficileassociated<br />
disease. These toxins contain a cysteine protease domain (CPD) that<br />
autoproteolytically releases a cytotoxic effector domain upon binding the eukaryotic-specific<br />
small molecule inositol hexakisphosphate (InsP6) in target cells. Although CPD-mediated<br />
processing is necessary for optimal toxin function, the mechanism by which the InsP6 signal<br />
is sensed by the CPD remains poorly characterized. In order to study the regulation <strong>and</strong><br />
function of CPD-mediated processing of glucosylating toxins, we rationally designed covalent<br />
small molecule inhibitors of TcdB CPD that inactivate TcdB holotoxin function in cells. By<br />
converting the inhibitors into activity-based probes, we have developed tools that sensitively<br />
detect InsP6-induced activation of TcdB CPD. Using this probe, we demonstrate that,<br />
although InsP6 dramatically shifts the conformational equilibrium of the CPD towards an<br />
active conformer, apo-CPD unexpectedly adopts an active conformer at low frequency.<br />
Structural analyses combined with disulfide bond engineering studies identify residues that<br />
functionally <strong>and</strong> mechanically couple the InsP6 binding site to the active site. Collectively, our<br />
results define an allosteric circuit that allows the glucosylating toxins to sense <strong>and</strong> respond to<br />
the eukaryotic environment. This work highlights the value of using chemical tools to study<br />
mechanisms of enzyme regulation, validate the CPD as a druggable target, <strong>and</strong> provides<br />
general insight into the nature of protein allostery. These tools may additionally facilitate<br />
analyses of glucosylating toxins in other Clostridium spp.
O8<br />
A NEUTRALIZING INTRABODY TO STUDY AUTOCLEAVAGE OF CLOSTRIDIUM<br />
DIFFICILE TOXIN B<br />
S. Li*, L. Shi, D. Schmidt, J. M. Tremblay, X. Sun, S. Tzipori, K. Chen, Y. Zhang, C. B.<br />
Shoemaker, H. Feng. Infectious Disease Division, Department of Biomedical Sciences, Tufts<br />
Cummings School of Veterinary Medicine, N. Grafton MA 01536, USA.<br />
Clostridium difficile induces a serious <strong>and</strong> potentially fatal inflammatory disease of the colon<br />
<strong>and</strong> is the most prevalent cause of antibiotic-associated diarrhea <strong>and</strong> colitis in hospitals. The<br />
disease is mainly caused by two exotoxins, TcdA <strong>and</strong> TcdB. TcdB is a 269 KD protein<br />
containing at least 2 enzymatic domains, the glucosyltransferase domain (GTD) <strong>and</strong> the<br />
cysteine protease domain (CPD). After internalization, the GTD is released into the cytosol<br />
<strong>and</strong> enzymatically inactivates Rho GTPases, leading to the intoxication of host cells. The<br />
cytosolic liberation of GTD is thought to be mediated through auto-cleavage by CPD, a<br />
notion that is primarily based on observations from in vitro studies. Direct evidence<br />
generated using intact cells is necessary to prove this concept. In this study, we identified a<br />
VHH antibody, 7F, which is capable of neutralizing the cytotoxicity of TcdB on cultured cells.<br />
In vitro studies demonstrate that 7F blocks CPD-mediated auto-cleavage <strong>and</strong> release of<br />
GTD. Epitope mapping found that 7F binds to the GTD sequence immediately adjacent to<br />
the auto-catalytic site, so its binding likely blocks access to the protease cleavage site. To<br />
study whether blocking CPD-mediated auto-cleavage in TcdB-exposed intact cells inhibits<br />
the intoxication process, 7F was expressed as an intrabody in the mammalian cell cytosol.<br />
The 7F VHH extracted from transiently transfected mammalian cells bound to TcdB <strong>and</strong><br />
neutralized the toxin in cell assays. Cells expressing 7F intrabody were resistant to<br />
intoxication of TcdB, but not those expressing C6, a non-neutralizing VHH antibody that also<br />
recognizes GTD of TcdB. Our data thus provide direct evidence that CPD-mediated autocleavage<br />
is crucial for the cellular activity of TcdB.
09<br />
IDENTIFICATION OF NOVEL ENDOCYTOSIS PATHWAY FOR TcdB OF CLOSTRIDIUM<br />
DIFFICILE THROUGH shRNAmir LIBRARY SCREENING AND HIGH-THROUGHPUT<br />
SEQUENCING ANALYSIS<br />
C. Li, C. Cai, R. He, X. Wu, Y. Wang, W. Wei*. College of Life Sciences, Peking University,<br />
Beijing China 100871.<br />
As the leading cause of healthcare associated diarrhea in North America <strong>and</strong> Europe,<br />
Clostridium difficile infection has drawn great attention in research. TcdA <strong>and</strong> TcdB are two<br />
essential virulence determinants. By employing human shRNAmir library screening, in<br />
combination with deep sequencing analysis, we have identified a number of novel host<br />
pathways that are important for both toxins‘ endocytosis <strong>and</strong> damaging mechanism. These<br />
screenings have been carried out in three cell lines: HeLa, HT29 <strong>and</strong> KBM7 (a human<br />
myeloid leukemia cell), by using TcdB, TcdA, or chimeric DTA-TcdB toxins. Bioinformatic<br />
analysis of these data reveals that TcdB employs a novel mechanism to gain its access to<br />
host cells. In addition, a few cell surface membrane proteins have been identified to play<br />
important roles in TcdB toxicity. More interestingly, two of these receptor/co-receptor<br />
c<strong>and</strong>idates belong to the same family, <strong>and</strong> their normal functions include the activation of<br />
signaling molecule RhoA. Further characterization of these cell surface proteins as well as<br />
other essential host targets are still in process. Details of this work will be presented.
O10<br />
THE MECHANISM OF CONJUGATION IN CLOSTRIDIUM PERFRINGENS<br />
J. I. Rood* 1 , T. L. Bannam 1 , R. Bantwal 1 , J. Wisniewski 1 , C. J Porter 2 <strong>and</strong> J. C Whisstock 2 .<br />
ARC Centre of Excellence in Structural <strong>and</strong> Functional Microbial Genomics, 1 Department of<br />
Microbiology <strong>and</strong> 2 Department of Biochemistry <strong>and</strong> Molecular Biology, Monash University,<br />
Clayton, Victoria 3800, Australia.<br />
In Clostridium perfringens antibiotic resistance genes <strong>and</strong> genes encoding extracellular<br />
toxins are often carried on conjugative plasmids that have the tcp conjugation locus. We<br />
have mutated each of the eleven genes in this locus <strong>and</strong> examined their role in the<br />
conjugation process. Five tcp genes are essential for conjugative transfer; they encode a<br />
putative FtsK-like coupling protein (TcpA), two integral membrane proteins of unknown<br />
function (TcpD <strong>and</strong> TcpE), a potential hexameric ATPase (TcpF) <strong>and</strong> TcpH, which appears<br />
to be the major structural component of the transfer complex. Three proteins are required for<br />
maximal transfer efficiency, since mutation of their structural genes leads to greatly reduced<br />
transfer frequencies. These proteins include IntP, which appears to be an unusual type of<br />
DNA relaxase, TcpC, a transmembrane protein that localises to the cell membrane<br />
independently of the other Tcp proteins, <strong>and</strong> TcpG, a peptidoglycan hydrolase with two<br />
functional hydrolase domains. The remaining genes are not required for conjugative transfer.<br />
The crystal structure of the soluble C-terminal component of TcpC (TcpC99-354) has been<br />
determined to 1.8-Å resolution. It crystallised as a homotrimer, with each monomer<br />
comprising two independent domains that have very similar structures, even though they<br />
only have 16% sequence identity. Comparative structural analysis showed that TcpC was<br />
member of the NTF-2 superfamily <strong>and</strong> that each of the closely related structural domains<br />
was related to VirB8-like conjugation proteins, which are important components of type IV<br />
secretion systems from Gram-negative bacteria. Both of the C-terminal domains of TcpC<br />
were required for its function, with the second domain (aa 239-354) having a major role in<br />
protein-protein interactions. Based on these studies, detailed site-directed mutagenesis<br />
experiments, bacterial two-hybrid analysis <strong>and</strong> protein-protein interaction experiments we<br />
have developed a model that provides insight into the conjugation process in C. perfringens.
O11<br />
CHARACTERIZATION OF THE TOXIN PLASMIDS OF CLOSTRIDIUM PERFRINGENS<br />
Bruce McClane* 1 , Jihong Li 1 , Kazuaki Miyamoto 2 , Sameera Sayeed 1 , Derek Fisher 1 , Abhijit<br />
Gurjar 1 , Meredith Hughes 3 , Rachel Poon 3 , Vicki Adams 3 <strong>and</strong> Julian Rood 3 . 1 Department of<br />
Microbiology <strong>and</strong> Molecular Biology, University of Pittsburgh School of Medicine, Pittsburgh,<br />
USA, 2 Department of Microbiology, Wakayama Medical University, Wakayama, Japan <strong>and</strong><br />
3 Department of Microbiology, Monash University, Clayton, Victoria, Australia.<br />
Clostridium perfringens is a major pathogen of humans <strong>and</strong> livestock due to its prolific toxinproducing<br />
ability. The ability to produce alpha, beta, epsilon <strong>and</strong> iota toxins is used to classify<br />
C. perfringens isolates into types A-E. With the exception of alpha toxin <strong>and</strong> perfringolysin O,<br />
all other C. perfringens toxins can be encoded by genes on large plasmids that range in size<br />
from ~40 kb to >150 kb. Molecular Koch‘s postulates analyses have demonstrated that<br />
some of these plasmid-encoded toxins are essential when C. perfringens causes diseases<br />
originating in the intestines. There is considerable diversity amongst C. perfringens isolates<br />
with respect to toxin plasmid carriage; a single strain can carry up to three different plasmids<br />
<strong>and</strong> a single plasmid can encode up to three different toxin genes. Some toxin plasmids are<br />
related <strong>and</strong> share a common backbone. Some, if not all, C. perfringens toxin plasmids can<br />
conjugatively transfer, probably due to their carriage of a tcp locus, which could convert<br />
normal intestinal flora C. perfringens strains to virulence, thereby enhancing disease. In<br />
addition, most C. perfringens plasmid-borne toxin genes are flanked by IS elements that can<br />
apparently mediate excision of these toxin genes from their plasmids. The association of<br />
many toxin genes with both conjugative plasmids <strong>and</strong> IS elements allows toxin gene mobility<br />
that likely impacts C. perfringens toxin plasmid diversity, evolution <strong>and</strong> virulence. Recently,<br />
some C. perfringens isolates were identified that carry variant plasmid-borne enterotoxin <strong>and</strong><br />
iota toxin genes. These isolates initially genotyped as type A because their variant iota toxin<br />
gene did not amplify with current PCR toxin genotyping assays, indicating that some PCRbased<br />
diagnoses of C. perfringens infections could be incorrectly attributed to type A strains.<br />
Lastly, PFGE Southern blot studies identified only limited diversity of beta toxin <strong>and</strong> epsilon<br />
toxin plasmids in type B isolates. This observation could suggest that only certain toxin<br />
plasmid combinations are compatibly maintained in a single C. perfringens cell. If true, this<br />
may help to explain the limited number of C. perfringens types found in nature.
O12<br />
THE DEVELOPMENT OF CLOSTRIDIAL GENE SYSTEMS AND THEIR EXPLOITATION<br />
N.P. Minton*, S.T. Cartman, S.A. Kuehne, J. T. Heap, R. Ng, D. Walker, M. Ehsaan, M.L.<br />
Kelly, S. Baban, A. Kubiak, K. Winzer <strong>and</strong> A. Cockayne. School of Molecular Medical<br />
Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park,<br />
Nottingham NG7 2RD, United Kingdom.<br />
In recent years, tools for genetic modification of Clostridium difficile have been considerably<br />
exp<strong>and</strong>ed by the development of both directed (Cambell-like plasmid integration <strong>and</strong> the<br />
ClosTron) <strong>and</strong> r<strong>and</strong>om (transposon mariner) insertional systems for ‗knock-out‘ <strong>and</strong> ‗knockin‘.<br />
However, as insertional mutagens, their deployment can cause polar effects. Moreover,<br />
they are limited in the amount of DNA that can be inserted into the genome. We have now<br />
developed a new series of systems, based on allelic exchange, with which it is possible both<br />
to routinely make in-frame deletions of target genes, using negative selection markers, <strong>and</strong><br />
to insert DNA fragments of any size or complexity into the genome, a prerequisite for<br />
synthetic biology. The former may also be used to make subtle changes (ie., the introduction<br />
of SNPs) to specific genes by allelic exchange. The latter system, termed Allele-Coupled<br />
Exchange (ACE) technology, provides the facility to develop in-frame deletion/ allelic<br />
exchange methodology for virtually any clostridial species. It may additionally be deployed to<br />
introduce wildtype alleles into the chromosome, dispensing with the need to use autonomous<br />
plasmids for complementation studies. Moreover, it has provided a simple assay to confirm<br />
the essentiality of genes suggested to be essential on the basis of under-representation in<br />
transposon-directed insertion site sequencing (TraDIS) of mariner transposon mutant pools.<br />
The basis of these systems will be discussed, together with examples of their deployment.<br />
This work was supported by the UK Medical Research Council (G0601176) <strong>and</strong> the<br />
European Union (HEALTH-F3-2008-223585).
O13<br />
ANALYSIS OF THE GENOME-WIDE TEMPORAL EXPRESSION OF A CLOSTRIDIUM<br />
DIFFICILE 027 HYPERVIRULENT STRAIN IN MONOXENIC MICE<br />
A. Barketi-Klai 1 , M. Monot 2 , S. Hoys 1 , S. Lambert 1 , B. Dupuy 2 , I. Kansau 1 , <strong>and</strong> A.<br />
Collignon* 1 . 1 EA 4043, USC INRA Faculté de Pharmacie, Université Paris-Sud 11, 92296<br />
Châtenay-Malabry, France; 2 Institut Pasteur, 28, rue du Docteur Roux, 75015, Paris, France.<br />
Clostridium difficile is a frequent cause of recurrent post-antibiotic diarrhea <strong>and</strong><br />
pseudomembranous colitis. Recently, new hypervirulent epidemic strains, such as the NAP1<br />
027 strain, have emerged <strong>and</strong> have been responsible for several outbreaks worldwide. The<br />
hypervirulence of the Clostridium difficile 027 strains seems related to several factors<br />
including high-toxin A <strong>and</strong> B production, presence of the binary toxin <strong>and</strong> high sporulation<br />
rate. However, other factors could be involved in its hypervirulence. In this work, we focused<br />
our interest on the colonization mechanism of a C. difficile 027 strain by an in vivo<br />
transcriptomic approach. We analyzed the C. difficile 027 transcriptome during the early<br />
stages of intestinal colonization in our monoxenic mouse model. Bacterial mRNA was<br />
extracted from the caeca at 4, 6, 8, 14 <strong>and</strong> 38 h post-infection, then transcribed into cDNA by<br />
RT-PCR <strong>and</strong> finally labeled for hybridization to DNA microarrays (Agilent ). The overall<br />
analysis of data (by software Ma2HTLM) showed a differential expression of 707 genes in<br />
the early stage (4-6 h) <strong>and</strong> late stage (14-38h) of colonization. 285 genes were significantly<br />
up-regulated, while 456 were down-regulated. Genes encoding known virulence factors such<br />
as the toxins were up-regulated in the late phase (38 h), whereas genes encoding surface<br />
proteins likely involved in the colonization process showed little or no variation. In addition,<br />
genes involved in sporulation, membrane transport, metabolism <strong>and</strong> fermentation, underwent<br />
significant changes during the infection process suggesting a potential role for the encoded<br />
proteins in the early stages of C. difficile intestinal colonization. This analysis will be<br />
completed by RT-qPCR on the regulated genes during the in vivo kinetics of colonization.<br />
These results will help to elucidate the mechanisms of intestinal colonization by C. difficile<br />
027 strain <strong>and</strong> to better underst<strong>and</strong> the hypervirulence of this strain.
O14<br />
PHYLOGENOMIC ANALYSIS OF CLOSTRIDIUM BOTULINUM STRAINS<br />
E. A. Johnson* 1 , L. Papazisi 2 , M. J. Jacobson 1 , G. Lin 1 , <strong>and</strong> S. N. Peterson 2 . 1 Department of<br />
Bacteriology, Food Research Institute, University of Wisconsin-Madison, Madison, WI USA;<br />
2 The J. Craig Venter Institute (JCVI), Rockville, MD USA.<br />
Clostridium botulinum is a heterogenous group of bacteria that have the common property of<br />
producting botulinum neurotoxin (BoNT). There is a growing body of evidence to suggest that<br />
genomic diversity within this taxon is relatively large <strong>and</strong> that exchange of genetic elements<br />
among members of this group is commonplace. There is considerable genetic variation even<br />
among the seven major seven BoNT serotypes, as demonstrated by the recognition of at<br />
least many BoNT subtypes. The most studied serotype, BoNT/A, has been found in a large<br />
<strong>and</strong> diverse group of clostridia, most of which express the subtype BoNT/A1. Nearly 50% of<br />
C. botulinum strains producing BoNT/A1 have been shown to also encode unexpressed<br />
variants of BoNT/B with a distinct cluster arrangement. The flow of genetic information within<br />
the C. botulinum group motivated us to identify novel genes for the purpose of creating a<br />
―species‖ DNA microarray to better underst<strong>and</strong> the ancestral relationships among its<br />
members. Based on preliminary genotyping (MLST, <strong>and</strong> CGH using a single-genome-based<br />
array), 20 diverse C. botulinum strains were selected for targeted sequencing. We developed<br />
a strategy we refer to as Gene Discovery (GD) that enables the identification <strong>and</strong> rapid<br />
characterization of strain-specific sequences present in microbial genomes. Sequence<br />
information obtained from this project, <strong>and</strong> from other publicly available sources, led to the<br />
development of a comprehensive species microarray containing ca. 40,000 genomic markers<br />
The availability of the C. botulinum species DNA microarray has allowed us to carry out a<br />
CGH genotyping project to validate this microarray as well as underst<strong>and</strong> the phylogenomic<br />
relationships among members of this very diverse group. The putative role of horizontal<br />
gene transfer in C. botulinum phylogeny is also discussed.<br />
Acknowledgments: This work was supported by the NIAID contract No. N01-AI-15447 to Pathogen Functional<br />
Genomics Resource Center at JCVI. Studies in EAJ‘s laboratory was partly sponsored by the NIH/NIAID Regional<br />
Center of Excellence for Bio-defense <strong>and</strong> Emerging Infectious Diseases Research (RCE) Program. The authors<br />
wish to acknowledge membership within <strong>and</strong> support from the Pacific Southwest Regional Center of Excellence<br />
grant U54 AI065359 <strong>and</strong> from the Region V ‗Great Lakes‘ RCE (NIH award 1-U54-AI-057153).
O15<br />
CLOSTRIDIUM DIFFICILE CDT AND CLOSTRIDIUM PERFRINGENS IOTA TOXIN:<br />
IDENTIFICATION OF THEIR MEMBRANE RECEPTORS AND ACTIONS ON THE<br />
CYTOSKELETON OF TARGET CELLS<br />
Klaus Aktories. Institute of Experimental <strong>and</strong> Clinical Pharmacology <strong>and</strong> Toxicology,<br />
University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany.<br />
Clostridium difficile infection (CDI) causes antibiotic-associated diarrhea <strong>and</strong><br />
pseudomembranous colitis. Hypervirulent strains of the pathogen, which are responsible for<br />
increased morbidity <strong>and</strong> mortality of CDI, produce the actin-ADP-ribosylating toxin C. difficile<br />
transferase (CDT) in addition to the Rho glucosylating toxins A <strong>and</strong> B. CDT is a binary actin-<br />
ADP-ribosylating toxin, which consists of an enzymatic component <strong>and</strong> a separated<br />
binding/translocation component. The toxin ADP-ribosylates G-actin at Arg-177. Thereby, the<br />
actin cytoskeleton is depolymerized <strong>and</strong> formation of microtubule protrusions is induced. The<br />
protrusions form a network on the cell surface of host target cells <strong>and</strong> increase adherence<br />
<strong>and</strong> colonization of clostridia, indicating a role as virulence factors.<br />
We have identified the cell surface receptor for CDT by gene trapping using a haploid genetic<br />
screen. We found that C. perfringens iota toxin, a related binary actin-ADP-ribosylating toxin,<br />
enters target cells via the same membrane receptor. Nature <strong>and</strong> properties of the receptor<br />
will be discussed.
O16<br />
IDENTIFICATION OF HOST FACTORS CONTRIBUTING TO CLOSTRIDIUM<br />
PERFRINGENS EPSILON-TOXIN-INDUCED CELL DEATH<br />
C.M. Fennessey 1 , S.E. Ivie 1 , J.Sheng 1 , D.H. Rubin 1,2,3 , <strong>and</strong> M.S. McClain* 1 . 1 Division of<br />
Infectious Disease, Department of Medicine, V<strong>and</strong>erbilt University School of Medicine,<br />
Nashville, Tennessee, USA; 2 Department of Microbiology <strong>and</strong> Immunology, V<strong>and</strong>erbilt<br />
University School of Medicine, Nashville, Tennessee, USA; 3 Research Medicine, VA<br />
Tennessee Valley Healthcare System, Nashville, Tennessee, USA.<br />
Results of recent studies indicate that the mechanism by which pore-forming toxins mediate<br />
cell death is more complicated than previously thought <strong>and</strong> that inhibition of cellular<br />
responses can protect from cell death induced by pore-forming toxins. Thus, identifying host<br />
factors contributing to the activity of pore-forming toxins may identify c<strong>and</strong>idates for new<br />
therapeutic approaches. We have used gene trap selection to identify mammalian factors<br />
contributing to Clostridium perfringens epsilon-toxin-induced cytotoxicity. One gene identified<br />
encodes caveolin 2 (CAV2), a membrane protein involved in endocytosis, signal<br />
transduction, lipid metabolism, cellular growth control <strong>and</strong> apoptosis. RNA interference was<br />
used to disrupt CAV2 expression <strong>and</strong> decreased CAV2 correlated with decreased toxin<br />
sensitivity. Immunoprecipitation of CAV2 from toxin-treated cells resulted in co-purification of<br />
epsilon-toxin. Similarly, epsilon-toxin was co-purified when caveolin 1 (CAV1, a protein<br />
known to interact with CAV2) was immunoprecipitated from toxin treated cells. Together,<br />
these results suggest that interaction between epsilon-toxin <strong>and</strong> the caveolins is an important<br />
step in the process by which the toxin induces cell death, <strong>and</strong> a new target for possible<br />
therapeutic intervention.
O17<br />
EXPRESSION AND FUNCTIONAL CHARACTERISATION OF CLOSTRIDIUM<br />
PERFRINGENS NETB TOXIN<br />
S. P. Fern<strong>and</strong>es da Costa* 1 , C. G. Savva 2 , M. Bokori-Brown 1 , C. E. Naylor 2 , A. K. Basak 2 , D.<br />
S. Moss 2 , R. W. Titball 1 . 1 College of Life <strong>and</strong> Environmental Sciences, University of Exeter,<br />
Exeter, United Kingdom; 2 Institute of Structural <strong>and</strong> Molecular Biology, Birkbeck College,<br />
London, United Kingdom.<br />
Clostridium perfringens is able to cause several diseases as a consequence of the<br />
differential production of a variety of toxins, some of them known to be the most powerful<br />
naturally occurring poisons. NetB (Necrotic enteritis toxin B) is a novel β-pore-forming toxin<br />
produced by C. perfringens toxinotype A, <strong>and</strong> to a lesser extent by strains of type C. NetB<br />
has been associated with avian necrotic enteritis, a severe gastro-intestinal disease that<br />
manifests in gross lesions within the intestines of poultry. However, the molecular basis of<br />
toxicity is still little understood <strong>and</strong> remains to be revealed. In this study, we present an<br />
expression system for netB in E. coli <strong>and</strong> show that the recombinant protein is able to form<br />
oligomeric complexes. Cytotoxicity assays on LMH cells (chicken hepatocellular carcinoma<br />
cell line) showed that the toxin is capable of forming functional pores <strong>and</strong> to cause cell lysis.<br />
Moreover, cryo-electron microscopy of NetB in the presence of liposomes confirm that the<br />
NetB is able to form multimeric ring-like structures. Based on sequence similarities with<br />
related pore-forming toxins a monomeric/heptameric model of NetB has been designed <strong>and</strong><br />
been used to identify potentially important amino acids for oligomerisation, cell binding, <strong>and</strong><br />
pore formation. Several site specific mutants have been created so far <strong>and</strong> their role on NetB<br />
functionality is currently being investigated. Non-toxic NetB variants might have the potential<br />
to be used as a toxoid to protect chicken against necrotic enteritis.
O18<br />
STRUCTURAL RELATIONSHIPS IN CLOSTRIDIAL BETA-PORE-FORMING TOXINS<br />
A. K. Basak*, C. E. Naylor, C. Savva, J. Huyet, <strong>and</strong> T. Yell<strong>and</strong>. Department of Biological<br />
Sciences, Birkbeck College, Malet St, London, WC1E 7HX, UK.<br />
Clostridium perfringens is a facultative anaerobe that secretes a large panel of toxins <strong>and</strong> is<br />
associated with a broad range of diseases in man <strong>and</strong> animals. A number of these toxins,<br />
including the epsilon, delta <strong>and</strong> beta-toxins, enterotoxin <strong>and</strong> the recently discovered NetB<br />
have been identified as pore-forming toxins. We have determined the 3D-structures of a<br />
number of these pore-forming toxins by X-ray crystallography, <strong>and</strong> are investigating others<br />
by Electron Microscopy, Biophysical techniques <strong>and</strong> Crystallography. Our work has<br />
uncovered interesting, <strong>and</strong> often unexpected, relationships between these toxins <strong>and</strong> also<br />
with toxins from other organisms. Our recently published enterotoxin (CPE) structure is a<br />
causative agent of type-A food poisoning <strong>and</strong> is linked to hospital- <strong>and</strong> community-acquired<br />
antibiotic-associated <strong>and</strong> sporadic diarrhoea. We will discuss the structural homology to the<br />
epsilon-toxin <strong>and</strong> other members of the aerolysin-like beta-pore-forming family. This<br />
relationship has enabled us to develop several models for oligomeric pore-formation. In<br />
addition, we have recently determined the structure of the soluble form of the delta-toxin to<br />
2.4 Å. This toxin is implicated in haemolysis in animals. The structure is part of the alphahaemolysin<br />
like family of beta-pore-forming toxins, <strong>and</strong> is closely related to the recently<br />
discovered NetB toxin, which is responsible for enteric disease in farmed poultry. Our<br />
structure has enabled us to build models for the pore-inserted forms of both delta-toxin <strong>and</strong><br />
NetB, which we have validated by electron microscopy <strong>and</strong> biophysical studies. The<br />
mechanisms of pore-formation of these proteins are different, as the receptors they<br />
recognize are diverse <strong>and</strong> the regions of the proteins associated with these functions also<br />
differ markedly. These aspects of the proteins will be presented.
O19<br />
ROLE OF CARBON CATABOLITE REPRESSION (CCR) IN THE PATHOGENICITY OF<br />
CLOSTRIDIUM DIFFICILE<br />
A. Antunes, E. Camiade, M. Monot, I. Martin-Verstraete <strong>and</strong> B. Dupuy*. Laboratoire<br />
Pathogenèse des Bactéries Anaérobies, Institut Pasteur, 25 rue du Dr Roux, 75724, Paris,<br />
France.<br />
Many bacterial pathogens produce virulence factors to overcome the drastic changes in<br />
environment that they encounter during infection. Consequently, global regulation of<br />
virulence genes must be considered as an important step of the pathogenicity process. In<br />
Clostridium difficile, it is now well establish that toxin production is regulated in response to<br />
various environmental signals. Thus, in the presence of glucose or other rapidly<br />
metabolizable sugars, C. difficile toxin synthesis is repressed involving the Carbon Catabolite<br />
Repression (CCR) mechanism. CcpA is the global transcriptional regulator of the CCR,<br />
which generally binds a cis-acting catabolite response element (cre) in the promoter region of<br />
the controlled genes. Furthermore its DNA binding activity is enhanced by its interaction with<br />
HPr-Ser-P, a component of the phosphotransferase system (PTS). We have shown by in<br />
vivo <strong>and</strong> in vitro approaches that CCR is implicated in the glucose-dependent repression of<br />
the toxin gene. CcpA binds to regulatory regions of tcdA <strong>and</strong> tcdB genes but not through the<br />
cre motif. Moreover, only FBP enhances CcpA binding, establishing a novel mode of action<br />
of CcpA. In order to underst<strong>and</strong> the impact of glucose <strong>and</strong> the regulatory role of CcpA in C.<br />
difficile physiology, we performed a transcriptome analysis during the growth of JIR8094<br />
strain <strong>and</strong> its isogenic ccpA mutant, in the presence or absence of glucose. 18% of C. difficile<br />
genes are regulated in response to glucose availability <strong>and</strong> half of them are controlled by<br />
glucose in a CcpA-dependent manner. CcpA plays a central role in several metabolic<br />
pathways <strong>and</strong> cellular processes like stress response <strong>and</strong> sporulation. We have recently<br />
identified more than 60 CcpA direct targets by ChIP to chip analysis <strong>and</strong> we proposed a<br />
CcpA binding motif. Finally, we showed in the hamster model that virulence is delayed in the<br />
ccpA mutant indicating that CcpA controls the infectious process of C. difficile. To look for<br />
processes potentially related to the CDI, co-ordinately regulated by CcpA, we used a axenic<br />
mouse model to determine the CcpA regulon in vivo. 183 genes are specifically controlled by<br />
CcpA in vivo including genes involved in ethanolamine utilization, an abundant compound of<br />
the intestinal tract that can be used as carbon <strong>and</strong> nitrogen sources during the C. difficile<br />
gastrointestinal lifestyle.
O20<br />
A NEW ROLE FOR C. DIFFICILE GLUTAMATE DEHYDROGENASE<br />
R. Govind*. Division of Biology, Kansas <strong>State</strong> University, Manhattan, KS, 66506 USA.<br />
Clostridium difficile produces an NAD-specific glutamate dehydrogenase (GDH), which<br />
converts L- glutamate into alpha-ketoglutarate through an irreversible reaction. The enzyme<br />
GDH is detected in the stool samples of the patients with C. difficile-associated disease <strong>and</strong><br />
serves as one of the diagnostic tools to detect C. difficile infection. Initial experiments<br />
detected GDH in the supernatant fluids of C. difficile cultures. To underst<strong>and</strong> the role of this<br />
extracellular GDH, a mutation was introduced into the GDH encoding gene, gdhA. The<br />
presence of the gdhA mutation in the C. difficile chromosome was confirmed by PCR <strong>and</strong> the<br />
absence of GDH in the mutant was documented by Western blot analysis. Various<br />
phenotypic assays were performed to underst<strong>and</strong> the effect of GDH on C. difficile growth.<br />
Higher sensitivity of the mutant to H 2 O 2 , as compared to the parent strain, led me to<br />
hypothesize that GDH might play an important role in fighting against reactive oxygen<br />
species (ROS) released as a part of host defense against C. difficile infection. Results that<br />
suggest the role of extracellular GDH in C. difficile defense against ROS will be discussed.
O21<br />
NOVEL REGULATORY MECHANISM FOR SPORE FORMATION AND ENTEROTOXIN<br />
PRODUCTION IN CLOSTRIDIUM PERFRINGENS<br />
K. Ohtani* 1 , H. Hirakawa 2 , D. Pareds-Sabja 3 , K. Tashiro 4 , S. Kuhara 4 , M. Sarker 5 <strong>and</strong> T.<br />
Shimizu 1 . 1 Kanazawa Univ., Japan; 2 Kazusa DNA Research Institute, Japan; 3 Universidad<br />
Andres Bello, Chile, 4 Kyushu Univ., Japan; 5 Oregon <strong>State</strong> University, USA.<br />
C. perfringens causes clostridial myonecrosis (gas gangrene) <strong>and</strong> gastrointestinal (GI)<br />
diseases in humans. The most common cause of C. perfringens associated food poisoning is<br />
the consumption of C. perfringens vegetative cells followed by sporulation <strong>and</strong> production of<br />
enterotoxin in the gut. However, C. perfringens-associated gas gangrene <strong>and</strong> other nonfoodborne<br />
GI illnesses occur when C. perfringens spores come in contact with the host,<br />
where spores undergo germination followed by cell proliferation <strong>and</strong> toxin secretion. Despite<br />
the importance of spore formation in C. perfringens pathogenesis, the detailed regulation of<br />
sporulation has not yet been defined. Although C. perfringens lacks some sporulation-related<br />
genes compared to Bacillus subtilis, the most important regulatory genes encoding the<br />
master regulator of sporulation (Spo0A) <strong>and</strong> sigma factors are highly conserved between<br />
these two species. Despite the identification of regulators for sporulation that are common to<br />
both Clostridium <strong>and</strong> Bacillus species, no unique regulators for this process have been<br />
identified in C. perfringens. In this context, our microarray <strong>and</strong> bioinformatic analyses<br />
identified a novel c<strong>and</strong>idate gene (virX) that might be involved in repression of genes<br />
encoding positive regulators (i.e., Spo0A <strong>and</strong> sigma factors) of C. perfringens sporulation.<br />
When a virX mutant was constructed in food poisoning strain SM101 <strong>and</strong> the sporulation<br />
phenotype of the isogenic strains was compared, the virX mutant had a higher sporulation<br />
efficiency compared to wild-type. Northern blot analyses demonstrated that the transcription<br />
of sigE, sigF <strong>and</strong> sigK was strongly induced in a 2.5h - culture of the virX mutant. Moreover<br />
the transcription of the enterotoxin gene (cpe) was also strongly induced in a virX mutant.<br />
Western blotting analyses confirmed much higher levels of enterotoxin production in the virX<br />
mutant compared to wild-type. Importantly, the virX mutant‘s phenotype could be restored by<br />
complementing the mutant with wild-type virX, indicating that the higher levels of sporulation<br />
<strong>and</strong> enterotoxin production in the virX mutant are specifically due to inactivation of the virX<br />
gene. Collectively, our current study, for the first time, identified a novel negative regulator for<br />
sporulation <strong>and</strong> enterotoxin production in C. perfringens, which might explain why two<br />
different morphotypes of C. perfringens cells are required to cause gas gangrene<br />
(vegetative) <strong>and</strong> food poisoning (sporulation).
O22<br />
CLOSTRIDIUM DIFFICILE Spo0A REGULATES SPORULATION, BUT NOT TOXIN<br />
PRODUCTION, BY DIRECT BINDING TO TARGET DNA<br />
K.E. Rosenbusch 1 , D. Bakker 1 , E.J. Kuijper 1 , W.K. Smits* 1 . 1 Department of Medical<br />
Microbiology, Leiden University Medical Center, Leiden, the Netherl<strong>and</strong>s<br />
Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant<br />
endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the<br />
symptoms can range from a mild diarrhea to potentially fatal toxic megacolon <strong>and</strong><br />
pseudomembranous colitis. Endospore formation in Firmicutes, including C. difficile, is<br />
governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription<br />
factor is also directly or indirectly involved in various other cellular processes. Here, we<br />
report that C. difficile Spo0A shows a high degree of similarity to the well characterized B.<br />
subtilis protein <strong>and</strong> recognizes a similar binding sequence, with two guanine residues that<br />
are crucial for specific binding. We find that our laboratory strain C. difficile 630Deltaerm<br />
contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain<br />
630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile<br />
Spo0A protein demonstrate direct binding to DNA upstream of spo0A <strong>and</strong> sigH, early<br />
sporulation genes, <strong>and</strong> several other putative targets. In silico analyses <strong>and</strong> in vitro binding<br />
assays suggest that the genes encoding the major clostridial toxins TcdA <strong>and</strong> TcdB, the<br />
binary toxin locus CdtAB, <strong>and</strong> their regulators TcdR <strong>and</strong> CdtR are not direct targets of<br />
Spo0A. These results identify, for the first time, putative targets of the Spo0A protein in C.<br />
difficile <strong>and</strong> make a direct effect of Spo0A on toxin production in this organism highly unlikely.
O23<br />
REGULATOR-MEDIATED MODULATION OF TOXIN PRODUCTION AND SPORULATION<br />
IN AN EPIDEMIC CLINICAL ISOLATE OF CLOSTRIDIUM DIFFICILE<br />
K. Mackin* 1 , G. Carter 1 , P. Howarth 1 , R. Govind 2 , B. Dupuy 2 , G. Douce 3 , J. I. Rood 1 , <strong>and</strong> D.<br />
Lyras 1 . 1 Department of Microbiology, Monash University, Victoria, Australia; 2 Unité de<br />
Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France; 3 Division of Infection <strong>and</strong><br />
Immunity, FBLS Glasgow Biomedical Research Centre, University of Glasgow, UK.<br />
Epidemic ribotype 027 isolates of Clostridium difficile have emerged throughout the world<br />
over the last decade. These strains are reported to produce significantly more toxin than<br />
other clinical isolates; this is thought to be due to a nonsense mutation within tcdC, which<br />
encodes a negative regulator of toxin production. The emergence of ribotype 078 isolates in<br />
the human population is also of concern as these strains also are associated with more<br />
severe disease. Various hypotheses have been proposed for the emergence of these strains,<br />
<strong>and</strong> for their persistence <strong>and</strong> increased virulence, but supportive experimental data are<br />
lacking. To study these isolates at the molecular level, we have developed a new <strong>and</strong> more<br />
efficient plasmid transfer system which has facilitated the genetic manipulation of both<br />
ribotype 027 <strong>and</strong> 078 strains. We have carried out two separate series of proof-of-principle<br />
experiments. Firstly, we have used it to inactivate the gene encoding the global regulator<br />
Spo0A in different strain backgrounds, including ribotypes 027 <strong>and</strong> 078. Spo0A controls the<br />
initiation of sporulation, <strong>and</strong> the mutants constructed in this study were unable to sporulate;<br />
complementation restored this phenotype. In other bacteria Spo0A is also involved in the<br />
regulation of other cell behaviour such as competence, motility, biofilm formation <strong>and</strong> toxin<br />
production. Interestingly, our results show that Spo0A appears to be involved in TcdA <strong>and</strong><br />
TcdB production in 027 strains, but not in the other strain backgrounds tested. Secondly, we<br />
have used the new plasmid transfer system to complement an 027 epidemic isolate with an<br />
intact copy of tcdC. This complementation resulted in a significant repression of toxin<br />
production. Most importantly, using the hamster infection model we showed that<br />
complementation of the defective tcdC with an intact copy of the gene attenuated the<br />
hypervirulent 027 phenotype. These data provide definitive evidence that TcdC is a negative<br />
regulator of toxin production in C. difficile <strong>and</strong>, for the first time, evidence that mutation of<br />
tcdC in epidemic 027 isolates is an important factor in the development of hypervirulence.
O24<br />
ANIMAL MODELS TO STUDY CLOSTRIDIUM PERFRINGENS DISEASES<br />
F.A. Uzal *1 , B.A. McClane 2 , J. Rood 3 , J. Saputo 1 , J.P. Garcia 1 , J. Caserta 2 , S. Robertson 2 , M.<br />
Ma 2 , J.E. Vidal 2 , A. Shrestha 2 , M. Hughes 3 , R. Poon 3 <strong>and</strong> V. Adams 3 . 1 University of<br />
California Davis, USA; 2 University of Pittsburgh, USA; 3 Monash University, Australia.<br />
Several animal models have been developed recently to study Clostridium perfringens<br />
diseases of animals <strong>and</strong> humans. Models in rabbits, mice, sheep, <strong>and</strong> goats have allowed us<br />
to conduct molecular Koch‘s postulates analyses for several toxins <strong>and</strong> to study the<br />
pathogenicity of several C. perfringens diseases. Examples of these models include a mouse<br />
ligated intestinal loop model to study systemic effects <strong>and</strong> lethality of enterotoxin (CPE).<br />
Lethality was observed after inoculation of CPE into ligated intestinal loops, <strong>and</strong> a correlation<br />
was noted between intestinal histological damage <strong>and</strong> lethality. A dose-dependent increase<br />
in serum CPE <strong>and</strong> serum potassium levels was observed, as was CPE binding to the liver<br />
<strong>and</strong> kidney. These data suggest that CPE can be absorbed from the intestine into the<br />
circulation, followed by binding of the toxin to internal organs to induce potassium leakage,<br />
which can cause death. Rabbit small intestinal loops were used to study the use of a soluble<br />
claudin-4 receptor decoy as a defense against CPE. Intestinal loops incubated with claudin-4<br />
<strong>and</strong> then with CPE for 6 hours did not show the typical fluid accumulation <strong>and</strong> histological<br />
damage seen in loops incubated with CPE alone. These results suggest that claudin receptor<br />
decoys could have application in preventing or treating CPE-associated disease. Rabbit<br />
intestinal loops <strong>and</strong> mouse intraduodenal models can be used to demonstrate that the<br />
VirS/VirR two-component system regulates intestinal pathogenicity <strong>and</strong> lethality of C.<br />
perfringens type C. An isogenic virR null mutant lost the ability to cause necrotic enteritis in<br />
rabbit small intestinal loops <strong>and</strong> showed strongly attenuated lethality in a mouse<br />
intraduodenal model. Mouse, sheep, <strong>and</strong> goat models are useful in demonstrating that<br />
epsilon toxin (ETX) is essential for disease in sheep, goats, <strong>and</strong> mice. Cultures of a wild-type<br />
D isolate inoculated intraduodenally caused acute clinical disease <strong>and</strong> death in sheep, goats,<br />
<strong>and</strong> mice, while no disease was observed after inoculation of an isogenic ETX toxin null<br />
mutant. These results provide evidence that ETX has a key role in type D disease<br />
pathogenesis. Overall, our studies indicate the importance of combining genetic studies <strong>and</strong><br />
the use of animal disease models to study the pathogenesis of diseases caused by C.<br />
perfringens.
025<br />
SYSTEMIC DISSEMINATION OF CLOSTRIDIUM DIFFICILE TOXINS A AND B IS<br />
ASSOCIATED WITH SEVERE FATAL DISEASE IN THE PIGLET AND MOUSE MODELS<br />
J. Steele 1 , K. Chen* 1 , X. Sun 1 , Y. Zhang 1,2 , H. Wang 1,3 , S. Tzipori 1 , <strong>and</strong> H. Feng 1 . 1 Tufts<br />
Cummings School of Veterinary Medicine, North Grafton, MA USA; 2 School of<br />
Bioengineering, East China University of Science <strong>and</strong> Technology, Shanghai, China; 3 School<br />
of Bioscience <strong>and</strong> Biotechnology, South China University of Technology, Guangzhou, China.<br />
Clostridium difficile infection (CDI), the most common cause of antibiotic-associated diarrhea<br />
<strong>and</strong> colitis in developed countries, can cause a wide range of disease, from mild diarrhea to<br />
fulminant systemic disease. Disease is mainly caused by two exotoxins, TcdA <strong>and</strong> TcdB.<br />
Although the incidence of systemic CDI <strong>and</strong> fatality has increased rapidly in recent years, the<br />
causes for the development of systemic disease are not well understood but may be due to<br />
the toxins liberated into the circulation, despite a current lack of such evidence. Using an<br />
ultrasensitive cytotoxicity assay developed by our laboratory, we measured TcdA <strong>and</strong> TcdB<br />
in serum <strong>and</strong> body fluids of piglets <strong>and</strong> mice exposed to CDI to investigate the relationship<br />
between presence of toxins in body fluids <strong>and</strong> systemic manifestations of CDI. We found that<br />
TcdA <strong>and</strong> TcdB are frequently liberated into the blood <strong>and</strong> body fluids in animals with<br />
systemic CDI. The presence of toxins in the circulation caused significant systemic<br />
manifestations <strong>and</strong> was also associated with fatal consequences. Both piglets <strong>and</strong> mice may<br />
develop lesions of the respiratory tract <strong>and</strong> ascites, <strong>and</strong> in the piglet model, cardiac lesions<br />
<strong>and</strong> pancreatitis were also noted in a few cases. Pro-inflammatory cytokines IL-1β <strong>and</strong> IL-6<br />
were elevated in the serum of mice <strong>and</strong> piglets with systemic CDI, highlighting the potential<br />
for inflammation induced tissue damage systemically. Interestingly, IL-4, an antiinflammatory<br />
cytokine was elevated in piglets with non-systemic CDI, indicating a potential<br />
protective effect when anti-inflammatory mediators are induced. Expectedly, systemic<br />
administration of neutralizing antibodies against both toxins to mice blocked the development<br />
of systemic disease. Our study demonstrates the existence of a strong correlation between<br />
toxemia <strong>and</strong> the occurrence of systemic disease, supporting the hypothesis that systemic<br />
CDI is most likely due to the toxicity of TcdA <strong>and</strong> TcdB <strong>and</strong> the induction of pro-inflammatory<br />
cytokines by the toxins.
O26<br />
ASPECTS OF THE HAMSTER MODEL OF INFECTION.<br />
A.M. Buckley* 1 , J. Spencer 1 , L.F. Dawson 2 , E.J. Beards 3 , J.M. Ketley 3 , B.W. Wren 2 , <strong>and</strong><br />
G.R. Douce 1 . 1 Dept of Microbiology, University of Glasgow, Glasgow, UK G12 8TA;<br />
2 Pathogen molecular biology unit, LSHTM, London, UK WC1E 7HT; 3 Department of<br />
Genetics, University of Leicester, Leicester, UK LE1 7RH.<br />
Clostridium difficile is an important cause of antibiotic associated disease, which is of high<br />
socio-economical importance that has recently been compounded by the emergence of<br />
epidemiological strains of different toxinotypes. C. difficile cases attributed to these strains<br />
show increased disease severity <strong>and</strong> high recurrence rates (up to 36%), which is increasing<br />
the financial burden faced by hospitals. The hamster model of infection is widely accepted as<br />
an appropriate model for studying aspects of C. difficile host-pathogen interactions. Infection<br />
of hamsters with toxigenic C. difficile manifests as hemorrhagic caecitis (wet tail) followed by<br />
death. Using this model we characterised the virulence of naturally occurring isolates which<br />
vary in toxin expression (A+B+, 630 & R20291; A-B+, M68 & CF5; A-B-, CD1342). 100% of<br />
hamsters challenged with A+B+ strains succumbed to the infection by ~47 hours, whilst<br />
those challenged with A-B+ strains showed ~60% survival rate. Hamsters challenged with<br />
the A-B- strain remained well. Bacterial colonisation, in vivo toxin production <strong>and</strong> caecal<br />
histology with these strains will be discussed. As with humans, the majority of clinical<br />
symptoms observed in the hamster are thought to be due to the action of TcdA <strong>and</strong> TcdB;<br />
making these proteins potential vaccine targets.<br />
Vaccination using recombinant proteins encoding the repeat regions from the binding domain<br />
of each toxin protected animals against severe disease. Vaccination with either antigen<br />
individually could not prevent death in the hamster model, however, in combination these<br />
antigens could protect animals challenged either 630 (100%), B1 (83%) or R20291 (62%).<br />
Surviving animals still shed C. difficile spores up to 14 days post challenge, were colonised<br />
at low levels, <strong>and</strong> showed adaptations to gut morphology. Ribosomal 16S sequencing of<br />
faecal samples taken throughout the infection process showed gross microbiota changes<br />
post clindamycin <strong>and</strong> C. difficile challenge, which recovered in the vaccinated model to pretreatment<br />
levels from 6 days post challenge onwards. Taken together these data give<br />
valuable insights into the infection profiles of different C. difficile strains <strong>and</strong> the use of toxin<br />
fragment vaccines as a potential prophylactic intervention.
O27<br />
A MOUSE RELAPSE MODEL OF CLOSTRIDIUM DIFFICILE INFECTION AND ITS<br />
APPLICATION IN EVALUATING IMMUNOTHERAPIES AGAINST THE DISEASE<br />
X. Sun*, H. Wang, Y. Zhang, K. , B. Davis, H. Feng. Tufts University, Cummings School of<br />
Veterinary Medicine, North Grafton, MA USA.<br />
Clostridium difficile is the causative agent of primary <strong>and</strong> recurrent antibiotic-associated<br />
diarrhea <strong>and</strong> colitis in hospitalized patients. The disease is mainly caused by two exotoxins<br />
TcdA <strong>and</strong> TcdB produced by the bacteria. Recurrent C. difficile infection (CDI) constitutes<br />
one of the most significant clinical issues in this disease, <strong>and</strong> occurs in more than 20% of<br />
patients after the first episode, <strong>and</strong> may be increasing in frequency. We recently established<br />
a mouse model of CDI relapse (Sun et al, 2011, IAI, 79:2856-64) enabling us to evaluate<br />
immunotherapies against recurrent CDI. In this study we found that the primary episode of<br />
CDI induced little or no protective antibody responses against the two toxins <strong>and</strong> mice<br />
continued shedding C. difficile spores. Antibiotic treatment of the surviving mice induced a<br />
second episode of diarrhea while a simultaneous re-exposure of animals with C. difficile<br />
bacteria or spores elicited a full spectrum of CDI symptom similar to that of the primary<br />
infection. Moreover, mice treated with immunosuppressive agents were prone to more<br />
severe <strong>and</strong> fulminant recurrent disease, indicating a protective role of the host innate<br />
immune response against CDI recurrence. We have used this model to test both prophylactic<br />
<strong>and</strong> therapeutic effects of neutralizing antitoxin polysera on recurrent CDI. We found that<br />
vancomycin treatment only delayed disease recurrence, whereas, polysera against both<br />
TcdA <strong>and</strong> TcdB completely protected mice against CDI relapse. Furthermore, we evaluated<br />
protection of a novel vaccine against recurrent CDI in the mouse disease model. We<br />
demonstrated that prophylactic vaccination induced potent neutralizing antitoxin antibodies<br />
<strong>and</strong> conferred a complete protection against C. difficile spore-induced recurrent disease<br />
activities, including diarrhea, weight loss <strong>and</strong> death. Our data indicate that antitoxin<br />
neutralizing antibodies may have great therapeutic potential against recurrent CDI.
O28<br />
LESSONS FROM NATURAL DISEASE IN PIGS: PATHOGENESIS OF CLOSTRIDIUM<br />
PERFRINGENS TYPE C ENTERITIS<br />
H. Posthaus* 1 , M. Wyder 1 , D. Authemann 2 , S. Christen 2 , M. Popoff 3 , F. Van Immerseel 4 .<br />
1 Institute of Animal Pathology, Vetsuisse Faculty, University of Berne, Switzerl<strong>and</strong>; 2 Institute<br />
of Infectious diseases, Medical School, University of Bern, Switzerl<strong>and</strong>; 3 Institut Pasteur,<br />
Paris, France; 4 Department of Pathology, Bacteriology <strong>and</strong> Avian Medicine, Gent University,<br />
Belgium.<br />
Clostridium perfringens is an important enteric pathogen in veterinary <strong>and</strong> human medicine.<br />
Toxinotypes of the organism are responsible for several distinct diseases in various host<br />
species. The pathogenesis of most enteric diseases associated with these pathogens is<br />
incompletely understood. C. perfringens type C strains induce a rapidly fatal necrotizing<br />
enteritis in pigs, humans, <strong>and</strong> other mammalian species. One essential virulence factor of<br />
these strains is beta toxin (CPB), a beta-barrel pore-forming toxin. Its cellular <strong>and</strong> molecular<br />
mode of action on natural target cells are not known in detail. We studied the effects of<br />
purified CPB <strong>and</strong> C. perfringens type C culture supernatant fluids on porcine small intestinal<br />
mucosa <strong>and</strong> intestinal epithelial <strong>and</strong> endothelial cells using different in vitro <strong>and</strong> in vivo<br />
approaches. Type C strains rapidly induced necrohemorrhagic lesions in ligated neonatal<br />
porcine small intestinal loops. Purified CPB was highly toxic to primary porcine <strong>and</strong> human<br />
endothelial cells, inducing rapid programmed necrosis by membrane pore-formation.<br />
Moreover, it bound to endothelial cells in small intestinal mucosal explants. Porcine small<br />
intestinal epithelial cells were not affected by CPB. Nevertheless, culture supernatants<br />
induced morphological damage in intestinal epithelial cells. In conclusion, our results suggest<br />
that endothelial cells are the primary target of beta toxin. Epithelial damage, required for<br />
penetration of the toxin into the tissue, is most likely induced by additional virulence factors.<br />
Identification of these factors will be important to underst<strong>and</strong> the pathogenesis of enteric<br />
diseases caused by C. perfringens strains.
029<br />
THE MOLECULAR EPIDEMIOLOGY OF CLOSTRIDIUM DIFFICILE IN THE UNITED<br />
STATES<br />
D. MacCannell, L. Anderson, J. Cohen, F. Lessa, <strong>and</strong> B. Limbago. CDC‘s Emerging<br />
Infections Program. CDC, Atlanta, GA USA..<br />
Abstract unavailable.
O30<br />
ELUCIDATING THE ROLE AND REQUIREMENT FOR SELENOENZYMES IN GROWTH<br />
OF CLOSTRIDIUM DIFFICILE<br />
K. Cobaugh 1 , M. Srivastava 1 , L. Bouillaut 2 , A. L. Sonenshein 2 , <strong>and</strong> W. T. Self* 1 . 1 Department<br />
of Molecular Biology <strong>and</strong> Microbiology, University of Central Florida; 2 Department of<br />
Molecular Biology <strong>and</strong> Microbiology, Tufts University.<br />
The incorporation of the metalloid selenium into proteins requires the activation of selenium<br />
into selenophosphate prior to translational insertion of the amino acid selenocysteine into the<br />
polypeptide chain. We have previously shown that C. difficile expresses at least two<br />
selenoenzymes, glycine reductase <strong>and</strong> D-proline reductase, when cultured in rich media. In<br />
addition, the presence of selenium <strong>and</strong> the substrates for these enzymes (proline <strong>and</strong><br />
glycine) significantly improves growth. We also have shown that exposure of cells to<br />
auranofin, a drug that blocks the metabolism of selenium, inhibits growth of C. difficile,<br />
suggesting that these enzymes are required for growth. To test the hypothesis that C.<br />
difficile requires one or both of these selenoenzymes for growth, we isolated mutant strains<br />
that carry deletions in genes encoding one subunit of glycine reductase (grdA), D-proline<br />
reductase (prdB) or selenophosphate synthetase (selD). All three mutant strains are viable,<br />
although the selD mutant grows at a slower rate. The level of D-proline reductase enzyme is<br />
significantly increased in the grdA mutant. Likewise the level of glycine reductase is<br />
increased in the prdB mutant. These results indicate that the cell balances its need for ATP<br />
synthesis through concerted regulation of these two enzymes. To our surprise the selD<br />
mutant is viable, <strong>and</strong> radiolabeling studies confirm that selenium is not incorporated into<br />
either enzyme in the mutant strain, but growth is noticeably slower than with the wild type.<br />
Our ongoing studies aim to elucidate the role that these selenoenzymes play in the<br />
physiology <strong>and</strong> bioenergetics of C. difficile, <strong>and</strong> to determine whether strains that lack one or<br />
both of these enzymes can trigger infection in animal models of disease.
O31<br />
INTEGRATION OF METABOLISM AND VIRULENCE IN CLOSTRIDIUM DIFFICILE.<br />
A. L. Sonenshein* 1 , L. Bouillaut 1 , S. S. Dineen 1 , S. M. McBride 1 <strong>and</strong> W. Self 2 . 1 Department of<br />
Molecular Biology <strong>and</strong> Microbiology, Tufts University School of Medicine <strong>and</strong> 2 Department of<br />
Molecular Biology <strong>and</strong> Microbiology, University of Central Florida.<br />
A broad linkage between nutrient availability <strong>and</strong> toxin production in C. difficile has been<br />
documented through the contributions of many laboratories. When cells are grown in rich<br />
medium, toxin gene expression <strong>and</strong> toxin production are repressed during rapid exponential<br />
growth <strong>and</strong> induced in stationary phase. Glucose, proline, glycine, branched-chain amino<br />
acids, cysteine <strong>and</strong> biotin have all been found to affect toxin production. The mechanistic<br />
bases for some of these effects are now becoming known. Two global regulators have been<br />
shown to repress toxin gene expression. CodY, a protein found in virtually all low G+C Grampositive<br />
bacteria, is activated by the accumulation of branched-chain amino acids (BCAAs;<br />
Ile, Leu, Val) <strong>and</strong> GTP, controls dozens of metabolic genes <strong>and</strong> also represses transcription<br />
of tcdR, the gene that encodes the alternative RNA polymerase sigma factor that recognizes<br />
the promoters of the toxin genes (tcdA <strong>and</strong> tcdB). CcpA links toxin gene expression to<br />
metabolism by regulating a vast array of carbon metabolism genes <strong>and</strong> directly repressing<br />
tcdA <strong>and</strong> tcdB when glucose is in excess. The role of proline can now be attributed at least in<br />
part to its role as an activator of PrdR. C. difficile, like many Clostridium spp., uses the<br />
Stickl<strong>and</strong> reaction (co-fermentation of pairs of amino acids) to generate ATP. The key<br />
enzymes, D-proline reductase (Prd) <strong>and</strong> glycine reductase (Grd), are encoded in multi-gene<br />
loci whose transcription responds to the presence of proline <strong>and</strong> glycine. The prdR gene<br />
located upstream of the prd genes was shown to encode a positive regulator of the prd<br />
genes <strong>and</strong> a negative regulator of the grd genes. The effect of PrdR on prd gene<br />
transcription appeared to be direct, while the effect of a prdR null mutation on grd<br />
transcription was shown to be due to lack of Prd activity <strong>and</strong> the consequent failure to<br />
produce an inhibitor of Grd gene expression. The inhibitor is postulated to be 5-<br />
aminovalerate. Moreover, a null mutation in prdB, which led to high proline accumulation,<br />
greatly decreased toxin gene expression, suggesting that the effect of proline on toxin<br />
production is mediated at least in part through activation of PrdR. The BCAAs provide a<br />
second potential interaction between the Stickl<strong>and</strong> pathways <strong>and</strong> toxin synthesis. Since the<br />
BCAAs serve as electron donors for the Stickl<strong>and</strong> reactions, increased grd expression in a<br />
prdB mutant may cause a sufficient reduction in the BCAA pool that the ability of CodY to<br />
repress the tcdR gene is compromised.
O32<br />
CLOSTRIDIUM DIFFICILE HAS TWO PARALLEL AND ESSENTIAL SEC SECRETION<br />
SYSTEMS<br />
R. P. Fagan* <strong>and</strong> N. F. Fairweather. Division of Cell <strong>and</strong> Molecular Biology, Centre for<br />
Molecular Microbiology <strong>and</strong> Infection, Imperial College London, London SW7 2AZ, United<br />
Kingdom.<br />
Protein translocation across the cytoplasmic membrane is an essential process in all<br />
bacteria. The Sec system, comprising at its core an ATPase, SecA, <strong>and</strong> a membrane<br />
channel, SecYEG, is responsible for the majority of this protein transport. Recently, a second<br />
parallel Sec system has been described in a number of Gram-positive species. This<br />
accessory Sec system is characterized by the presence of a second copy of the energizing<br />
ATPase, SecA2; where it has been studied, SecA2 is responsible for the translocation of a<br />
subset of Sec substrates. In common with many pathogenic Gram-positive species,<br />
Clostridium difficile possesses two copies of SecA. Here we describe the first<br />
characterization of the Clostridium difficile accessory Sec system <strong>and</strong> the identification of its<br />
major substrates. Using inducible antisense RNA expression <strong>and</strong> dominant negative alleles<br />
of SecA1 <strong>and</strong> SecA2, we demonstrate that export of the S-layer proteins (SLPs) <strong>and</strong> an<br />
additional cell wall protein (CwpV) is dependent on SecA2. Accumulation of the cytoplasmic<br />
precursor of the S-layer protein SlpA <strong>and</strong> other cell wall proteins was observed in cells<br />
expressing dominant negative SecA1 or SecA2 alleles, concomitant with a decrease in the<br />
levels of mature SLP proteins in the cell wall. Furthermore, expression of either dominant–<br />
negative allele or antisense RNA knock-down of SecA1 or SecA2 dramatically impaired<br />
growth, indicating that both Sec systems are essential in C. difficile.
O33<br />
THE MOLECULAR MECHANISM of CLOSTRIDIUM PERFRINGENS SPORE<br />
GERMINATION<br />
M. R. Sarker* 1 , D. Paredes-Sabja 2 , <strong>and</strong> P. Setlow 3 . 1 Department of Biomedical Sciences,<br />
Oregon <strong>State</strong> University, Corvallis, OR97331, USA; 2 Departmento de Ciencias Biológicas,<br />
Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile; 3 Department of<br />
Molecular, Microbial <strong>and</strong> Structural Biology, University of Connecticut Health Center,<br />
Farmington, CT06030, USA.<br />
Clostridium perfringens is a Gram-positive, spore-forming, anaerobic pathogen that causes<br />
diseases in animals <strong>and</strong> humans. The virulence of this bacterium is largely attributed to its<br />
capability to produce a large number of toxins. However, in addition to toxin production, C.<br />
perfringens has the ability to form metabolically dormant spores that are extremely resistant<br />
to heat <strong>and</strong> other environmental stresses. To cause deleterious effects, C. perfringens<br />
dormant spores must first go through germination <strong>and</strong> then outgrowth to be converted to<br />
vegetative cells capable of producing toxins. In Bacillus subtilis, spore germination is initiated<br />
when nutrient germinants bind to germinant receptors (GRs) in the spore's inner membrane<br />
triggering the release of the spore core's depot of dipicolinic acid as a 1:1 chelate with Ca2+<br />
(Ca-DPA), <strong>and</strong> replacement of Ca-DPA by water. These events trigger the hydrolysis of the<br />
spore's peptidoglycan cortex by cortex-lytic enzymes, <strong>and</strong> subsequent germ cell wall<br />
expansion allowing full spore core hydration, resumption of metabolism <strong>and</strong> macromolecular<br />
synthesis. Although spore germination in Clostridium species is less well studied than in<br />
Bacillus species, significant progress on the molecular mechanism of C. perfringens spore<br />
germination has been made recently in our laboratory. First, C. perfringens spores can<br />
germinate with the nutrient germinants L-asparagine, KCl, <strong>and</strong> inorganic phosphate (pH 6.0),<br />
<strong>and</strong> with non-nutrients such as dodecylamine <strong>and</strong> Ca-DPA. GerKA <strong>and</strong>/or GerKC are the<br />
main GR for these germinants, while GerAA <strong>and</strong> GerKB play auxiliary roles in germination.<br />
Second, release of Ca-DPA is not required for the initiation of hydrolysis of the spore‘s<br />
peptidoglycan cortex. Third, among two cortex-lytic enzymes (CLEs) (i.e., SleC <strong>and</strong> SleM),<br />
SleC is the single essential CLE for spore‘s peptidoglycan (PG) cortex hydrolysis during C.<br />
perfringens spore germination, with SleM playing only an auxiliary role. Finally, the serine<br />
protease CspB is essential to generate active SleC <strong>and</strong> allow DPA release <strong>and</strong> PG cortex<br />
hydrolysis early in C. perfringens spore germination. The ongoing research towards<br />
determining the mechanism of activation of CspB should help in underst<strong>and</strong>ing further the<br />
molecular mechanism of spore germination in C. perfringens.
O34<br />
THE KEY SIGMA FACTOR OF TRANSITION PHASE, SigH, CONTROLS SPORULATION,<br />
METABOLISM, AND VIRULENCE FACTOR EXPRESSION IN CLOSTRIDIUM DIFFICILE<br />
L. Saujet*, M. Monot, B. Dupuy, O. Soutourina, <strong>and</strong> I. Martin-Verstraete. Laboratoire<br />
Pathogenèse des Bactéries Anaérobies, Institut Pasteur, 25 rue du Dr Roux 75724 Paris<br />
Cedex, 15 <strong>and</strong> Université Paris 7-Denis Diderot, 75205 Paris, France.<br />
Toxin synthesis in Clostridium difficile is subject to multiple forms of environmental regulation<br />
<strong>and</strong> increases as cells enter stationary phase. The regulatory network controlling postexponential<br />
events in C. difficile is still poorly characterized. However, among the regulators<br />
that trigger this transition, CodY <strong>and</strong> Spo0A were shown to modulate toxin gene expression.<br />
So, we decided to test the role of SigH, an alternative sigma factor involved in the transition<br />
to post-exponential phase in Bacillus subtilis. We inactivated sigH in C. difficile <strong>and</strong><br />
compared the expression profiles of strain 630E after 4 h <strong>and</strong> 10 h of growth as well as the<br />
630E <strong>and</strong> the sigH mutant at the onset of stationary phase (10h). About 60% of the genes<br />
differentially expressed between exponential <strong>and</strong> stationary phases including genes involved<br />
in motility, sporulation, cellular division, <strong>and</strong> metabolism were regulated by SigH appearing<br />
as a key regulator of transition phase in C. difficile. SigH positively controls genes required<br />
for sporulation. Accordingly, sigH inactivation results in an asporogeneous phenotype. The<br />
spo0A <strong>and</strong> CD2492 genes encoding the master regulator of sporulation <strong>and</strong> one of its<br />
associated kinases <strong>and</strong> the spoIIA operon were transcribed from a SigH-dependent<br />
promoter. The expression of tcdA <strong>and</strong> tcdB encoding the toxins <strong>and</strong> of tcdR encoding the<br />
sigma factor required for toxin production increased in a sigH mutant. So, the sigH mutant is<br />
unable to sporulate but still produces toxins demonstrating that toxin synthesis is a stationary<br />
phase event. Finally, SigH regulated the expression of genes encoding surface-associated<br />
proteins like the Cwp66 adhesin, the S-layer precursor <strong>and</strong> the flagellum components.<br />
Among the 286 genes positively regulated by SigH, about 40 transcriptional units presenting<br />
a SigH consensus in their promoter regions are good c<strong>and</strong>idates for direct SigH targets. We<br />
have recently performed a genome wide determination of transcriptional start sites of the<br />
630E strain using RNA-seq. Among the 40 predicted SigH direct targets, we confirmed the<br />
existence of a SigH promoter for 22 of them <strong>and</strong> proposed a SigH-dependent promoter<br />
consensus.
O35<br />
A CHIMERIC TOXIN VACCINE PREVENTS PRIMARY AND RECURRENT CLOSTRIDIUM<br />
DIFFICILE INFECTION<br />
H. Wang *1, 2 , X. Sun *1 , Y. Zhang 1, 3 , S. Li 1 , K. Chen 1 , G.P. Cordon 1 , L.Shi 1 , W. Nie 1 , R.<br />
Kumar 4 , S. Tzipori 1 , J. Wang 2 , T. Savidge 5 , <strong>and</strong> H. Feng. 1 1 Tufts University, Cummings<br />
School of Veterinary Medicine, North Grafton, Massachusetts 01536, US. 2 School of<br />
Bioscience <strong>and</strong> Biotechnology, South China University of Technology, Guangzhou, China.<br />
3 School of Bioengineering, East China University of Science <strong>and</strong> Technology, Shanghai,<br />
China. 4 Department of Basic Sciences, The Commonwealth Medical College, Scranton, US.<br />
5 Department of Gastroenterology & Hepatology, The University of Texas Medical Branch,<br />
Galveston, Texas 77555, US.<br />
*Authors contributed equally to this work.<br />
The global emergence of hypervirulent Clostridium difficile infection (CDI) has contributed to<br />
the recent surge in severe antibiotic-associated colonic disease. C. difficile produces two<br />
homologous glucosylating exotoxins, TcdA <strong>and</strong> TcdB, both of which require neutralization to<br />
prevent disease. Antitoxin immunization is widely recognized to be the preferred vaccination<br />
strategy against CDI. However, because of their large size <strong>and</strong> complex multifunctional<br />
domain structure, it has been a challenge to produce recombinant toxin vaccines. As a<br />
consequence, clinical immunization has been limited to toxoids that show suboptimal<br />
efficacy. Here we describe a novel chimeric toxin vaccine that confers complete disease<br />
protection in animal models of CDI <strong>and</strong> characterize the underlying antitoxin protective<br />
mechanism. Using a non-pathogenic Bacillus megaterium expression system, we generated<br />
glucosyltransferase-deficient holotoxins <strong>and</strong> demonstrated their loss of toxicity. These<br />
holotoxins induced markedly greater antitoxin neutralizing responses than toxoid, but showed<br />
little cross-protection. To facilitate simultaneous protection against both toxins, we generated<br />
an active clostridial toxin chimera by replacing the receptor binding domain of TcdB with<br />
TcdA, thus conserving immunodominant, conformation-dependent epitopes. Parenteral<br />
immunization with the avirulent toxin chimera, cTxAB, induced rapid <strong>and</strong> complete disease<br />
protection against oral challenge with laboratory or epidemic C. difficile strains. Moreover,<br />
prophylactic cTxAB-vaccination prevented spore-induced disease relapse, which constitutes<br />
one of the most significant clinical issues in CDI. Systemic <strong>and</strong> local mucosa-associated IgG<br />
immunity conferred this disease protection. Thus, the rational design of recombinant chimeric<br />
toxins provides a novel approach for rapidly protecting individuals at high risk of developing<br />
infectious disease such as CDI.
O36<br />
OPIOID-BASED ANALGESICS BLOCK THE PROGRESSION AND DEVELOPMENT OF C.<br />
PERFRINGENS MEDIATED MYONECROSIS<br />
A. Chakravorty*, M.M. Awad, T.J. Hiscox, J.K. Cheung, J.M. Choo, D. Lyras <strong>and</strong> J.I. Rood.<br />
Monash University, Department of Microbiology, Melbourne, Australia, 3800.<br />
Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that is the<br />
causative agent of traumatic gas gangrene. The key pathological characteristic of this<br />
disease is a paucity of polymorphonuclear leukocytes (PMNs) within sites of infection, which<br />
allows the infection to spread unhindered. This process involves the formation of plateletplatelet<br />
<strong>and</strong> platelet-leukocyte aggregates within the blood vessels, which in turn blocks<br />
blood flow into the sites of infection, the resultant development of an ischemic environment.<br />
The end result is rapid bacterial growth <strong>and</strong> widespread necrosis of the infected tissues.<br />
Current treatment of gas gangrene involves the use of antibiotics, primarily penicillin, <strong>and</strong><br />
debridement of necrotic tissues. However, the disease develops rapidly <strong>and</strong> the amputation<br />
of infected limbs is often necessary to stop the spread of infection. We have recently shown<br />
that mice pre-treated with buprenorphine, a morphine-based opioid analgesic, do not<br />
succumb to experimental C. perfringens-mediated myonecrosis. Independent of their role in<br />
the neuroendorcrine system, previous studies have shown that opioids can repress the<br />
immune response <strong>and</strong> increase the host‘s susceptibility to infection. Our study shows the<br />
reverse effect. Infected mice that have been pre-treated with buprenorphine do not develop<br />
the characteristic pathology associated with gas gangrene, especially blackening of the thigh<br />
<strong>and</strong> footpad. A similar effect was observed with morphine, suggesting that it has a similar<br />
mechanism of action. These data provide evidence that treatment with opiates retards the<br />
progression of clostridial myonecrosis <strong>and</strong> provides valuable insights into the mechanisms by<br />
which these effects are mediated.
O37<br />
SUB-INHIBITORY CONCENTRATIONS OF TIGECYCLINE INHIBIT SPORULATION OF<br />
CLOSTRIDIUM DIFFICILE IN VITRO<br />
J. R. Garneau, L. Valiquette <strong>and</strong> L.C. Fortier*. Department of Microbiology <strong>and</strong> Infectious<br />
Diseases, Faculty of Medicine <strong>and</strong> Health Sciences, Université de Sherbrooke, 3001, 12th<br />
Ave North, Sherbrooke, QC, Canada, J1H 5N4.<br />
Sub-inhibitory concentrations of certain antibiotics were previously shown to affect the<br />
expression of several genes in Clostridium difficile, but how antibiotics affect sporulation is<br />
not well understood. Spores are insensitive to antibiotics <strong>and</strong> sporulation of C. difficile in the<br />
colon during infection could possibly explain treatment failures <strong>and</strong> relapses reported with<br />
metronidazole <strong>and</strong> vancomycin. The objective of our study was to evaluate the impact of<br />
sub-inhibitory concentrations of metronidazole (MTZ), vancomycin (VAN), ciprofloxacin (CIP),<br />
<strong>and</strong> tigecycline (TIGE) on the in vitro sporulation of C. difficile. The aim was to determine<br />
whether these antibiotics could promote or inhibit sporulation. The reference strains ATCC<br />
9689, 630 <strong>and</strong> VPI 10463, as well as seven other clinical isolates of C. difficile were<br />
characterized by PCR ribotyping, t<strong>and</strong>em repeat sequence typing (TRST), binary toxin<br />
detection, <strong>and</strong> sequencing of the tcdC gene. Three clinical isolates were PCR ribotype 027<br />
(R027) <strong>and</strong> had common characteristics of the epidemic NAP1/027 strain. Minimum<br />
inhibitory concentrations (MIC) were determined for MTZ, VAN, CIP <strong>and</strong> TIGE <strong>and</strong> all strains<br />
were sensitive, except the R027 isolates that were resistant to CIP (MIC=128 µg/mL).<br />
Sporulation was assessed on TY agar in the absence or presence of 0.5X MIC of each<br />
antibiotic. Colonies were picked after 24, 48, <strong>and</strong> 96 h of growth <strong>and</strong> Gram stains were<br />
observed under the microscope. The number of spores <strong>and</strong> vegetative cells from 10 different<br />
fields was used to calculate the sporulation rate. We found that MTZ <strong>and</strong> VAN did not<br />
significantly modify the sporulation rate of C. difficile, but CIP inhibited sporulation up to 18-<br />
fold, but only for R027 strains <strong>and</strong> VPI 10463. On the other h<strong>and</strong>, TIGE systematically<br />
inhibited sporulation of all strains tested by a factor of up to 254-fold. In concentration range<br />
assays with a subset of 3 strains, we observed that the inhibition of sporulation with TIGE<br />
<strong>and</strong> CIP was greater as the concentration of antibiotic approached the MIC. In summary,<br />
TIGE consistently inhibited sporulation of C. difficile, <strong>and</strong> this inhibition may in part explain<br />
the recent success of TIGE in treating recurrent CDI cases.
O38<br />
IMMUNIZATION WITH BACILLUS SPORES EXPRESSING TOXIN A PEPTIDE REPEATS<br />
PROTECTS AGAINST INFECTION WITH CLOSTRIDIUM DIFFICILE STRAINS<br />
PRODUCING TOXIN A AND B<br />
P. Permpoonpattana* 1 , H. A. Hong 1 , J. Phetcharaburanin 1 , J. Huang 1 , J. Cook 1 , N.F.<br />
Fairweather 2 , <strong>and</strong> S. M. Cutting* 1 . 1 School of Biological Sciences, Royal Holloway,<br />
University of London, Egham, Surrey, TW20 0EX United Kingdom; 2 Department of Life<br />
Sciences, Imperial College London, London, SW7 2AZ United Kingdom.<br />
Clostridium difficile is a leading cause of nosocomial infection in the developed world. Two<br />
toxins, A <strong>and</strong> B, produced by most strains of C. difficile are implicated as virulence factors,<br />
yet only recently has the requirement of these for infection been investigated by genetic<br />
manipulation. Current vaccine strategies are focused mostly on parenteral delivery of<br />
toxoids. In this work, we have used bacterial spores (Bacillus subtilis) as a delivery vehicle to<br />
evaluate the carboxy-terminal repeat domains of toxins A <strong>and</strong> B as protective antigens. Our<br />
findings are important <strong>and</strong> show that oral immunization of the repeat domain of toxin A is<br />
sufficient to confer protection in a hamster model of infection designed to closely mimic the<br />
human course of infection. Importantly, neutralizing antibodies to the toxin A repeat domain<br />
were shown to be cross-reactive with the analogous domain of toxin B <strong>and</strong>, being of high<br />
avidity, provided protection against challenge with a C. difficile strain producing toxins A <strong>and</strong><br />
B (A+B+). Thus, although many strains produce both toxins, antibodies to only toxin A can<br />
mediate protection. Animals vaccinated with recombinant spores were fully able to survive<br />
reinfection, a property that is particularly important for a disease with which patients are<br />
prone to relapse. We show that mucosal immunization, not parenteral delivery, is required to<br />
generate secretory IgA <strong>and</strong> that production of these neutralizing polymeric antibodies<br />
correlates with protection. This work demonstrates that an effective vaccine against C.<br />
difficile can be designed around two attributes, mucosal delivery <strong>and</strong> the repeat domain of<br />
toxin A.
O39<br />
THE C. DIFFICILE CPR LOCUS MEDIATES RESISTANCE TO ANTIMICROBIAL<br />
PEPTIDES THROUGH MOLECULAR MIMICRY<br />
S.M. McBride <strong>and</strong> A.L. Sonenshein. Department of Molecular Biology <strong>and</strong> Microbiology,<br />
Tufts University School of Medicine, Boston, MA 02111 USA.<br />
Clostridium difficile is the major causative agent of antibiotic-associated diarrhea. To persist<br />
in the intestine the bacteria must cope with innate defenses within the host, such as cationic<br />
antimicrobial peptides (CAMPs) made by the host <strong>and</strong> indigenous flora. We previously<br />
showed that C. difficile responds to CAMPs by inducing expression of genes that lead to<br />
CAMP resistance. The first such C. difficile gene cluster identified (cprABCK) encodes an<br />
ABC-type transporter <strong>and</strong> a sensor kinase typical of bacterial two-component systems (TCS).<br />
These genes are highly related to genes that encode immunity proteins in lantibiotic producer<br />
species. Using qRT-PCR <strong>and</strong> directed mutagenesis, we showed that the transporter <strong>and</strong><br />
kinase genes are directly involved in resistance to CAMPs. However, no apparent response<br />
regulator is encoded in the vicinity of cprABCK. Here, we identify an orphan response<br />
regulator, CD3320, which encodes the response regulator that controls the cprABCK genes<br />
in response to antimicrobial peptides. We cloned the CD3320 gene <strong>and</strong> upstream region in<br />
a vector that replicates in C. difficile <strong>and</strong> demonstrated that cells adapted faster to CAMPs<br />
<strong>and</strong> expression of the cprABCK operon increased. By expressing these regulators <strong>and</strong> a<br />
Pcpr::lacZ reporter fusion in a heterologous host, Bacillus subtilis, we were able to show how<br />
CprK <strong>and</strong> CD3320 (renamed CprR) interact to activate expression of the cpr operon. In<br />
addition, we were able to identify putative residues of both the lantibiotics <strong>and</strong> of CprK that<br />
are involved in sensing <strong>and</strong> activation of this system. These results demonstrate how the cpr<br />
ABC-transporter <strong>and</strong> regulators allow for substrate recognition that is broader than the typical<br />
narrow-substrate recognition of lantibiotic producer systems, resulting in resistance to<br />
multiple bacterial CAMPs through a single mechanism. The cpr lantibiotic immunity system<br />
represents a novel antimicrobial peptide resistance mechanism that has not been described<br />
in any other species.
O40<br />
DIFFERENTIAL SENSITIVITY OF CLOSTRIDIUM DIFFICILE CLINICAL ISOLATES TO<br />
MAMMALIAN CATIONIC ANTIMICROBIAL PEPTIDES<br />
R. McQuade* 1 , M. Mallozzi 1 , B. Roxas 1 , G. Vedantam 1,2,3 , <strong>and</strong> V.K. Viswanathan 1,2 . 1 Dept.<br />
of Veterinary Science <strong>and</strong> Microbiology, University of Arizona, Tucson, AZ; 2 The BIO5<br />
Institute, University of Arizona, Tucson, AZ, USA; 3 The Southern Arizona VA Healthcare<br />
System, Tucson, AZ, USA.<br />
Background <strong>and</strong> Rationale: Clostridium difficile is a leading cause of hospital-acquired<br />
diarrhea. Recent outbreaks of C.difficile infection (with increased morbidity <strong>and</strong> mortality)<br />
have involved ―hypervirulent‖ (HV) strains, including those belonging to the 027 <strong>and</strong> 078<br />
ribotypes. Cationic antimicrobial peptides (CAMPs; e.g. human LL37 & sheep SMAP29)<br />
contribute to gut innate immunity by interacting with, <strong>and</strong> disrupting the negatively-charged<br />
bacterial cell-membrane. Some pathogens have evolved specific defense mechanisms to<br />
evade CAMP-mediated killing. We hypothesized that increased resistance to CAMPs<br />
contributes to the more persistent infections associated with HV C. difficile strains.<br />
Methods: Minimum inhibitory concentrations (MIC) of LL37 <strong>and</strong> SMAP29 were determined<br />
for various HV <strong>and</strong> non-HV C. difficile strains using a st<strong>and</strong>ard broth microdilution. To<br />
visualize the action of CAMPs on C. difficile, LL37-exposed bacteria were imaged by electron<br />
microscopy. The kinetics of CAMP killing was evaluated by determining the number of<br />
surviving C. difficile throughout an 8h exposure to various concentrations of LL37. To<br />
determine whether C. difficile has inducible CAMP-resistance mechanisms, the sensitivity of<br />
strain 630 to LL37 was assessed with <strong>and</strong> without pre-exposure to a sub-inhibitory<br />
concentration of the peptide. Changes in the proteome of LL37-exposed C. difficile were<br />
determined using mass spectrometry.<br />
Results: Compared to non-epidemic strains, HV C. difficile isolates display a consistent<br />
increase in resistance to both LL37 <strong>and</strong> SMAP29. Electron microscopy of LL37-exposed<br />
bacteria revealed surface distortion characteristic of CAMP-mediated killing. LL37 inhibited<br />
C. difficile growth in a dose-dependent manner, with bacterial death evident as early as 0.5h<br />
post-exposure. Exposure of bacteria to a sub-inhibitory concentration of LL37 resulted in<br />
increased resistance to the CAMP. Mass-spectrometry analysis revealed proteome<br />
alterations, including the increased expression of putative ABC transporters, in bacteria<br />
exposed to sub-inhibitory concentrations of LL37. Discussion: C. difficile strains display<br />
variable sensitivity to CAMPs, <strong>and</strong> the increased resistance of HV strains likely contributes to<br />
their persistence in the gut. CAMPs appear to act on C. difficile by disrupting the bacterial<br />
membrane. Pre-exposure of C. difficile to LL37 results in altered protein expression, as well<br />
as increased resistance to LL37, suggesting adaptability of the bacteria to CAMPs.
O41<br />
BOTULINUM TOXIN STRUCTURE/FUNCTION RELATIONSHIPS<br />
J.T. Barbieri, Department of Microbiology <strong>and</strong> Molecular Genetics, Medical College of<br />
Wisconsin, Milwaukee, WI 53226 USA.<br />
The botulinum neurotoxins (BoNT) are the most potent protein toxins for humans. There are<br />
seven serotypes of BoNTs termed (A-G) which are defined by a lack of cross anti-sera<br />
neutralization. Several of the BoNT serotypes (A, B, E, F, <strong>and</strong> G) utilize dual receptors, a<br />
ganglioside <strong>and</strong> protein, to enter neurons. Initially, these serotypes of BoNT bind ganglioside<br />
on resting neurons which supports cell association until a synaptic vesicle fuses to the cell<br />
membrane, exposing the protein receptor <strong>and</strong> facilitating entry into the neuron via synaptic<br />
vesicle internalization. In contrast, tetanus toxin utilizes dual-gangliosides as functional<br />
receptors <strong>and</strong> enters through an endosome-based mechanism. BoNT serotypes C <strong>and</strong> D<br />
include mosaic toxins that are organized as D-C <strong>and</strong> C-D toxins. One BoNT D-C mosaic<br />
toxin, BoNT-D(South Africa), was not fully neutralized by immunization with vaccines derived<br />
from either BoNT serotype C or D, which prompted characterization of their biological <strong>and</strong><br />
cellular properties. Structural studies showed that the receptor binding domains of BoNT-C,<br />
BoNT-D, <strong>and</strong> BoNT-D(SA) possessed overall structural similarity with other BoNTs<br />
serotypes. However, a structural alignment of the primary amino acid sequence showed that<br />
BoNT-C, BoNT-D, <strong>and</strong> BoNT-D(SA) lacked residues that were components of the<br />
ganglioside binding domain (GBP) <strong>and</strong> that was conserved in BoNT serotypes A,B,E, F, <strong>and</strong><br />
G. This alignment also identified a unique loop that was present in BoNT-C, BoNT-D, <strong>and</strong><br />
BoNT-D(SA) that was distanced from the conserved GBP of the other BoNT serotypes<br />
termed the ganglioside binding loop (GBL). The GBL included a tryptophan for BoNT-C <strong>and</strong><br />
BoNT-D(SA) <strong>and</strong> a phenylalanine for BoNT-D. Mutation of this aromatic amino acid<br />
eliminated the ability to bind ganglioside without perturbing protein structure. These studies<br />
showed that BoNT-C, BoNT-D, <strong>and</strong> BoNT-D(SA) recognized <strong>and</strong> bound gangliosides<br />
differently than the other serotypes of BoNTs, implicating a unique mechanism for entry <strong>and</strong><br />
intoxication of neurons. Continued studies may provide new information on the action of<br />
these neurotoxins that may extend their clinical utility.
O42<br />
A TWO-COMPONENT SYSTEM NEGATIVELY REGULATES BOTULINUM NEUROTOXIN<br />
EXPRESSION<br />
Z. Zhang 1 , H. Korkeala 1 , E. Dahlsten 1 , J. T. Heap 2 , N. P. Minton 2 , M. Lindström* 1 .<br />
1 Department of Food Hygiene <strong>and</strong> Environmental Health, Centre of Excellence in Microbial<br />
Food Safety Research, Faculty of Veterinary Medicine, University of Helsinki, Finl<strong>and</strong>;<br />
2 School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of<br />
Nottingham, University Park, Nottingham NG7 2RD, United Kingdom.<br />
Two-component signal transduction systems (TCSs) play an important regulatory role in<br />
virulence in many pathogenic bacteria. However, little is known about the role of TCSs in<br />
Clostridium botulinum. We identified a TCS involved in neurotoxin regulation in C. botulinum<br />
type A strain ATCC3502. Using the ClosTron mutagenesis system 1 , we inactivated tcsR,<br />
encoding a response regulator, <strong>and</strong> tcsK, encoding a sensor histidine kinase. Inactivation of<br />
tcsR <strong>and</strong> tcsK resulted in a significantly higher level of neurotoxin gene (botA) transcripts <strong>and</strong><br />
neurotoxin production, as measured with ELISA. Complementation of tcsR mutant cells with<br />
a plasmid 2 expressing tcsKR restored neurotoxin production to the wild-type level. Further<br />
experiments suggested that TcsKR also down-regulates the transcription of botR, the<br />
alternative sigma factor which activates botA transcription 3 . The regulatory role of TcsKR was<br />
confirmed by protein-DNA binding assays. To our knowledge, this is the first report on<br />
negative regulation of botulinum neurotoxin production <strong>and</strong> a role of TCS in C. botulinum.<br />
Profound underst<strong>and</strong>ing of the negative regulation of the neurotoxin production may provide<br />
tools to control the risk of botulism in foods.<br />
1. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. 2007. The ClosTron: a universal gene knock-out system for<br />
the genus Clostridium. J Microbiol Methods 70: 452-464<br />
2. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, Minton NP. 2010. The ClosTron: mutagenesis in<br />
Clostridium refined <strong>and</strong> streamlined. J Microbiol Methods 80: 49-55<br />
3. Marvaud JC, Gibert M, Inoue K, Fujinaga Y, Oguma K, <strong>and</strong> Popoff MR. 1998. botR/A is a positive regulator of botulinum<br />
neurotoxin <strong>and</strong> associated non-toxin protein genes in Clostridium botulinum A. Mol Microbiol 29: 1009-1018
O43<br />
DISRUPTION OF sigK IN CLOSTRIDIUM BOTULINUM ATCC3502 PREVENTS<br />
SPORULATION.<br />
D. Kirk*, H. Korkeala, Z. Zhang, E. Dahlsten, <strong>and</strong> M. Lindström. Department of Food<br />
Hygiene <strong>and</strong> Environmental Health, Centre of Excellence in Microbial Food Safety Research,<br />
Faculty of Veterinary Medicine, University of Helsinki, 00014 Finl<strong>and</strong>.<br />
Clostridium botulinum is a spore-forming obligate anaerobe. The Spo0A protein is thought to<br />
be responsible for initiating sporulation in clostridia, triggering the sporulation pathway which<br />
involves different sigma factors at each stage. C. botulinum also produces potent<br />
neurotoxins, the causative agents of botulism. Spore contamination is a key factor in cases<br />
of infant <strong>and</strong> wound botulism as spores germinate <strong>and</strong> neurotoxin is produced in vivo. It is<br />
unknown how the activation of Spo0A is regulated in C. botulinum, as those mechanisms<br />
found in Bacillus are not present. The subsequent stages of sporulation have homologues in<br />
most Bacillus species, though there may be differences in target genes <strong>and</strong> their expression.<br />
Using the ClosTron tool developed by Heap et al. (2007), which utilizes a group II intron<br />
insertion to disrupt gene function, we created a mutation in the late-stage sporulation sigma<br />
factor gene sigK. This mutant failed to produce spores after seven days‘ incubation at 37°C<br />
in a st<strong>and</strong>ard nutrient medium. Upon spore staining <strong>and</strong> observation with light microscopy,<br />
no spores were visible. In addition, the individual mutant cells‘ morphology appeared more<br />
elongated than that of the wild type, occasionally forming long strings of growth. By creating<br />
a plasmid containing sigK, we were able to complement spore formation. The wildtype strain,<br />
with <strong>and</strong> without the empty plasmid vector, formed spores as expected. The results suggest<br />
that successful sporulation in C. botulinum ATCC 3502, a Group I type A toxin producing<br />
strain, is dependent upon the late-stage sporulation sigma factor SigK. Expression analysis<br />
of spo0A <strong>and</strong> other sporulation related genes is ongoing in the sigK mutant <strong>and</strong> with type<br />
ATCC 3502.<br />
References: Heap et al. (2007). The ClosTron: A universal gene knock-out system for the genus Clostridium. Journal of<br />
Microbiological Methods 70: 452–464.
O44<br />
THE ROLE OF TOXINS IN CLOSTRIDIUM DIFFICILE PATHOGENESIS<br />
G. Carter 1 , A. Chakravorty 1 , K. Mackin 1 , M. Kelly 1 , G. Douce 2 , R. Govind 3 , B. Dupuy 4 , S.<br />
Johnson 5 , D. Gerding 5 , J. Rood 1 , T. Lawley 6 <strong>and</strong> D. Lyras* 1 . 1 Department of Microbiology,<br />
Monash University, Victoria, Australia; 2 Division of Infection <strong>and</strong> Immunity, FBLS Glasgow<br />
Biomedical Research Centre, University of Glasgow, UK; 3 Division of Biology, Kansas <strong>State</strong><br />
University, Manhattan, KS, USA; 4 Unité de Génétique Moléculaire Bactérienne, Institut<br />
Pasteur, Paris, France; 5 Hines Veterans Affairs Hospital, Hines, IL, USA; 6 Pathogen<br />
Genomics, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,<br />
Cambridge, UK.<br />
Clostridium difficile is an important cause of nosocomial disease worldwide. Most diseasecausing<br />
C. difficile isolates produce two large clostridial cytotoxins, A <strong>and</strong> B, encoded by<br />
tcdA <strong>and</strong> tcdB, respectively. Studies using derivatives of the historical strain 630 have shown<br />
that toxin B plays a major role in virulence while the role of toxin A is not as clear. Recently,<br />
―hypervirulent‖ NAP1/027 isolates have been associated with more severe disease <strong>and</strong><br />
higher rates of mortality. These strains appear to produce more toxin than other clinical<br />
isolates, which is thought to be the result of a nonsense mutation within a gene encoding a<br />
putative negative regulator of toxin production, tcdC. This hypothesis is contentious <strong>and</strong><br />
there are conflicting reports on the role of TcdC. To study NAP1/027 epidemic strains at the<br />
molecular level, <strong>and</strong> to clarify the role of toxins in disease, we developed a new <strong>and</strong> more<br />
efficient plasmid transfer system which facilitated the construction of toxin gene mutants <strong>and</strong><br />
the complementation of a NAP1/027 epidemic isolate with an intact copy of tcdC. We have<br />
constructed isogenic toxin gene mutants of a virulent C. difficile NAP1/027 strain <strong>and</strong><br />
assessed these mutants for their virulence properties. The data obtained by studying these<br />
strains provide new insights into the role of toxins in disease <strong>and</strong> the contribution of TcdC to<br />
the development of hypervirulence in C. difficile.
O45<br />
THE INDIVIDUAL ROLES OF TOXIN A AND TOXIN B IN CLOSTRIDIUM DIFFICILE<br />
INFECTION<br />
S.A. Kuehne*, M.M. Collery, M.L. Kelly, S.T. Cartman, A. Cockayne, <strong>and</strong> N.P. Minton.<br />
Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular<br />
Sciences, University of Nottingham, Nottingham, NG7 2RD, UK.<br />
Clostridium difficile is the major cause of antibiotic associated diarrhoea in Europe <strong>and</strong> North<br />
America. It leads to higher mortality rates than MRSA <strong>and</strong> imposes a significant financial<br />
burden on our healthcare systems. The two large toxins, TcdA <strong>and</strong> TcdB, are the main<br />
virulence factors in Clostridium difficile infection (CDI). Despite their undoubted importance<br />
there has been much controversy over the years with regards to their individual contributions<br />
to the disease. In recent years, two papers have been published, one by Lyras et al, 2009<br />
indicating that in strain 630 only toxin B is essential for CDI, <strong>and</strong> the other one by our<br />
laboratory (Kuehne et al, 2010) showing that the same strain producing either toxin A or toxin<br />
B alone can cause fulminant disease. The reason for the different outcome of the two studies<br />
is not clear, but may be a consequence of the presence of ancillary mutations in the isolates<br />
used in the two laboratories. This is currently being investigated through genome resequencing.<br />
In the meantime, we have extended our analysis to another strain, R20291.<br />
This is a PCR ribotype 027/NAP1/B1 (‗hypervirulent‘) strain. Hypervirulent strains are linked<br />
to more severe CDI, <strong>and</strong> produce a third toxin, which is a binary toxin called Cdt. We have<br />
created stable toxin mutants in strain R20291 using ClosTron technology, to allow<br />
examination of each individual toxin <strong>and</strong> combinations of the three toxins during the disease<br />
process. In common with our early study using strain 630 (Kuehne et al, 2010), experiments<br />
have shown that R20291 derivatives producing either of the two main toxins (A or B) alone<br />
can cause fatal disease in the hamster infection model. These findings re-establish the<br />
importance of TcdA <strong>and</strong> TcdB in CDI <strong>and</strong> emphasize a need to consider both when<br />
developing effective countermeasures.
O46<br />
THE AGR QUORUM SENSING SYSTEM IS A GLOBAL REGULATOR OF CLOSTRIDIUM<br />
PERFRINGENS TOXIN PRODUCTION AND VIRULENCE<br />
J. Chen* 1 , M. Ma 1 , J. Li 1 , J. Vidal 1 , J. Garcia 3 , J. Saputo 3 , J. Rood 2 , F. Uzal 3 , B. McClane 1 .<br />
1 Department of Microbiology <strong>and</strong> Molecular Genetics, University of Pittsburgh School of<br />
Medicine, Pittsburgh, PA, USA; 2 Department of Microbiology, Monash University, Clayton,<br />
Australia; 3 California Animal Health <strong>and</strong> Food Safety Laboratory, University of California<br />
Davis, San Bernardino, CA, USA.<br />
Clostridium perfringens, an important pathogen of humans <strong>and</strong> livestock, causes wound<br />
infections (e.g. gas gangrene) <strong>and</strong> diseases originating in the intestines (e.g. food poisoning,<br />
necrotic enteritis <strong>and</strong> enterotoxemia). C. perfringens virulence is largely dependent upon<br />
prolific toxin production, with this bacterium capable of producing at least 17 different toxins.<br />
The agr system is a well-characterized regulator, in a quorum-sensing (QS) manner, of toxin<br />
genes in Staphylococcus aureus. Recently an Agr-like system was shown to regulate the<br />
production of alpha toxin <strong>and</strong>, particularly, perfringolysin O, by C. perfringens type A strain<br />
13. We later demonstrated that the Agr-like locus also regulates sporulation <strong>and</strong> enterotoxin<br />
production by C. perfringens type A strain F5603. C. perfringens epsilon toxin (ETX) is<br />
considered the third most potent of all clostridial toxins <strong>and</strong> thus has been classified by the<br />
CDC as a class B select agent toxin. ETX is also a major virulence factor in several<br />
important, <strong>and</strong> often fatal, natural veterinary enterotoxemias caused by type B <strong>and</strong> D strains.<br />
Similarly, when C. perfringens type C isolates cause necrotic enteritis in humans or livestock,<br />
beta toxin (CPB) is considered the most important toxin. There has been limited information<br />
available regarding the regulation of ETX production by type D strains or CPB production by<br />
type C strains. To obtain a better underst<strong>and</strong>ing of how toxin production is regulated in these<br />
strains, agrB null mutants were generated by Targetron insertional mutagenesis technology<br />
in C. perfringens type C strain CN3685 <strong>and</strong> type D strain CN3718. We found that the<br />
CN3685::agrB mutant exhibits strongly reduced CPB production <strong>and</strong> the CN3718::agrB<br />
mutant makes much less ETX toxin compared to the wild-type parents. Initial animal testing<br />
has shown that the CN3685::agrB mutant is attenuated in its ability to cause hemorrhagic<br />
necrotic enteritis in rabbits or fatal enterotoxemia in mice. Mouse lethality experiments<br />
demonstrated that the CN3718::agrB mutant also exhibits less lethality compared with wildtype<br />
CN3718. The findings support the Agr-like QS system‘s global importance for regulating<br />
C. perfringens toxin production <strong>and</strong> pathogenicity.
O47<br />
GLOBAL GENE EXPRESSION OF EPITHELIAL CELLS FROM AN IN VIVO MODEL OF C.<br />
DIFFICILE TOXIN A AND B INTOXICATION<br />
K.M. D'Auria* 1 , G.L. Kolling 2 , G.M. Donato 2 , C.A. Warren 2 , M.C. Gray 2 , J.A. Papin †1 , E.L.<br />
Hewlett †2 . 1 Department of Biomedical Engineering, 2 Division of Infectious Diseases <strong>and</strong><br />
International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia,<br />
22908 USA.<br />
† Equal contribution<br />
Clostridium difficile Toxins A (TcdA) <strong>and</strong> B (TcdB) cause differing degrees of inflammation in<br />
the same mammalian host, <strong>and</strong> the potency or cytotoxicity of each individual toxin differs<br />
across many hosts <strong>and</strong> cell types. In our previous analysis of gene expression in human<br />
ileocecal cells (HCT-8), the toxins elicited very similar transcriptional responses, with TcdB<br />
inducing greater changes at earlier times. Unexpectedly, very few genes that contribute to<br />
inflammation had altered expression, suggesting that signaling involved in pathology is due<br />
to the interplay of multiple cell types rather than simply an epithelial cell response. To<br />
investigate these effects in vivo, we injected 20 μg of one or both toxins directly into the<br />
cecum of C57 black mice <strong>and</strong> harvested ceca after 2, 6 <strong>and</strong> 16 h. Pathology scores were<br />
significantly higher for TcdA-treated compared to sham mice, <strong>and</strong> this difference increased<br />
with time. Myeloperoxidase staining revealed increased infiltration of granulocytes 6 <strong>and</strong> 16<br />
hours after TcdA <strong>and</strong>/or TcdB injection. Hence, TcdB elicits pathology though not to the<br />
extent of TcdA; TcdA plus TcdB did not cause a significantly greater change than TcdA alone<br />
in any measurement. Cecal epithelial cells were dissociated with EDTA <strong>and</strong> gene expression<br />
quantified using transcriptome-wide microarrays. The number of differentially expressed<br />
genes is more than 5-fold higher in TcdA- vs. TcdB-treated mice. The expression profiles of<br />
TcdA-treated, TcdB-treated, <strong>and</strong> sham mice are all distinct from one another <strong>and</strong> expression<br />
in TcdA/TcdB-treated mice is highly correlated with TcdA-treated mice. Computational<br />
analyses revealed increased expression of many transcription factors <strong>and</strong> many of the genes<br />
with altered expression at 2h are associated with intracellular organelles, such as the<br />
endosome, mitochondria, or Golgi apparatus. At 6 hours, the most significantly affected<br />
functions in TcdA-treated mice are inflammation, defense response, <strong>and</strong> apoptosis. TcdBtreated<br />
mice have fewer changes in inflammation-associated genes, but affected genes are<br />
also associated with intracellular organelles (nuclear lumen <strong>and</strong> Golgi apparatus).<br />
Contrasting TcdA <strong>and</strong> TcdB treatments, several differentially expressed genes include small<br />
GTPases, the targets of toxin-mediated glucosylation. At 16 hours, several TcdA-affected<br />
genes are linked to mitochondrial membrane, peroxisome, <strong>and</strong> apoptosis. Overall, TcdA<br />
induces greater changes in gene expression than TcdB, especially regarding the<br />
inflammatory response, but TcdB does alter physiology <strong>and</strong> distinctly change gene<br />
expression. A more in-depth analysis of the factors <strong>and</strong> affected pathways is currently<br />
leading us to studies clarifying the role of epithelial cells in the development of C. difficileassociated<br />
diarrhea <strong>and</strong> colitis.
O48<br />
COMPARATIVE PROTEOMIC ANALYSES OF EPIDEMIC-ASSOCIATED CLOSTRIDIUM<br />
DIFFICILE STRAINS: IDENTIFICATION OF PROTEINS WITH LIKELY ROLES IN<br />
INTESTINAL COLONIZATION AND DISEASE<br />
G. Vedantam. Deptartment of Veterinary Science <strong>and</strong> Microbiology, University of Arizona,<br />
Tucson, AZ USA; <strong>and</strong> Southern Arizona VA Healthcare System, Tucson, AZ USA.<br />
Clostridium difficile (CD) causes diarrheic disease in humans <strong>and</strong> non-human mammals.The<br />
past decade has seen the emergence of CD ―variant‖ strains that are associated with<br />
outbreaks/epidemics, severe disease, increased recurrence rate(s) <strong>and</strong> community onset of<br />
disease. There is a renewed sense of urgency to define specific virulence traits underlying<br />
the robustness of CD epidemic-associated strains. CD clinical isolates can be resistant to<br />
genetic manipulation; therefore, we employed proteomic analyses to rapidly identify<br />
molecules altered in epidemic-associated CD strains. A mass spectrometry-based<br />
quantitative approach was used to identify differentially expressed proteins in BI-17, an<br />
epidemic-associated CD strain, as compared with BI-1, a "historic" non-epidemic-associated<br />
CD strain, phylogenetically related to BI-17. Once quantitation parameters were optimized,<br />
similar experiments were performed with 13 different epidemic- <strong>and</strong> non-epidemic associated<br />
CD clinical isolates. For all strains, both secreted <strong>and</strong> total cellular proteomes were analyzed<br />
at three growth phases. 2D LC MALDI-TOF/TOF analyses revealed that approximately 550<br />
proteins (~15% of the genome; consistent with other bacteria) were expressed by BI-1 <strong>and</strong><br />
BI-17 in each growth phase. Further analyses, using stringent fold-change cut-off<br />
computations, identified a total of 121 proteins that were differentially abundant between BI-1<br />
<strong>and</strong> BI-17. Consistent with our published studies, epidemic-associated strains showed only<br />
marginal increases, if at all, in toxin (A & B) levels. In contrast, functional classification<br />
analyses revealed that non-toxin CD proteins constituted the most differentially abundant<br />
molecules; these included surface proteins, transporters <strong>and</strong> host innate immunity<br />
modulators. Similar, <strong>and</strong> extremely consistent results, were obtained for the other epidemicassociated<br />
CD strains tested. Alteration of phenotypes known to be dependent on some of<br />
the specifically dysregulated molecules was also experimentally confirmed. Epidemicassociated<br />
CD strains showed increased resistance to host innate immune system<br />
molecules, increased host-cell adherence, altered surface morphology, <strong>and</strong> altered<br />
sporulation/germination profiles compared with the genetically-related non-epidemicassociated<br />
strain. Our proteomic-based approach may thus be useful in identifying attractive<br />
targets for CDI intervention focused on preventing GI-tract establishment of C. difficile.
O49<br />
MOTILITY AND ADHERENCE TO MYOBLASTS<br />
Andrea Hartman, Brittany Gianetti, <strong>and</strong> Stephen Melville*. Department of Biological<br />
Sciences, Virginia Tech, Blacksburg, VA 24061, USA.<br />
The anaerobic pathogen Clostridium perfringens possesses a unique type IV pilus (TFP)-<br />
mediated gliding motility. We have recently shown that TFP are necessary for adherence of<br />
C. perfringens to rodent myoblasts (muscle cells), which is likely an important feature of this<br />
bacterium‘s ability to cause rapidly spreading gas gangrene. On the surface of agar plates,<br />
elongated C. perfringens move in a group, aligned end-to-end <strong>and</strong>-side to-side, pushing<br />
themselves away from the mother colony as they grow. The filaments must remain intact for<br />
motility to occur. We used immunofluorescence to identify the two major pilins as colocalizing<br />
on the surface at the poles of the cells. We also used fluorescently tagged proteins<br />
responsible for TFP assembly to track their movement <strong>and</strong> organization in the cells as they<br />
grow <strong>and</strong> divide. C. perfringens possesses two PilA (the pilin monomer), two PilB (the<br />
assembly ATPase), <strong>and</strong> two PilC (an essential assembly protein) homologs, but has just one<br />
PilT (a pilus retraction ATPase) homolog. We created translational fusions of yellow<br />
fluorescent protein (YFP) or cyan fluorescent protein (CFP) to both PilB proteins <strong>and</strong> PilT,<br />
expressed pairs of these fusions in C. perfringens <strong>and</strong> viewed them with fluorescent<br />
microscopy in time-lapse anaerobic videos. We found the two PilB proteins co-localized at<br />
the poles of cells <strong>and</strong> at points which later develop into division septa. PilT frequently<br />
localized to the same locations as the PilB proteins, but also moved independently <strong>and</strong><br />
rapidly throughout the cells in a helical pattern, localizing to sites that correspond with CFPtagged<br />
FtsA, a septal division protein. We have shown previously that the pilT gene is found<br />
in an operon with the ftsA <strong>and</strong> ftsZ genes in C. perfringens. These findings were used to<br />
develop a comprehensive model for the roles <strong>and</strong> locations of the TFP proteins as the cells<br />
grow <strong>and</strong> divide, in which the division septa proteins FtsA <strong>and</strong> FtsZ provide the localization<br />
signal for TFP proteins to assemble so they can synthesize pili, which keep the filaments<br />
intact <strong>and</strong> anchor them to the surface the cells are gliding on.
O50<br />
ANTIGENIC AND PHASE VARIATION IN CELL WALL PROTEINS OF CLOSTRIDIUM<br />
DIFFICILE<br />
N. F. Fairweather*. Department of Life Sciences <strong>and</strong> Centre for Molecular Microbiology <strong>and</strong><br />
Infection, Imperial College London, London SW7 2AZ, UK.<br />
Clostridium difficile, a Gram-positive spore-forming anaerobe, is the main cause of<br />
nosocomial antibiotic-associated diarrhea in Europe <strong>and</strong> the US. Two toxins, TcdA <strong>and</strong><br />
TcdB, are produced by most strains <strong>and</strong> mediate damage to host cells through glucosylation<br />
of host GTPases. The epidemiology of C. difficile has changed over the last 10 years with<br />
the emergence of several new endemic strains. Spores of C. difficile are highly infectious<br />
<strong>and</strong> are difficult to eradicate from the environment. Spores germinate within the intestine <strong>and</strong><br />
the vegetative cells proliferate producing toxins <strong>and</strong> other virulence factors. However, little is<br />
known of the mechanisms employed by C. difficile to colonize the enteric system.<br />
Colonization would seem to be essential for longevity of infection <strong>and</strong> for efficient production<br />
of toxins. C. difficile expresses a proteinaceous S-layer array on its cell surface consisting<br />
primarily of the major S-layer protein, SlpA. A family of SlpA paralogs is also found,<br />
collectively known as cell wall proteins (CWPs). We have investigated the function of several<br />
CWPs <strong>and</strong> have thrown light on their putative roles in cell wall biogenesis <strong>and</strong> virulence.<br />
One example is the cysteine protease Cwp84; this cleaves the SlpA precursor to yield the<br />
mature S-layer proteins that then form a complex on the cell surface. Mutants in cwp84 are<br />
viable but produce an altered cell wall that is defective in retention of some CWPs. CwpV is<br />
the largest member of the CWP family <strong>and</strong> is expressed in a phase variable manner;<br />
approximately 5% of cells in culture express CwpV. Phase variation is mediated by DNA<br />
inversion that requires the recombinase RecV. We have shown that CwpV promotes C.<br />
difficile aggregation, <strong>and</strong> that this activity is mediated by the C-terminal repetitive domain.<br />
This domain varies markedly between strains; five distinct repeat types have been identified<br />
<strong>and</strong> are antigenically distinct. Thus, CwpV function, regulation, <strong>and</strong> processing are highly<br />
conserved across C. difficile strains, whilst the functional domain exists in at least five<br />
antigenically distinct forms. Conservation of CwpV function <strong>and</strong> its phase-variable<br />
expression hint at a role for this protein in pathogenesis.
O51<br />
SIALIDASES AFFECT THE HOST CELL ADHERENCE AND EPSILON TOXIN-INDUCED<br />
CYTOTOXICITY OF CLOSTRIDIUM PERFRINGENS TYPE D STRAIN CN3718<br />
J. Li* 1 , S. Sayeed 2 , S. Robertson 1 , J. Chen 1 , <strong>and</strong> B. A. McClane 1 . 1 Department of<br />
Microbiology <strong>and</strong> Molecular Genetics, University of Pittsburgh School of Medicine,<br />
Pittsburgh, PA, USA; 2 Department of Environmental <strong>and</strong> Occupational Health, University of<br />
Pittsburgh, PA, USA.<br />
Clostridium perfringens type B or D isolates, which cause natural enterotoxemias in livestock,<br />
produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class<br />
B select toxin. These C. perfringens strains also express sialidases, although the possible<br />
contributions of those enzymes to type D pathogenesis remain unclear. Type D isolate<br />
CN3718 carries two genes (nanI <strong>and</strong> nanJ) encoding secreted sialidases <strong>and</strong> one gene<br />
(nanH) encoding a cytoplasmic sialidase. Construction of single nanI, nanJ <strong>and</strong> nanH null<br />
mutants, as well as a nanI/nanJ double null mutant <strong>and</strong> a triple sialidase null mutant, in<br />
CN3718 identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells<br />
with NanI sialidase, or with C. perfringens culture supernatants containing NanI, enhanced<br />
the binding <strong>and</strong> cytotoxic effects of ETX. Contact of CN3718 with Caco-2 cells induced more<br />
rapid sialidase production. Inactivation of nanI reduced ETX production by CN3718; this<br />
effect was fully reversible by complementation with the wildtype nanI. It was also partially<br />
reversible by supplementation of cultures with sialic acid, suggesting that sialic acid could<br />
signal upregulation of ETX production. NanI production also substantially increased CN3718<br />
adherence to enterocyte-like Caco-2 cells. Finally, the sialidase activity of NanI (but not NanJ<br />
or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that,<br />
during type B/D diseases originating in the intestines, trypsin may activate NanI, which (in<br />
turn) could contribute to intestinal colonization by C. perfringens type D isolates <strong>and</strong> also<br />
increase ETX production <strong>and</strong> action.
O52<br />
EXTRA-CYTOPLASMIC FUNCTION SIGMA FACTOR CSFV REGULATES LYSOZYME<br />
RESISTANCE OF CLOSTRIDIUM DIFFICILE<br />
T. D. Ho <strong>and</strong> C.D. Ellermeier*. University of <strong>Iowa</strong>, Department of Microbiology, <strong>Iowa</strong> City, IA<br />
USA.<br />
Clostridium difficile is an anaerobic, Gram-positive, spore-forming opportunistic pathogen<br />
which is the most common cause of hospital-acquired infectious diarrhea. In numerous<br />
pathogens, stress response mechanisms are required for survival within the host. Extra-<br />
Cytoplasmic Function sigma (σ) factors (ECF σ factors) are a major family of signal<br />
transduction systems which sense <strong>and</strong> respond to extracellular stresses. Activity of C.<br />
difficile ECF σ CsfV is induced in response to lysozyme, a substantial component of innate<br />
immunity. To further characterize the role of CsfV in responding to lysozyme, we generated<br />
a C. difficile mutant in csfV. The csfV mutant exhibited a marked decrease in lysozyme<br />
resistance when compared to the wild type C. difficile parent. Interestingly, transmission<br />
electron micrographs revealed that csfV mutant cells were irregularly shaped with<br />
impaired/damaged cell envelope only in the presence of sub-inhibitory levels of lysozyme. In<br />
in vivo hamster experiments, the C. difficile csfV mutant was significantly attenuated<br />
compared to the wild type, indicating that CsfV is critical during C. difficile infection. Using<br />
quantitative RT-PCR we established that CsfV regulates expression of a putative<br />
polysaccharide deacetylase (pdaV) <strong>and</strong> a presumed post-translational secreted protein<br />
chaperone (prsA2). When pdaV was over-expressed in Bacillus subtilis, PdaV conferred an<br />
increase in lysozyme resistance. These data suggest that, in C. difficile, PdaV may play a<br />
role in lysozyme resistance which is regulated by the CsfV signal transduction system.<br />
Taken together, our data imply that the CsfV signal transduction system is important during<br />
C. difficile infection, likely as a means to control mechanisms for surviving host immune<br />
system assaults such as lysozyme.
POSTER OVERVIEW
Last Name<br />
Title<br />
Auchtung, Jennifer P1 HIGH THROUGHPUT ANALYSIS OF INTERACTIONS<br />
BETWEEN THE HUMAN FECAL MICROBIOME AND<br />
CLOSTRIDIUM DIFFICILE<br />
Babakhani, Farah P2 FIDAXOMICIN INHIBITS PRODUCTION OF TOXIN A<br />
AND TOXIN B IN CLOSTRIDIUM DIFFICILE<br />
Babakhani, Farah P3 FIDAXOMICIN INHIBITS SPORE PRODUCTION IN<br />
CLOSTRIDIUM DIFFICILE<br />
Bakker, Dennis P4 MUTATION OF tcdC IN C. DIFFICILE 630∆ERM DOES<br />
NOT INFLUENCE TOXIN EXPRESSION<br />
Bouillaut, Lauren P5 REGULATION OF AMINO ACID FERMENTATION IN<br />
CLOSTRIDIUM DIFFICILE<br />
Carlson, Paul P6 PHENOTYPIC CHARACTERIZATION OF<br />
CLOSTRIDIUM DIFFICILE CLINICAL ISOLATES<br />
Cartman, Stephen P7 PRECISE MANIPULATION OF THE CLOSTRIDIUM<br />
DIFFICILE CHROMOSOME TO EXPLORE THE ROLE<br />
OF TCDC IN TOXIN PRODUCTION<br />
Collignon, Anne P8 FLAGELLIN OF CLOSTRIDIUM DIFFICILE 027<br />
HYPERVIRULENT STRAIN ACTIVATES ERK1/2<br />
MAPK IN AN EPITHELIAL TLR-5-EXPRESSING CELL<br />
LINE<br />
Collignon, Anne P9 ANALYSIS OF THE GENOME-WIDE EXPRESSION<br />
OF A CLOSTRIDIUM DIFFICILE FLIC MUTANT IN<br />
MONOXENIC MICE<br />
Collignon, Anne P10 PROTEOMIC ANALYSIS OF THE CLOSTRIDIUM<br />
DIFFICILE 630 STRAIN SECRETOME ACCORDING<br />
TO GROWTH KINETICS<br />
Corver, Jeroen P11 ANALYSIS OF A CLOSTRIDIUM DIFFICILE PCR<br />
RIBOTYPE 078 100 KILOBASE ISLAND REVEALS<br />
THE PRESENCE OF A NOVEL TRANSPOSON<br />
Curry, Scott P12 A PILOT STUDY OF CLOSTRIDIUM DIFFICILE IN<br />
RAW RETAIL MEATS FROM WESTERN<br />
PENNSYLVANIA
Figueroa, Iris P13 RELAPSE VERSUS REINFECTION: TIMING AND<br />
INFLUENCE OF THE INFECTING STRAIN ON<br />
RECURRENT CLOSTRIDIUM DIFFICILE INFECTION<br />
Heeg, Daniela P14 SPORULATION RATES AND THE<br />
'HYPERVIRULENCE' OF CLOSTRIDIUM DIFFICILE:<br />
STANDARDISING EXPERIMENTAL PROCEDURES<br />
FOR ACCURATE STRAIN COMPARISON<br />
Ho, Theresa P15 EXTRA-CYTOPLASMIC FUNCTION SIGMA FACTOR<br />
CSFV REGULATES LYSOZYME RESISTANCE OF<br />
CLOSTRIDIUM DIFFICILE<br />
Janezic, S<strong>and</strong>ra P16 CLOSTRIDIUM DIFFICILE 16S-23S rRNA<br />
INTERGENIC SPACER REGION (ISR) AS A MARKER<br />
FOR PHYLOGENETIC STUDIES<br />
Kõljalg, Siiri P17 SPORULATION LEVEL OF CLOSTRIDIUM DIFFICILE<br />
INTEGRN POSITIVE AND NEGATIVE STRAINS IN<br />
THE PRESENCE OF LACTOBACILLI<br />
Lei, Xiang P18 USING PHENOTYPE MICROARRAYS TO<br />
DETERMINE CULTURE CONDITIONS THAT INDUCE<br />
OR REPRESS TOXIN PRODUCTION BY<br />
CLOSTRIDIUM DIFFICILE<br />
Liu, Mingyu P19 INCREASED BILE SALTS IN THE CECA OF<br />
ANTIBIOTIC-TREATED MICE BY FLAGELLIN<br />
ADMINISTRATION INHIBIT VEGETATIVE<br />
REPLICATION OF CLOSTRIDIUM DIFFICILE<br />
Mallozzi, Michael P20 SURVEILLANCE AND MOLECULAR<br />
CHARACTERIZATION OF CLOSTRIDIUM DIFFICILE<br />
INFECTIONS IN TUCSON<br />
Marsh, Jane P21 CHARACTERIZATION OF RECURRENT<br />
CLOSTRIDIUM DIFFICILE DISEASE ISOLATES BY<br />
MULTI-LOCUS VARIABLE NUMBER TANDEM<br />
REPEAT ANALYSIS AND TOXIN SEQUENCING<br />
Marsh, Jane P22 GENETIC DIVERSITY OF NON-TOXIGENIC<br />
CLOSTRIDIUM DIFFICILE DETERMINED BY MULTI-<br />
LOCUS VARIABLE NUMBER TANDEM REPEAT<br />
ANALYSIS AND MULTI-LOCUS SEQUENCE TYPING
Miezeiewski,<br />
Matthew<br />
P23 AN IN VITRO MODEL TO STUDY COLONIC<br />
MICROBIOTA DISRUPTION IN RELATION TO<br />
INFECTION WITH CLOSTRIDIUM DIFFICILE IN<br />
SYRIAN GOLDEN HAMSTERS<br />
Moura, Hercules P24 PROTEOMIC ANALYSIS OF THE Clostridium difficile<br />
LARGE TOXINS<br />
Naaber, Paul P25 STRAIN-SPECIFIC SUSCEPTIBILITY OF<br />
CLOSTRIDIUM DIFFICILE TO INHIBITORY ACTIVITY<br />
OF LACTOBACILLUS PLANTARUM IN VITRO<br />
Olling, Alex<strong>and</strong>ra P26 ROLE OF THE CROP DOMAIN OF CLOSTRIDIUM<br />
DIFFICILE TOXINS A AND B IN BINDING AND<br />
UPTAKE<br />
Paredes-Sabja,<br />
Daniel<br />
Quesada-Gómez,<br />
Carlos<br />
P27<br />
P28<br />
ADHERENCE OF CLOSTRIDIUM DIFFICILE SPORES<br />
TO HUMAN COLONIC ENTEROCYTE-LIKE CACO-2<br />
CELLS<br />
FLUOROQUINOLONE RESISTANCE, BUT NOT<br />
TOXIN HYPERPRODUCTION, CORRELATES WITH<br />
THE INCREASED FINDING OF A NOVEL<br />
PULSOTYPE OF VIRULENT CLOSTRIDIUM<br />
DIFFICILE STRAINS<br />
Reeves, Angela P29 ROLE OF MEMBERS OF THE RESIDENT GUT<br />
MICROBIOTA IN LIMITING CLOSTRIDIUM DIFFICILE<br />
GROWTH AND TOXIN PRODUCTION<br />
Robinson, Cathy P30 IDENTIFICATION AND CHARACTERIZATION OF<br />
GENES SPECIFIC TO HYPERVIRULENT NAP1<br />
CLOSTRIDIUM DIFFICILE STRAINS<br />
Scholl, Dean P31 PHAGE TAIL-LIKE BACTERIOCINS OF<br />
CLOSTRIDIUM DIFFICILE<br />
Schoster, Angelika P32 EPIDEMIOLOGIC INVESTIGATION OF<br />
CLOSTRIDIUM DIFFICILE AND CLOSTRIDIUM<br />
PERFRINGENS IN HEALTHY HORSES<br />
Shin, Bo-Moon P33 THE PREVALENCE OF Clostridium difficile AND<br />
Clostridium perfringens AS PATHOGENS OF ACUTE<br />
DIARRHEA IN PATIENTS VISITING A TERTIARY<br />
CARE HOSPITAL IN KOREA
Shin, Bo-Moon P34 COMPARISON OF TWO PCR ASSAYS FOR TOXIN B<br />
FOR DIRECT DETECTION OF TOXIN PRODUCING<br />
Clostridium difficile IN FECAL SPECIMENS<br />
Sorg, Joseph P35 EFFECT OF INCREASED FECAL<br />
CHENODEOXYCHOLIC ACID LEVELS ON C.<br />
DIFFICILE VIRULENCE<br />
Squire, Michele P36 DETECTION OF CLOSTRIDIUM DIFFICILE IN<br />
PIGGERY EFFLUENT AFTER TREATMENT IN A 2-<br />
STAGE POND SYSTEM<br />
Theriot, Casey P37 COMPARATIVE PATHOGENICITY OF CLOSTRIDIUM<br />
DIFFICILE STRAINS IN CEFOPERAZONE-TREATED<br />
MICE<br />
von Eichel-Streiber,<br />
Christoph<br />
P38<br />
MONOCLONAL ANTIBODIES TO SPECIFICALLY<br />
IDENTIFY TOXIN B OF CLOSTRIDIUM DIFFICILE<br />
RIBOTYPE 027<br />
Walk, Seth P39 A PHYLOGENETIC ANALYSIS OF THE NEGATIVE<br />
TOXIN REGULATOR (TcdC) OF CLOSTRIDIUM<br />
DIFFICILE<br />
Wright, Lorinda P40 EFFECT OF PROTON PUMP INHIBITORS WITH AND<br />
WITHOUT ANTIBIOTICS ON CLOSTRIDIUM<br />
DIFFICILE INFECTION IN HAMSTERS<br />
Blasi, Juan P41 HIGH AFFINITY BINDING OF CLOSTRIDIUM<br />
PERFRINGENS EPSILON TOXIN TO THE RENAL<br />
SYSTEM<br />
Butel, Marie-José P42 CLOSTRIDIAL GUT COLONIZATION IN PRETERM<br />
NEONATES<br />
Derman, Yagmur P43 csps PLAY A ROLE IN NaCl AND pH STRESS<br />
RESPONSE OF CLOSTRIDIUM BOTULINUM ATCC<br />
3502<br />
Farias, Luana P44 MOLECULAR DIAGNOSIS OF BLACKLEG FROM<br />
COMMON FILTER PAPER<br />
Garcia, Jorge P45 THE EFFECT OF CLOSTRIDIUM PERFRINGENS<br />
TYPE C AND ITS BETA TOXIN MUTANT IN GOATS<br />
Goossens, Evy P46 CLOSTRIDIUM PERFRINGENS STRAINS OF<br />
VARIOUS ORIGIN CAN CAUSE HEMORRHAGIC<br />
ENTERITIS IN A CALF INTESTINAL LOOP MODEL.
Govoni, Gregory P47 A FUNCTIONAL, HIGH MOLECULAR WEIGHT<br />
BACTERIOCIN (―DIFFOCIN‖) FROM CLOSTRIDIUM<br />
DIFFICILE CLONED AND EXPRESSED IN BACILLUS<br />
SUBTILIS<br />
Kelly, Michelle P48 IMPROVING THE REPRODUCIBILITY OF INFECTION<br />
OF THE NAP1/B1/027 HYPERVIRULENT STRAIN<br />
R20291 IN THE HAMSTER MODEL OF INFECTION.<br />
Keto-Timonen,<br />
Riikka<br />
P49<br />
AMPLIFIED FRAGMENT LENGTH POLYMORPHISM<br />
ANALYSIS IN STRAIN TYPING AND<br />
IDENTIFICATION OF CLOSTRIDIUM SPECIES<br />
Lahti, Päivi P50 COMPARATIVE GENOMIC HYBRIDIZATION<br />
ANALYSIS SHOWS DIFFERENT EPIDEMIOLOGY OF<br />
CHROMOSOMAL AND PLASMID-BORNE cpe-<br />
CARRYING C. PERFRINGENS TYPE A STRAINS<br />
Lambert, Dominic P51 CHARACTERIZATION OF SURFACE-LAYER<br />
PROTEINS OF CLOSTRIDIUM BOTULINUM GROUP I<br />
AND II<br />
Lepp, Dion P52 IDENTIFICATION OF C. PERFRINGENS GENES<br />
ASSOCIATED WITH AVIAN NECROTIC ENTERITIS<br />
BY MICROARRAY COMPARATIVE GENOMIC<br />
HYBRIDIZATION<br />
Moura, Hercules P53 APPLYING TOXIN PROTEOMICS TO MEASURE<br />
RELATIVE QUANTITIES OF PROTEINS WITHIN THE<br />
BOTULINUM NEUROTOXIN COMPLEX<br />
Nowell, Victoria P54 GENOMIC AND PROTEOMIC ANALYSIS OF A<br />
BOVINE HEMORRHAGIC ABOMASITIS TYPE A<br />
CLOSTRIDIUM PERFRINGENS ISOLATE<br />
Shi, Lianfa P55 A FRAGMENT OF 97 AMINO ACIDS (D97) WITHIN<br />
THE TRANSMEMBRANE DOMAIN IS ESSENTIAL<br />
FOR THE CELLULAR ACTIVITY OF CLOSTRIDIUM<br />
DIFFICILE TOXIN B<br />
Vargas, Agueda P56 NECROTIZING ENTERITIS ASSOCIATED WITH<br />
CLOSTRIDIUM PERFRINGENS TYPE B IN<br />
CHINCHILLAS (CHINCHILLA LANIGERA)<br />
Yan, Xuxia P57 DETERMINATION OF FUNCTIONAL RESIDUES ON<br />
NETB TOXIN FROM CLOSTRIDIUM PERFRINGENS
Yumine, Natsuko P58 SEAFOOD AS A RESERVOIR AND A SUPPLIER OF<br />
THE PROTOTYPE PLASMID ENCODING<br />
CLOSTRIDIUM PERFRINGENS ENTEROTOXIN<br />
Rupnik, Maja P59 COMPARISON OF CHANGES IN HUMAN AND<br />
POULTRY INTESTINAL MICROBIOTA DURING<br />
COLONISATION WITH CLOSTRIDIUM DIFFICILE<br />
Ardis, Tara C. P60 DEVELOPMENT OF A QUANTITATIVE IMMUNO-PCR<br />
ASSAY FOR THE DETECTION OF GROUP III<br />
CLOSTRIDIUM BOTULINUM NEUROTOXINS IN<br />
CATTLE<br />
Sadighi Akha, Amir P61 THE LOCAL AND SYSTEMIC IMMUNE RESPONSE IN<br />
A MOUSE MODEL OF ACUTE CLOSTRIDIUM<br />
DIFFICILE INFECTION<br />
Zemljič, Majeta P62 PORCELLIO SCABER – A NEW NONVERTEBRATE<br />
MODEL FOR C. DIFFICILE COLONIZATION
ABSTRACTS OF POSTER PRESENTATIONS<br />
SESSION I: P1 TO P20<br />
WEDNESDAY, OCTOBER 26, 2011
P1<br />
HIGH THROUGHPUT ANALYSIS OF INTERACTIONS BETWEEN THE HUMAN FECAL<br />
MICROBIOME AND CLOSTRIDIUM DIFFICILE<br />
J. Auchtung* 1 , C. Robinson 1 , R. Stedtfeld 2 , S. Hashsham 2 , <strong>and</strong> R. Britton 1 . 1 Department of<br />
Microbiology <strong>and</strong> Molecular Genetics <strong>and</strong> 2 Department of Civil <strong>and</strong> Environmental<br />
Engineering, Michigan <strong>State</strong> University, East Lansing, MI 48824, USA.<br />
We have developed continuous-flow microbioreactors that allow us to study the interaction<br />
between human fecal microbial communities <strong>and</strong> Clostridium difficile in vitro. The small size<br />
of these reactors (15 ml culture volume) allows up to 36 individual reactors to operate in<br />
parallel, providing more flexibility to examine multiple conditions simultaneously. Preliminary<br />
experiments have shown that when a pool of human fecal samples is used to inoculate<br />
microbioreactors containing complex media, stable microbial communities composed of<br />
species typically found in the human gut microbiome are established. Furthermore, these<br />
communities can inhibit the growth of C. difficile. Ongoing <strong>and</strong> future experiments will<br />
examine the how the perturbation of the microbiota with antibiotics makes them susceptible<br />
to C. difficle invasion. Specifically, we plan to analyze how changes in microbial community<br />
structure (as assayed through 16S rDNA <strong>and</strong> metagenomic sequencing) <strong>and</strong> function (as<br />
assayed through metatranscriptomics <strong>and</strong> metabolomics) correlate with the ability of C.<br />
difficile to invade <strong>and</strong> express virulence functions.
P2<br />
FIDAXOMICIN INHIBITS PRODUCTION OF TOXIN A AND TOXIN B IN CLOSTRIDIUM<br />
DIFFICILE<br />
L. Bouillaut 1 , C. Sims 2 , A. Gomez 2 , P. Sears 2 , J. Seddon 2 , A.L. Sonenshein 1 , <strong>and</strong> F.<br />
Babakhani* 2 . 1 Tufts University School of Medicine, Boston, MA 021111, USA; 2 Optimer<br />
Pharmaceuticals, Inc., San Diego, CA 921212, USA.<br />
Fidaxomicin, recently approved for the treatment of C. difficile associated diarrhea,<br />
possesses bactericidal activity against C. difficile <strong>and</strong> was superior in clinical trials to<br />
vancomycin (VAN) in its sustained clinical response. In this study, we showed its mechanism<br />
of antimicrobial activity to be via inhibition of RNA polymerase (RNAP). Since this<br />
mechanism may also lead to inhibition of virulence factors, we examined toxin levels <strong>and</strong><br />
toxin gene expressions following exposure to FDX. Transcription inhibition studies were<br />
performed with purified C. difficile RNAP in presence of FDX. Impact of FDX on toxin<br />
production was determined by adding drugs to C.difficile ATCC 43255 (a high toxin<br />
producing strain) <strong>and</strong> UK-14 (NAP1/BI/027 epidemic strain) at early stationary phase of<br />
growth followed by incubation ~7 days. Culture supernatants were tested by commercial<br />
ELISA-tgcBiomics for the presence of toxins A <strong>and</strong> B. Toxin gene expression was assessed<br />
in presence of drugs (qRT-PCR) in C. difficile UK1 (NAP1/BI/027 type epidemic strain) <strong>and</strong> in<br />
its close non-epidemic relative, CD196. Toxin gene transcript levels were normalized to<br />
levels of 16s rRNA <strong>and</strong> rpoC mRNA. Toxin production in the absence of drugs peaked<br />
between 3 <strong>and</strong> 5 days in C. difficile ATCC 43255, however, in the presence of FDX <strong>and</strong> its<br />
major metabolite OP-1118 (both at 1/4xMIC), production of TcdB <strong>and</strong> TcdA levels were<br />
strongly suppressed. In contrast, VAN did not inhibit toxin production. Similar inhibitory<br />
effects on TcdA <strong>and</strong> TcdB expression by FDX <strong>and</strong> OP-1118 but not VAN, or metronidazole<br />
(MTZ) were seen in the UK-14 strain. Addition of FDX (2xMIC) or OP-1118 (2.5xMIC) at the<br />
end of exponential growth phase led to nearly complete inhibition of subsequent<br />
accumulation of transcripts from the pathogenicity locus, i.e. tcdR, tcdA, tcdB <strong>and</strong> tcdC. By<br />
contrast, addition of vancomycin ( 2.5xMIC) at the end of exponential growth phase had little<br />
or no significant effect on subsequent toxin production. Transcription inhibition studies with<br />
C. difficile<br />
in toxin production may be mediated via the inhibition of RNAP.<br />
Results demonstrate that both FDX <strong>and</strong> OP-1118, but not VAN or MTZ, block C. difficile toxin<br />
synthesis even when added to stationary phase cells.
P3<br />
FIDAXOMICIN INHIBITS SPORE PRODUCTION IN CLOSTRIDIUM DIFFICILE<br />
A. Gomez 1 , L. Bouillaut 2 , A.L. Sonenshein 2 , P. Sears 1 , L. Nguyen 1 , <strong>and</strong> F. Babakhani* 1 .<br />
1 Optimer Pharmaceuticals, Inc., San Diego, CA 921211, USA; 2 Tufts University School of<br />
Medicine, Boston, MA 021112, USA.<br />
Introduction: Fidaxomicin was recently approved for the treatment of Clostridium difficile<br />
associated diarrhea. This is a novel narrow spectrum antibacterial agent that demonstrates<br />
potent bactericidal activity against C. difficile. In recent clinical trials with over 1100 CDI<br />
subjects, FDX was shown to be superior to vancomycin in providing sustained clinical<br />
response (response without recurrence of disease) at 25 days following therapy. A possible<br />
mechanism for reduced recurrences is that FDX may exert an inhibitory effect on sporulation.<br />
Methods: FDX <strong>and</strong> its major metabolite, OP-1118, were evaluated with comparator drugs<br />
vancomycin, metronidazole, <strong>and</strong> rifaximin for their effects on kinetics of C. difficile (UK-14, an<br />
epidemic NAP1/BI/027 strain <strong>and</strong> ATCC 43255, a high toxin producer strain) growth <strong>and</strong><br />
sporulation. Drugs were added to cells at early stationary phase of growth followed by<br />
collection of cells, at various time intervals, for quantitation of total viable cell count <strong>and</strong> spore<br />
counts (by heating to kill the vegetative cells) on media containing taurocholate. The effect of<br />
drugs on sporulation gene expression in the C. difficile NAP1/BI/027 type epidemic strain<br />
UK1 was also compared via qRT-PCR using Roche LightCycler 480 <strong>and</strong> SYBR Green<br />
fluorescence. Transcript levels for test genes were normalized to levels of 16s rRNA <strong>and</strong><br />
rpoC mRNA. Results: Both FDX <strong>and</strong> OP-1118 at sub-MIC concentrations (1/4xMIC) inhibited<br />
sporulation when added to early stationary phase cells in C. difficile strains UK-14 <strong>and</strong> ATCC<br />
432455. In contrast, vancomycin, metronidazole <strong>and</strong> rifaximin (at similar sub-MIC levels) did<br />
not inhibit sporulation. The number of spores following treatment with comparator drugs<br />
increased to the same level as the no-drug control treatment. Expression of mother cellspecific<br />
(spoIIID) <strong>and</strong> forespore-specific (spoIIR) sporulation genes was also inhibited by<br />
fidaxomicin <strong>and</strong> OP-1118, but not by vancomycin. Conclusion: Both FDX <strong>and</strong> OP-1118,<br />
unlike vancomycin, rifaximin, or metronidazole, effectively inhibited sporulation by C. difficile.<br />
Fidaxomicin‘s inhibitory effect on C. difficile sporulation may contribute to its superior<br />
performance in sustaining clinical response <strong>and</strong> reducing recurrences <strong>and</strong> may also be<br />
beneficial in decreasing shedding <strong>and</strong> transmission of this pathogen.
P4<br />
MUTATION OF tcdC IN C. DIFFICILE 630∆ERM DOES NOT INFLUENCE TOXIN<br />
EXPRESSION<br />
D. Bakker*, W.K. Smits, E.J. Kuijper <strong>and</strong> J. Corver. Department of Medical Microbiology,<br />
Center of Infectious Diseases, Leiden University Medical Center, the Netherl<strong>and</strong>s<br />
The main virulence factors of the enteropathogen Clostridium difficile are toxins A <strong>and</strong> B. The<br />
pathogenicity locus (PaLoc) contains the genes coding for toxin A (tcdA) <strong>and</strong> toxin B (tcdB),<br />
flanked by the genes for the positive regulator TcdR <strong>and</strong> the negative regulator TcdC.<br />
Currently, there is a debate about the role of TcdC as a regulator of toxin expression. Our<br />
aim is to clarify the role of tcdC. For this purpose we generated a Clostron-based mutant of<br />
tcdC in Clostridium difficile strain 630∆Erm. Clostridium difficile 630∆Erm (wt) <strong>and</strong> the C.<br />
difficile CT::tcdC (∆tcdC) mutant were grown under anaerobic conditions in pre-reduced<br />
Brain Heart Infusion broth supplemented with Yeast extract <strong>and</strong> L-cysteine. Samples for RNA<br />
preparation <strong>and</strong> toxin detection were taken 1, 2, 5, 7, 24, <strong>and</strong> 48 h post inoculation.<br />
Synthesized cDNA was used in a real-time PCR for detecting transcription levels of the<br />
PaLoc genes <strong>and</strong> compared to the levels of toxin expression in a cytotoxicity/MTS assay<br />
measuring the viability of the cells. We find that transcription levels of the PaLoc genes are<br />
growth dependent. The early logarithmic phase is associated with high transcription levels of<br />
tcdC <strong>and</strong> low level expression of transcription levels of tcdA, tcdB <strong>and</strong> tcdR. The late<br />
logarithmic <strong>and</strong> stationary growth phases are associated with low transcription levels of tcdC<br />
<strong>and</strong> high transcription levels of tcdA, tcdB, <strong>and</strong> tcdR. The transcription levels of the PaLoc<br />
genes in the ∆tcdC strain are similar to the transcription levels in the wt strain. Furthermore,<br />
we detected increasing levels of toxin expression in time <strong>and</strong> the toxin expression levels<br />
were similar in both strains. We demonstrated an inverse correlation between tcdC <strong>and</strong> the<br />
other PaLoc genes. The increasing transcription levels of tcdA, tcdB, <strong>and</strong> tcdR correlates<br />
with increased toxin expression. These data are consistent with prevailing models. However,<br />
the similar transcription levels of the PaLoc genes <strong>and</strong> toxin expression levels in both wt <strong>and</strong><br />
tcdC strains indicate that TcdC is not a major (negative) regulator of toxin expression under<br />
the conditions tested.
P5<br />
REGULATION OF AMINO ACID FERMENTATION IN CLOSTRIDIUM DIFFICILE<br />
L. Bouillaut* 1 , W. Self 2 , <strong>and</strong> A.L. Sonenshein 1 . Molecular Biology <strong>and</strong> Microbiology, Tufts<br />
University; 2 Molecular Biology <strong>and</strong> Microbiology, University of Central Florida.<br />
Stickl<strong>and</strong> fermentation reactions are key to growth of C. difficile. In Stickl<strong>and</strong> reactions, pairs<br />
of amino acids donate <strong>and</strong> accept electrons, generating ATP <strong>and</strong> reducing power in the<br />
process. Reduction of the electron acceptors proline <strong>and</strong> glycine requires the proline<br />
reductase (Prd) <strong>and</strong> the glycine reductase (Grd) enzyme complexes, respectively. Addition of<br />
proline to the medium increases the level of Prd protein but decreases the level of Grd<br />
protein <strong>and</strong> synthesis of toxins. Upstream of the prd operon, we identified a putative<br />
regulatory gene, prdR. To investigate PrdR function, we constructed a C. difficile prdR<br />
mutant using Targetron technology. Our analysis revealed that PrdR is responsible for<br />
activation of the transcription of the Prd-encoding genes in the presence of proline. We<br />
conclude that PrdR is a proline-activated activator. Furthermore, transcripts of Grd genes<br />
were more abundant in the prdR mutant strain compared to the wild type, suggesting a role<br />
for PrdR in the glycine utilization pathway as well. The results suggest that PrdR is a central<br />
metabolism regulator that controls preferential utilization <strong>and</strong> fermentation of proline <strong>and</strong><br />
glycine to produce energy via the Stickl<strong>and</strong> reaction. Given the important role of proline in<br />
toxin synthesis, we are investigating the role of PrdR in toxin gene expression.
P6<br />
PHENOTYPIC CHARACTERIZATION OF CLOSTRIDIUM DIFFICILE CLINICAL ISOLATES<br />
P. E. Carlson Jr*, <strong>and</strong> P. C. Hanna. Department of Microbiology <strong>and</strong> Immunology, University<br />
of Michigan, Ann Arbor, MI 48104 USA.<br />
Clostridium difficile (Cd) causes between 1 <strong>and</strong> 3 million cases of diarrhea <strong>and</strong> colitis in the<br />
United <strong>State</strong>s each year at an annual cost of $1.1B dollars for nosocomial infections alone. It<br />
is likely that Cd spores, <strong>and</strong> not the vegetative bacilli, are the direct contagion, similar to<br />
other pathogenic Clostridium sp. Spores are hardy, not easily cleared from the body, <strong>and</strong><br />
resistant to antibiotics. The robust physical resistance properties of spores necessitate<br />
prolonged periods of antibiotic treatment <strong>and</strong> a high risk of developing serious drug<br />
intolerances, <strong>and</strong> residual spores may cause infection to recur after initial treatment is<br />
stopped. We hypothesize that there is a correlation between Cd germination/ sporulation <strong>and</strong><br />
human pathogenesis. Cd was isolated from sick patients at multiple U.S. hospitals. To date,<br />
more than 40 isolates have been examined. Spore stocks were produced by growing each<br />
isolate on BHIS plates for seven days at 37°C under anaerobic conditions. Cultures were<br />
exposed to oxygen <strong>and</strong> washed repeatedly to purify spores. Germination assays were<br />
performed by incubating spores with germinant <strong>and</strong> monitoring loss of heat resistance over<br />
time. Sporulation was measured by observing increase in heat resistant CFU in a culture<br />
over 3 days. Finally, spore viability was measured by examining the ratio between spores<br />
present <strong>and</strong> those able to outgrow <strong>and</strong> produce colonies. Cd clinical isolates exhibited a<br />
range of phenotypes. These isolates exhibited a wide range of spore viability, with some<br />
spore preps containing fewer than 25% viable spores. Interestingly, for nearly all isolates<br />
tested, the spores that were viable were capable of rapid germination, with 100%<br />
germination observed within 10 minutes. Sporulation rates were even more variable. While<br />
most isolates were capable of sporulation to some level, the total number of spores observed<br />
for each isolate was significantly different. Clinical isolates of Cd exhibit clear differences in<br />
sporulation <strong>and</strong> spore viability. While this fact alone is interesting, the effect of these<br />
differences on clinical outcome remains to be elucidated. Patient information will be<br />
examined to determine if any of these bacterial phenotypes lead to an altered disease course<br />
in humans.
P7<br />
PRECISE MANIPULATION OF THE CLOSTRIDIUM DIFFICILE CHROMOSOME TO<br />
EXPLORE THE ROLE OF TCDC IN TOXIN PRODUCTION<br />
S. T. Cartman* 1 , M. L. Kelly 1 , D. Heeg 1 , D. A. Burns 1 , S. A. Kuehne 1 , J. T. Heap 1 <strong>and</strong> N. P.<br />
Minton 1 . 1 Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7<br />
2RD, United Kingdom.<br />
TcdC is widely considered to be a negative regulator of toxin production. However, this<br />
notion has never been tested rigorously using isogenic strains of Clostridium difficile. We<br />
have developed a new 2-step allele exchange procedure which permits precise manipulation<br />
of the C. difficile chromosome. Using this approach we have constructed recombinant strains<br />
of C. difficile with different tcdC genotypes. Here we will describe the construction of these<br />
strains <strong>and</strong> the subsequent analysis, which suggests that tcdC genotype does not affect toxin<br />
production.
P8<br />
FLAGELLIN OF CLOSTRIDIUM DIFFICILE 027 HYPERVIRULENT STRAIN ACTIVATES<br />
ERK1/2 MAPK IN AN EPITHELIAL TLR-5-EXPRESSING CELL LINE<br />
P-A. Jolivot 1 , A. Collignon* 1 , <strong>and</strong> I. Kansau 1 . 1 EA 4043, USC INRA Faculté de Pharmacie,<br />
Université Paris-Sud 11, 92296 Châtenay-Malabry, France.<br />
Clostridium difficile is a Gram-positive bacillus responsible for antibiotic-associated diarrhea<br />
<strong>and</strong> pseudomembranous colitis. Recently, emerging hypervirulent ribotype 027 strain was<br />
associated with a higher mortality rate. The pathogenesis of C. difficile is essentially based<br />
on the production of two toxins: tcdA <strong>and</strong> tcdB that disrupt cellular cytoskeleton <strong>and</strong> cause<br />
severe tissue damage. To date, the role of flagella of C. difficile in its pathogenesis is not<br />
understood. The aim of this study was to characterize the signaling pathways elicited in<br />
epithelial cells by the flagella of this opportunistic pathogen through Toll-Like Receptor-5<br />
(TLR-5), which recognizes flagellin monomers. We focused our work on a potential activation<br />
of Mitogen-Activated Protein Kinases (MAPK) <strong>and</strong> more specifically the activation of<br />
Extracellular signal-Regulated kinases 1 <strong>and</strong> 2 (ERK1/2). To reach this aim, first the<br />
epithelial polarized MDCK (Madin-Darby Canine Kidney) cell line were transfected with TLR-<br />
5 so that it expresses this receptor of the innate immune response in a homogenous <strong>and</strong><br />
stable way. Then, the TLR-5-expressing MDCK cells (MDCK-TLR5) <strong>and</strong> the control MDCK<br />
cell line were infected during different times (from 5 to 60 minutes) by two C. difficile strains:<br />
the wild-type hypervirulent 027 strain (R20291, WT) <strong>and</strong> the isogenic strain mutated in the<br />
fliC gene that did not express flagellin (ΔFliC). Furthermore, a recombinant His-tagged FliC<br />
flagellin from C. difficile was purified by immobilized metal ion affinity chromatography. This<br />
recombinant FliC was incubated during various times (30 <strong>and</strong> 60 min) <strong>and</strong> at different<br />
concentrations (from 1 to 10 µg/ml) with MDCK-TLR5 <strong>and</strong> MDCK cells. The ERK1/2<br />
activation was analyzed on cell lysates by Western blot. The MDCK-TLR5 infection by the<br />
WT hypervirulent strain triggered a time-dependent activation of ERK1/2 whereas no<br />
activation was observed with the control cell line. On both MDCK-TLR5 <strong>and</strong> MDCK cell lines,<br />
the ΔfliC strain did not activate ERK1/2. The recombinant flagellin, regardless of<br />
concentration <strong>and</strong> during 30 <strong>and</strong> 60 min incubation, also activated ERK1/2 in MDCK-TLR5<br />
cells but not in MDCK cells. The ERK1/2 activation by a flagellated C. difficile strain <strong>and</strong><br />
purified flagellin suggests that the C. difficile flagellin might play a role in the initiation of an<br />
innate immune response <strong>and</strong> contribute to the pathogenesis of this bacterium.
P9<br />
ANALYSIS OF THE GENOME-WIDE EXPRESSION OF A CLOSTRIDIUM DIFFICILE FLIC<br />
MUTANT IN MONOXENIC MICE<br />
A. Barketi-Klai 1 , M. Monot 2 , S. Hoys 1 , S. Lambert 1 , B. Dupuy 2 , A. Collignon* 1 , I. Kansau 1 . 1 EA<br />
4043, USC INRA, Faculté de Pharmacie, Université Paris-Sud 11, 92296 Châtenay-Malabry,<br />
France; 2 Institut Pasteur, 28, rue du Docteur Roux, 75015, Paris, France.<br />
The hypervirulence of the Clostridium difficile 027 strains seems to be due to several factors<br />
including high-toxin production, presence of binary toxin, <strong>and</strong> high sporulation rate. However,<br />
other factors such as colonization factors could also be involved. We are therefore interested<br />
in the flagellar proteins <strong>and</strong> in particular the structural flagellin protein FliC.<br />
We previously constructed a fliC mutant in a C. difficile 027 strain by the Clostron technique.<br />
The mutant showed: 1) inability to synthesize FliC <strong>and</strong> flagella, 2) a greater adherence on<br />
human colonic cell line Caco-2 as compared to the parental strain, <strong>and</strong> 3) ability to colonize<br />
the mouse intestine either in a monoxenic or a dixenic model in competition with the wild type<br />
strain. Growth competition between the wild type <strong>and</strong> the fliC mutant strains showed the<br />
dominance of the wild type. Surprisingly, the fliC mutant was more virulent than the wild type<br />
strain. Actually, all monoxenic mice challenged with the fliC mutant died 48 h post-infection in<br />
contrast to mice colonized with the wild type strain, which all survived. Thus, the absence of<br />
FliC increases the virulence of the C. difficile 027 strain suggesting that FliC could play a role<br />
in virulence. We then analyzed <strong>and</strong> compared the bacterial transcriptomes of the fliC mutant<br />
<strong>and</strong> the parental strains at the early stage of intestinal colonization in the monoxenic mouse<br />
model. The global analysis of the fliC mutant data (by Ma2HTLM) showed a differential<br />
expression of 310 genes. High regulations were particularly observed for genes involved in<br />
motility, membrane transport systems (PTS, ABC transporters), carbon metabolism,<br />
regulation (Agr-2, s54, PadR system) <strong>and</strong> sporulation. Significant regulation was also<br />
observed for genes involved in the synthesis of toxins (TcdC down-regulated), the cell wall<br />
(cwp66 up-regulated), cell growth, fermentation, metabolism (AA, nucleic acids, lipids), stress<br />
(antibiotic resistance) <strong>and</strong> anaerobic respiration. This in vivo transcriptomic analysis will<br />
provide important data to better underst<strong>and</strong> the hypervirulence of C. difficile.
P10<br />
PROTEOMIC ANALYSIS OF THE CLOSTRIDIUM DIFFICILE 630 STRAIN SECRETOME<br />
ACCORDING TO GROWTH KINETICS<br />
C. Viala 1 , C. Boursier 2 , A. Collignon* 1 , <strong>and</strong>, S. Péchiné 1 . 1 EA 4043, USC INRA, Faculté de<br />
Pharmacie, Université Paris-Sud 11, Châtenay-Malabry, France; 2 Plate-forme TransProt,<br />
IFR141-IPSIT, Faculté de Pharmacie, Université Paris-Sud 11, Châtenay-Malabry, France.<br />
Clostridium difficile, a Gram-positive spore forming anaerobe, is a major cause of antibioticassociated<br />
diarrhea <strong>and</strong> pseudomembranous colitis. Toxins TcdA <strong>and</strong> TcdB are the primary<br />
virulence factors <strong>and</strong> are secreted late in the exponential phase <strong>and</strong> during the stationary<br />
phase. However, other aspects of C. difficile virulence are poorly understood. In particular,<br />
little is known about the mechanisms of gastrointestinal colonization, an undoubtedly<br />
essential step in pathogenesis. Different adhesins <strong>and</strong> probably also some hydrolytic <strong>and</strong><br />
proteolytic enzymes are implicated. They could be considered as secondary virulence<br />
factors. Indeed, bacterial pathogens generally secrete a subset of proteins mediating<br />
essential functions during pathogenesis such as adhesion to host tissues <strong>and</strong> modulation of<br />
the host immune response. The identification of such proteins <strong>and</strong> the mechanisms of<br />
secretion employed by C. difficile will give new clues regarding the pathogenic process. By a<br />
proteomic approach that combines bidimensional electrophoresis with nano-liquid<br />
chromatography-t<strong>and</strong>em mass spectrometry (nanoLC-MS/MS), we studied the secretome of<br />
the C. difficile 630 strain. This work was based on an in vitro growth kinetic at five different<br />
times. Secreted proteins were collected <strong>and</strong> precipitated from the culture supernatant fluids<br />
at T=0, T=4h, T=6h, T=8h <strong>and</strong> T=10h. Culture <strong>and</strong> extraction was done for each point in<br />
triplicate. Then, they were analyzed by two-dimensional gel electrophoresis. The gels were<br />
compared two by two: T=0 was compared to T=4h, T=4h to T=6h, T=6h to T=8h <strong>and</strong> T=8h to<br />
T=10h. Appearance or disappearance of spots, as well as significant variation in spot<br />
intensity was noted (software ImageMaster 2-D Platinum®). 202 differential protein spots<br />
were of interest. Some of these spots have been analyzed <strong>and</strong> identified by nanoLC-MS/MS.
P11<br />
ANALYSIS OF A CLOSTRIDIUM DIFFICILE PCR RIBOTYPE 078 100 KILOBASE ISLAND<br />
REVEALS THE PRESENCE OF A NOVEL TRANSPOSON<br />
J. Corver* 1 , D. Bakker 1 , M. Brouwer 2 , C. Harmanus 1 , M. P. Hensgens 1 , A.P. Roberts 2 , E. J.<br />
Kuijper 1 <strong>and</strong> H. C. van Leeuwen 1 . 1 Department of Medical Microbiology, Leiden University<br />
Medical Center, Leiden, The Netherl<strong>and</strong>s; 2 UCL Eastman Dental Institute, Division of<br />
Microbial Diseases, London, United Kingdom<br />
A collection of 229 Clostridium difficile PCR ribotype 078 isolates from human <strong>and</strong> porcine<br />
origin was tested for the presence of a 100 kb insert, previously described to be unique for<br />
PCR ribotype 078. The insert contained over 90 open reading frames, encoding mainly<br />
proteins from transposons, phages <strong>and</strong> plasmids. Indeed, through PCR, the presence of a<br />
circular transposon DNA, containing the 5‘<strong>and</strong> 3‘ends of the whole insert, could be shown.<br />
The transposon was named Tn6164 <strong>and</strong> was present in 9 human isolates. All 9 isolates were<br />
resistant to tetracycline <strong>and</strong> they originated from various countries in Europe. Moreover, 9<br />
isolates, all from humans, contained only half of the transposon, suggesting multiple insertion<br />
steps yielding the full Tn6164. MLVA analysis clearly revealed genetic relatedness between<br />
transposon- containing isolates, but not all isolates. The presence of Tn6164 did not result in<br />
a readily identifiable phenotype but clinical data suggest a possible increase in virulence<br />
linked to presence of the transposon.
P12<br />
A PILOT STUDY OF CLOSTRIDIUM DIFFICILE IN RAW RETAIL MEATS FROM WESTERN<br />
PENNSYLVANIA<br />
S.R. Curry * 1 , J.W. Marsh 1 , M.M. Tulenko 1 , L.H. Harrison 1 . 1 Infectious Diseaess<br />
Epidemiology Research Unit, University of Pittsburgh, Pittsburgh, PA, 15261, USA.<br />
Background: The prevalence of C. difficile in retail meat samples has varied widely, with<br />
North American studies reporting 12-45% prevalence in contrast to European studies<br />
reporting 0-4.5%.The food supply may be a source for C. difficile infections. Methods: In<br />
phase I, 102 raw ground meat <strong>and</strong> sausage samples (n=20 beef, 2 buffalo, 22 chicken, 2<br />
lamb, 40 pork, 10 turkey, <strong>and</strong> 5 veal) were purchased from 3 grocers in Pittsburgh, PA<br />
between 2/28/2011 <strong>and</strong> 4/28/2011. Where available, the USDA establishment number was<br />
recorded. All products were processed in batches of no more than 30 samples. Samples<br />
were broth amplified using 10g sample in 100 mL cycloserine cefoxitin mannitol broth with<br />
taurocholate <strong>and</strong> lysozyme <strong>and</strong> incubated anaerobically for five days. A negative broth<br />
control was carried through food sample processing. Meat spiked with C. difficile of known<br />
genotype was included as positive control. Fermenting samples were sub-cultured to 5%<br />
sheep blood agar <strong>and</strong> cycloserine cefoxitin fructose agar with horse blood <strong>and</strong> taurocholate.<br />
Colonies with morphologies consistent with C. difficile were confirmed by L-proline<br />
aminopeptidase <strong>and</strong> typed using tcdC genotyping <strong>and</strong> multilocus variable number of t<strong>and</strong>em<br />
repeats analysis (MLVA). For phase II, an additional 12 samples of br<strong>and</strong> A pork sausages<br />
were purchased 5/14/2011 <strong>and</strong> sampled as above. Results: 2/102 (2.0%) raw meat products<br />
sampled were positive for C. difficile. Both were from br<strong>and</strong> A pork sausage products. The<br />
products were purchased on separate dates <strong>and</strong> cultured in different batches. Both products<br />
were from the same processing facility (facility A). Another br<strong>and</strong> A sample from a different<br />
processing facility was negative for C. difficile. Of the additional 12 samples of br<strong>and</strong> A, 4/5<br />
samples from processing facility A were positive for C. difficile. None of the 7 samples from<br />
facilities B, C, <strong>and</strong> D were positive for C. difficile. The isolates in phase I were both tcdC<br />
genotype A (inferred ribotype 078) but were distinct by MLVA with a summed t<strong>and</strong>em repeat<br />
difference of 21. The positive control strain was tcdC genotype 2. Conclusions: The<br />
prevalence of C. difficile in retail meats in western Pennsylvania is 2%. These data suggest<br />
that the prevalence of C. difficile in retail meat may not be as high as previously reported in<br />
North America.
P13<br />
RELAPSE VERSUS REINFECTION: TIMING AND INFLUENCE OF THE INFECTING<br />
STRAIN ON RECURRENT CLOSTRIDIUM DIFFICILE INFECTION<br />
I. Figueroa* 1 , S.P. Sambol 1,2 , E.J.C. Goldstein 3,4 , S. Johnson 1,2 , <strong>and</strong> D.N. Gerding 1,2 . 1 Hines<br />
VA Hospital, Hines, IL, USA; 2 Loyola University Medical Center, Maywood, IL, USA; 3 RM<br />
Alden Research Laboratory, Culver City, CA, USA; 4 David Geffen School of Medicine at<br />
UCLA, Los Angeles, CA, USA.<br />
Recurrences of Clostridium difficile infection (CDI) following successful treatment are<br />
common <strong>and</strong> may be due to the same strain that caused initial infection (relapse) or a<br />
different strain (reinfection). The frequency of relapse <strong>and</strong> reinfection <strong>and</strong> the timing <strong>and</strong><br />
influence of the initial infecting strain on these events are not well defined. Therefore, we<br />
identified <strong>and</strong> compared CD isolates from initial <strong>and</strong> recurrent episodes of CDI in 93 patients<br />
from a large clinical treatment trial comparing vancomycin <strong>and</strong> fidaxomicin. Culture of stool<br />
specimens was performed anaerobically on selective media for CD <strong>and</strong> strain typing was<br />
performed by HindIII Restriction Endonuclease Analysis (REA). Early recurrences after<br />
successful treatment (within 14 days of treatment completion) were relapses in 87.3% <strong>and</strong><br />
reinfections in 12.7%. Late recurrences (15-30 days after treatment) were relapses in 76.7%<br />
<strong>and</strong> reinfections in 23.3%. The average time to recurrence was 11.9 days for relapses <strong>and</strong><br />
14.9 days for reinfections (positive trend, but P=NS). The most common subtype of the<br />
current epidemic strain in North America, REA type BI 6/8/17, was the initial infecting strain<br />
in 26 patients of whom 25 had relapses <strong>and</strong> one who was reinfected with a different strain.<br />
Reinfection with new strains were more common following initial infection by strains other<br />
than BI 6/8/17. Reinfection with new strains may occur early or late after treatment<br />
completion, but tend to occur somewhat later than relapses with the same strain. Most<br />
recurrences following infection with the epidemic BI 6/8/17 strain were relapses of the same<br />
strain. The influence of the treatment agent on relapse <strong>and</strong> reinfection is currently being<br />
analyzed.
P14<br />
SPORULATION RATES AND THE 'HYPERVIRULENCE' OF CLOSTRIDIUM DIFFICILE:<br />
STANDARDISING EXPERIMENTAL PROCEDURES FOR ACCURATE STRAIN<br />
COMPARISON<br />
D. A. Burns, D. Heeg*, S. T. Cartman, <strong>and</strong> N. P.Minton. School of Molecular Medical<br />
Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park,<br />
Nottingham, NG7 2RD UK.<br />
Clostridium difficile is the leading cause of antibiotic-associated diarrhoea <strong>and</strong> a major<br />
burden to healthcare services worldwide. In recent years, C. difficile strains belonging to the<br />
BI/NAP1/027 type have become highly represented among clinical isolates. These so-called<br />
‗hypervirulent‘ strains are associated with outbreaks of increased disease severity, higher<br />
relapse rates <strong>and</strong> an exp<strong>and</strong>ed repertoire of antibiotic resistance. Spores, formed during<br />
sporulation, play a pivotal role in disease transmission <strong>and</strong> it has been suggested that<br />
BI/NAP1/027 strains are more prolific in terms of sporulation in vitro than ‗non-epidemic‘ C.<br />
difficile types. Work in our laboratory has since provided credible evidence to the contrary<br />
suggesting that the strain-to-strain variation in C. difficile sporulation characteristics is not<br />
type-associated. However, the BI/NAP1/027 type is still widely stated to have an increased<br />
rate of sporulation. On analysis of the other studies currently in the literature, it is apparent<br />
that sample sizes have remained small <strong>and</strong>, perhaps most importantly, the methods used to<br />
quantify sporulation have severe limitations. In this study, we analysed the sporulation rates<br />
of 53 C. difficile strains, the largest sample size used to-date in such a study, including 28<br />
BI/NAP1/027 isolates. Our data confirm that significant variation exists in the rate at which<br />
different C. difficile strains form spores. However, we clearly show that the sporulation rate of<br />
the BI/NAP1/027 type is no higher than that of non-BI/NAP1/027 strains. In addition, we<br />
observed substantial variation in sporulation characteristics within the BI/NAP1/027 type.<br />
Predictably, given the clinical importance of C. difficile BI/NAP1/027 a considerable amount<br />
of time <strong>and</strong> money is now being utilised to investigate the molecular basis of virulence in this<br />
type. This work highlights the danger of assuming that all strains of one type behave similarly<br />
without studying adequate sample sizes. Furthermore, we stress the need for a st<strong>and</strong>ard set<br />
of experimental procedures in order to quantify C. difficile sporulation more accurately in the<br />
future.
P15<br />
DETECTION OF CELL ENVELOPE STRESS IN C. DIFFICILE<br />
T.D. Ho <strong>and</strong> C. Ellermeier. Department of Microbiology, University of <strong>Iowa</strong>, <strong>Iowa</strong> City, IA<br />
52242 USA.<br />
Extra-Cytoplasmic Function sigma (σ) factors (ECF σ factors) are a major family of signal<br />
transduction systems which sense <strong>and</strong> respond to extracellular stresses. We have identified<br />
three C. difficile ECF σ factors. These ECF σ factors, CsfT, CsfU, <strong>and</strong> CsfV, induce their own<br />
expression <strong>and</strong> are negatively regulated by their cognate anti-σ factors RsiT, RsiU <strong>and</strong> RsiV,<br />
respectively. The expression of csfT <strong>and</strong> csfU are induced following exposure to the<br />
antimicrobial peptide bacitracin <strong>and</strong>/or lysozyme. In contrast, csfV is specifically induced by<br />
lysozyme stress. The activity of many ECF σ factors is controlled by site-1 <strong>and</strong> site-2<br />
proteases which cleave anti-σ factors. The C. difficile prsW mutant exhibited decreased<br />
expression of CsfT <strong>and</strong> CsfU but not CsfV. When expressed in a heterologous host, C.<br />
difficile PrsW induces degradation of RsiT but not RsiU. When the prsW mutant was tested in<br />
competition assays against its isogenic parent in the hamster model of C. difficile infection,<br />
we found that the prsW mutant was 30-fold less virulent than the wild type. The prsW mutant<br />
was also significantly more sensitive to bacitracin <strong>and</strong> lysozyme than the wild type in in vitro<br />
competition assays. Taken together, these data suggest that PrsW likely regulates activation<br />
of ECF σ factor CsfT in C. difficile <strong>and</strong> controls resistance of C. difficile to antimicrobial<br />
peptides which are important for survival in the host.
P16<br />
CLOSTRIDIUM DIFFICILE 16S-23S rRNA INTERGENIC SPACER REGION (ISR) AS A<br />
MARKER FOR PHYLOGENETIC STUDIES<br />
S. Janezic* 1 , T. Weinmaier 2 , T. Rattei 2 , A. Indra 3 <strong>and</strong> M. Rupnik 1,4,5 . 1 Institute of Public Health<br />
Maribor, Centre for Microbiology, Maribor, Slovenia; 2 University of Vienna, Faculty of Life<br />
Sciences, Vienna, Austria; 3 Austrian Agency for Health <strong>and</strong> Food Safety (AGES), Vienna,<br />
Austria; 4 University of Maribor, Faculty of Medicine, Maribor, Slovenia; 5 Centre of Excellence<br />
CIPKeBIP, Ljubljana, Slovenia.<br />
For molecular epidemiological investigation of Clostridium difficile infections, a PCR<br />
ribotyping method based upon size variation in 16S-23S rRNA intergenic spacer region<br />
(ISR), is widely used in European reference laboratories. The size variability seen in ISR is<br />
due to the presence or absence of tRNA genes <strong>and</strong> spacers of different lengths separated<br />
with direct repeats which, as suggested could result from slipped-str<strong>and</strong> mispairing <strong>and</strong>/or<br />
homologous recombinations (Indra et al., 2010). To investigate the extent of diversity of ISRs<br />
in C. difficile, <strong>and</strong> to see if ISRs could be a marker for phylogenetic studies, ISRs of a total of<br />
31 strains of 26 different PCR ribotypes were amplified using primers described by Bidet et<br />
al., (1999). PCR products or fragments excised from the gel were then cloned <strong>and</strong><br />
sequenced. More than 300 sequences were obtained (ISR sequences of published C.<br />
difficile genomes; strains 630, R20291 <strong>and</strong> CD196, were also included in the analysis). To<br />
remove the sequence variability that could have resulted from sequencing errors <strong>and</strong> to<br />
reduce the number of sequences <strong>and</strong> facilitate analysis, sequence clustering with 97%<br />
identity was performed. The representatives of non-redundant sets were then aligned using<br />
M-Locarna software which first calculates the secondary structure for all ISRs using the<br />
Vienna RNA package <strong>and</strong> then generates multiple alignment that keeps as many structural<br />
features as possible. It has been demonstrated previously that ISRs are organized in mosaiclike<br />
structure with sequence blocks that are present or absent in different rDNA copies with<br />
alignment done only on the basis of sequence similarity. Sequences aligned on the basis of<br />
the secondary structure also show many conserved blocks that could be for example used<br />
for phylogeny calculations <strong>and</strong> would help us to better underst<strong>and</strong> the evolution of the<br />
virulent strains. Moreover, our results show that ISR sequences of different ribotypes that are<br />
of the same or comparable sizes are very similar <strong>and</strong> hence the PCR ribotyping reflects this<br />
relatedness.
P17<br />
SPORULATION LEVEL OF CLOSTRIDIUM DIFFICILE INTEGRN POSITIVE AND<br />
NEGATIVE STRAINS IN THE PRESENCE OF LACTOBACILLI<br />
S. Kõljalg* 1 , M. Rätsep 1 , J. Stsepetova 1 , I. Smidt 1 , E. Shkut 1 , P. Naaber 1,2 , E. Sepp 1 .<br />
1 Department of Microbiology, University of Tartu, Tartu, Estonia; 2 Department of Medical<br />
Microbiology, Stavanger University Hospital, Stavanger, Norway.<br />
Clostridium difficile (CD) is the leading cause of nosocomial diarrheas in adults <strong>and</strong> also the<br />
most important agent of antibiotic-associated diarrhea. Sporulation is important for disease<br />
transmission, but also for surviving in the intestinal tract in the presence of indigenous<br />
bacteria such as lactobacilli. The aim of this work was to clarify sporulation level (SL) <strong>and</strong><br />
integrons in CD strains <strong>and</strong> the possible influence on SL of Lactobacillus plantarum (LP)<br />
strains. Materials <strong>and</strong> methods. Eighty-two CD isolates were used, including 64 from Estonia<br />
[22 from 1994 (EE1) <strong>and</strong> 42 from 2008 (EE2)] <strong>and</strong> 18 from Norway in 2008 (NO)]. Five LP<br />
strains were used. The presence of class I integrons was studied by PCR. SL at room<br />
temperature (initial conc 8x10 8 CFU/ml) was estimated. SL at body temperature was studied<br />
at 0, 10, 24, <strong>and</strong> 48 hours of growth of CD alone or with LP. Results. Class I integrons were<br />
found in 29/82 (35%) strains. Five sizes of integrons were detected (200 to 600bp). The most<br />
common size was 300 bp (14/29). The majority of integron-containing strains (n = 27) were<br />
isolates from EE2 <strong>and</strong> it differed from data of EE1 (27/42 vs 1/21, p < 0.001) <strong>and</strong> NO (27/42<br />
vs 1/17, p < 0.001). SL at room temperature varied from 6.1 to 10.7 log 10 CFU/ml. EE2<br />
strains exhibited more spores than NO strains (median 10 vs 8 log 10 CFU/ml, p < 0.001).<br />
Integron-containing strains had higher SL than integron-negative ones (median 9.7 vs 8.3<br />
log 10 CFU/ml, p = 0.02) at room temperature, but differences were not found at body<br />
temperature. Compared to CD alone, co-growth with LP caused a decrease in SL at 24h<br />
(median 3.0 vs 0 log 10 CFU/ml, p = 0.016) <strong>and</strong> 48h (median 4.8 vs 1.3 log 10 CFU/ml, p <<br />
0.001). The influence of LP was more expressed to integron-positive than negative strains<br />
(median at 10 h: 0 vs 1.6 log 10 CFU/ml, p = 0.014; at 24h: 0 vs 1.5 log 10 CFU/ml, p < 0.001;<br />
at 48h: 0 vs 1.9 log 10 CFU/ml, p < 0.001). Conclusions. We revealed the putative influence of<br />
probiotic LP strains on CD sporulation level. The presumed linkage between sporulation<br />
capacity <strong>and</strong> integrons needs to be clarified.
P18<br />
USING PHENOTYPE MICROARRAYS TO DETERMINE CULTURE CONDITIONS THAT<br />
INDUCE OR REPRESS TOXIN PRODUCTION BY CLOSTRIDIUM DIFFICILE<br />
X.H. Lei*, B.R. Bochner. Biolog, Inc., Hayward, CA, USA.<br />
Background: Toxins A <strong>and</strong> B are important determinants of C. difficile virulence. In this study<br />
we developed a simple but reliable <strong>and</strong> efficient cytotoxicity assay for C. difficile toxin to<br />
systematically examine the effects of culture conditions on toxin production in Phenotype<br />
MicroArray (PM) panels.<br />
Methods: C. difficile type strain ATCC 9689 was cultured in an anaerobic chamber (5% H2,<br />
5% CO2, 90% N2) at 36ºC for 24 hours <strong>and</strong> then inoculated into PM panels <strong>and</strong> incubated<br />
for 72 hours. Toxins in the microplate well supernatants were harvested by filtration with a<br />
96 well Filter Plate. Mammalian cells CHO-k1 <strong>and</strong> Vero from ATCC were grown in flasks at<br />
37ºC with 5% CO 2 in RPMI 1640 plus 10% FBS <strong>and</strong> Pen/Strep <strong>and</strong> then plated into 96-well<br />
tissue-culture treated microplates in an assay medium. Five to 10ul aliquots of filtered<br />
supernatant containing toxins were transferred into each well of the plated cells. Cell<br />
morphologies were observed <strong>and</strong> photos were taken after 18-20 hours of exposure. Then<br />
viability of the cells was determined using a dye reduction assay with Biolog Dye Mix MB <strong>and</strong><br />
OmniLog instrument. The plates were also read at 590 nm with a microplate reader.<br />
Results: Strong effects on C. difficile toxin production were seen with certain nitrogen<br />
sources. Dipeptides <strong>and</strong> a few tripeptides had stronger stimulatory effects on toxin<br />
production than single amino acids. Arginine peptides gave among the highest levels of<br />
toxin production. Some carbon sources also had strong inducing effects. D-threonine gave<br />
highest toxin production followed by L-serine, L-alanine, D-serine, <strong>and</strong> a few other amino<br />
acids. Acetyl amino sugars or amino sugars as carbon or nitrogen sources (e.g., N-acetyl-Dglucosamine)<br />
also showed a strong or above average stimulation effect. Fumaric acid <strong>and</strong><br />
bromosuccinic acid gave highest toxin production among the carboxylic acid carbon sources.<br />
Conclusions: We demonstrated an effective approach for studying toxin production by C.<br />
difficile under hundreds of different culture conditions. Our results provide valuable insights<br />
into metabolic regulation of toxin production by C. difficile. The combination of Phenotype<br />
MicroArray technology with a cytotoxicity assay, a toxin neutralization assay employing a<br />
specific antibody, <strong>and</strong> a cell viability assay based on cellular dye reduction, makes it possible<br />
to reliably, quantitatively, effectively, <strong>and</strong> efficiently study regulation of toxin production by C.<br />
difficile <strong>and</strong> other bacteria.
P19<br />
INCREASED BILE SALTS IN THE CECA OF ANTIBIOTIC-TREATED MICE BY FLAGELLIN<br />
ADMINISTRATION INHIBIT VEGETATIVE REPLICATION OF CLOSTRIDIUM DIFFICILE<br />
M. Liu* 1 , I. Jarchum 1 , <strong>and</strong> E.G. Pamer 1 . 1 Immunology Program, Sloan-Kettering Institute,<br />
Department of Medicine (Division of Infectious Disease), Memorial Sloan-Kettering Cancer<br />
Center, New York, NY 10065, USA.<br />
Clostridium difficile (C. difficile), a spore-forming enteric bacterium, is often acquired by oral<br />
ingestion of spores <strong>and</strong> can cause severe antibiotic-associated diarrhea in humans. To<br />
cause disease, oral acquired spores must germinate <strong>and</strong> undergo vegetative growth in host.<br />
Antibiotic treatment of mice increases the concentrations of small intestinal bile salts that can<br />
stimulate germination of C. difficile. However, a few bile salt compounds inhibit vegetative<br />
growth of C. difficile in vitro. Administration of Salmonella-derived purified flagellin, a Toll-like<br />
receptor 5 (TLR5) lig<strong>and</strong>, delays the vegetative replication of C. difficile in mice <strong>and</strong> protects<br />
mice from acute C. difficile colitis. We found that small intestinal extracts from mice without<br />
antibiotic treatment, but administered with flagellin, can enhance germination of C. difficile<br />
spores. Cholestyramine, a bile salt sequesterer, abolishes the enhanced germination,<br />
suggesting that bile salts are involved in the process. Further analyses of bile salts in<br />
intestinal extracts from either antibiotic-treated or untreated mice show that flagellin<br />
administration increases the concentrations of bile salts in small intestinal extracts from<br />
untreated mice but not antibiotic-treated mice. By contrast, the concentrations of bile salts in<br />
cecal extracts from antibiotic-treated mice but not untreated mice are elevated by flagellin.<br />
The effects of flagellin administration on bile salts are dependent on TLR5. Compared with<br />
mice only treated with antibiotics, cecal extracts from mice treated with both flagellin <strong>and</strong><br />
antibiotics support vegetative replication of C. difficile much worse. Treatment of cecal<br />
extracts with cholestyramine augments the vegetative growth of C. difficile in extracts <strong>and</strong><br />
abolishes the difference between extracts from mice with <strong>and</strong> without flagellin administration.<br />
Addition of deoxycholate, a bile salt compound, to cecal extracts from mice only treated with<br />
antibiotics, restores the suppression of vegetative replication of C. difficile to the extent<br />
observed in the extracts from mice treated with both flagellin <strong>and</strong> antibiotics. Our results<br />
support the idea that flagellin administration causes an elevated level of bile salts in the ceca<br />
of antibiotic-treated mice, which is associated with enhanced germination, but more likely<br />
suppressing vegetative replication of C. difficile.
P20<br />
SURVEILLANCE AND MOLECULAR CHARACTERIZATION OF CLOSTRIDIUM DIFFICILE<br />
INFECTIONS IN TUCSON<br />
F. Anwar 1 , M.J. Mallozzi* 1,6 , D. Wolk 3,5 , N. Ampel 5 , A. Noon 4 , V.K. Viswanathan 1,5 , <strong>and</strong> G.<br />
Vedantam 1,2,5,6 . 1 Dept. of Veterinary Science <strong>and</strong> Microbiology, 2 Dept. of Immunobiology,<br />
3 Dept. of Pathology, 4 Dept. of Medicine, 5 BIO5 Research Institute, University of Arizona,<br />
Tucson AZ, USA; 6 Southern Arizona VA Healthcare System, Tucson AZ, USA.<br />
BACKGROUND: Clostridium difficile (CD) is a leading bacterial cause of nosocomial<br />
infections, with over 400,000 cases reported annually in the US. Risk factors for C. difficile<br />
infection (CDI) include age, immune-suppression <strong>and</strong> the use of antibiotics. Since the year<br />
2000, new ―hypervirulent‖ CD strains have emerged, that are associated with CDI<br />
epidemics/outbreaks, severe disease, increased mortality, <strong>and</strong> increased risk of recurrent<br />
infection. Thus CDI surveillance studies are urgently required to document the various<br />
virulent types of CD. The last CDI surveillance study in Arizona was approximately<br />
seventeen years ago. METHODS AND RESULTS: 83 stool specimens were collected from<br />
three Tucson-area hospitals. CD isolates were recovered through the use of selective media.<br />
The molecular types of these isolates were determined by PCR-based ribotyping. Semiquantitative<br />
toxin testing was done on a subset of these isolates. We identified multiple CD<br />
ribotypes, including several which are epidemic-associated (ribotypes 001, 027, 078 <strong>and</strong><br />
106). Low toxin-producing CD was also recovered from multiple specimens. Epidemicassociated<br />
CD accounted for up to 50% of CD-positive samples at one facility (private<br />
hospital). Two facilities had one predominant ribotype: 027 (private hospital) <strong>and</strong> 001 (VA<br />
Medical Center) respectively, while a third (University Medical Center) had a mix of various<br />
ribotypes. Toxin analysis revealed that the samples from the various hospitals produced<br />
varying amounts of toxin, <strong>and</strong> high toxin levels<br />
did not necessarily correlate with epidemic-associated types. CONCLUSIONS: Epidemicassociated<br />
molecular types of CD are prevalent <strong>and</strong> frequently isolated from Southern<br />
Arizona hospitals. Interestingly, we found a preponderance of ribotype 001 strains at a<br />
hospital previously associated with a CDI outbreak (17 years ago) that was also dominated<br />
by ribotype 001 strains. Further, our toxin data indicate that the toxin-producing capability<br />
does not vary drastically among the isolated ribotypes. Thus, other mechanisms (besides<br />
toxin-production) likely play equally important roles in pathogenesis in vivo. These results<br />
underscore the importance of surveillance for this important pathogen, <strong>and</strong> substantiate the<br />
use of highly-sensitive molecular methods to identify CD.
ABSTRACTS OF POSTER PRESENTATIONS<br />
SESSION II: P21 TO P40<br />
THURSDAY, OCTOBER 27, 2011
P21<br />
CHARACTERIZATION OF RECURRENT CLOSTRIDIUM DIFFICILE DISEASE ISOLATES<br />
BY MULTI-LOCUS VARIABLE NUMBER TANDEM REPEAT ANALYSIS AND TOXIN<br />
SEQUENCING<br />
J. Marsh*, J. Gee, K. Shutt, S. Curry, L. Harrison. Epidemiology Research Unit, University of<br />
Pittsburgh, Pittsburgh, PA, 15261, USA.<br />
Background. Recurrent Clostridium difficile infection (CDI) occurs in ~25% of CDI cases.<br />
Host immune responses against C. difficile are protective <strong>and</strong> vaccines that target toxin<br />
peptides are under development for prevention of CDI. Discriminatory genotyping tools are<br />
required to distinguish relapse from re-infection in patients with recurrence. In this study,<br />
multi-locus variable number t<strong>and</strong>em repeat analysis (MLVA) was used to classify recurrent<br />
CDI as either relapse or re-infection. C-terminal antigenic variation of toxins A <strong>and</strong> B was<br />
examined in relapsing isolates of individual patients. Methods. MLVA <strong>and</strong> tcdC genotyping<br />
were performed on 355 C. difficile isolates from 148 in-patients with recurrent CDI at UPMC<br />
from 2001-2009. The summed t<strong>and</strong>em repeat difference (STRD) among isolates from<br />
individual patients was determined from MLVA data. Serial patient isolates with STRD < 2<br />
were defined as relapse while those with STRD > 3 were defined as reinfection. Sequence<br />
analysis <strong>and</strong> peptide alignments of the C-terminal domains of toxins A <strong>and</strong> B were performed<br />
on 242 isolates using SeqMan <strong>and</strong> MegAlign software in LaserGene DNAstar v.8. Results.<br />
Among 108 patients with a single recurrence, 69 (64%) were classified as relapse with a<br />
median interval between episodes of 36 days. Of relapsing isolates, 62% had the tcdC-1<br />
allele characteristic of BI/NAP1. The 39 patients with a single recurrence due to re-infection<br />
had a median interval between episodes of 92 days. Among 40 patients with >1 recurrence,<br />
14 (35%) were relapse, 5 (13%) were re-infections <strong>and</strong> 21 (53%) were a combination of<br />
relapses <strong>and</strong> re-infections. Among 242 selected isolates, there were 16 different TcdA <strong>and</strong><br />
10 different TcdB C-terminal peptides identified generating 20 different TcdA/TcdB peptide<br />
combinations. No antigenic variation among toxin peptides was observed in relapsing CDI<br />
cases. Of 104 relapsing cases, 60 (58%) were attributable to BI/NAP1. Conclusions. Based<br />
on MLVA genotyping, the majority of recurrent CDI was due to relapse <strong>and</strong> many of these<br />
were attributable to the BI/NAP1 hypervirulent clone. C-terminal toxin variation among serial<br />
isolates from patients with relapsing disease was not observed. Further investigation of<br />
t<strong>and</strong>em repeat mutation rates combined with tcdC <strong>and</strong> toxin genotypes will help define useful<br />
metrics of genetic relatedness based on MLVA.
P22<br />
GENETIC DIVERSITY OF NON-TOXIGENIC CLOSTRIDIUM DIFFICILE DETERMINED BY<br />
MULTI-LOCUS VARIABLE NUMBER TANDEM REPEAT ANALYSIS AND MULTI-LOCUS<br />
SEQUENCE TYPING<br />
J. W. Marsh* 1 , T. K. Henderson 1 , S. P. Sambol 2 , D. N. Gerding 2,3 , L.H. Harrison 1 .<br />
1 Infectious Diseases Epidemiology Research Unit, University of Pittsburgh, Pittsburgh, PA<br />
15261, USA; 2Hines Veterans Affairs Hospital, Hines, IL, USA; 3 Loyola University Chicago<br />
Stritch School of Medicine, Maywood, IL, USA.<br />
Background. Colonization by non-toxigenic Clostridium difficile (NTCD) prevents CD infection<br />
in hamsters. Since asymptomatic carriage of NTCD in humans is common, these strains may<br />
be of therapeutic benefit. Multi-locus variable number t<strong>and</strong>em repeat analysis (MLVA) <strong>and</strong><br />
multi-locus sequence typing (MLST) were performed to determine the genetic diversity of<br />
NTCD strains. A parallel serial passage experiment (PSPE) was performed to examine the<br />
stability of MLVA loci for discrimination of NTCD. Methods. MLVA <strong>and</strong> MLST were performed<br />
on 121 NTCD isolates from 6 different restriction enzyme analysis (REA) groups<br />
representing 71 REA types. Minimum spanning tree (MST) was generated using the<br />
summed t<strong>and</strong>em repeat difference (STRD) based on the MLVA data. The PSPE was<br />
performed on a NTCD representing the most common REA type by inoculating 96 agar<br />
plates from a single colony <strong>and</strong> performing 10 serial passages on each lineage. MLVA was<br />
performed on the 10th passage of each lineage <strong>and</strong> the rate of mutation at each t<strong>and</strong>em<br />
repeat locus was calculated. Results. Overall, NTCD isolates clustered on the MST<br />
according to REA groups <strong>and</strong> sequence type. There were 115 MLVA types identified. REA<br />
type M3, the most common NTCD in this collection could be discriminated by a 6 locus<br />
MLVA typing scheme. T<strong>and</strong>em repeat loci CDR4 <strong>and</strong> CDR6 had the highest mutation rates<br />
while CDR5 <strong>and</strong> CDR48 were invariant in the PSPE. Nine sequence types (ST) were<br />
identified. All of the REA group M strains belonged to ST-15 <strong>and</strong> the majority of REA group<br />
T were ST-3. Conclusions. Asymptomatically carried NTCD are relatively clonal by MLST.<br />
MLVA identifies NTCD strains according to REA groups but is substantially more<br />
discriminatory than REA.
P23<br />
AN IN VITRO MODEL TO STUDY COLONIC MICROBIOTA DISRUPTION IN RELATION<br />
TO INFECTION WITH CLOSTRIDIUM DIFFICILE IN SYRIAN GOLDEN HAMSTERS<br />
M. Miezeiewski* 1 , S. Wang 1 , T. Schnaufer 2 , M. Muravsky 2 , T. Mitchell 2 , J. Zorman 1 , K.<br />
Soring 1 , A. Xie 1 , J. Heinrichs 1 , J. ter Meulen 1 , J. Shiver 1 , <strong>and</strong> J.L. Bodmer 1 . 1 Merck Research<br />
Laboratories, Vaccine Basic Research, West Point, PA, USA; 2 Merck Research Laboratories,<br />
Bioanalytics & Pathology, West Point, PA, USA.<br />
Clostridium difficile is the pathogen commonly associated with antibiotic-associated diarrhea<br />
in humans. Clostridium difficile infections (CDI) are caused by the outgrowth of toxigenic<br />
strains of C. difficile in the colon of individuals whose microbiota has previously been<br />
perturbed by antimicrobial therapy. The lethal enterocolitis model in Syrian golden hamsters<br />
(Mesocricetus auratus) is the most commonly accepted animal model mimicking CDI in<br />
humans. The critical components of this model are the disruption of the hamster‘s GI flora by<br />
antibiotic treatment <strong>and</strong> the challenge with C. difficile spores. The relationship between<br />
colonization, the status of the gut microbiota <strong>and</strong> ultimately toxin production <strong>and</strong> disease is<br />
an ongoing area of research. Here we present an in vitro method to determine the in vivo<br />
susceptibility of Syrian hamsters to colitis <strong>and</strong> allow for the quantitative measurement of C.<br />
difficile growth <strong>and</strong> toxin production in a convenient format. In brief, fecal samples were<br />
collected from Syrian hamster prior <strong>and</strong> post administration of a single dose of clindamycin.<br />
C. difficile spores of strain VPI 10463 were then cultured in broth in the presence or absence<br />
of the filtered soluble fraction of the fecal material for 72hr at 37C under anaerobic<br />
conditions. Cultures were analyzed by qPCR to determine C. difficile genome copy numbers<br />
<strong>and</strong> toxin production levels by EIA. The data demonstrated that (a) normal fecal extracts<br />
inhibit C. difficile growth, (b) fecal extracts from animals treated with clindamycin are<br />
permissive to C. difficile growth <strong>and</strong> toxin production, <strong>and</strong>. (c) the restoration of clindamycin<br />
treated microbiota is gradual <strong>and</strong> closely matches recovery kinetics observed in the<br />
challenge itself. Overall, the timing of disruption <strong>and</strong> subsequent recovery in this in vitro<br />
system is highly suggestive of the proposed pathogenesis in humans. It is, therefore, a<br />
central tool in the underst<strong>and</strong>ing of the hamster model <strong>and</strong> its applications for<br />
prevention/treatment of CDI in humans.
P24<br />
PROTEOMIC ANALYSIS OF THE CLOSTRIDIUM DIFFICILE LARGE TOXINS<br />
H. Moura 1 *, R.R. Terilli 1,2 , A.R. Woolfitt 1 , Y.M. Williamson 1 , T.A. Blake 1 , M.I. Solano 1 , <strong>and</strong><br />
J.R. Barr 1 . 1 Division of Laboratory Sciences, National Center for Environmental Health;<br />
Centers for Disease Control <strong>and</strong> Prevention (CDC), Atlanta, GA USA; 2 Oak Ridge Institute<br />
for Scientific Education, Oak Ridge, TN USA.<br />
Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitals<br />
worldwide, caused by hypervirulent epidemic strains which have the ability to produce<br />
greater quantities of the toxins TcdA <strong>and</strong> TcdB. The expression level of these toxins from<br />
different toxinotypes is not completely known. Additionally, accurate quantification of TcdA<br />
<strong>and</strong> TcdB using small samples has not yet been reported. Proteomics strategies can be used<br />
for accurate quantification of TcdA <strong>and</strong> TcdB <strong>and</strong> the information obtained can be applied to<br />
new toxin detection methods, investigating toxic mechanisms, <strong>and</strong> developing new<br />
therapeutics. In the present study, we describe the analyses of both purified TcdA <strong>and</strong> TcdB<br />
<strong>and</strong> a st<strong>and</strong>ard culture filtrate, using two different proteomics platforms. Three biological<br />
samples of each analyte were analyzed in triplicate using gel-based <strong>and</strong> gel-free<br />
approaches. The proteins were separated by SDS-PAGE <strong>and</strong> the gel b<strong>and</strong>s were digested<br />
<strong>and</strong> analyzed using a mass spectrometer (MS) instrument. In addition, purified TcdA <strong>and</strong><br />
TcdB were digested using five different enzymes followed by analysis on two different MS<br />
instruments. Discovery-type proteomics experiments were applied to the culture filtrate. The<br />
toxins were further quantified using label-free MS <strong>and</strong> the method was applied to measure<br />
TcdA <strong>and</strong> TcdB in the st<strong>and</strong>ard culture filtrate. The use of two different proteomics platforms<br />
provided unique results. First, the gel-based approach revealed basic information on toxin<br />
integrity <strong>and</strong> provided sequence confirmation. Secondly, gel-free in-solution digestion of both<br />
toxins generated broad amino acid sequence coverage with 84% for TcdA <strong>and</strong> 90% for<br />
TcdB. This data coupled to in silico sequencing analysis suggested that the method has the<br />
potential to distinguish different C. difficile toxinotypes based on protein sequence.<br />
Proteomics analysis of the culture filtrate uncovered a number of proteins, among them<br />
TcdA, TcdB, <strong>and</strong> S-layer protein. Lastly, MS-based label-free proteomics analysis was used<br />
to estimate the amounts of TcdA <strong>and</strong> TcdB in small (
P25<br />
STRAIN-SPECIFIC SUSCEPTIBILITY OF CLOSTRIDIUM DIFFICILE TO INHIBITORY<br />
ACTIVITY OF LACTOBACILLUS PLANTARUM IN VITRO<br />
P. Naaber* 1;2 , I. Smidt 1 , M. Rätsep 1 , S. Kõljalg 1 , E. Shkut 1 <strong>and</strong> E. Sepp 1 . 1 Department of<br />
Microbiology, University of Tartu, Tartu, Estonia; 2 Department of Medical Microbiology,<br />
Stavanger University Hospital, Stavanger, Norway.<br />
Clostridium difficile (CD) causes diarrhea <strong>and</strong> colitis in patients with disrupted intestinal<br />
microbiota. Our previous studies have shown the possible role of L. plantarum (LP) in<br />
resistance to colonization with CD. The aim of our study was to evaluate the in vitro<br />
antagonistic effect of indigenous LP strains on the growth of CD strains. Materials <strong>and</strong><br />
methods. In total, 11 LP strains (different genotypes) isolated from antibiotic-associated<br />
diarrhea patients‘ fecal samples were initially screened for antagonistic properties against<br />
two CD strains. Five LP strains potentially active against CD were co-cultivated with 12 CD<br />
strains of different ribotypes. The presence of integrons <strong>and</strong> resistance patterns, counts of<br />
CD, <strong>and</strong> spore production after 10, 24, <strong>and</strong> 48 hours of co-cultivation were measured.<br />
Results. The highest inhibition of CD strains by lactobacilli as compared to controls was<br />
found after 24 h co-cultivation (median inhibition 4.1 log 10 CFU/mL vs. 1.0 after 10 h, p <<br />
0.0001; <strong>and</strong> vs. 2.85 after 48 h, p = 0.26). The CD strain-related variations in inhibition were<br />
high (range 0 – 8.7 log 10 at 24h), while no significant differences in inhibition activity between<br />
different LP strains were found. CD strains showed different degrees of susceptibility to LP<br />
inhibition: highly-sensitive (inhibition > 5 log 10 by all LP strains) – 3 strains (medians 6.7; 6.9<br />
<strong>and</strong> 8.3), moderately sensitive (inhibition 2.5 - 5) – 2 strains (medians 4.1 <strong>and</strong> 4.3) <strong>and</strong> weak<br />
(inhibition < 2.5) – 2 strains (medians 1.3 <strong>and</strong> 1.4). In the case of 5 CD strains, high variation<br />
of sensitivity (from highly-sensitive to insensitive; range 0-6.0) was found, depending on the<br />
particular LP strain. There was no correlation between CD strain sensitivity to inhibition <strong>and</strong><br />
their PCR-ribotype, presence of integrons, spore production, or quinolone resistance.<br />
However, these 3 CD strains that were highly-sensitive to LP were resistant to clindamycin,<br />
whereas the other 9 strains were clindamycin sensitive. Conclusions. Our in vitro results<br />
suggest that antagonistic activity of L. plantarum against CD is CD strain-specific, rather than<br />
LP strain-specific. High sensitivity of CD to inhibition could be related to clindamycin<br />
resistance. This strain-specific sensitivity to indigenous intestinal bacteria can partly explain<br />
the clinical variation in signs <strong>and</strong> symptoms of CD infection <strong>and</strong> contradictory results of effect<br />
of probiotics against CD infection.
P26<br />
ROLE OF THE CROP DOMAIN OF CLOSTRIDIUM DIFFICILE TOXINS A AND B IN<br />
BINDING AND UPTAKE<br />
A. Olling*, S. Goy, E. Frenzel, H. Tatge, I. Just <strong>and</strong> R. Gerhard. Institute of Toxicology,<br />
Hannover Medical School, 30625 Hannover, Germany.<br />
The Clostridium difficile toxins TcdA <strong>and</strong> TcdB are intracellular acting toxins that inhibit small<br />
GTP-binding proteins of the Rho subfamily after entering host cells via receptor-mediated<br />
endocytosis. The C-terminally combined repetitive oligopeptides (CROPs) function as<br />
receptor binding domain. Antibodies directed against the CROPs are able to neutralize TcdA<br />
<strong>and</strong> TcdB. Based on previous findings we hypothesized that the CROPs are not essential for<br />
function of TcdA <strong>and</strong> TcdB. To re-evaluate the role of the CROPs we investigated binding<br />
properties <strong>and</strong> potency towards different cells of full length <strong>and</strong> CROP-truncated TcdA<br />
(TcdA1-1874) <strong>and</strong> TcdB (TcdB1-1852). TcdA1 1874 <strong>and</strong> TcdB1-1852 induced time <strong>and</strong><br />
concentration dependent cell rounding <strong>and</strong> Rac1-glucosylation; however, depending on the<br />
cell line, with up to 10-fold reduced potency compared to the respective full length toxin.<br />
FACS <strong>and</strong> Western blot analyses revealed a correlation between CROP-binding to the cell<br />
surface <strong>and</strong> toxin potency. This was supported by the observation of a faster internalization<br />
process of full length TcdA compared to CROP- truncated TcdA1 1874. These findings refute<br />
the accepted opinion of a solely CROP- mediated toxin uptake <strong>and</strong> led to the assumption of<br />
an additional alternative uptake process at least for TcdA. This process might be based<br />
either on the recognition of different receptor structures or on various endocytotic pathways.<br />
To substantiate this hypothesis, we analyzed toxin potency following apical or basolateral<br />
uptake into the polarized intestinal epithelial cell line CaCo-2 as model for different receptor<br />
structures. In fact, cells were hardly susceptible towards apically applied TcdA1-1874<br />
whereas basolateral treatment caused a pronounced cytopathic effect. In contrast, potency<br />
of full length TcdA was almost identical <strong>and</strong> independent on the side of application.<br />
Furthermore, competition experiments indicated that TcdA <strong>and</strong> TcdA1 1874 predominantly<br />
use different receptor structures corroborating the notion of alternative toxin internalization<br />
processes. Thus, different routes for cellular uptake might enable the toxins to enter a<br />
broader repertoire of cell types leading to the observed multifarious pathogenesis of C.<br />
difficile. The characterization of specific receptor structures <strong>and</strong> alternative endocytotic<br />
pathways used by the C. difficile toxins might therefore be the basis to investigate the<br />
opportunity of toxin uptake inhibition as a therapeutic option.
P27<br />
ADHERENCE OF CLOSTRIDIUM DIFFICILE SPORES TO HUMAN COLONIC<br />
ENTEROCYTE-LIKE CACO-2 CELLS<br />
D. Paredes-Sabja 1 *, M.R. Sarker 2,3 . 1 Universidad Andrés Bello, Chile;<br />
2 Oregon <strong>State</strong> University, USA.<br />
Clostridium difficile is a Gram positive, anaerobic, spore-forming, entero-pathogenic<br />
bacterium, <strong>and</strong> is the main causative agent of antibiotic-associated diarrhea. There is general<br />
consent that C. difficile spores are the morphotype of transmission, infection <strong>and</strong> persistence<br />
of Clostridium difficile infections (CDI). Antibiotic treatments disrupt the normal colonic<br />
microbiota, allowing C. difficile spores to germinate, proliferate, secrete toxins <strong>and</strong> cause<br />
CDI. During the course of CDI, the appearance of high level C. difficile spores in the patient‘s<br />
stools suggest that C. difficile vegetative cells sporulate inside the host leading to persistence<br />
of C. difficile spores in the intestinal tract. The persistence of C. difficile spores in the colon of<br />
CDI-patients complicates effective treatments, as C. difficile spores exhibit resistance to all<br />
currently available treatments <strong>and</strong> can, therefore, survive in the colon until suppression of<br />
CDI treatments. The latter observations suggest that C. difficile spores might have unique<br />
adherence properties to the host‘s epithelial surfaces. In this context, our current study<br />
shows that, despite the similarities in spore-surface hydrophobicity between spores of C.<br />
difficile <strong>and</strong> C. perfringens (another enteric pathogen that also sporulates in the gut), spores<br />
of C. difficile adhere better to Caco-2 cells. The adherence of C. difficile spores to Caco-2<br />
cells was localized to the apical microvilli, <strong>and</strong> adherence was significantly reduced when C.<br />
difficile spores were trypsin-treated. A competitive inhibition assay between fluorescently<br />
labeled <strong>and</strong> unlabeled C. difficile spores revealed that adherence of C. difficile spores to<br />
Caco-2 cells is mediated by unknown spore-specific receptors on Caco-2 cells. Sonication of<br />
C. difficile spores altered the ultrastructure of the outermost exosporium-like structure,<br />
released two protein species of ~ 40-kDa, significantly reduced spore-hydrophobicity <strong>and</strong><br />
adherence to Caco-2 cells. Using a trifunctional crosslinker, we were able to coimmunoprecipitate<br />
four protein species from the surface of Caco-2 cells. In conclusion, this<br />
study provides, for the first time, evidence that C. difficile spores are well suited to adhere to<br />
human colonic enterocyte-like cells <strong>and</strong> that the mechanism of adherence might be mediated<br />
through specific spore-surface <strong>and</strong> enterocytic-surface receptors(s) <strong>and</strong>/or lig<strong>and</strong>(s).
P28<br />
FLUOROQUINOLONE RESISTANCE, BUT NOT TOXIN HYPERPRODUCTION,<br />
CORRELATES WITH THE INCREASED FINDING OF A NOVEL PULSOTYPE OF<br />
VIRULENT CLOSTRIDIUM DIFFICILE STRAINS<br />
C. Quesada-Gómez 1 , C. Rodríguez 1 , M. del Mar Gamboa-Coronado 1 , C. Guzmán-Verri 2 , T.<br />
Du 3 , M.R. Mulvey 3 , E.Chaves-Olarte 1,2 *, E. Rodríguez-Cavallini* 1 . 1 Centro de Investigación<br />
en Enfermedades Tropicales <strong>and</strong> Facultad de Microbiología, Universidad de Costa Rica, San<br />
José, Costa Rica; 2 Programa de Investigación en Enfermedades Tropicales, Escuela de<br />
Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica; 3 National Microbiology<br />
Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada. *Contact:<br />
esteban.chaves@ucr.ac.cr, evelyn.rodriguez@ucr.ac.cr<br />
During a recent outbreak of C. difficile infection, we found 35 PFGE macrorestriction patterns<br />
among 105 isolates. While nearly one third of these isolates were classified as the<br />
hypervirulent NAP1 (33%), a similar proportion of isolates (34%) were grouped in a novel<br />
pulsotype designated as NAP-CR1. The remaining isolates corresponded to NAP4 (6.7%),<br />
NAP9 (2.8%), NAP6 (1.9%), NAP2 (1.9%) <strong>and</strong> other 21 PFGE patterns without NAP<br />
designation (19.7%). To gain insight into the high abundance of the NAP-CR1 strains, we<br />
compared the pulsotypes to their toxin genotype (PCR), toxin expression (Western Blot, real<br />
time PCR, <strong>and</strong> cytotoxicity), <strong>and</strong> resistance to several antibiotics (E-test). The genes tcdA,<br />
tcdB, <strong>and</strong> tcdC were detected in all the pulsotypes. By contrast, cdtB was only found among<br />
the NAP1 strains. The NAP-CR1 strains contained the 18 bp deletion in tcdC previously<br />
described in toxin hyperproducers like NAP1, but lacked the 1 bp deletion in the upstream<br />
region of this gene. Accordingly, the hyperproduction of TcdA <strong>and</strong> TcdB was confirmed for<br />
the NAP1 strains but not for the NAP-CR1 strains. The NAP1 <strong>and</strong> NAP-CR1 strains were<br />
highly resistant to the fluoroquinolones (ciprofloxacin <strong>and</strong> moxifloxacin), whereas almost all<br />
the other strains were highly sensitive to these antibiotics. The resistance to clindamycin of<br />
the NAP-CR1 strains was distinctive because they exhibited elevated MIC for this<br />
antimicrobial (MIC50 >256 µg ml-1) <strong>and</strong> were positive for ermB. All the other pulsotyopes,<br />
including NAP1, had a lower MIC for clindamycin (MIC 50 = 8 µg ml-1) <strong>and</strong> were negative for<br />
the aforementioned gene.<br />
Altogether, our results indicate that the higher incidence of NAP1 <strong>and</strong> NAP-CR1 strains in<br />
this outbreak correlates more with the antibiotic resistance profile than with the level of<br />
production of TcdA <strong>and</strong> TcdB. Thus, we postulate that resistance to antibiotics plays a major<br />
role in the intranosocomial dissemination of C. difficile strains.
P29<br />
ROLE OF MEMBERS OF THE RESIDENT GUT MICROBIOTA IN LIMITING CLOSTRIDIUM<br />
DIFFICILE GROWTH AND TOXIN PRODUCTION<br />
A. Reeves* 1 , <strong>and</strong> V. Young 1,2 . 1 Department of Microbiology <strong>and</strong> Immunology; 2 Department of<br />
Internal Medicine/Division of Infectious Diseases, University of Michigan, Ann Arbor, MI<br />
48109, USA.<br />
Clostridium difficile is a pathogen that causes nosocomial antibiotic-associated diarrhea <strong>and</strong><br />
colitis. The indigenous gut microbiota has an important role in protecting the host against<br />
infection with C. difficile. Administration of antibiotics disrupts the gut microbiota, thus<br />
allowing C. difficile to cause disease. In this study we employed in vivo <strong>and</strong> in vitro systems<br />
to examine the role of members of the normal gut microbiota in limiting C. difficile growth <strong>and</strong><br />
toxin production. WT C57BL/6 mice were treated for 3 days with 5 antibiotics (kanamycin,<br />
gentamicin, vancomycin, colistin <strong>and</strong> metronidazole) in drinking water. Following a two-day<br />
recovery period without antibiotics, animals received a single dose of clindamycin <strong>and</strong> were<br />
then challenged with C. difficile (VPI 10463) one day later. Control animals received no<br />
treatment. Clinical signs of disease were monitored, <strong>and</strong> at necropsy, tissue was harvested<br />
for histopathologic <strong>and</strong> culture-independent analysis of the gut community. Specific members<br />
of the gut community were isolated by selective culture <strong>and</strong> spent culture supernatants from<br />
each isolate were tested for the ability to inhibit C. difficile growth <strong>and</strong> toxin production in<br />
vitro. Antibiotic-treated mice challenged with C. difficile either developed rapidly lethal CDI or<br />
were stably colonized with mild disease. The gut community of animals with mild disease<br />
was dominated by Lachnospiraceae, resembling the control community. Moribund animals<br />
had high levels of Enterobacteriaceae. Treatment with only antibiotics resulted in a<br />
predominance of Lactobacillus. Based on this information, we isolated E.coli, Lactobacillus<br />
spp, <strong>and</strong> Lachnospiraceae spp. We also obtained a short-chain fatty acid producing<br />
Lachnospiraceae, Butyrivibrio fibrisolvens (ATCC 19171). Spent culture supernatants from B.<br />
fibrisolvens <strong>and</strong> the Lachnospiraceae isolate significantly decreased levels of C. difficile<br />
growth. Correspondingly, a cytotoxin assay using Vero cells also showed decreased toxin<br />
activity. Lactobacillus spp <strong>and</strong> E. coli showed lower levels of C. difficile growth inhibition.<br />
Lachnospiraceae members had the greatest inhibitory effect on C. difficile growth <strong>and</strong> toxin<br />
activity. It is likely that these organisms secrete inhibitory substances such as short-chain<br />
fatty acids that limit C. difficile growth <strong>and</strong> toxin production. Underst<strong>and</strong>ing how members of<br />
the indigenous microbiota interfere with C. difficile could lead to new modalities for<br />
prevention <strong>and</strong> treatment of this important infection.
P30<br />
IDENTIFICATION AND CHARACTERIZATION OF GENES SPECIFIC TO<br />
HYPERVIRULENT NAP1 CLOSTRIDIUM DIFFICILE STRAINS<br />
C. Robinson*, J. P. van Pijkeren <strong>and</strong> R. Britton. Department of Microbiology <strong>and</strong> Molecular<br />
Genetics, Michigan <strong>State</strong> University, East Lansing, MI 48824, USA.<br />
Clostridium difficile, the leading cause of antibiotic-associated diarrhea, is a prevalent<br />
nosocomial pathogen. Growth of this enteric pathogen is normally inhibited by mechanisms<br />
of colonization resistance by the intestinal microbiota; however, growth can ensue following a<br />
perturbation of the microbiota, usually initiated by administration of antibiotics. Many<br />
aspects of the physiology of this pathogen <strong>and</strong> its interactions with the microbiota are poorly<br />
understood. In the past decade a hypervirulent strain of C. difficile has emerged<br />
(NAP1/ribotype 027), generating an even greater need to underst<strong>and</strong> this organism <strong>and</strong> its<br />
pathology. Using Phylogenetic Profiler we compared sequenced C. difficile genomes <strong>and</strong><br />
identified fifty genes unique to NAP1 strains. A cluster of four genes was found that<br />
interrupts the thymidylate synthase gene (thyX), including an alternative thymidylate<br />
synthase (thyA), dihydrofolate reductase (dhfR), a hypothetical protein, <strong>and</strong> thiamine<br />
synthase (thiC), respectively. This is of interest because it has been demonstrated in recent<br />
literature that ThyA is catalytically more active than ThyX, promoting up to 10-fold faster DNA<br />
replication rates, which has physiological implications including faster growth rates <strong>and</strong><br />
maintenance of larger genome sizes. Current experiments are being conducted to<br />
investigate growth differences by competing NAP1 <strong>and</strong> non-NAP1 strains in continuous<br />
culture competitions. A cluster of CRISPR (clustered regularly interspersed palindromic<br />
repeats)-associated proteins was also found to be unique to NAP1 strains. CRISPR genes,<br />
which play a role in phage resistance, may confer a competitive advantage to hypervirulent<br />
strains over historical strains that are susceptible to phage infection. PCR detection of the<br />
thyA <strong>and</strong> CRISPR loci in over 80 recent clinical isolates of various NAP lineages from<br />
Michigan verified that these gene clusters are unique to, <strong>and</strong> conserved among, NAP1<br />
hypervirulent strains. In addition to the genetic investigations, we have also conducted<br />
phenotypic screens of several NAP1 <strong>and</strong> non-NAP1 strains using Phenotypic MicroArrays<br />
(Biolog Inc.); these are 96-well plates used to screen preselected compounds for their effects<br />
on growth, sporulation, <strong>and</strong> germination. Using this technology, we have identified several<br />
compounds that increase growth yield of C. difficile, including ones that are selectively<br />
efficacious to just the NAP1 strains screened. We expect that these investigations will<br />
elucidate differences between hypervirulent <strong>and</strong> non-hypervirulent strains on the<br />
physiological <strong>and</strong> ecological levels.
P31<br />
PHAGE TAIL-LIKE BACTERIOCINS OF CLOSTRIDIUM DIFFICILE<br />
D. Gebhart, S. Williams, L. Fortier, G. Govoni, <strong>and</strong> D. Scholl*. AvidBiotics Corp., South San<br />
Francisco, CA 94080 USA.<br />
Some strains of C. difficile produce high molecular weight phage tail-like particles, ―diffocins,‖<br />
upon induction of the SOS response. We have purified these particles <strong>and</strong> showed for the<br />
first time that they have bactericidal activity against other C. difficile strains, <strong>and</strong> can be<br />
classified as bacteriocins. The gene cluster that encodes diffocin structural <strong>and</strong> regulatory<br />
proteins has been identified. Diffocins produced from different strains possess unique<br />
bactericidal spectra. One of the most interesting features of these particles is a very large<br />
protein of ~200 kDa, encoded by Orf 1374. This large protein determines the killing spectrum<br />
of the particles <strong>and</strong> is likely the receptor-binding protein. We plan to develop diffocins as<br />
antimicrobial agents for both therapeutic <strong>and</strong> prophylactic use.
P32<br />
EPIDEMIOLOGIC INVESTIGATION OF CLOSTRIDIUM DIFFICILE AND CLOSTRIDIUM<br />
PERFRINGENS IN HEALTHY HORSES.<br />
A. Schoster* 1 , L.G. Arroyo 1 , H.R. Staempfli 1 , P.E. Shewen 1 , J.S. Weese 1 . 1 Ontario Veterinary<br />
College, University of Guelph, Guelph, ON Canada.<br />
Clostridium difficile <strong>and</strong> Clostridium perfringens are important causes of equine colitis but can<br />
also be found in healthy individuals. Epidemiologic information is restricted to cross-sectional<br />
studies of fecal shedding with little information on prevalence in gastrointestinal<br />
compartments other than feces <strong>and</strong> variability in shedding over time. The objectives were to<br />
investigate the presence of C. difficile <strong>and</strong> C. perfringens in healthy horses over time <strong>and</strong><br />
assess prevalence in different gastrointestinal compartments. Feces were collected monthly<br />
from 25 horses for one year. Ingesta were collected from nine GI compartments of a<br />
separate group of 15 euthanized horses. Selective enrichment culture was performed,<br />
followed by toxin gene detection <strong>and</strong> ribotyping (C. difficile) <strong>and</strong> multiplex PCR (C.<br />
perfringens). Toxigenic C. difficile was isolated from 15/275 (5.5%) samples from 10/25<br />
(40%) horses over one year. Three horses were positive in consecutive months, but different<br />
ribotypes were found in 2/3. Ribotypes included 078 (n=6), 001 (n=5) <strong>and</strong> an A+B+CDT+<br />
ribotype previously identified as toxinotype IX in this laboratory (n=4). C. perfringens was not<br />
recovered despite a detection threshold of 9cfu/g feces. Toxigenic C. difficile was isolated<br />
from 14/135 (10.3%) ingesta samples from 8/15 (50.3%) horses, from both the small <strong>and</strong><br />
large intestine. Multiple compartments were positive in four horses but more than one<br />
ribotype was present in all. In 3/8 colonized horses (38%) the rectal sample was negative but<br />
a proximal site was positive. Ribotypes included 078 (n=5), 001 (n=4) <strong>and</strong> three A+B+CDTribotypes<br />
not previously identified in this laboratory. C. perfringens type A (CPE- <strong>and</strong> β2-)<br />
was recovered from the colon of 1/15 horses (6.6%). In the longitudinal study only one horse<br />
shed the same strain for more than one month, suggesting horses are short-term carriers or<br />
that C. difficile spores are passively moving through the intestinal tract. The presence of<br />
multiple ribotypes in different gastrointestinal compartments in the same horse further<br />
supports transient passage. Agreement between feces <strong>and</strong> proximal compartments was<br />
moderate to good (κ=0.61), suggesting limitations with studies using feces. The<br />
predominance of ribotype 078, in contrast to earlier reports, suggests recent emergence in<br />
this horse population, similar to reports from humans. The low prevalence of C. perfringens<br />
supports results of previous studies that indicate this organism is rare in healthy horses.
P33<br />
THE PREVALENCE OF CLOSTRIDIUM DIFFICILE AND CLOSTRIDIUM PERFRINGENS<br />
AS PATHOGENS OF ACUTE DIARRHEA IN PATIENTS VISITING A TERTIARY CARE<br />
HOSPITAL IN KOREA<br />
B. Shin*, S. Mun, E. Lee, E. Kuak. Department of Laboratory Medicine, Sanggye Paik<br />
Hospital, Inje University, Seoul, Korea.<br />
Object: Clostridium perfringens is one of the most common pathogens associated with acute<br />
diarrhea. Clostridium difficile is a common pathogen of health care associated infection, <strong>and</strong><br />
community associated CDI (CA-CDI) seems to be increasing as well. Both of these<br />
pathogens are difficult to culture in routine microbiology laboratories <strong>and</strong> the prevalence<br />
rates are not exactly known in Korea. Therefore, we need to investigate the prevalence rate<br />
of both C. difficile <strong>and</strong> C. perfrignens as community associated diarrheic pathogens. Method:<br />
We performed muliplex-PCR (Seeplex Diarrhea ACE Detection, Seegene, Seoul, South<br />
Korea) to detect C. difficile, tcdB <strong>and</strong> C. perfringens genes with 262 stool specimens from<br />
adult diarrheic patients visiting an emergency department in a tertiary hospital in Korea from<br />
July 2008 to June 2011. We also perfomed C. difficile culture <strong>and</strong> tcdA <strong>and</strong> tcdB gene PCR<br />
assay with C. difficile isolated from culture. Results: The number of C. difficile <strong>and</strong> C.<br />
perfringens were 34 (13.0%) <strong>and</strong> 58 (22.1%), respectively. Among 34 C. difficile isolates,<br />
tcdA+tcdB+, tcdA-tcdB+ <strong>and</strong> tcdA-tcdB- strains were 31 (91.2%), 2 (5.9%) <strong>and</strong> 1 (2.9%).<br />
Salmonella spp, Shigella spp, Campylobacter spp <strong>and</strong> Vibrio spp were also detected in 11,<br />
12, 32, <strong>and</strong> 9 specimens with multiplex PCR. Conclusion: C. perfringens was the most<br />
prevalent pathogen as etiologic agent causing acute diarrhea in patients visiting the<br />
emergency department in a tertiary care hospital in Korea. C. difficile was the second most<br />
prevalent pathogen causing acute diarrhea in patients visiting the ED, which means C.<br />
difficile is an emerging community associated diarrheic pathogen. Most C. difficile strains<br />
isolated from patients visiting the ED were tcdA+tcdB+, but tcdA-tcdB+ strains were also<br />
found. The isolation <strong>and</strong> identification of C. difficile <strong>and</strong> C. perfrignens are important,<br />
because the treatment regimens of C. difficile <strong>and</strong> C. perfringens are different, <strong>and</strong> more<br />
intensive infection control is needed for C. difficile.
P34<br />
COMPARISON OF TWO PCR ASSAYS FOR TOXIN B FOR DIRECT DETECTION OF<br />
TOXIN PRODUCING CLOSTRIDIUM DIFFICILE IN FECAL SPECIMENS<br />
B. Shin*, S. Mun, E. Lee, E. Kuak. Department of Laboratory Medicine, Sanggye Paik<br />
Hospital, Inje University, Seoul, Korea.<br />
Objectives: Clostridium difficile (C. difficile) is a spore-forming, gram-positive anaerobe,<br />
responsible for virtually all cases of pseudomembranous colitis (PMC), <strong>and</strong> for 15-25% of<br />
cases of antibiotic-associated diarrhea (AAD). Toxigenic C. difficile strains usually produce<br />
two toxins, enterotoxin (TcdA) <strong>and</strong> cytotoxin (TcdB), both involved in the pathogenic<br />
characteristics of the microorganism. The aim of this study was to evaluate the performance<br />
of the two new PCR assays (BD GeneOhm Cdiff assay <strong>and</strong> Seegene Diarrhea ACE ) <strong>and</strong><br />
compared the results with bacterial culture <strong>and</strong> in house PCR assay for tcdA <strong>and</strong> tcdB from<br />
C. difficile isolates for the detection of toxigenic C. difficile. Materials <strong>and</strong> Methods: Freshly<br />
collected samples (n = 215) were cultured on a Clostridium difficile selective media (BD) <strong>and</strong><br />
isolates were tested using in-house PCR assays for tcdA <strong>and</strong> tcdB. In this study, culture <strong>and</strong><br />
PCR assays were considered as the reference method. Each sample was analyzed using<br />
two commercial PCR assays (BD GeneOhm <strong>and</strong> Seegene ACE B ). BD GeneOhm<br />
(SanDiego, CA, US) Cdiff assay is a real-time PCR assay that amplifies tcdB <strong>and</strong> Seegene<br />
ACE B is a dual priming oligo multiplex PCR assay that contains primer to detect tcdB.<br />
Results: C. difficile was identified in 56 samples (26.0%), <strong>and</strong> 159 samples (74.0%) showed<br />
culture-negative results. PCR assays for tcdA <strong>and</strong> tcdB genes were conducted on the 56 C.<br />
difficile isolates. The number of tcdA+/tcdB+, tcdA-/tcdB+, <strong>and</strong> tcdA-/tcdB- strains are 36 , 5 ,<br />
15, respectively. The concordance rate between BD GeneOhm <strong>and</strong> Seegene ACE toxin B<br />
was 96.7% (208/215). The sensitivity <strong>and</strong> specificity <strong>and</strong> positive <strong>and</strong> negative predictability<br />
of BD GeneOhm <strong>and</strong> Seegene ACE Toxin B for tcdB gene was 95.1%/96.0%/ 84.8%/98.8%<br />
<strong>and</strong> 90.2%/96.6%/86.0%/97.7%, respectively. Conclusion: Both PCR assays (BD GeneOhm<br />
<strong>and</strong> Seegene ACE Toxin B for tcdB gene) represented good performances compared to<br />
toxigenic culture for the detection of toxigenic C. difficile directly in stool specimens.
P35<br />
EFFECT OF INCREASED FECAL CHENODEOXYCHOLIC ACID LEVELS ON C. DIFFICILE<br />
VIRULENCE<br />
C. Allen <strong>and</strong> J. Sorg*. Department of Biology, Texas A&M University, College Station, TX<br />
77843 USA.<br />
Clostridium difficile infections remain a significant burden to the hospital <strong>and</strong> long-term<br />
healthcare settings. C. difficile is spread in the environment in the form of a dormant spore.<br />
Upon inoculation into susceptible hosts, C. difficile spores germinate in response to certain<br />
bile acids <strong>and</strong> glycine. Bile acids are steroid-based compounds synthesized in the liver as<br />
cholic acid or chenodeoxycholic acid derivatives <strong>and</strong> help emulsify fat <strong>and</strong> cholesterol in the<br />
intestinal tract. Previously, we found that C. difficile spore germination is initiated upon<br />
exposure to cholic acid derivatives (taurocholic acid, glycocholic acid, cholic acid <strong>and</strong><br />
deoxycholic acid). Chenodeoxycholic acid competitively inhibits cholic acid-mediated<br />
germination <strong>and</strong> is toxic for growth. Inhibiting in vivo spore germination would be a powerful<br />
way in overcoming new or relapsing C. difficile infections. Thus, to test this hypothesis, we<br />
will feed chenodeoxycholic acid to Syrian hamsters <strong>and</strong> monitor the levels of fecal<br />
chenodeoxycholic acid by High Performance Liquid Chromatography. Hamsters will be<br />
treated with clindamycin <strong>and</strong> inoculated with C. difficile spores. Animals will be monitored for<br />
signs of disease <strong>and</strong> compared to animals fed chow without the compound. These<br />
experiments may highlight the importance of anti-germination therapy in preventing C.<br />
difficile infections.
P36<br />
DETECTION OF CLOSTRIDIUM DIFFICILE IN PIGGERY EFFLUENT AFTER TREATMENT<br />
IN A 2-STAGE POND SYSTEM<br />
M.M. Squire* 1 , S.C. Lim 1 , N.F. Foster 1 , <strong>and</strong> T.V. Riley 1,2 . 1 Microbiology <strong>and</strong> Immunology,<br />
School of Biomedical, Biomolecular <strong>and</strong> Chemical Sciences, The University of Western<br />
Australia, Perth, 6009, Australia; 2 Microbiology <strong>and</strong> Infectious Diseases, PathWest<br />
Laboratory Medicine, Perth, 6009, Australia.<br />
Clostridium difficile is associated with enteric disease in man <strong>and</strong> animals. It has recently<br />
emerged as an agent of neonatal enteritis in piglets, with high asymptomatic carriage rates<br />
(up to 75%). C. difficile may be part of a zoonosis; there is increasing overlap between<br />
molecular types of C. difficile causing human <strong>and</strong> animal disease. Transmission may be via<br />
environmental contamination or food-borne spores as C. difficile is found in retail meat <strong>and</strong><br />
ready-to-eat salads. The association with enteric disease is poorly understood, but it is clear<br />
that C. difficile is prevalent in Australian piggeries. Piggery effluent is treated in anaerobic<br />
ponds to remove pathogens <strong>and</strong> re-used to wash sheds or applied to agricultural l<strong>and</strong>. We<br />
hypothesized that C. difficile would also survive the effluent treatment process due to the<br />
resistant nature of its spores. We conducted a study to ascertain the presence, number <strong>and</strong><br />
molecular type of C. difficile at various stages of piggery effluent treatment <strong>and</strong> storage. Ten<br />
samples of effluent before <strong>and</strong> after treatment in a 2-stage pond system were collected,<br />
cultured, <strong>and</strong> C. difficile enumerated by direct spread plate counting. C. difficile was isolated<br />
from all 10 samples. Isolates were confirmed as PCR ribotype UK 237 which predominates<br />
in piglets at this farm. C. difficile (8.0 x 10 1 cfu/ml) was isolated from raw effluent prior to<br />
treatment. As expected, C. difficile numbers increased during anaerobic <strong>and</strong> facultative<br />
treatment phases (1.3 x 10 2 cfu/ ml <strong>and</strong> 2.3 x 10 2 cfu/ ml respectively), decreasing to 9.3 x<br />
10 1 cfu/ ml in the aerobic evaporative pond. Low numbers of C. difficile (3.5 x 10 1 cfu/ ml on<br />
average) also remained in treated effluent storage tanks, although these samples were taken<br />
from the surface of full tanks <strong>and</strong> may have been affected by spore settling. These results<br />
confirm our hypothesis that C. difficile survives effluent treatment. This is the first study to<br />
detect <strong>and</strong> count C. difficile in treated piggery effluent. Further work is planned to confirm<br />
these findings in other piggeries <strong>and</strong> investigate the risk that re-use of treated effluent poses<br />
to public health.
P37<br />
COMPARATIVE PATHOGENICITY OF CLOSTRIDIUM DIFFICILE STRAINS IN<br />
CEFOPERAZONE-TREATED MICE<br />
C.M. Theriot*, V.B. Young. Department of Internal Medicine, Division of Infectious Diseases,<br />
Department of Microbiology <strong>and</strong> Immunology, The University of Michigan, Ann Arbor, MI<br />
48109 USA.<br />
INTRODUCTION: The toxin-producing bacterium C. difficile is the leading cause of antibioticassociated<br />
colitis, with an estimated 1-3 million cases of C. difficile infection (CDI) each year<br />
in the U.S. Despite the significance of CDI, little is known about the pathogenesis of this<br />
infection. The recent development of tractable murine models of CDI that utilize antibiotic<br />
treatment prior to C. difficile infection will allow us to study determinants of C. difficile<br />
pathogenesis in vivo. We wished to determine if challenge of cefoperazone-treated mice<br />
could distinguish the relative pathogenicity of C. difficile strains. METHODS: 5-8 week old<br />
C57BL/6 WT mice were pretreated with a 10 day course of cefoperazone administered in the<br />
drinking water. Following a 2-day recovery period with plain water, the animals were orally<br />
challenged with C. difficile strains VPI 10463, NAP1/BI/027, 630 <strong>and</strong> a non-toxigenic strain.<br />
Animals were monitored for loss of weight <strong>and</strong> clinical signs of colitis. At the time of harvest,<br />
C. difficile strains were isolated from cecal contents <strong>and</strong> the severity of colitis was<br />
determined by histopathologic examination of the cecum <strong>and</strong> colon. RESULTS:<br />
Cefoperazone-treated mice challenged with the more historically virulent C. difficile strains<br />
(known to produce more toxin) VPI 10463 <strong>and</strong> NAP1/BI/027, developed clinically more<br />
severe disease than those infected with 630 <strong>and</strong> the non-toxigenic strain. This increased<br />
clinical severity was correlated with more severe microscopic lesions of disease, with<br />
significantly more severe edema, inflammation, <strong>and</strong> epithelial damage in animals infected<br />
with the more virulent strains. More severe histopathologic disease in cefoperazone-treated<br />
mice also correlated with increased cytotoxicity <strong>and</strong> elevated white blood cell counts in vivo.<br />
CONCLUSIONS: Cefoperazone-treated mice exhibit varying degrees of colitis when<br />
challenged with different C. difficile strains. This murine model, along with clinically-relevant<br />
strains of C. difficile, will help us better underst<strong>and</strong> the pathogenesis <strong>and</strong> virulence of CDI. It<br />
will also allow us to explore the host-pathogen relationship along with the role that the gut<br />
microbiota plays in colonization resistance against C. difficile.
P38<br />
MONOCLONAL ANTIBODIES TO SPECIFICALLY IDENTIFY TOXIN B OF CLOSTRIDIUM<br />
DIFFICILE RIBOTYPE 027<br />
C. von Eichel-Streiber* 1 , K. Gisch 1 , A. Lange 1 , C. Oester 1 . R. Murillio 2 , M. Villacampa 2 ,<br />
T.Toribio 2 . 1 tgcBIOMICS GmbH, Carl Zeiss. Str. 51, D-55129 Mainz, Germany; 2 Oporon<br />
S.A., Camino del Plano 19, 50410 Cuarte de Huerva (Zaragoza), Spain.<br />
Toxin B genes of Clostridium difficile are known to be genetically diverse. tgcBIOMICS has<br />
developed monoclonal antibodies that differentially recognize TcdB molecules of different<br />
isolates. Partial characterization of such antibodies will be presented. Some of the antibodies<br />
specifically recognize TcdB-027 of C. difficile ribotype 027. The antibodies were used to<br />
establish a CE-certified lateral flow test <strong>and</strong> an ELISA for the identification of C. difficile<br />
infections with hypervirulent isolates.
P39<br />
A PHYLOGENETIC ANALYSIS OF THE NEGATIVE TOXIN REGULATOR (TcdC) OF<br />
CLOSTRIDIUM DIFFICILE<br />
S. Walk* 1 , V. Young 1 , <strong>and</strong> D. Aronoff 1 . 1 Department of Internal Medicine, Division of<br />
Infectious Diseases, <strong>and</strong> the Department of Microbiology, University of Michigan, Ann Arbor,<br />
MI USA.<br />
Epidemiologic observations suggest that the prevalence of severe cases of Clostridium<br />
difficile infection (CDI) is increasing. One hypothesis to explain this trend is that strains<br />
associated with severe disease produce higher levels of the C. difficile toxins TcdA <strong>and</strong><br />
TcdB. As a result, C. difficile toxin regulation has received much attention <strong>and</strong> a negative<br />
regulator of toxin expression, called TcdC, has been identified. In support of the above<br />
hypothesis, certain C. difficile isolates possess an allele of tcdC that encodes a premature<br />
stop codon, resulting in a truncated <strong>and</strong> presumably non-functional protein. However,<br />
multiple clinical studies have not found a link between specific tcdC alleles <strong>and</strong> severe CDI.<br />
To better examine the genetic diversity of tcdC <strong>and</strong> the abundance <strong>and</strong> distribution of<br />
different alleles among C. difficile pathogens, we sequenced this gene in 250 ribotyped<br />
isolates isolated from patients with C. difficile infection at the University of Michigan Health<br />
System. Alignment of these tcdC sequences with 44 previously identified alleles from online<br />
databases revealed that previously published alignments could be improved. An improved<br />
alignment based on all available alleles revealed 44 variable nucleotide sites <strong>and</strong> 19 variable<br />
amino acid sites throughout tcdC. A total of 19 unique alleles were represented among the<br />
clinical isolates in this study <strong>and</strong> 6 of them were novel. The most abundant allele, tcdC-3,<br />
was identical to a previously identified allele <strong>and</strong> was shared by 31% of all isolates. A<br />
phylogenetic analysis was conducted on the updated alignment <strong>and</strong> a model for the evolution<br />
of tcdC diversity was developed. We found evidence for intragenic recombination using<br />
break-point analysis as well as evidence for at least 4 different homologous recombination<br />
events (shared alleles between C. difficle ribotypes). Based on our results, we propose a<br />
st<strong>and</strong>ardized alignment <strong>and</strong> naming convention for tcdC alleles that can be used in future<br />
surveillance of C. difficile pathogens, thereby aiding the quantification of association between<br />
tcdC alleles <strong>and</strong> CDI severity.
P40<br />
EFFECT OF PROTON PUMP INHIBITORS WITH AND WITHOUT ANTIBIOTICS ON<br />
CLOSTRIDIUM DIFFICILE INFECTION IN HAMSTERS<br />
L. Wright* 1 , K. Aleksoniene 2 , S. Sambol 2 , L. Petrella 1 , D.N. Gerding 1 , S. Johnson 1,2 . 1 Hines<br />
VA Hospital, Hines, IL USA; 2 Loyola University Medical Center, Maywood, IL USA.<br />
The most important risk factor for Clostridium difficile infection (CDI) is exposure to<br />
antibiotics. Controversy exists over whether proton pump inhibitors (PPIs) increase the risk of<br />
CDI. We sought to determine whether PPI administration to hamsters renders these animals<br />
susceptable to colonization <strong>and</strong>/or disease following challenge with toxigenic CD.<br />
Esomeprazole in bicarbonate buffer was administered orally to groups of 10 hamsters<br />
starting on day 1 <strong>and</strong> oral challenge with CD spores occurred on day 5. Pilot studies were<br />
conducted to optimize the PPI dose, duration, <strong>and</strong> CD challenge inoculum. Initial<br />
experiments did not include clindamycin treatment, but in subsequent experiments,<br />
clindamycin was given to hamsters on day 0. In order to determine the effect of PPIs alone<br />
(no antibiotic) on CDI, we used a high inoculum (1 million spores) of a highly virulent CD<br />
strain (BI17) to challenge hamsters given continuous PPI (50 mg/kg esomeprazole daily by<br />
oral gavage for 7 days). 10/10 hamsters had fecal pellet cultures positive for CD up to 48<br />
hours after spore challenge vs. 2/10 up to 24 hours in controls given bicarbonate diluent but<br />
no PPI (P
ABSTRACTS OF POSTER PRESENTATIONS<br />
Session III: P41 to P61<br />
Friday, October 28, 2011
P41<br />
HIGH AFFINITY BINDING OF CLOSTRIDIUM PERFRINGENS EPSILON TOXIN TO THE<br />
RENAL SYSTEM<br />
J. Dorca-Arévalo 1,2 , M. Martín-Satué 1,2 , <strong>and</strong> J. Blasi* 1,2 . 1 Department of Pathology <strong>and</strong><br />
Experimental Therapeutics. School of Medicine. Campus of Bellvitge. Health Universitat de<br />
Barcelona Campus (HUBc). University of Barcelona. c/ Feixa Llarga s/n 08907. L‘Hospitalet<br />
de Llobregat. Spain; 2 Institut d‘Investigació Biomèdica de Bellvitge (IDIBELL).<br />
Epsilon toxin (ε-toxin), produced by Clostridium perfringens types B <strong>and</strong> D, causes fatal<br />
enterotoxaemia in livestock. In the renal system, the toxin binds to target cells before<br />
oligomerization, pore formation, <strong>and</strong> cell death. Still, there is little information about the<br />
cellular <strong>and</strong> molecular mechanism involved in the initial steps of the cytotoxic action of ε-<br />
toxin, including the specific binding to the target sensitive cells. In the present work, the<br />
binding step of ε-toxin to the MDCK cell line, a very sensitive cellular model for ε-toxin<br />
cytotoxic effect, is characterized by means of an ELISA based assay <strong>and</strong> using ε-toxin-green<br />
fluorescence protein (ε-toxin-GFP) <strong>and</strong> ε-prototoxin-GFP. In addition, different treatments<br />
with Pronase E, detergents, N-glycosidase F <strong>and</strong> beta-elimination on MDCK cells <strong>and</strong> renal<br />
tissue cryosections have been performed to further characterize the ε-toxin binding. ELISA<br />
assays revealed a single binding site with a similar dissociation constant (Kd) for ε-toxin-GFP<br />
<strong>and</strong> ε-prototoxin-GFP, but a three-fold increase in Bmax levels in the case of ε-toxin-GFP.<br />
Double staining on kidney cryoslices with lectins <strong>and</strong> ε-prototoxin-GFP revealed specific<br />
binding to distal <strong>and</strong> collecting tubule cells. Moreover, experiments on kidney <strong>and</strong> bladder<br />
cryoslices demonstrated the specific binding to distal tubules <strong>and</strong> urothelium of a range of<br />
mammalian renal systems, including mouse, cow, goat, sheep <strong>and</strong> human kidneys <strong>and</strong><br />
mouse, cow, goat <strong>and</strong> sheep bladders. Pronase E <strong>and</strong> beta-elimination treatments on kidney<br />
cryoslices <strong>and</strong> MDCK cells showed that the binding of epsilon-toxin in the renal system is<br />
mediated by an O-glycoprotein. In addition, detergent treatments revealed that the integrity of<br />
the plasma membrane is required for the binding of ε-toxin to its receptor. This work has<br />
been supported by the grant SAF2008/00732 <strong>and</strong> FIS 00305 from the Ministerio de Ciencia e<br />
Innovación of the Spanish Government.
P42<br />
CLOSTRIDIAL GUT COLONIZATION IN PRETERM NEONATES<br />
L. Ferraris 1 , M. Butel* 1 , F. Campeotto 1 , I. Nicolis 2 , M. Vodovar 3 , J.C. Rozé 4 , <strong>and</strong> J. Aires 1 .<br />
1 EA4065 <strong>and</strong> 2 EA2498, Sorbonne Paris Cité, Université Paris Descartes, Paris, France;<br />
3 Néonatologie, Institut de Puériculture, Paris, France; 4 Médecine Néonatale, INSERM<br />
CIC004, Nantes, France.<br />
Clostridia are part of the normal gut microbiota in humans <strong>and</strong> have been associated with<br />
endogenous infections. Several reports have suggested their role in necrotizing enterocolitis<br />
(NEC), a devastating gastrointestinal disease in preterm infants. However, little data are<br />
available on the gut colonization at birth by this genus. The aim of our study was to report the<br />
distribution of clostridial species colonizing the gut of preterm neonates, <strong>and</strong> to investigate<br />
the colonization factors. Moreover, since preterm infants are often treated with broad<br />
spectrum antibiotics, antibiotic susceptibility patterns of the clostridial isolates have been<br />
studied. Clostridial colonization has been investigated in the feces of 76 preterm neonates<br />
(median gestational age 32.8 wk [range 24-35.9 wk]) collected each week throughout their<br />
hospital stay (median 4 wk [range 2-11 wk]). The incidence of colonization was high: 78.9%<br />
at hospital discharge (mean level of 7.4 CFU/g of feces [range 3.3-9.2 CFU/g]). This<br />
incidence increased throughout the hospitalization from 27.5% at wk 1 to 100% at the end of<br />
the follow-up. The most frequently isolated species were C perfringens, C butyricum, C<br />
difficile, <strong>and</strong> C paraputrificum. No differences in the microbiota between neonates either<br />
colonized or not by clostridia were observed. The main factor of colonization was antibiotic<br />
courses. Ante natal antibiotic therapy decreased the level of colonization (p=0.006). Neonatal<br />
antibiotic therapy decreased both the incidence (p=0.002) <strong>and</strong> the level (p= 0.001) of<br />
colonization only when antibiotic treatment lasted more than 10 days. Overall, clostridia<br />
strains were found susceptible to the usual anti-anaerobe agents. However, as already<br />
known, all C. difficile isolates were resistant to ertapemen, cefoxitin <strong>and</strong> cefotaxim <strong>and</strong> 97%<br />
to clindamycin. Resistance to amoxicillin <strong>and</strong> piperacillin was observed for C. butyricum<br />
strains (12.5%). This study showed clostridial colonization as part of the microbial community<br />
of preterm neonates; this was in contrast to other anaerobes, for which gut colonization is<br />
sharply delayed. The increase in the incidence throughout the hospital stay suggests<br />
colonization from the hospital environment by this spore-forming genus. The study gives new<br />
insights on the factors of clostridial colonization in preterm infants <strong>and</strong> may participate to<br />
better underst<strong>and</strong>ing of neonatal infections involving clostridia such as NEC.
P43<br />
csps PLAY A ROLE IN NACL AND PH STRESS RESPONSE OF CLOSTRIDIUM<br />
BOTULINUM ATCC 3502<br />
Y. Derman* 1 , M. Lindström 1 , H. Söderholm 1 , <strong>and</strong> H. Korkeala 1 .<br />
1 Department of Food Hygiene <strong>and</strong> Environmental Health, the Centre of Excellence in<br />
Microbial Food Safety Research, Faculty of Veterinary Medicine, University of Helsinki,<br />
Finl<strong>and</strong>.<br />
Growth <strong>and</strong> toxin production of Clostridium botulinum in foods may lead to life-threatening<br />
food poisoning called botulism. Some C. botulinum strains can adapt to <strong>and</strong> survive in stress<br />
conditions such as low pH <strong>and</strong> presence of salt <strong>and</strong> therefore they pose a risk for food<br />
safety. Mutations in the cold shock protein coding genes (csps) cspB or cspC, but not cspA,<br />
resulted in a cold-sensitive phenotype in C. botulinum ATCC 3502 (Söderholm H., M.<br />
Lindström, P. Somervuo, J. Heap, N. Minton, J. Lindén, <strong>and</strong> H. Korkeala. 2011. cspB<br />
encodes a major cold shock protein in Clostridium botulinum ATCC 3502. Int. J. Food<br />
Microbiol. 146:23-30). In this study, the growth of csp mutants was compared to wild type<br />
strain in different concentrations of added NaCl <strong>and</strong> various pH conditions. The mutant <strong>and</strong><br />
wild type strains were anaerobically grown in tryptone-peptone-glucose-yeast extract broth<br />
with up to 4% NaCl <strong>and</strong> pH of 4 to 8 in the Bioscreen turbidity reader (BCDE Group, Helsinki,<br />
Finl<strong>and</strong>) placed in anaerobic conditions at 37°C. No growth differences between the csp<br />
mutants <strong>and</strong> the wild type strain were observed in native broth, in the presence of 1% NaCl,<br />
or at pH 5.5 to 8.0. In the presence of 2% to 4% NaCl, the cspB mutant showed an increased<br />
lag phase compared to the wild type strain, with the difference being greatest at 4% NaCl.<br />
None of the strains grew in broth with pH of 4.0 or 4.5. At pH 5.0, only the wild type strain,<br />
but none of the csp mutants, grew. The data suggest that cspB is involved in NaCl stress,<br />
whereas all csps seem to play a role in pH stress at 37°C. Further studies on the role of the<br />
csp genes in the ethanol <strong>and</strong> hydrogen peroxide stress response are ongoing.
P44<br />
MOLECULAR DIAGNOSIS OF BLACKLEG FROM COMMON FILTER PAPER<br />
L. Farias* 1 , S. Botton, A.C. Vargas 1 . 1 Laboratory of Bacteriology, UFSM, CEP 97105-900<br />
Santa Maria/RS, Brazil.<br />
The absence of conclusive diagnosis of blackleg usually happens for practical <strong>and</strong><br />
economical infeasibility of sending sample for laboratory diagnosis. Seeking to facilitate the<br />
collection, storage <strong>and</strong> material shipment to the laboratory, the objective of this work was to<br />
verify the viability of Clostridium chauvoei in common filter paper for subsequent molecular<br />
identification by using PCR (Polymerase chain reaction). For this we tested in vitro the<br />
sensitivity, specificity <strong>and</strong> feasibility of this method over different storage periods at room<br />
temperature. For the viability test, we used twelve bovine liver tissues which ten samples<br />
were impregnated with suspension containing C. chauvoei inoculums <strong>and</strong> the tests were<br />
performed at 24h, 48h, 72h, <strong>and</strong> one week after impregnation on common filter paper. The<br />
sensitivity of the technique was not affected with the use of common filter paper. There were<br />
no cross-reaction <strong>and</strong> no amplification from the negative control samples. Direct PCR by<br />
using common filter paper showed amplification in 50% of the samples in 24 hours, 100% in<br />
48h, 70% in 72h <strong>and</strong> 90% within one week of storage at room temperature. Therefore, the<br />
common filter paper can be used as practical <strong>and</strong> economical alternative for collection,<br />
storage <strong>and</strong> material shipment for molecular diagnosis of blackleg.
P45<br />
THE EFFECT OF CLOSTRIDIUM PERFRINGENS TYPE C AND ITS BETA TOXIN<br />
MUTANT IN GOATS<br />
J. P. Garcia* 1 , J. Saputo 1 , D. J. Fisher 2,3 , S. Sayeed 2 , B. A. McClane 2,3 , H. Posthaus 4 , <strong>and</strong> F.<br />
A. Uzal 1 . 1 California Animal Health <strong>and</strong> Food Safety Laboratory System, School of<br />
Veterinary Medicine, University of California Davis, San Bernardino, CA, USA; 2 Department<br />
of Microbiology <strong>and</strong> Molecular Genetics; 3 Molecular Virology <strong>and</strong> Microbiology Graduate<br />
Program, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 4 Institute of<br />
Animal Pathology, Vetsuisse Faculty, University of Bern, Bern, Switzerl<strong>and</strong>.<br />
Clostridium perfringens type C is an important cause of enteritis <strong>and</strong> enterocolitis in several<br />
animal species including pigs, sheep, goats, horses, <strong>and</strong> humans. The disease is a classic<br />
enterotoxemia <strong>and</strong> the enteric lesions <strong>and</strong> associated systemic effects are thought to be<br />
caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens<br />
type C. This has been demonstrated recently by fulfilling molecular Koch‘s postulates in<br />
rabbits <strong>and</strong> mice. We present here an experimental study to fulfill these postulates in goats, a<br />
natural host of type C disease. Nine healthy male or female Anglo Nubian goat kids were<br />
inoculated with a virulent C. perfringens type C wild-type strain, an isogenic CPB null mutant,<br />
or a strain where the CPB mutation had been reversed (complement strain). Three goats<br />
inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea,<br />
necrotizing enterocolitis, pulmonary edema, hydropericardium, <strong>and</strong> death within 24 h of<br />
inoculation. Two goats inoculated with the CPB null mutants <strong>and</strong> two goats inoculated with<br />
sterile culture media (negative controls) remained clinically healthy during 24 h after<br />
inoculation <strong>and</strong> no gross or histological abnormalities were observed in any of their tissues.<br />
Reversal of the null mutation restored CPB production <strong>and</strong> virulence; 2 goats inoculated with<br />
this reversed mutant presented clinical <strong>and</strong> pathological changes similar to those observed in<br />
goats inoculated with the wildtype, except that spontaneous death was not observed. These<br />
results indicate that CPB is both required <strong>and</strong> sufficient for C. perfringens type C to induce<br />
disease in goats, supporting a key role for this toxin in natural type C disease pathogenesis.
P46<br />
CLOSTRIDIUM PERFRINGENS STRAINS OF VARIOUS ORIGIN CAN CAUSE<br />
HEMORRHAGIC ENTERITIS IN A CALF INTESTINAL LOOP MODEL<br />
B. Valgaeren 1 , E. Goossens* 2 , S. Verherstraeten 2 , B. Pardon 1 , L. Timbermont 2 , S.<br />
Schauvlieghe 3 , R. Ducatelle 2 , F. Van Immerseel 2 , P. Deprez 1 . 1 Department of Large Animal<br />
Internal Medicine, Ghent University, B-9820 Merelbeke, Belgium; 2 Department of Pathology,<br />
Bacteriology <strong>and</strong> Avian Diseases, Ghent University, B-9820 Merelbeke, Belgium;<br />
3 Department of Surgery <strong>and</strong> anaesthesiology of domestic animals, Ghent University, B-9820<br />
Merelbeke, Belgium.<br />
Hemorrhagic enteritis is one of numerous pathologies caused by Clostridium perfringens in<br />
cattle. It is an acute syndrome with a case fatality rate close to 100%, that affects mainly<br />
suckling <strong>and</strong> veal calves in good to excellent body condition up to four months of age. In<br />
Belgian Blue cattle, losses due to hemorrhagic enteritis may be responsible for up to 20% of<br />
total mortality. The pathogenesis <strong>and</strong> the toxins involved in bovine hemorrhagic enteritis<br />
remain to be elucidated. Therefore, an intestinal loop model was developed to test a<br />
collection of Clostridium perfringens strains for their ability to reproduce the pathology. Loops<br />
were injected with logarithmic C. perfringens cultures in combination or not with commercial<br />
milk replacer for veal calves. The tested set of strains consisted of bovine strains isolated<br />
from both hemorrhagic enteritis cases <strong>and</strong> healthy animals, as well as human, NetB-positive<br />
<strong>and</strong> -negative chicken strains, <strong>and</strong> beta toxin positive porcine isolates. Also a VirR, α toxin,<br />
<strong>and</strong> a θ toxin mutant were tested. All tested strains were capable of eliciting hemorrhagic<br />
enteritis in this experimental setup when inoculated together with commercial milk replacer. A<br />
time course experiment showed that the pathogenesis is characterized by early capillary<br />
congestion, followed by hemorrhages <strong>and</strong> necrosis of the lamina propria, originating from the<br />
tips of the villi. No edema was present. C. perfringens bacteria were often attached to cellular<br />
debris in the lumen. These observations indicate that any Clostridium perfringens strain,<br />
independent of the isolation source, may be capable of eliciting hemorrhagic enteritis, <strong>and</strong><br />
that this syndrome is not caused by host-specific strains or toxins. In addition, the<br />
pathological process seems to be independent of α- or θ-toxin or VirR-regulated<br />
mechanisms.
P47<br />
A FUNCTIONAL, HIGH MOLECULAR WEIGHT BACTERIOCIN (―DIFFOCIN‖) FROM<br />
CLOSTRIDIUM DIFFICILE CLONED AND EXPRESSED IN BACILLUS SUBTILIS<br />
G. Govoni*, D. Gebhart, S. Williams, <strong>and</strong> D. Scholl. AvidBiotics Corp., South San Francisco,<br />
CA 94080 USA.<br />
Several C. difficile isolates have been observed to produce phage tail-like particles with no<br />
capsid structure. Recently, it was shown that these particles can act as high molecular<br />
weight bacteriocins (termed ―diffocins‖) <strong>and</strong> kill other C.difficile isolates. We have cloned the<br />
22kb diffocin gene cluster, which contains 25 ORFs <strong>and</strong> multiple predicted transcripts, <strong>and</strong><br />
integrated it into the amyE gene in the B. subtilis chromosome via homologous<br />
recombination. As in C. difficile, expression of diffocin can be induced by addition of<br />
mitomycin C during exponential phase. Preliminary studies suggest we can also express<br />
diffocins in B. subtilis by inducing expression of a constitutively active form of E. coli RecA.<br />
Using a bioassay on sensitive C. difficile isolates, we were able to confirm that the diffocins<br />
produced by these B. subtilis strains were functionally active. Interestingly, the spectrum of<br />
killing C. difficile isolates could be altered by exchanging orthologues of the putative receptor<br />
binding protein encoded by ORF CD1374. This platform provides the opportunity to engineer<br />
the spectrum of killing <strong>and</strong> to produce diffocins for clinical applications.
P48<br />
IMPROVING THE REPRODUCIBILITY OF INFECTION OF THE NAP1/B1/027<br />
HYPERVIRULENT STRAIN R20291 IN THE HAMSTER MODEL OF INFECTION<br />
M. Kelly*, R. Ng, S. Cartman, A. Cockayne <strong>and</strong> N.P. Minton.<br />
Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular<br />
Science, University Park, University of Nottingham, Nottingham, NG7 2RD U.K.<br />
Clostridium difficile has become the leading cause of diarrhoea in hospitalized patients over<br />
the past three decades in North America <strong>and</strong> Europe, placing a substantial financial burden<br />
on healthcare systems. The situation has been exacerbated by the emergence of so-called<br />
‗hypervirulent‘ strains (NAP1/BI/027). As its genome sequence is available, we have been<br />
using the strain R20291 as a model of hypervirulence. This strain was isolated from the<br />
2003-2004 outbreak at the Stoke M<strong>and</strong>eville Hospital in the UK, where 334 cases of C.<br />
difficile associated disease (CDAD) <strong>and</strong> 38 mortalities were recorded. The golden Syrian<br />
hamster has been the model of choice for in vivo investigations of CDAD for the past three<br />
decades as it mimics the progression of the disease in humans. CDAD in the hamster is<br />
brought about by oral infection following administration of the antibiotic clindamycin. Studies<br />
using the 630 wild type <strong>and</strong> 630Δerm strains of C. difficile have shown that the onset of<br />
disease is observed routinely between 48 <strong>and</strong> 72 hours post infection. Investigation of strain<br />
R20291 using the hamster model of infection is, however, hampered by the strains‘<br />
sensitivity to clindamycin (MIC= 16µg/ml) which leads to irreproducible infection (between 52<br />
<strong>and</strong> 240 hours post infection). This complicates experiments being undertaken to assess the<br />
comparative virulence of directed mutants, which, in contrast to the parental control, have<br />
become resistant to clindamycin as a consequence of the ClosTron-mediated insertion of<br />
ermB. We have, therefore, created an R20291 strain that is erythromycin resistant through<br />
the insertion of an equivalent copy of the ClosTron-derived ermB gene into the genome using<br />
Allele Coupled Exchange (ACE) technology. This new strain shows an increased resistance<br />
to clindamycin that is comparable to the 630 wild type strain. Data on the reproducibility of<br />
infection in the hamster model will be presented.
P49<br />
AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS IN STRAIN TYPING AND<br />
IDENTIFICATION OF CLOSTRIDIUM SPECIES<br />
R. Keto-Timonen* <strong>and</strong> H. Korkeala. Department of Food Hygiene <strong>and</strong> Environmental Health,<br />
Faculty of Veterinary Medicine, FI-00014 University of Helsinki, Finl<strong>and</strong>.<br />
Amplified fragment length polymorphism (AFLP) is a PCR-based DNA fingerprinting method,<br />
which inspects the entire genome for polymorphism <strong>and</strong> can be used both for strain<br />
characterization <strong>and</strong> species identification. The AFLP technique was applied to 129 strains<br />
representing 24 different Clostridium species to assess the potential of AFLP for identification<br />
of clostridia. The ability of the same AFLP protocol to type Clostridium perfringens at the<br />
strain level was also assessed. All Clostridium strains were typeable by AFLP <strong>and</strong>, thus, the<br />
method seemed to overcome the problem of extracellular DNase production. AFLP<br />
differentiated all Clostridium spp tested, except for C. ramosum <strong>and</strong> C. limosum, which<br />
clustered together with a 45% similarity level. Other Clostridium spp were divided into<br />
species-specific clusters or occupied separate positions. Wide genetic diversity was<br />
observed among C. botulinum strains, which were divided into seven species-specific<br />
clusters. With a 93% cut-off value, a total of 29 different AFLP types were identified for 37<br />
strains of C. perfringens. AFLP analysis of unrelated C. perfringens strains resulted in<br />
divergent fingerprints, whereas identical b<strong>and</strong>ing patterns were observed for strains<br />
originating from the same isolate or from the same food poisoning outbreak. AFLP showed<br />
potential to subtype C. perfringens strains, <strong>and</strong> due to the high throughput of samples, the<br />
AFLP approach is especially well-suited for screening large numbers of isolates. In addition,<br />
AFLP may be used as a valuable additional tool in identification of Clostridium spp if an<br />
exp<strong>and</strong>able identification library with several AFLP profiles of well-defined strains for each<br />
species is established.
P50<br />
COMPARATIVE GENOMIC HYBRIDIZATION ANALYSIS SHOWS DIFFERENT<br />
EPIDEMIOLOGY OF CHROMOSOMAL AND PLASMID-BORNE cpe-CARRYING C.<br />
PERFRINGENS TYPE A STRAINS<br />
P. Lahti* 1 , M. Lindström 1 , P. Somervuo 1 , A. Heikinheimo 1 , <strong>and</strong> H. Korkeala 1 . 1 Department of<br />
Food Hygiene <strong>and</strong> Environmental Health, Faculty of Veterinary Medicine, University of<br />
Helsinki, Helsinki, Finl<strong>and</strong>.<br />
The reservoirs <strong>and</strong> the routes of contamination of a common food poisoning pathogen,<br />
enterotoxin-producing C. perfringens, remain unknown, which complicates the prevention of<br />
C. perfringens food poisonings. cpe, encoding the enterotoxin, can be chromosomal or<br />
plasmid-borne. The chromosomal cpe-carrying strains are a more common cause of food<br />
poisonings than the plasmid-borne cpe-genotypes, perhaps due to their better heat<br />
tolerance. However, the plasmid-borne cpe-positive genotypes are more commonly found in<br />
human feces <strong>and</strong> in environmental samples than are chromosomal cpe-positive genotypes.<br />
The different prevalence of the cpe-positive genotypes in environmental niches <strong>and</strong> the<br />
differences in heat tolerance raise a question, whether the epidemiology of C. perfringens<br />
type A food poisonings caused by plasmid-borne <strong>and</strong> chromosomal cpe-carrying strains is<br />
different. A DNA microarray was designed for analysis of genetic relatedness between the<br />
different cpe-positive <strong>and</strong> cpe-negative genotypes of C. perfringens strains isolated from<br />
human, animal, environmental, <strong>and</strong> food samples. The DNA microarray contained two<br />
probes for all protein coding sequences in the three genome sequenced strains (C.<br />
perfringens type A strains 13, ATCC13124, <strong>and</strong> SM101). The analysis of the variable gene<br />
pool was complemented with the growth studies <strong>and</strong> the growth of the cpe-positive C.<br />
perfringens strains were examined in the minimal medium using myo-inositol, cellobiose or<br />
ethanolamine as the sole source of energy. The chromosomal <strong>and</strong> plasmid-borne C.<br />
perfringens genotypes were grouped into two distinct clusters, one consisting of the<br />
chromosomal cpe-genotypes <strong>and</strong> the other consisting of plasmid-borne cpe-genotypes. The<br />
plasmid-borne cpe-carrying strains carried operons encoding for utilization of myo-inositol<br />
<strong>and</strong> ethanolamine, <strong>and</strong> were able to utilize these as a sole source of energy. Most<br />
chromosomal strains carried a gene cluster encoding the utilization of cellobiose <strong>and</strong> were<br />
able to utilize cellobiose as a sole source of energy. The epidemiology of chromosomal <strong>and</strong><br />
plasmid-borne cpe-carrying C. perfringens type A food poisonings seem to be different. The<br />
plasmid-borne cpe-carrying strains are suggested to contaminate food mainly by humans,<br />
whereas the epidemiology of the chromosomal cpe-carrying strains is suggested to be<br />
connected to decomposing plant material. Further research should be done to elucidate the<br />
habitat of these strains.
P51<br />
CHARACTERIZATION OF SURFACE-LAYER PROTEINS OF CLOSTRIDIUM BOTULINUM<br />
GROUP I AND II<br />
D. Lambert* 1 , S. M. Twine 2 <strong>and</strong> J. W. Austin 1 . 1 Microbiology Research Division, Bureau of<br />
Microbial Hazards, HPFB, Health Canada, Ottawa, Ontario, Canada; 2 Institute for Biological<br />
Sciences, National Research Council, Ottawa, Ontario, Canada.<br />
Interactions between bacteria <strong>and</strong> their environments, including their hosts in the case of<br />
pathogens, regularly involve surface proteins. For Gram-positive bacteria like Clostridium<br />
botulinum, such surface proteins are putative virulence factors involved in the colonization of<br />
wounds or gastrointestinal tracts of adults or infants. These infections lead to bacterial<br />
growth <strong>and</strong> the subsequent production of botulism neurotoxin (BoNT) in vivo, causing rare<br />
forms of the disease, viz. wound botulism, adult gut colonization botulism, or infant botulism.<br />
The surface proteins of C. botulinum can also potentially be used in detection assays as<br />
surrogates for the presence of BoNT in contaminated foods during an outbreak or possible<br />
bio-terrorist attacks. Other C. botulinum surface proteins have now been characterized, but<br />
little is known about its surface (S-) layer. It comprises a planar array of regularly ordered<br />
often self-assembling proteinaceous subunits, representing some of the most abundant<br />
cellular proteins. We thus performed an initial characterization of the S-layer proteins from C.<br />
botulinum groups I <strong>and</strong> II. We isolated putative S-layer proteins using various mechanical<br />
<strong>and</strong> chemical extraction techniques, followed by electrophoresis. Protein identities were<br />
confirmed using mass spectrometry analysis using MASCOT. We observed signs of posttranslational<br />
glycosylation using glycan-specific dyes, but not with commercially available<br />
lectins. Using colony <strong>and</strong> genomic DNA PCR, followed by amplicon sequencing <strong>and</strong><br />
sequence alignments (Blast), we determined various levels of homology between S-layer<br />
protein genes of selected group I <strong>and</strong> II strains. We are now exploring the possibility of using<br />
these findings to develop new detection methods for C. botulinum contamination.
P52<br />
IDENTIFICATION OF C. PERFRINGENS GENES ASSOCIATED WITH AVIAN NECROTIC<br />
ENTERITIS BY MICROARRAY COMPARATIVE GENOMIC HYBRIDIZATION<br />
D. Lepp* 1,2 , V. Parreira 1 , J. G. Songer 3 , A.Kropinski 4 , P. Boerlin 1 , J. Gong 2 , J. Prescott 1 .<br />
1 Department of Pathobiology, University of Guelph; 2 Guelph Food Research Centre,<br />
Agriculture <strong>and</strong> Agri-Food Canada; 3 College of Veterinary Medicine, <strong>Iowa</strong> <strong>State</strong> University;<br />
4 Laboratory for Foodborne Zoonoses, Public Health Agency of Canada.<br />
Clostridium perfringens type A causes poultry necrotic enteritis (NE), an enteric disease of<br />
considerable economic importance. A recently-identified novel pore-forming toxin, NetB, is<br />
critical to NE pathogenesis <strong>and</strong> closely associated with virulent strains. More recently, we<br />
demonstrated that netB resides on a large, plasmid-borne pathogenicity locus (NELoc-1)<br />
that, in conjunction with two other loci (NELoc-2 <strong>and</strong> 3), is highly conserved in poultry-virulent<br />
Cp strains. To determine the prevalence of these loci in a larger group of poultry isolates <strong>and</strong><br />
to identify other genes associated with NE-causing strains, we have developed a C.<br />
perfringens microarray for comparative genomic hybridization analyses (CGH). Based on the<br />
genome sequences of two NE-causing strains, a microarray consisting of 3,335<br />
oligonucleotide probes, also representing 85% - 92% of the three publicly available C.<br />
perfringens genome sequences (ATCC13124, SM101 <strong>and</strong> Str13) was constructed.<br />
Hybridization of the array with two sequenced strains (ATCC13124 <strong>and</strong> Str13) <strong>and</strong><br />
comparison with the predicted gene content based on sequence analysis revealed it to be<br />
accurate <strong>and</strong> sensitive (sensitivity >99%, specificity >95%). A set of 57 C. perfringens<br />
strains, 28 from birds with NE <strong>and</strong> 29 from healthy birds, were chosen for analysis by array<br />
CGH. Pulsed field gel electrophoresis (PFGE) of the strains confirmed that they were<br />
genetically distinct. The three NE loci were more prevalent in isolates from diseased than<br />
from healthy birds. In particular, NELoc-2 was present in 85.7% of isolates from diseased<br />
<strong>and</strong> only 41.3% of isolates from healthy birds. Several other smaller loci were also<br />
significantly associated with isolates from diseased birds, which include an alternative sigma<br />
factor, a two-component signal transduction system, <strong>and</strong> a ferric iron siderophore transport<br />
system. These findings suggest that the three NE loci are important for pathogenesis, in<br />
particular NELoc-2. Additionally, several other fitness-related chromosomal genes may<br />
contribute to a strain‘s ability to cause disease.
P53<br />
APPLYING TOXIN PROTEOMICS TO MEASURE RELATIVE QUANTITIES OF PROTEINS<br />
WITHIN THE BOTULINUM NEUROTOXIN COMPLEX<br />
H. Moura*, A.R Woolfitt, R.R. Terilli, M.I. Solano & J.R. Barr. Division of Laboratory Sciences,<br />
National Center for Environmental Health; Centers for Disease Control <strong>and</strong> Prevention<br />
(CDC), Atlanta, GA USA.<br />
Botulinum neurotoxins (BoNT) are specific endopeptidases that block the neurotransmitter<br />
release in peripheral nerve endings, causing botulism. There are seven known types of<br />
BoNTs (A through G). BoNTs are large proteins (~150 kDa) <strong>and</strong> exist as non-covalent<br />
complexes with neurotoxin-associated proteins (NAPs). Accurate quantification of BoNT <strong>and</strong><br />
NAPs (composed of NTNH <strong>and</strong> hemagglutinin) within complexes has not been<br />
accomplished. Accurate quantification of BoNT <strong>and</strong> NAPs within the BoNT complex would<br />
reveal key information with applications on toxin detection, toxic mechanisms, <strong>and</strong><br />
therapeutics. To determine the ratio of BoNT <strong>and</strong> NAPs present in different lots of<br />
commercially available BoNT complexes, we applied a label-free mass spectrometry<br />
quantification method based on nanoscale ultra-performance liquid chromatography coupled<br />
to electrospray-ionization mass spectrometry with data-independent acquisition (UPLC-ESI-<br />
MSE). A rapid (3-minute) digestion method was used to obtain tryptic peptides from each lot<br />
of BoNT complex. The digests were run in triplicate <strong>and</strong> analyzed by UPLC-ESI-MSE; the<br />
proteomics data sets were further analyzed using commercial software (PLGS, IdentityE)<br />
<strong>and</strong> custom software written in-house. All BoNTs <strong>and</strong> NAPs were detected with high protein<br />
sequence coverage in the commercial complexes along with other proteins such as flagellin<br />
<strong>and</strong> ORFs. The relative quantity of proteins in the BoNT complexes was accurately<br />
determined. The BoNT:NAPs ratios were higher for BoNT /E <strong>and</strong> /F complexes (1:1) than for<br />
BoNT /A, /B, /C, <strong>and</strong> /D (1:5 – 1:7); hemagglutinins were the most abundant proteins<br />
detected in the complexes in the latter. The label-free method brings accurate quantification<br />
to the study of BoNT, <strong>and</strong> will contribute to a better underst<strong>and</strong>ing of BoNT complexes, their<br />
effects <strong>and</strong> applications.
P54<br />
GENOMIC AND PROTEOMIC ANALYSIS OF A BOVINE HEMORRHAGIC ABOMASITIS<br />
TYPE A CLOSTRIDIUM PERFRINGENS ISOLATE<br />
V. J. Nowell* 1 , J. F. Prescott 1 , J. G. Songer 2 , A. M. Kropinski 3 , J. I. MacInnes 1 <strong>and</strong> V. R.<br />
Parreira 1 . 1 Department of Pathobiology, University of Guelph, Guelph, ON, Canada. 2 <strong>Iowa</strong><br />
<strong>State</strong> University, Ames, IA, USA. 3 Public Health Agency of Canada, Laboratory for<br />
Foodborne Zoonoses, Guelph, ON, Canada.<br />
Type A Clostridium perfringens is a cause of hemorrhagic abomasitis in calves, a serious<br />
<strong>and</strong> often fatal infection in milk-fed animals. The disease has been reproduced<br />
experimentally, but the basis of its virulence is not known. To underst<strong>and</strong> the characteristics<br />
of C. perfringens associated with hemorrhagic abomasitis, an isolate of type A C. perfringens<br />
obtained from a small outbreak in calves was analyzed using whole genome sequencing.<br />
The outbreak strain sequenced was shown by PFGE to be identical to that isolated from<br />
another calf that died of hemorrhagic abomasitis, supporting evidence that the correct strain<br />
was sequenced. The genome was sequenced using the Roche 454 GS FLX Titanium<br />
massively parallel shotgun sequencing technology with ~69X coverage total. The genome<br />
was annotated <strong>and</strong> compared to other C. perfringens sequences to identify genes unique to<br />
the isolate. A hypothesized novel toxin gene was not identified, but interesting findings<br />
included a chromosomally-integrated plasmid fragment (~25 kb), three circular plasmids (~5<br />
kb, ~9 kb <strong>and</strong> ~55 kb) <strong>and</strong> a large plasmid fragment (50 kb) of unknown location.<br />
Approximately 300 kb of DNA <strong>and</strong> 261 ORFs were unique to this strain, including a number<br />
of recombination-associated elements, phage sequences <strong>and</strong> CRISPR elements. The most<br />
surprising finding was a frameshift in the virS gene that would result in the truncation of ~100<br />
amino acids at the C-terminal end. Given this, it is likely that the entire VirR/VirS system<br />
would be non-functional <strong>and</strong> therefore that VirR would be unable to activate transcription of<br />
many virulence genes. Despite this, the strain expressed perfringolysin O, alpha-toxin <strong>and</strong><br />
beta2-toxin. We were unable to identify any novel toxin genes by genome analysis or using<br />
mass spectrometry of the supernatant. It is possible that hemorrhagic abomasitis caused by<br />
type A C. perfringens is the result of a combination of factors unrelated to the presence of<br />
novel toxins or that novel virulence factors remain hidden in the 60% of hypothetical proteins<br />
found among the unique ORFs.
P55<br />
A FRAGMENT OF 97 AMINO ACIDS (D97) WITHIN THE TRANSMEMBRANE DOMAIN IS<br />
ESSENTIAL FOR THE CELLULAR ACTIVITY OF CLOSTRIDIUM DIFFICILE TOXIN B<br />
Y. Zhang 1,2 , L.Shi* 1 , X. Sun 1 , R. Kumar 3 , T. Savidge 3 , R. Zhuge 4 , Z. Yang 2 , X. Wang 2 , <strong>and</strong> H.<br />
Feng 1 . 1 Tufts Cummings School of Veterinary Medicine, North Grafton, MA, 01536, USA;<br />
2 School of Bioengineering, East China University of Science <strong>and</strong> Technology, Shanghai<br />
200237, China; 3 The University of Texas Medical Branch, Galveston, TX, 77555, USA;<br />
4 University of Massachusetts School of Medicine, Worcester.<br />
Clostridium difficile toxin B (TcdB) is one of the major virulence factors associated with C.<br />
difficile-associated disease, which intoxicates target cells by glucosylating Rho GTPases.<br />
TcdB is a 269 kDa protein containing at least 4 functional domains including a<br />
glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a transmembrane<br />
domain (TD), <strong>and</strong> a receptor binding domain (RBD). In this study, we have found that a 97-<br />
amino acid fragment (D97) located in the C-terminus of the TD is essential for the cellular<br />
activity of TcdB. We deleted this fragment <strong>and</strong> expressed a truncated protein designated as<br />
TxB-D97, <strong>and</strong> found that this deletion did not significantly alter the conformation of the toxin<br />
or the functions of the other domains. The toxin cell binding <strong>and</strong> uptake were similar between<br />
the mutant <strong>and</strong> wild type TcdB. Both wildtype <strong>and</strong> mutant toxins released their GTDs<br />
similarly in the presence of inositol hexakisphosphate (InsP6), <strong>and</strong> showed similar activity in<br />
a cell-free glucosylating assay. Despite these similarities, the cytotoxic activities of TxB-D97<br />
were reduced by more than 5 logs compared to wild type toxin. Cells exposed to TxB-D97<br />
also had an undetectable level of glucosylated Rac1. Unlike wild type TcdB, the mutant toxin<br />
failed to induce macrophages to produce tumor necrosis factor alpha (TNF-α), an outcome<br />
dependent on the GT activity of the toxin. Cellular fractionation of toxin-exposed cells<br />
demonstrated that the TxB-D97 was unable to release its GT domain efficiently into cytosol.<br />
Thus, the D97 fragment, located in the C-terminus of the TD, adjacent to the RBD, appears<br />
to play an essential role in the delivery of the GT domain into cell cytosol.
P56<br />
NECROTIZING ENTERITIS ASSOCIATED WITH CLOSTRIDIUM PERFRINGENS TYPE B<br />
IN CHINCHILLAS (CHINCHILLA LANIGERA)<br />
R. Lucena 1 , L. Farias 2 , F. Libardoni 2 , A.C. Vargas* 2 , P. Giaretta 1 , C.S. L. Barros 1 .<br />
1 Laboratory of Veterinary Pathology, UFSM,CEP 97105-900 Santa Maria/RS, BRAZIL;<br />
2 Laboratory of Bacteriology, UFSM,CEP 97105-900 Santa Maria/RS, BRAZIL.<br />
Four 3-4 month-old chinchillas (Chinchilla lanigera) from a commercial flock of 395<br />
chinchillas, were found dead with evidence of previous diarrhea <strong>and</strong> prolapsed rectum. A fifth<br />
8 month-old chinchilla died 8 hours after being found recumbent, apathetic, diarrheic, <strong>and</strong><br />
with a prolapsed rectum. Two chinchillas were necropsied <strong>and</strong> gross lesions consisted of<br />
extensive hemorrhagic enteritis, mild pulmonary edema, <strong>and</strong> enlarged <strong>and</strong> yellow liver; this<br />
latter finding was particularly prominent in the chinchilla presenting with the longer clinical<br />
course. Histologically, there was necrotizing enteritis associated with abundant aggregates of<br />
bacterial rods on the intestinal epithelial surface <strong>and</strong> within the lamina propria. In the lungs,<br />
there were small amounts of pink proteinaceous material (edema) in the interstitium <strong>and</strong><br />
marked vacuolar hepatocellullar degeneration (lipidosis) in the liver. Anaerobic cultures from<br />
the intestinal contents of one of the affected chinchillas yielded Clostridium perfringens.<br />
Genotyping of this C. perfringens isolate was achieved by multiplex polymerase chain<br />
reaction (mPCR), which revealed that the isolates was genotype B, due to detection of alpha,<br />
beta, <strong>and</strong> epsilon-toxin genes. These findings suggest C. perfringens type B as a cause of<br />
sudden or acute death in chinchillas.
P57<br />
DETERMINATION OF FUNCTIONAL RESIDUES ON NETB TOXIN FROM CLOSTRIDIUM<br />
PERFRINGENS<br />
X. Yan* 1 , C. J. Porter 2 , A. L. Keyburn 1,3 , D. Steer 2 , A. I. Smith 2 , N. Quinsey 2 , V. Hughes 2 , J.<br />
K. Cheung 1 , J. C. Whisstock 2 , R. J. Moore 1,3 , T. L. Bannam 1 <strong>and</strong> J. I. Rood 1 . Australia<br />
Research Council Centre of Excellence in Structural <strong>and</strong> Functional Microbial Genomics,<br />
Departments of 1 Microbiology <strong>and</strong> 2 Biochemistry <strong>and</strong> Molecular Biology, Monash University,<br />
VIC, Australia; 3 CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong,<br />
VIC, Australia.<br />
The novel toxin NetB is a putative β-barrel pore-forming toxin with 31% amino acid sequence<br />
identity to α-hemolysin (Hla) from Staphylococcus aureus. NetB is almost exclusively found<br />
in avian isolates of Clostridium perfringens type A <strong>and</strong> is associated with necrotic enteritis in<br />
chickens. The objective of this study was to determine NetB residues essential for its<br />
function. Both site-directed <strong>and</strong> r<strong>and</strong>om mutagenesis were used to generate netB point<br />
mutations that led to single amino acid substitutions in NetB. Based on the similarity of NetB<br />
to Hla, two residues that were equivalent to amino acids known to be important for Hla<br />
function were substituted by site-directed mutagenesis. One of the resultant NetB<br />
derivatives, R230Q, was no longer functional, whereas the other, D186N, had wild-type<br />
activity. Subsequently, a non-haemolytic C. perfringens strain was used as the host to screen<br />
for netB mutants after passage of a complementation plasmid through the mutator strain XL-<br />
1 Red. A library of over 200 mutants was obtained; subsequent analysis led to the<br />
determination of 14 unique NetB amino acid substitutions that resulted in a non-haemolytic<br />
phenotype on horse blood agar. Seven of these substituted NetB proteins were overexpressed<br />
<strong>and</strong> purified. Analysis of their CD spectra showed that they all adopted a<br />
predominant β-sheet fold that was similar to wild-type NetB. Functional analysis of these<br />
proteins led to the identification three residues that were essential for the lysis of chicken <strong>and</strong><br />
duck red blood cells. These results represent the first structure-function data generated for<br />
NetB <strong>and</strong> will have significant implications for our underst<strong>and</strong>ing of the mode of action of this<br />
novel toxin.
P58<br />
SEAFOOD AS A RESERVOIR AND A SUPPLIER OF THE PROTOTYPE PLASMID<br />
ENCODING CLOSTRIDIUM PERFRINGENS ENTEROTOXIN<br />
N. Yumine*, K. Mimura, K. Miyamoto. Wakayama Medical University, Department of<br />
Microbiology, Wakayama, Japan.<br />
Clostridium perfringens is a ubiquitous bacterium in the environment <strong>and</strong> in the<br />
gastrointestinal (GI) tract of human <strong>and</strong> domestic animals. This bacterium can produce up to<br />
16 different toxins. Of these toxins, C. perfringens enterotoxin (CPE) is the most important for<br />
human GI diseases. C. perfringens isolates carrying the CPE gene (cpe) are rarely found in<br />
retail seafood, although freshwater sediments contain many cpe-positive isolates. Therefore,<br />
the current study investigated the frequency of cpe-positive C. perfringens in raw retail<br />
seafood <strong>and</strong> then performed a genetic analysis of those isolates. PCR survey using DNA<br />
prepared from enrichment culture of seafood samples revealed that 80% of 70 surveyed<br />
samples showed a positive reaction in a cpe-PCR assay. For the investigated seafood<br />
samples, 49 of 50 retail oyster samples without shells contained cpe-positive C. perfringens.<br />
In estimating the amount of iron-reducing Clostridia present in oyster samples with a three<br />
tube MPN method, iron-reducing Clostridia including C. perfringens were 1,000 colonies/gram. Moreover, only 28 of 908 lecito-vetelline-positive colonies<br />
(mostly C. perfringens) showed a positive reaction with cpe-PCR assay. From these findings,<br />
seafood might be a reservoir for cpe-positive C. perfringens. However, retail seafood should<br />
not be a food vehicle for food poisoning, if prepared promptly. To further characterize<br />
seafood isolates, genetic assays were performed. Toxin genotype PCR assay indicated that<br />
all 34 cpe-positive <strong>and</strong> 11 cpe-negative isolates classify as type A C. perfringens. To identify<br />
the cpe location by cpe-genotyping PCR assay, 33 of 34 cpe-positive isolates carry their cpe<br />
gene on plasmid; 27 have IS1151 near their cpe gene, 6 have IS1470-like near their cpe<br />
gene, 1 has an unknown cpe locus. The genetic relationship among cpe-positive seafood<br />
<strong>and</strong> human clinical isolates using groEL sequence typing revealed that those cpe-positive<br />
isolates might be closely related. Collectively, these results suggest that retail seafood might<br />
be a major reservoir of plasmid borne cpe-positive C. perfringens.
P59<br />
COMPARISON OF CHANGES IN HUMAN AND POULTRY INTESTINAL MICROBIOTA<br />
DURING COLONISATION WITH CLOSTRIDIUM DIFFICILE<br />
J. Škraban 1 , M. Medved 2 , M. Rupnik* 1,2,3 . 1 University of Maribor, Faculty of Medicine, 2000<br />
Maribor, Slovenia; 2 Institute of Public Health Maribor, Centre for Microbiology, 2000 Maribor,<br />
Slovenia; 3 Centre of Excellence CIPKeBiP, 1000 Ljubljana, Slovenia.<br />
Although the changes in intestinal microbiota during colonization of C. difficile have become<br />
an important part of research in humans, the data are still very limited. Few published studies<br />
describe changes of the bacterial microbiota in humans only <strong>and</strong> there are virtually no data<br />
on changes/effects of gut microbiota during colonisation with C. difficile in animals or on<br />
changes in non-bacterial microbial groups. The aim of our study was to analyse the changes<br />
in bacterial, fungal <strong>and</strong> archaeal gut microbiota in C. difficile colonised <strong>and</strong> non – colonised<br />
individuals. We used a new molecular high-throughput method for DNA fragment analysis<br />
called denaturing high performance liquid chromatography (DHPLC; Wave system,<br />
Transgenomic). The level of the analysis is comparable to gradient gels (DGGE), with the<br />
advantage of high automatisation <strong>and</strong> repeatability. In the study we have analysed 200<br />
human <strong>and</strong> 143 poultry faecal samples from individuals colonised (n (humans) = 105, n<br />
(poultry) = 86) <strong>and</strong> non-colonised (n (humans) = 95, n (poultry) = 57) with C. difficile. In<br />
humans the main differences were observed in the species diversity. The results show that<br />
C. difficile colonised subjects have a simpler bacterial <strong>and</strong> archaeal microbiota <strong>and</strong> more<br />
complex fungal microbiota. The colonisation status correlates to the presence/absence of<br />
certain bacterial groups like clostridia, Bacteroides spp, <strong>and</strong> bifidobacteria. In poultry, we<br />
have followed also the development of the gut microbiota over time. As the bacterial<br />
microbiota complexity increased with age, the C. difficile colonisation rates dropped. The<br />
colonisation status was associated to the presence/absence of certain enterococci. While the<br />
archaeal microbiota developed between 2 <strong>and</strong> 4 months of age, the fungal microbiota<br />
seemed to be relatively diverse from the beginning. Our results show a change in diversity<br />
<strong>and</strong> a shift in the composition of the bacterial, but also in the fungal <strong>and</strong> archaeal, gut<br />
microbiota in correlation to C. difficile colonisation.
P60<br />
DEVELOPMENT OF A QUANTITATIVE IMMUNO-PCR ASSAY FOR THE DETECTION OF<br />
GROUP III CLOSTRIDIUM BOTULINUM NEUROTOXINS IN CATTLE<br />
T.C. Ardis* 1 , C.E. Brooks 1 , D. Fairley 2 , <strong>and</strong> H.J. Ball 1 . 1 Agri-food & Bioscience Institute,<br />
Veterinary Science Division, Stoney Road, Belfast, BT4 3SD UK; 2 Regional Virus<br />
Laboratory, Belfast Health & Social Care Trust, Kelvin Building, Grosvenor Road, Belfast,<br />
BT12 6BA UK.<br />
Introduction: Cattle botulism typically involves Group III neurotoxins, types C <strong>and</strong> D,<br />
produced by Clostridium botulinum. The ‗gold st<strong>and</strong>ard‘ diagnostic method for detection of<br />
botulinum neurotoxin (BoNT) is the mouse bioassay. This assay has greater sensitivity than<br />
any in vitro assay available, but it also has certain disadvantages. The quantitative Immuno-<br />
PCR (qIPCR) technique, which combines PCR with the st<strong>and</strong>ard ELISA method, can<br />
reportedly lead to a 10- to 1000-fold increase in the limit of detection as compared to the<br />
original ELISA. The aim of this work was to increase the sensitivity of a previously<br />
developed monoclonal antibody-based sELISA that detects both types C <strong>and</strong> D BoNTs, by<br />
developing a qIPCR assay which could provide a possible alternative to the st<strong>and</strong>ard mouse<br />
bioassay. Method: A sequential qIPCR method was employed using the same capturedetection<br />
MAbs as the original sELISA. Recombinant streptavidin was used to bridge the<br />
biotinylated detection antibody with a biotinylated 438 bp DNA marker. Quantitation of BoNT<br />
type D was achieved by real-time PCR amplification of the DNA marker. Results: The<br />
sequential qIPCR protocol allowed for approximately a 40-fold (98.4 pg/ml) increase in<br />
sensitivity as compared to the conventional sELISA. Detection of BoNT type D was linear<br />
over a range of 10 – 0.078 ng/ml (R 2 = 0.986). Conclusions: While a considerable<br />
improvement on the sensitivity of the original sELISA, this level of sensitivity is not yet<br />
sufficient to provide an in vitro replacement for the mouse bioassay, which detects BoNT at<br />
approximately 20 pg/ml. Alternative qIPCR methods, direct <strong>and</strong> modular, are currently being<br />
investigated. These methods are reported to be more sensitive than the sequential method.
P61<br />
THE LOCAL AND SYSTEMIC IMMUNE RESPONSE IN A MOUSE MODEL OF ACUTE<br />
CLOSTRIDIUM DIFFICILE INFECTION<br />
A.A. Sadighi Akha *1 , C.M. Theriot 2 , A.J. McDermott 3 , A.E. Reeves 3 , V.B. Young 2,3 <strong>and</strong> G.B.<br />
Huffnagle 1,3 . 1 Divisions of Pulmonary <strong>and</strong> Critical Care Medicine <strong>and</strong> 2 Infectious Diseases,<br />
3 Department of Internal Medicine, <strong>and</strong> Department of Microbiology <strong>and</strong> Immunology, The<br />
University of Michigan Medical School, Ann Arbor, MI 48109 USA.<br />
Clostridium difficile infection (CDI) is a significant cause of morbidity <strong>and</strong> mortality in<br />
hospitals <strong>and</strong> hospices. While the higher susceptibility of immuno-compromised individuals<br />
<strong>and</strong> the elderly to CDI underscore the protective role of the immune response to this<br />
organism, the topography, kinetics, <strong>and</strong> mechanisms of the host response that mediate this<br />
protection remain largely unknown. Here we report our initial findings in an acute mouse<br />
model of CDI. 5-8 week old male C57BL/6 mice were either left untreated or made<br />
susceptible to C. difficile infection <strong>and</strong> colitis by administering a cocktail of 5 antibiotics in<br />
drinking water for 3 days followed by a single intra-peritoneal dose of clindamycin. The<br />
antibiotic-treated mice were then infected with 10 5 CFUs of C. difficile strain VPI 10463, <strong>and</strong><br />
sacrificed 42 hours after the infection. Flow-cytometric study of the spleens, mesenteric<br />
lymph nodes (MLN), colons <strong>and</strong> cecums of C. difficile-infected mice showed a significant<br />
increase in the number of CD45 + cells in their colons <strong>and</strong> ceca, but not their spleens or MLN,<br />
in comparison to non-antibiotic-treated, uninfected controls. This in turn was due to a<br />
significant across-the-board increase in the numbers of B cells, CD4 <strong>and</strong> CD8 T cells,<br />
FOXP3 + CD4 T cells, NK cells, neutrophils, macrophages, <strong>and</strong> dendritic cells in the colons<br />
<strong>and</strong> ceca of the infected mice. The spleens <strong>and</strong> MLN of the infected mice did not display any<br />
such increase. A fraction of the recruited neutrophils in the colons <strong>and</strong> ceca of the infected<br />
mice displayed up-regulated levels of CD11b, an indication of their activated state. By<br />
contrast, a similar fraction of CD4 <strong>and</strong> CD8 T cells in the colons <strong>and</strong> ceca of uninfected <strong>and</strong><br />
infected mice showed an up-regulated level of the activation marker CD69. These data<br />
demonstrate the diversity of the hematopoetic cells recruited to the colons <strong>and</strong> ceca of mice<br />
acutely infected with C. difficile, provide no indication of overwhelming activation in the<br />
recruited lymphocytes to these organs, <strong>and</strong> underscore the local nature of the response.
P62<br />
PORCELLIO SCABER – A NEW NONVERTEBRATE MODEL FOR C. DIFFICILE<br />
COLONIZATION<br />
M. Zemljič* 1,2 , M. Rupnik 1,2,3 , <strong>and</strong> D.Drobne 4 . 1 University of Maribor, Faculty of Medicine,<br />
Maribor, Slovenia; 2 Centre of Excellence for Integrated Approaches in Chemistry <strong>and</strong> Biology<br />
of Proteins, Ljubljana, Slovenia; 3 Institute of Public Health Maribor, Centre for Microbiology,<br />
Department for Microbiology Research, Maribor, Slovenia; 4 University of Ljubljana,<br />
Biotechnical Faculty, Department of Biology,Večna pot 111, Ljubljana, Slovenia.<br />
The terrestrial isopod Porcellio scaber (Crustacea) is widely used as a model for toxicity<br />
testing of pollutants. The aim of this study was to test P. scaber as a possible model for<br />
Clostridium difficile colonization. P. scaber is a decomposer that preferentially feeds on<br />
decayed plant material colonised by microorganisms, which are also utilized as a source of<br />
nutrients <strong>and</strong> enzymes. Whereas some undigested microorganisms are passive transients,<br />
others proliferate in the gut. The gut‘s simple tube-like anatomy, the rapid passage of the<br />
food <strong>and</strong> frequent renewal of the gut cuticle appeared to be unsuitable for development of<br />
resident <strong>and</strong> anaerobic microbiota. However, the reports on isolation of strictly anaerobic<br />
bacteria from P. scaber confirmed the presence of anaerobic microniches. As in a st<strong>and</strong>ard<br />
P. scaber experiment, animals were divided into groups of ten <strong>and</strong> were fed with hazelnut<br />
tree leaves (Corylus avellana) for 30 days. Test groups were exposed to leaves with C.<br />
difficile spores, <strong>and</strong> the control group to leaves without C. difficile. The selected endpoints<br />
were the following: i) feeding activity between control <strong>and</strong> tested animals, ii) colonization with<br />
C. difficile <strong>and</strong> iii) survival of the animals. In the first set of experiments, we compared two<br />
control groups exposed to C. difficile 630, one of them previously exposed also to<br />
clindamycin. Strain 630 caused death of animals in a very broad time interval from 3 to 21<br />
days after exposure. Use of antibiotic did not affect the survival rates. In the second set of<br />
experiments, animals were exposed to a combination of two different strains. Strain pairs<br />
included a) toxigenic <strong>and</strong> nontoxigenic strains, b) toxigenic strain with high <strong>and</strong> toxigenic<br />
strain with low virulence potential, <strong>and</strong> c) two strains with high virulence potential. All<br />
combinations resulted in 50 to 100% colonization <strong>and</strong> in 30 to 100% mortality. Survival was<br />
lowest in the group exposed to two strains with high virulence. These results suggest that P.<br />
scaber could be a potentially useful model for assessing C. difficile virulence or to compare<br />
fitness among strains under in vivo conditions.
<strong>CLOSPATH</strong> 7 PARTICIPANTS
ClosPath<br />
Institution<br />
Participants Country E-mail Abstract<br />
Aktories, Klaus Germany University of Freiburg O15<br />
klaus.aktories@pharmakol.uni-freiburg.de<br />
Ardis, Tara United Kingdom Agri-Food <strong>and</strong> Biosciences Institute P60<br />
jenna.donaldson@afbini.gov.uk<br />
Aronoff, David United <strong>State</strong>s The University of Michigan O2<br />
daronoff@umich.edu<br />
Aubry, Annie Canada National Research Council<br />
annie.aubry@nrc-cnrc.gc.ca<br />
Auchtung, Jennifer United <strong>State</strong>s Michigan <strong>State</strong> University P1<br />
auchtun3@msu.edu<br />
Babakhani Farah Optimer Pharmaceuticals, Inc. P2, P3<br />
fbabakhani@optimerpharma.com<br />
Bakker, Dennis Netherl<strong>and</strong>s Leiden University Medical Center P4<br />
d.bakker@lumc.nl<br />
Ballard, Jimmy United <strong>State</strong>s OUHSC O5<br />
jimmy-ballard@ouhsc.edu<br />
Barbieri, Joseph United <strong>State</strong>s Medical College of Wisconsin O41<br />
jtb01@mcw.edu<br />
Basak, Ajit United Kingdom Birkbeck College O18<br />
a.basak@mail.cryst.bbk.ac.uk<br />
Blasi, Juan Spain University of Barcelona P41<br />
blasi@ub.edu<br />
Bordeleau, Eric Canada University de Sherbrooke<br />
eric.bordeleau@usherbrooke.ca<br />
Bouillaut, Laurent United <strong>State</strong>s Tufts University P5<br />
laurent.bouillaut@tufts.edu<br />
Bradshaw, Marite United <strong>State</strong>s University of Wisconsin-Madison<br />
mbradsha@wisc.edu
ClosPath<br />
Institution<br />
Participants Country E-mail Abstract<br />
Buckley, Anthony United Kingdom University of Glasgow O26<br />
Anthony.Buckley@Glasgow.ac.uk<br />
Burrus, Vincent Canada University de Shebrooke<br />
Vincent.burrus@usherbrooke.ca<br />
Buss, Janice USA TechLab, Inc.<br />
jebuss2@gmail.com<br />
Butel, Marie-José France University Paris Descartes/Faculty of<br />
Pharmacy<br />
marie-jose.butel@parisdescartes.fr<br />
P42<br />
Carlson, Paul United <strong>State</strong>s University of Michigan P6<br />
pecarl@umich.edu<br />
Cartman, Stephen United Kingdom University of Nottingham P7<br />
stephen.cartman@nottingham.ac.uk<br />
Chakravorty, Anjana Australia Monash University O36<br />
anjana.chakravorty@monash.edu<br />
Chen, Jianming United <strong>State</strong>s University of Pittsburgh O46<br />
jic40@pitt.edu<br />
Chen, Kevin United <strong>State</strong>s Tufts University O25<br />
Chun-Nian.Chen@tufts.edu<br />
Cheng, Ying China National Institute for Communicable Disease<br />
Control <strong>and</strong> Prevention<br />
chengying@icdc.cn<br />
Collignon, Anne France University Paris Sud 11 O13, P8,<br />
anne.collignon@u-psud.fr<br />
P9, P10<br />
Coppe, Philippe Belgium Bio-X Diagnostics<br />
p.coppe@biox.com<br />
Corver, Jeroen Netherl<strong>and</strong>s Leiden University Medical Center P11<br />
j.corver@lumc.nl<br />
Curry, Scott United <strong>State</strong>s University of Pittsburgh P12<br />
currysr@upmc.edu<br />
O4
ClosPath<br />
Institution<br />
Participants Country E-mail Abstract<br />
Cuttingm, Simon United Kingdom Royal Holloway, University of London O38<br />
S.Cutting@rhul.ac.uk<br />
D'Auria, Kevin United <strong>State</strong>s University of Virginia O47<br />
kd3jd@virginia.edu<br />
Derman, Yagmur Finl<strong>and</strong> University of Helsinki, Faculty of Veterinary<br />
Medicine<br />
yagmur.derman@helsinki.fi<br />
Di Paolo, Emmanuel Beligum GlaxoSmithKline Biologicals R & D<br />
Emmanuel.di-paolo@gskbio.com<br />
Downing, Mariann United <strong>State</strong>s ViroPharma, Inc.<br />
mariann.downing@viropharma.com<br />
Dupuy, Bruno France Institut Pasteur O19<br />
bdupuy@pasteur.fr<br />
Dyson, Walter United <strong>State</strong>s Merck<br />
walter.dyson@merck.com<br />
Ellermeier, Craig United <strong>State</strong>s University of <strong>Iowa</strong> O52<br />
craig-ellermeier@uiowa.edu<br />
Etzel, Lisa United <strong>State</strong>s <strong>Iowa</strong> <strong>State</strong> University<br />
iaponygirl@aol.com<br />
Fagan, Robert United Kingdom Imperial College London O32<br />
r.fagan@imperial.ac.uk<br />
Fairweather, Neil United Kingdom Imperial College London O50<br />
n.fairweather@imperial.ac.uk<br />
Farias, Luana Brazil UFSM P44<br />
luana.vett@gmail.com<br />
Feng, Hanping United <strong>State</strong>s Tufts University O35<br />
hanping.feng@tufts.edu<br />
Fern<strong>and</strong>es Da Costa, United Kingdom University of Exeter O17<br />
Sérgio<br />
s.p.fern<strong>and</strong>es-da-costa@exeter.ac.uk<br />
Figueroa, Iris United <strong>State</strong>s Hines VA Hospital P13<br />
iristfig@gmail.com
ClosPath<br />
Institution<br />
Participants Country E-mail Abstract<br />
Fortier, Louis-Charles Canada Universite de Sherbrooke O37<br />
Louis-Charles.Fortier@USherbrooke.ca<br />
Garcia, Jorge United <strong>State</strong>s University of California, Davis P45<br />
jorgepampa@yahoo.con.ar<br />
Gaskin, Duncan United Kingdom Institute of Food Research<br />
duncan.gaskin@ifr.ac.uk<br />
Gergen, Linda United <strong>State</strong>s Merck Animal Health<br />
linda.gergen@merck.com<br />
Gibert, Xavier Spain Laboratorios Hipra, S.A.<br />
xgp@hipra.com<br />
Ginter, Annita Belgium Bio-X Diagnostics<br />
a.ginter@biox.com<br />
Goossens, Evy Belgium Ghent University P46<br />
evy.goossens@UGent.be<br />
Govind, Revathi United <strong>State</strong>s Kansas <strong>State</strong> University O20<br />
rgovind@ksu.edu<br />
Govoni, Gregory United <strong>State</strong>s AvidBiotics Corporation P47<br />
greg.govoni@avidbiotics.com<br />
Hanna, Philip United <strong>State</strong>s University of Michigan Medical School<br />
pchanna@umich.edu<br />
Heeg, Daniela United Kingdom University of Nottingham P14<br />
daniela.heeg@nottingham.ac.uk<br />
Heinrichs, Jon United <strong>State</strong>s Merck & Co., Inc.<br />
jon_heinrichs@merck.com<br />
Henderson, Abigail United <strong>State</strong>s Hipra Scinetific USA, LLC<br />
xgp@hipra.com<br />
Ho, Theresa United <strong>State</strong>s University of <strong>Iowa</strong> P15<br />
theresa-ho@uiowa.edu<br />
Huang, Haihui China Fudan University Hospital Huashan<br />
huanghaihui@fudan.edu.cn
ClosPath<br />
Institution<br />
Participants Country E-mail Abstract<br />
Huynh, Hong United Kingdom Royal Holloway University<br />
hong.huynh@rhul.ac.uk<br />
Jabbari, Sara United Kingdom University of Nottingham<br />
sara.jabbari@nottingham.ac.uk<br />
Janezic, S<strong>and</strong>ra Slovenia Institute of Public Health Maribor P16<br />
s<strong>and</strong>ra.janezic@zzv-mb.si<br />
Johnson, Eric United <strong>State</strong>s University of Wisconsin O14<br />
eajohnso@wisc.edu<br />
Johnson, Stuart United <strong>State</strong>s Hines VA Hospital<br />
sjohnson@lumc.edu<br />
Kelly, Michelle United Kingdom University of Nottingham P48<br />
michelle.kelly@nottingham.ac.uk<br />
Kempker, Jennifer United <strong>State</strong>s Boehringer Ingelheim Vetmedica<br />
jennifer.kempker@boehringeringelheim.com<br />
Keto-Timonen, Riikka Finl<strong>and</strong> University of Helsinki P49<br />
riikka.keto-timonen@helsinki.fi<br />
Kirk, David Finl<strong>and</strong> University of Helsinki O43<br />
david.kirk@helsinki.fi<br />
Knetsch, Wilco Netherl<strong>and</strong>s Leiden University Medical Center O3<br />
c.w.knetsch@lumc.nl<br />
Kõljalg, Siiri Estonia University of Tartu P17<br />
siiri.koljalg@ut.ee<br />
Korkeala, Hannu Finl<strong>and</strong> University of Helsinki<br />
hannu.korkeala@helsinki.fi<br />
Kuehne, Sarah United Kingdom University of Nottingham O45<br />
sarah.kuehne@nottingham.ac.uk<br />
Lacy, Dana United <strong>State</strong>s V<strong>and</strong>erbilt University School of Medicine O6<br />
borden.lacy@v<strong>and</strong>erbilt.edu
ClosPath<br />
Institution<br />
Participants Country E-mail Abstract<br />
Lahti, Päivi Finl<strong>and</strong> University of Helsinki P50<br />
paivi.lahti@helsinki.fi<br />
Lambert, Dominic<br />
Canada<br />
Health Canada, Health Product <strong>and</strong> Food<br />
Branch<br />
dominic.lambert@hc-sc.gc.ca<br />
Lei, Xiang United <strong>State</strong>s Biolog, Inc. P18<br />
xlei@biolog.com<br />
Leite, Fern<strong>and</strong>o USA <strong>Iowa</strong> <strong>State</strong> University<br />
fleite@iastate.edu<br />
Lepp, Dion Canada University of Guelph P52<br />
dlepp@uoguelph.ca<br />
Li, Jihong United <strong>State</strong>s University of Pittsburgh O51<br />
jihongli@pitt.edu<br />
Li, Shan United <strong>State</strong>s Tufts University O8<br />
lishan.li@tufts.edu<br />
Lindström, Miia Finl<strong>and</strong> University of Helsinki O42<br />
miia.lindstrom@helsinki.fi<br />
Liu, Mingyu United <strong>State</strong>s Memorial Sloan-Kettering Cancer Center P19<br />
mymingyuliu@gmail.com<br />
Liu, Shie-Chau United <strong>State</strong>s Stanford University<br />
liusc@stanford.edu<br />
Lu, Jinxing China National Institute for Communicable<br />
Disease Control <strong>and</strong> Prevention<br />
lujinxing@icdc.cn<br />
Lu, Shan United <strong>State</strong>s University of Massachusetts<br />
Cindi.callaghan@umassmed.edu<br />
Lyras, Dena Australia Monash University O44<br />
dena.lyras@monash.edu<br />
MacCannell, Duncan United <strong>State</strong>s Centers for Disease Control <strong>and</strong> Prevention O29<br />
fms2@cdc.gov<br />
P51
ClosPath<br />
Institution<br />
Participants Country E-mail Abstract<br />
Mackin, Kate Australia Monash University O23<br />
kate.mackin@monash.edu<br />
Mallozzi, Michael United <strong>State</strong>s University of Arizona P20<br />
mallozzi@email.arizona.edu<br />
Marsh, Jane United <strong>State</strong>s University of Pittsburgh P21, P22<br />
jwmarsh@pitt.edu<br />
McBride, Shonna United <strong>State</strong>s Tufts University School of Medicine O39<br />
shonna.mcbride@tufts.edu<br />
McClain, Mark United <strong>State</strong>s V<strong>and</strong>erbilt University O16<br />
mark.mcclain@v<strong>and</strong>erbilt.edu<br />
McClane, Bruce United <strong>State</strong>s University of Pittsburgh O11<br />
bamcc@pitt.edu<br />
McQuade, Rebecca United <strong>State</strong>s University of Arizona O40<br />
rmcquade@email.arizona.edu<br />
Melnyk, Roman Canada University of Toronto<br />
roman.melnyk@sickkids.ca<br />
Melville, Stephen United <strong>State</strong>s Virginia Tech O49<br />
melville@vt.edu<br />
Miezeiewski, Matthew United <strong>State</strong>s Merck P23<br />
matthew_miezeiewski@merck.com<br />
Minton, Nigel United Kingdom University of Nottingham O12<br />
nigel.minton@nottingham.ac.uk<br />
Miyamoto, Kazuaki Japan Wakayama Medical University<br />
kazuaki@wakayama-med.ac.jp<br />
Moura, Hercules United <strong>State</strong>s Centers for Disease Control <strong>and</strong> Prevention P24, P53<br />
HMoura@cdc.gov<br />
Naaber, Paul Estonia University of Tartu P25<br />
paul.naaber@gmail.com<br />
Nowell, Vicki Canada University of Guelph P54<br />
vnowell@uoguelph.ca
ClosPath<br />
Institution<br />
Participants Country E-mail<br />
Ohtani, Kaori Japan Kanazawa University O21<br />
ohtanik@med.kanazawa-u.ac.jp<br />
Olling, Alex<strong>and</strong>ra Germany Hannover Medical School P26<br />
olling.alex<strong>and</strong>ra@mh-hannover.de<br />
Paredes-Sabja, Chile Universidad Andres Bello P27<br />
Daniel<br />
daniel.paredes.sabja@gmail.com<br />
Parreira, Valeria Canada University of Guelph<br />
vparreir@uoguelph.ca<br />
Posthaus, Horst Switzerl<strong>and</strong> University of Bern O28<br />
horst.posthaus@vetsuisse.unibe.ch<br />
Quesada-Gómez, Costa Rica Rica. San José, Costa Rica P28<br />
Carlos<br />
carlos.quesada@ucr.ac.cr<br />
Reeves, Angela United <strong>State</strong>s University of Michigan P29<br />
angelhop@umich.edu<br />
Riley, Thomas Australia University of Western Australia<br />
Thomas.riley@uwa.edu.au<br />
Robinson, Catherine United <strong>State</strong>s Michigan <strong>State</strong> University P30<br />
pohlcath@msu.edu<br />
Rood, Julian Australia Monash University O10<br />
julian.rood@monash.edu<br />
Abstract<br />
Rupnik, Maja Slovenia Institute of Public Health Maribor O1, P59<br />
maja.rupnik@zzv-mb.si<br />
Sadighi Akha, Amir United <strong>State</strong>s University of Michigan Medical School P61<br />
asadighi@umich.edu<br />
Sarker, Mahfuzur United <strong>State</strong>s Oregon <strong>State</strong> University O33<br />
sarkerm@oregonstate.edu<br />
Saujet, Laure France Institut Pasteur O34<br />
laure.saujet@pasteur.fr<br />
Schmidt, Diane United <strong>State</strong>s Tufts University<br />
diane.schmidt@tufts.edu
ClosPath<br />
Institution<br />
Participants Country E-mail<br />
Scholl, Dean United <strong>State</strong>s AvidBiotics Corporation P31<br />
dean@avidbiotics.com<br />
Schoster, Angelika Canada University of Guelph P32<br />
angelika_schoster@hotmail.com<br />
Secore, Susan United <strong>State</strong>s Merck & Co., Inc.<br />
susan_secore@merck.com<br />
Seibel, Janice United <strong>State</strong>s <strong>Iowa</strong> <strong>State</strong> University<br />
jseibel@iastate.edu<br />
Self, William United <strong>State</strong>s University of Central Florida O30<br />
wself@mail.ucf.edu<br />
Sepp, Epp Estonia University Tartu<br />
epp.sepp@ut.ee<br />
Shen, Aimee United <strong>State</strong>s University of Vermont O7<br />
aimee.shen@uvm.edu<br />
Shi, Lianfa United <strong>State</strong>s Tufts University P55<br />
lianfa.shi@tufts.edu<br />
Shimizu, Tohru Japan Kanazawa University<br />
tshimizu@med.kanazawa-u.ac.jp<br />
Abstract<br />
Shin, Bo-Moon Korea Sanggye Paik Hospital, Inje University P33, P34<br />
bmshin@unitel.co.kr<br />
Smits, Wiep Klaas Netherl<strong>and</strong>s Leiden University Medical Center O22<br />
w.k.smits@lumc.nl<br />
Sonenshein, Abraham United <strong>State</strong>s Tufts University School of Medicine O31<br />
linc.sonenshein@tufts.edu<br />
Songer, Glenn United <strong>State</strong>s <strong>Iowa</strong> <strong>State</strong> University<br />
jgsonger@iastate.edu<br />
Sorg, Joseph United <strong>State</strong>s Texas A&M University P35<br />
jsorg@bio.tamu.edu<br />
Springer, Sven Germany IDT Biologika GmbH<br />
sven.springer@idt-biologika.de
ClosPath<br />
Institution<br />
Participants Country E-mail<br />
Squire, Michele Australia University of Western Australia P36<br />
mspb@bigpond.net.au<br />
Staempfli, Henry Canada University of Guelph<br />
hstaempf@uoguelph.ca<br />
Stringer, S<strong>and</strong>ra United Kingdom Institute of Food Research<br />
s<strong>and</strong>ra.stringer@ifr.ac.uk<br />
Strong, Philippa Canada NRC<br />
philippa.strong@nrc.ca<br />
Sun, Xingmin United <strong>State</strong>s Tufts University O27<br />
xingmin.sun@tufts.edu<br />
Tadepalli, United <strong>State</strong>s Novartis Animal Health US, Inc.<br />
Sambasivarao<br />
sambasivarao.tadepalli@novartis.com<br />
Tamayo, Rita United <strong>State</strong>s University of North Carolina at Chapel Hill<br />
rita_tamayo@med.unc.edu<br />
Tangudu, Ch<strong>and</strong>ra United <strong>State</strong>s <strong>Iowa</strong> <strong>State</strong> University<br />
ctangudu@iastate.edu<br />
Tatarowicz, Walter United <strong>State</strong>s ViroPharma Incorporated<br />
walter.tatarowicz@viropharma.com<br />
Theile, Teri United <strong>State</strong>s USDA<br />
teri.l.theile@aphis.usda.gov<br />
Therien, Alex United <strong>State</strong>s Merck & Co., Ltd.<br />
alex_therien@merck.com<br />
Theriot, Casey United <strong>State</strong>s University of Michigan P37<br />
caseythe@med.umich.edu<br />
Abstract<br />
Tweten, Rodney K. United <strong>State</strong>s University of Oklahoma Keynote<br />
rod_tweten@ouhsc.edu<br />
Uzal, Francisco United <strong>State</strong>s University of California, Davis O24<br />
fuzal@cahfs.ucdavis.edu<br />
Vargas, Agueda Brazil UFSM P56<br />
agueda.vargas@gmail.com
ClosPath<br />
Institution<br />
Participants Country E-mail<br />
Vedantam, Gayatri United <strong>State</strong>s University of Arizona O48<br />
vkv@email.arizona.edu<br />
Viswanathan, Vish United <strong>State</strong>s University of Arizona<br />
vkv@email.arizona.edu<br />
von Eichel-Streiber, Germany tgcBIOMICS GmbH P38<br />
Christoph<br />
chv.eichel@tgcbiomics.de<br />
Wachtel, Marian United <strong>State</strong>s NIH<br />
waschtelm@niaid.nih.gov<br />
Walk, Seth United <strong>State</strong>s University of Michigan P39<br />
sethwalk@umich.edu<br />
Weeramantri, Lakmini Australia Monash University<br />
lakmini.weeramantri@monash.edu<br />
Wei, Wensheng China Peking University O9<br />
wswei@pku.edu.cn<br />
Wilson, Janet M. United <strong>State</strong>s USDA<br />
Janet.M.Wilson@aphis.usda.gov<br />
Wright, Lorinda United <strong>State</strong>s Edward Hines Jr. VA Hospital P40<br />
lorinda.wright2@va.gov<br />
Yan, Xuxia Australia Monash University P57<br />
xuxia.yan@monash.edu<br />
Yu, Brian United <strong>State</strong>s Loyola University Medical Center<br />
bryu@lumc.edu<br />
Yumine, Natsuko Japan Wakayama Medical University P58<br />
yu-mie.n-i0o.o0j.@docomo.ne.jp<br />
Zemljic, Mateja Slovenia University of Maribor P62<br />
mateja.zemljic@zzv-mb.si<br />
Zhang, Zhen Finl<strong>and</strong> University of Helsinki<br />
zhen.zhang@helsinki.fi<br />
Zhu, Jun United <strong>State</strong>s University of Pennsylvania<br />
junzhu@upenn.edu<br />
Abstract
AUTHOR INDEX
Adams, V. – O11, O24<br />
Monash University, Australia<br />
Aktories, K. – O15<br />
University of Freiburg, Germany<br />
Albrow, V.E. – O7<br />
St<strong>and</strong>ford University, USA<br />
Technische Universitat Munchen, Germany<br />
Aleksoniene, K. – P40<br />
Hines VA Hospital, USA<br />
Allen, C. – P35<br />
Texas A & M University, USA<br />
Almassalha, L.M. – O2<br />
University of Michigan, USA<br />
Aires, J. – P42<br />
Université Paris Descartes, France<br />
Ampel, N. – P20<br />
University of Arizona, USA<br />
Anderson, L. – O29<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Antunes, A. – O19<br />
Institut Pasteur, France<br />
Anwar, F. – P20<br />
University of Arizona, USA<br />
Ardis, T.C. – P60<br />
Agri-food & Bioscience Institute, UK<br />
Aronoff, D.M. – O2, P39<br />
University of Michigan, USA<br />
Arroyo, L.G. – P32<br />
University of Guelph, Canada<br />
Auchtung. J. – P1<br />
Michigan <strong>State</strong> University, USA<br />
Austin, J.W. – P51<br />
Institute for Biological Sciences, Canada<br />
Authemann, D. – O28<br />
University of Berne, Switzerl<strong>and</strong><br />
Awad, M.M. – O36<br />
Monash University, Australia<br />
Babakhani, F. – P2, P3<br />
Optimer Pharmaceuticals, Inc., USA<br />
Baban, S. – O12<br />
University of Nottingham, UK<br />
Bakker, D. – O22, P4, P11<br />
Leiden University, Netherl<strong>and</strong>s<br />
Ball, H.J. – P60<br />
Agri-food <strong>and</strong> Bioscience Institute, UK<br />
Ballard, J. – O5<br />
University of Oklahoma, USA<br />
Bannam, T.L. – O10, P57<br />
Monash University, Australia<br />
Bantwal, R. – O10<br />
Monash University, Australia<br />
Barbieri, J. T. – O41<br />
Medical College of Wisconsin, USA<br />
Barketi-Klai, A. – O13, P9<br />
Université Paris-Sud, France<br />
Barr, J.R. – P24, P53<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Barros, C.S.L. – P56<br />
UFSM, Brazil<br />
Basak, A.K. – O17, O18<br />
Birkbeck College, UK<br />
Beards, E.J. – O26<br />
University of Leicester, UK<br />
Blake, T.A. – P24<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Blasi, J. – P41<br />
University of Barcelona, Spain<br />
Bochner, B.R. – P18<br />
Biolog, Inc., USA<br />
Bodmer, J.L. – P23<br />
Merck Research Laboratories, USA<br />
Boerlin, P. – P52<br />
University of Guelph, Canada<br />
Bogyo, M. – O7<br />
Stanford University, USA<br />
University of Vermont, USA<br />
Bokori-Brown, M. – O17<br />
University of Exeter, UK<br />
Botton, S. – P44<br />
UFSM, Brazil<br />
Bouillaut, L. – O30, O31, P2, P3, P5<br />
Tufts University, USA<br />
Boursier, C. – P10<br />
Université Paris-Sud, France<br />
Britton, R. – P1, P30<br />
Michigan <strong>State</strong> University, USA<br />
Brooks, C.E. – P60<br />
Agri-food & Bioscience Institute, UK<br />
Brouwer, M. – P11<br />
UCL Eastman Dental Institute, UK<br />
Buckley, A.M. – O26<br />
University of Glasgow, UK<br />
Burns, D.A. – P7, P14<br />
University of Nottingham, UK<br />
Butel, M. – P42<br />
Université Paris Descartes, France<br />
Cai, C. – O9<br />
Peking University, China<br />
Camiade, E. – O19<br />
Institut Pasteur, France<br />
Campeotto, F. – P42<br />
Université Paris Descartes, France
Carlson Jr., P.E. – P6<br />
University of Michigan, USA<br />
Carter, G. – O23, O44<br />
Monash University, Australia<br />
Cartman, S.T. – O12, O45, P7, P14, P48<br />
University of Nottingham, UK<br />
Caserta, J. – O24<br />
University of Pittsburgh, USA<br />
Chakravorty, A. – O36, O44<br />
Monash University, Australia<br />
Chaves-Olarte, E. – P28<br />
Universidad de Costa Rica, Costa Rica<br />
Universidad Nacional, Costa Rica<br />
Chen, C. – O4<br />
Chinese CDC & Prevention, China<br />
Chen, J. – O46, O51<br />
University of Pittsburgh, USA<br />
Chen, K. – O8, O25, O35<br />
Tufts University, USA<br />
Cheng, Y. – O4<br />
Chinese CDC & Prevention, China<br />
Cheung, J.K. – O36, P57<br />
Monash University, Australia<br />
Choo, J.M. – O36<br />
Monash University, Australia<br />
Christen, S. – O28<br />
University of Berne, Switzerl<strong>and</strong><br />
Chumbler, N.M. O6<br />
V<strong>and</strong>erbilt University, USA<br />
Cobaugh, K. – O30<br />
University of Central Florida, USA<br />
Cockayne, A. – O12, O45, P48<br />
University of Nottingham, UK<br />
Cohen, J. – O29<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Collery, M.M. – O45<br />
University of Nottingham, UK<br />
Collignon, A. – O13, P8, P9, P10<br />
Université Paris-Sud, France<br />
Cook, J. – O38<br />
University of London, UK<br />
Cordon, G.P. – O35<br />
Tufts University, USA<br />
Corver, J. – O3, P4, P11<br />
Leiden University, UKCurry, S.R. – P12, P21<br />
University of Pittsburgh, USA<br />
Cutting, S.M. – O38<br />
Imperial College, UK<br />
Dahlsten, E. – O42, O43<br />
University of Helsinki, Finl<strong>and</strong><br />
D‘Auria, K.M. – O47<br />
University of Virginia, USA<br />
Davis, B. – O27<br />
Tufts University, USA<br />
Dawson, L.F. – O26<br />
University of Leicester, UK<br />
del Mar Gamboa-Coronado, M. – P28<br />
Universidad de Costa Rica, Costa Rica<br />
Deprez, P. – P46<br />
Ghent University, Belgium<br />
Derman, Y. – P43<br />
University of Helsinki, Finl<strong>and</strong><br />
Dineen, S. – O31<br />
Tufts University, USA<br />
Donato, G.M. – O47<br />
University of Virginia, USA<br />
Dorca-Arévalo. J. – P41<br />
University of Barcelona, Spain<br />
Douce, G. – O23, O26, O44<br />
University of Glasgow, UK<br />
Drobne, D. – P62<br />
University of Ljubljana, Slovenia<br />
Du, P. – O4<br />
Chinese CDC & Prevention, China<br />
Du, T. – P28<br />
Natinal Microbiology Laboratory, Canada<br />
Ducatelle, R. P46<br />
Ghent University, Belgium<br />
Dupuy, B. – O13, O19, O23, O34, O44, P9<br />
Institut Pasteur, France<br />
Ehsaan, M. – O12<br />
University of Nottingham, UK<br />
Ellermeier, C.D. – O52, P15<br />
University of <strong>Iowa</strong>, USA<br />
Ewing, S.A. – O2<br />
University of Michigan, USA<br />
Fagan, R.P. – O32<br />
Imperial College, UK<br />
Fairley, D. – P60<br />
Belfast Health & Social Care Trust, UK<br />
Fairweather, N. – O32, 038, O50<br />
Imperial College, UK<br />
Farias, L. – P44, P56<br />
UFSM, Brazil<br />
Farrow, M.A. O6<br />
V<strong>and</strong>erbilt University, USA<br />
Feng, H. – O8, O25, O27, O35, P55<br />
Tufts University, USA<br />
Fennessey, C.M. – O16<br />
V<strong>and</strong>erbilt University, USA<br />
Fern<strong>and</strong>es da Costa, S. – O17<br />
University of Exeter, UK<br />
Ferraris, L. – P42<br />
Université Paris Descartes, France
Figueroa, I. – P13<br />
Hines VA Hospital, USA<br />
Fisher, D. J.– O11, P45<br />
University of Pittsburgh<br />
Fortier, L.C. – O37, P31<br />
Universite de Sherbrooke, Canada<br />
Foster, N.F. – P36<br />
University of Western Australia, Australia<br />
Frenzel, E. – P26<br />
Hanover Medical School, Germany<br />
Friedman, D.B. – O6<br />
V<strong>and</strong>erbilt University, USA<br />
Galecki, A.T. – O2<br />
University of Michigan, USA<br />
Garcia, K.C. – O7<br />
Stanford University, USA<br />
Garcia, J.P. – O24, O46, P45<br />
University of California Davis, USA<br />
Garneau, J.R. – O37<br />
Université de Sherbrooke, Canada<br />
Gebhart, D. – P31, P47<br />
AvidBiotics Corp., USA<br />
Gee, J. – P21<br />
University of Pittsburgh, USA<br />
Gerding, D. – O44, P13, P22, P40<br />
Hines Veterans Affairs Hospital, USA<br />
Gerhard, R. – P26<br />
Hanover Medical School, Germany<br />
Gersch, M.M. – O7<br />
St<strong>and</strong>ford University, USA<br />
Gianetti, B. – O49<br />
Virginia Tech, USA<br />
Giaretta, P. – P56<br />
UFSM, Brazil<br />
Gisch, K. – P38<br />
tgcBIOMICS GmbH, Germany<br />
Goldstein, E.J.C. – P13<br />
RM Aiden Research Laboratory, USA<br />
UCLA, USA<br />
Gomez, A. – P2, P3<br />
Optimer Pharmaceuticals, Inc., USA<br />
Gong, J. – P52<br />
Guelph Food Research Centre, Canada<br />
Goossens, E. – P46<br />
Ghent University, Belgium<br />
Govind, R. – O20, O23, O44<br />
Kansas <strong>State</strong> University, USA<br />
Govoni, G. – P31, P47<br />
AvidBiotics Corp., USA<br />
Goy, S. – P26<br />
Hanover Medical School, Germany<br />
Gray, M.C. – O47<br />
University of Virginia, USA<br />
Gurjar, A. – O11<br />
University of Pittsburgh<br />
Guzmán-Verri, C. – P28<br />
Universidad Nacional, Costa Rica<br />
Hanna, P.C. – P6<br />
University of Michigan, USA<br />
Harmanus, C. – O3, P11<br />
Leiden University Medical Center, Netherl<strong>and</strong>s<br />
Harrison, L.H. – P12, P21, P22<br />
University of Pittsburgh, USA<br />
Hartman, A. – O49<br />
Virginia Tech, USA<br />
Hashsham, S. – P1<br />
Michigan <strong>State</strong> University, USA<br />
He, R. – O9<br />
Peking University, China<br />
Heap, J.T. – O12, O42, P7<br />
University of Nottingham<br />
Heeg, D. – P7, P14<br />
University of Nottingham, UK<br />
Henderson, T.K. – P22<br />
University of Pittsburgh, USA<br />
Heikinheimo, A. – P50<br />
University of Helsinki, Finl<strong>and</strong><br />
Heinrichs, J. – P23<br />
Merck Research Laboratories, USA<br />
Hensgens, M.P.M. – O3, P11<br />
Leiden University Medical Center, Netherl<strong>and</strong>s<br />
Hewlett, E.L. – O47<br />
University of Virginia, USA<br />
Hirakawa, H. – O21<br />
Kazusa DNA Research Institute, Japan<br />
Hiscox, T.J. – O36<br />
Monash University, Australia<br />
Ho, T.D. – O52, P15<br />
University of <strong>Iowa</strong>, USA<br />
Hong, H.A. – O38<br />
University of London, UK<br />
Howarth, P. – O23<br />
Monash University, Australia<br />
Hoys, S. – O13, P9<br />
Université Paris-Sud, France<br />
Huang, J. – O38<br />
University of London, UK<br />
Huffnagle, G.B.<br />
University of Michigan, USA<br />
Hughes, M. – O11, O24<br />
Monash University, Australia<br />
Hughes, V. – P57<br />
Monash University, Australia
Huyet, J. – O18<br />
Birkbeck College, UK<br />
Indra, A. – P16<br />
Austrian Agency for Health & Food Safety, Vienna<br />
Ivie, S.E. – O16<br />
V<strong>and</strong>erbilt University, USA<br />
Jacobson, M.J. – O14<br />
University of Wisconsin-Madison, USA<br />
Jain, R. – O2<br />
University of Michigan, USA<br />
Janezic, S. – P16<br />
Institute of Public Health Maribor, Slovenia<br />
Jarchum, I. – P19<br />
Sloan-Kettering Institute, USA<br />
Johnson, E. – O14<br />
University of Wisconsin, USA<br />
Johnson, S. – O44, P13, P40<br />
Hines Veterans Affairs Hospital, USA<br />
Jolivot, P-A. – P8<br />
Université Paris-Sud, France<br />
Just, I. – P26<br />
Hanover Medical School, Germany<br />
Kansau, I. – O13, P8, P9<br />
Université Paris-Sud, France<br />
Kelly, M.L. – O12, O44, O45, P7, P48<br />
University of Nottingham, UK<br />
Keto-Timonen, R. – P49<br />
University of Helsinki, Finl<strong>and</strong><br />
Keyburn, A.L. – P57<br />
Monash University, Australia<br />
CSIRO Livestock Industries, Australia<br />
Kirk, D. – O43<br />
University of Helsinki, Finl<strong>and</strong><br />
Knetsch, C.W. – O3<br />
Leiden University, Netherl<strong>and</strong>s<br />
Kõljalg, S. – P17, P25<br />
University of Tartu, Estonia<br />
Kolling, G.L. – O47<br />
University of Virginia, USA<br />
Korkeala, H. – O42, O43, P43, P49, P50<br />
University of Helsinki, Finl<strong>and</strong><br />
Kropinski, A. – P52, P54<br />
Public Health Agency of Canada, Canada<br />
Kuak, E. – P33, P34<br />
Inje Univesity, South Korea<br />
Kubiak, A. – O12<br />
University of Nottingham, UK<br />
Kuehne, S.A. – O12, O45, P7<br />
University of Nottingham, UK<br />
Kuhara, S. – O21<br />
Kyushu University, Japan<br />
Kuijper, E.J. – O3, O22, P4, P11<br />
Leiden University Medical Center, Netherl<strong>and</strong>s<br />
Kumar, R. – O35, P55<br />
The Commonwealth Medical College, USA<br />
Lacy, D.B. – O6<br />
V<strong>and</strong>erbilt University, USA<br />
Lahti, P. – P50<br />
University of Helsinki, Finl<strong>and</strong><br />
Lambert, D. – P9, P51<br />
Health Canada, Canada<br />
Lange, A. – P38<br />
tgcBIOMICS GmbH, Germany<br />
Lapp, D. – P52<br />
University of Guelph, Canada<br />
Lawley, T. – O44<br />
The Wellcome Trust Sanger Institute, UK<br />
Lee, E. – P33, P34<br />
Inje Univesity, South Korea<br />
Lei, X. – P18<br />
Biolog, Inc., USA<br />
Lepp, D.<br />
University of Guelph, Canada<br />
Lessa, F. – O29<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Li, C. – O9<br />
Peking University, China<br />
Li, J. – O11, O46, O51<br />
University of Pittsburgh, USA<br />
Li, S. – O8, O35<br />
Tufts University, USA<br />
Libardoni, F. – P56<br />
UFSM, Brazil<br />
Lim, S.C. – P36<br />
University of Western Australia, Australia<br />
Limbago, B. – O29<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Lindström, M. – O42, O43, P43, P50<br />
University of Helsinki, Finl<strong>and</strong><br />
Liu, E.W. – O2<br />
University of Michigan, USA<br />
Liu, M. – P19<br />
Sloan-Kettering Institute, USA<br />
Lu, J. – O4<br />
Chinese CDC & Prevention, China<br />
Lucena, R. – P56<br />
UFSM, Brazil<br />
Lupardus, P.J. – O7<br />
Stanford University, USA<br />
Lyras, D. – O23, O36, O44<br />
Monash University, Australia<br />
Ma, M. – O24, O46<br />
University of Pittsburgh, USA
MacCannell, D. – O29<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
MacInnes, J.I. – P54<br />
University of Guelph, Canada<br />
Mackin, K. – O23, O44<br />
Monash University, Australia<br />
Mallozzi, M. – O40, P20<br />
University of Arizona, USA<br />
Marsh, J.W. – P12, P21, P22<br />
University of Pittsburgh, USA<br />
Martín-Satué, M. – P41<br />
University of Barcelona, Spain<br />
Martin-Verstraete, I. – O19<br />
Institut Pasteur, France<br />
McBride, S.M. – O31, O39<br />
Tufts University, USA<br />
McClane, B. – O11, O24, O46, O51, P45<br />
University of Pittsburgh, USA<br />
McClain, M.S. – O16<br />
V<strong>and</strong>erbilt University, USA<br />
McDermott, A.J. – P61<br />
University of Michigan, USA<br />
McQuade, R. – O40<br />
University of Arizona, USA<br />
Medved, M. – P59<br />
Institute of Public Health Maribor, Slovenia<br />
Melville, S. – O49<br />
Virginia Tech, USA<br />
Micic, D. – O2<br />
University of Michigan, USA<br />
Miezeiewski, M. – P23<br />
Merck Research Laboratories, USA<br />
Mimura, K. – P58<br />
Wakayama Medical University, Japan<br />
Minton, N.P. – O12, O42, O45, P14, P48<br />
University of Nottingham, UK<br />
Mitchell, T. – P23<br />
Merck Research Laboratories, USA<br />
Miyamoto, K. – O11, P58<br />
Wakayama Medical University, Japan<br />
Monot, M. – O13, O19, O34, P9<br />
Institut Pasteur, France<br />
Moore, R.J. – P57<br />
Monash University, Australia<br />
CSIRO Livestock Industries, Australia<br />
Moss, D.S. – O17<br />
Birkbeck College, UK<br />
Moura, H. – P24, P53<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Mulvey, M.R. – P28<br />
National Microbiology Laboratory, Canada<br />
Mun, S. – P33, P34<br />
Inje Univesity, South Korea<br />
Muravsky, M. – P23<br />
Merck Research Laboratories, USA<br />
Murillio, R. – P38<br />
Oporon S.A., Spain<br />
Naaber, P. – P17, P25<br />
Stavanger University Hospital, Norway<br />
Naylor, C.E. – O17, O18<br />
Birbeck College, UK<br />
Newton, D.W. – O2<br />
University of Michigan, USA<br />
Ng, R. – O12, P48<br />
University of Nottingham, UK<br />
Nguyen, L. – P3<br />
Optimer Pharmaceuticals, Inc., USA<br />
Nicolis, I. – P42<br />
Université Paris Descartes, France<br />
Nie, W.<br />
Tufts University, USA<br />
Noon, A. – P20<br />
University of Arizona, USA<br />
Nowell, V.J. – P54<br />
University of Guelph, Canada<br />
Oester, C. – P38<br />
tgcBIOMICS GmbH, Germany<br />
Ohtani, K. – O21<br />
Kanazawa University, Japan<br />
Olling, A. – P26<br />
Hanover Medical School, Germany<br />
Pamer, E.G.– P19<br />
Sloan-Kettering Institute, USA<br />
Papazisi, L. – O14<br />
The J. Craig Venter Institute, USA<br />
Papin, J.A. – O47<br />
University of Virginia, USA<br />
Pardon, B. P46<br />
Ghent University, Belgium<br />
Paredes-Ssabja, D. – O21, O33, P27<br />
Universidad Andrés Bello, Chile<br />
Parreira, V. – P52, P54<br />
University of Guelph, Canada<br />
Péchiné, S. – P10<br />
Université Paris-Sud, France<br />
Permpoonpattana, P. – O38<br />
University of London, UK<br />
Petrella, L. – P40<br />
Hines VA Hospital, USA<br />
Peterson, S.N. – O14<br />
The J. Craig Venter Institute, USA<br />
Phetcharaburanin, J. – O38<br />
University of London, UK
Poon, R. – O11, O24<br />
Monash University, Australia<br />
Popoff, M. – O28<br />
Institut Pasteur, France<br />
Porter, C.J. – O10, P57<br />
Monash University, Australia<br />
Posthaus, H. – O28, P45<br />
University of Berne, Switzerl<strong>and</strong><br />
Prescott, J.F. – P52, P54<br />
University of Guelph, Canada<br />
Pruitt, R.N. – O6<br />
V<strong>and</strong>erbilt University, USA<br />
Puri, A.W. – O7<br />
St<strong>and</strong>ford University, USA<br />
Quesada-Gómez, C. – P28<br />
Universidad de Costga Rica, Costa Rica<br />
Quinsey, N. – P57<br />
Monash University, Australia<br />
Rätsep, M. – P17, P25<br />
University of Tartu, Estonia<br />
Rattei, T. – P16<br />
University of Vienna, Austria<br />
Reeves, A.E. – P29, P61<br />
University of Michigan, USA<br />
Riley, T.V. – P36<br />
University of Western Australia, Australia<br />
PathWest Laboratory Medicine, Australia<br />
Ring, C. – O2<br />
University of Michigan, USA<br />
Roberts, A.P. – P11<br />
UCL Eastman Dental Institute, UK<br />
Robertson, S. – O24, O51<br />
University of Pittsburgh, USA<br />
Robinson, C. – P1, P30<br />
Michigan <strong>State</strong> University, USA<br />
Rodríguez, C. – P28<br />
Universidad de Costa Rica, Costa Rica<br />
Rodríguez-Cavallini, E. – P28<br />
Universidad de Costa Rica, Costa Rica<br />
Rood, J. – O10, O11, O23, O24, O36, O44, O46,<br />
P57<br />
Monash University, Australia<br />
Rosenbusch, K.E. – O22<br />
Leiden University, Netherl<strong>and</strong>s<br />
Roxas, B. – O40<br />
University of Arizona, USA<br />
Rozé, J.C. – P42<br />
Médecine Néonatale, France<br />
Rubin, D.H. – O16<br />
V<strong>and</strong>erbilt University, USA<br />
Rupnik, M. – O1, P16, P59, P62<br />
University of Maribor, Slovenia<br />
Sadighi Akha, A. – P61<br />
University of Michigan, USA<br />
Sambol, S.P. – P13, P22 P40<br />
Hines VA Hospital, USA<br />
Loyola Univesity Medical Center, USA<br />
Saputo, J. – O24, O46, P45<br />
University of California Davis, USA<br />
Sarker, M.R. – O21, O33, P27<br />
Oregon <strong>State</strong> University, USA<br />
Saujet, L. – O34<br />
Institut Pasteur, France<br />
Savelkoul, P.H.M. – O3<br />
VU University Medical Center, Netherl<strong>and</strong>s<br />
Savidge, T. – O35, P55<br />
University of Texas, USA<br />
Savva, C.G. – O17, O18<br />
Birkbeck College, UK<br />
Sayeed, S. – O11, O51, P45<br />
University of Pittsburgh, USA<br />
Schauvlieghe, S. P46<br />
Ghent University, Belgium<br />
Schmidt, D. – O8<br />
Tufts University, USA<br />
Schnaufer, T. – P23<br />
Merck Research Laboratories, USA<br />
Scholl, D. – P31, P47<br />
AvidBiotics Corp., USA<br />
Schoster, A. – P32<br />
University of Guelph, Canada<br />
Sears, P. – P2, P3<br />
Optimer Pharmaceuticals, Inc., USA<br />
Seeback, S.A. O6<br />
V<strong>and</strong>erbilt University, USA<br />
Self, W.T. – O30, O31, P5<br />
University of Central Florida, USA<br />
Sepp, E. – P17, P25<br />
University of Tartu, Estonia<br />
Setlow, P. – O33<br />
University of Connecticut, USA<br />
Shen, A. – O7<br />
University of Vermont, USA<br />
Sheng, J. – O16<br />
V<strong>and</strong>erbilt University, USA<br />
Shewen, P.E. – P32<br />
University of Guelph, Canada<br />
Shi, L. – O8, P55<br />
Tufts University, USA<br />
Shimizu, T. – O21<br />
Kanazawa University, Japan<br />
Shin, B. – P33, P34<br />
Inje University, South Korea
Shiver, J. – P23<br />
Merck Research Laboratories, USA<br />
Shkut, E. – P17, P25<br />
University of Tartu, Estonia<br />
Shoemaker, C.B. – O8<br />
Tufts University, USA<br />
Shrestha, A. – O24<br />
University of Pittsburgh, USA<br />
Shutt, K. – P21<br />
University of Pittsburgh, USA<br />
Sims, C. – P2<br />
Optimer Pharmaceuticals, Inc., USA<br />
Škraban, J. – P59<br />
University of Maribor, Slovenia<br />
Smidt, I. – P17, P25<br />
University of Tartu, Estonia<br />
Smith, A.I. – P57<br />
Monash University, Australia<br />
Smits, W.K. – O22, P4<br />
Leiden University Medical Center, Netherl<strong>and</strong>s<br />
Solano, M.I. – P24, P53<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Somervuo, P. – P50<br />
University of Helsinki, Finl<strong>and</strong><br />
Sonenshein, A. L. – O30, O31, O39, P2, P3, P5<br />
Tufts University, USA<br />
Songer, J.G. – P52, P54<br />
<strong>Iowa</strong> <strong>State</strong> University, USA<br />
Sorg, J. – P35<br />
Texas A & M University, USA<br />
Soring, K. – P23<br />
Merck Research Laboratories, USA<br />
Söderholm, H. – P43<br />
University of Helsinki, Finl<strong>and</strong><br />
Soutourina, O. – O34<br />
Institut Pasteur, France<br />
Spencer, J. – O26<br />
University of Glasgow, UK<br />
Spiller, B.W. O6<br />
V<strong>and</strong>erbilt University, USA<br />
Squire, M.M. – P36<br />
University of Western Australia, Australia<br />
Srivastava, M. – O30<br />
University of Central Florida, USA<br />
Staempfli, H.R. – P32<br />
University of Guelph, Canada<br />
Stedtfield, R. – P1<br />
Michigan <strong>State</strong> University, USA<br />
Steele, J. – O25<br />
Tufts University, USA<br />
Steer, D. – P57<br />
Monash University, Australia<br />
Stsepetova, J. – P17<br />
University of Tartu, Estonia<br />
Sun, X. – O8, O25, O27, O35, P55<br />
Tufts University, USA<br />
Tashiro, K. – O21<br />
Kyushu University, Japan<br />
Tatge, H. – P26<br />
Hanover Medical School, Germany<br />
ter Meulen, J. – P23<br />
Merck Research Laboratories, USA<br />
Terilli, R.R. – P24, P53<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Oak Ridge Institute for Scientific Education, USA<br />
Theriot, C.M. – P37, P61<br />
University of Michigan, USA<br />
Timbermont, L. P46<br />
Ghent University, Belgium<br />
Titball, R.W. – O17<br />
University of Exeter, UK<br />
Toribio, T. – P38<br />
Oporon S.A., Spain<br />
Tremblay, J.M. – O8<br />
Tufts University, USA<br />
Tulenko, M.M. – P12<br />
University of Pittsburgh, USA<br />
Tweten, R.K. – Keynote<br />
University of Oklahoma, USA<br />
Twine, S.M. – P51<br />
HPFB Health Canada, Canada<br />
Tzipori, S. – O8, O25, O35<br />
Tufts University, USA<br />
Uzal, F.A. – O24, O46, P45<br />
University of California, Davis, USA<br />
Valgaeren, B. – P46<br />
Ghent University, Belgium<br />
Valiquette, L. – O37<br />
Université de Sherbrooke, Canada<br />
van der Bijl, M.W. – O3<br />
VU University Medical Center, Netherl<strong>and</strong>s<br />
Van Immerseel, F. – O28, P46<br />
Gent University, Belgium<br />
van Leeuwen, H.C. – O3, P11<br />
Leiden University Medical Center, Netherl<strong>and</strong>s<br />
van Pijkeren, J.P. – P30<br />
Michigan <strong>State</strong> University, USA<br />
Vargas, A.C. – P44, P56<br />
Universidade Federal de Santa Maria, Brazil<br />
Vedantam, G. – O40, O48, P20<br />
University of Arizona, USA<br />
Verherstraeten, S. – P46<br />
Ghent University, Belgium
Viala, C. – P10<br />
Université Paris-Sud, France<br />
Vidal, J.E. – O24, O46<br />
University of Pittsburgh, USA<br />
Villacampa, M. – P38<br />
Oporon S.A., Spain<br />
Viswanathan, V.K. – O40, P20<br />
University of Arizona, USA<br />
Vodovar, M. – P42<br />
Institut de Puériculture, France<br />
von Eichel-Streiber, C. – P38<br />
tgcBIOMICS GmbH, Germany<br />
Walk, S.T. – O2, P39<br />
University of Michigan, USA<br />
Walker, D. – O12<br />
University of Nottingham, UK<br />
Wang, H. – O25, O27, O35<br />
Tufts University, USA<br />
South China University of Science <strong>and</strong><br />
Technology, China<br />
Wang, J. – O35<br />
South China University of Technology, China<br />
Wang, S. – P23<br />
Merck Research Laboratories, USA<br />
Wang, X. – P55<br />
East China University of Science <strong>and</strong> Technology,<br />
China<br />
Wang, Y. – O9<br />
Peking University, China<br />
Washer, L. – O2<br />
University of Michigan, USA<br />
Wei, W. – O9<br />
Peking University, China<br />
Weese, J.S. – P32<br />
University of Guelph, Canada<br />
Weinmaier, T. – P16<br />
University of Vienna, Austria<br />
Whisstock, J.C. – O10, P57<br />
Monash University, Australia<br />
Williams, S. – P31, P47<br />
AvidBiotics Corp., USA<br />
Williamson, Y.M. – P24<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Winzer, K. – O12<br />
University of Nottingham, UK<br />
Wisniewski, J. – O10<br />
Monash University, Australia<br />
Wolk, D. – P20<br />
University of Arizona, USA<br />
Woolfitt, A.R.– P24, P53<br />
Centers for Disease Control <strong>and</strong> Prevention, USA<br />
Wren, B.W. – O26<br />
University of Leicester, UK<br />
Wright, L. – P40<br />
Hines VA Hospital, USA<br />
Wu, X. – O9<br />
Peking University, China<br />
Wyder, M. – O28<br />
University of Berne, Switzerl<strong>and</strong><br />
Xie, A. – P23<br />
Merck Research Laboratories, USA<br />
Yan, Q. – O4<br />
Chinese CDC & Prevention, China<br />
Yan, X. – P57<br />
Monash University, Australia<br />
Yang, Z. – P55<br />
East China University of Science <strong>and</strong> Technology,<br />
China<br />
Yell<strong>and</strong>, T. – O18<br />
Birkbeck College, UK<br />
Young, V.B. – O2, P29, P37, P39, P61<br />
University of Michigan, USA<br />
Yumine, N. – P58<br />
Wakayama Medical University, Japan<br />
Zemljič, M. – P62<br />
University of Maribor, Slovenia<br />
Zhang, Y. – O8, O25, O27, O35, O42, O43, P55<br />
Tufts University, USA<br />
Zhuge, R. – P55<br />
University of Massachusetts, USA<br />
Zorman, J. – P23<br />
Merck Research Laboratories, USA