ABMM/ABMLI Professional Recognition Award

ABMM/ABMLI Professional 

Recognition Award 

Patrick R. Murray, PhD 

May 22, 2011

Clinical Microbiology: 

The Future is in Good Hands 

• My mentors and their influence 

• Contributions of fellows working in 

my NIH lab 

• The future of clinical microbiology

John Washington 

1936-2010 

Henry Isenberg 

1922-2006 

My Mentors 

Alex Sonnenwirth 

1923-1984 

Al Balows 

1921-2006

NIH Microbiology Fellows 

• 2001-2003 Pattarachai Kiratisin, MD, PhD 

• 2003-2006 Adrian Zelazny, PhD 

• 2004-2006 Susan Harrington, PhD 

• 2006-2009 Clemente Montero, PhD 

• 2007-2009 Lindsay Stevenson, PhD 

• 2008-present Lilia Mijares, PhD 

• 2011-present Anna Lau, PhD 

• 2011-present Stella Antonara, PhD

• Bangkok, Thailand 

• MD, Mahidol University 

Pattarachai Kiratisin, MD, PhD 

• PhD, University of Rochester Medical 

Center, New York 

• NIH Microbiology Fellow (2001-03) 

• Recipient of ASM Dade MicroScan 

Young Investigator Award (2004) 

• Chairman of Clinical Microbiology 

Society of Thailand 

• Professor, Mahidol University and 

Deputy Chairman, Faculty of Medicine, 

Siriraj Hospital, Bangkok Thailand

Bacterial Identification by Gene Sequencing 

• Initiated the program for identification of bacteria by Sanger 

sequencing of 16S rRNA gene. 

• March 2002 – May 2003: 5% of all bacterial isolates identified 

by sequencing including – 

Abiotrophia defectiva Capnocytophaga sputigena Helicobacter cinaedi 

Achromobacter 

xylosoxidans 

Cardiobacterium hominis Herbaspirillum huttiense 

Brevundimonas diminuta Corynebacterium accolens Kingella denitrificans 

Campylobacter fetus Dysgonomonas capnocytophagoides Kytococcus schroeteri 

Campylobacter upsaliensis Haemophilus aphrophilus Tsukamurella pulmonis 

• Following year, determined that sequencing the housekeeping 

genes for glucose-6-phosphate dehydrogenase and glucose 

kinase could be used to identify members of the 

Streptococcus mitis group.

• Born Buenos Aires, Argentina 

• PhD, Weizmann Institute, Israel 

Adrian Zelazny, PhD 


• Recipient of NIH Fellows Award for 

Research Excellence (2005) 

• Fellow, ABMM (2006) 

• NIH Director’s Award for Research 

(2007) 

• Recipient of ASM Dade MicroScan 

Young Investigator Award (2008) 

• Staff Scientist, NIH Clinical 

Microbiology Laboratory

Identification of Mycobacterium 

and Nocardia Species by Gene Sequencing 

• A number of gene targets had been used to identify 

mycobacteria and nocardia with variable success including 

16S rRNA, 16S-23S internal transcribed spacer, heat shock 

protein, recA, rpoB, and gyrB genes. 

• Speculated that since SecA1 protein is essential for protein 

export, secA1 gene would be a good target for identification 

of these genera.

Identification of Mycobacterium Species 

• Identified the 700-bp gene sequence that codes the substrate 

specificity domain and protein translocation domain of the 

secA1 gene. 

• 47 reference strains representing 30 species and 59 clinical 

isolates representing 9 species were evaluated. 

• Variability of the target sequence allowed differentiation of all 

species except members of the M. tuberculosis complex 

which had identical sequences. 

• Strains belonging to the same species had high intraspecies 

similarity and all strains were correctly identified.

Identification of Nocardia Species 

• A similar study was performed with reference strains and 

clinical isolates of Nocardia. 

• 30 reference strains (30 species) and 40 clinical isolates 

representing 12 species were evaluated. 

• All 30 species could be differentiated by sequence analysis of 

the SecA1 gene target. Additionally, all clinical isolates were 

identified accurately using this gene target. 

• Subsequent studies have demonstrated that multiple gene 

targets may be necessary for the identification of some 

uncommonly isolated species.

Direct Detection and Identification 

of Mycobacteria and Nocardia in Clinical Specimens 

• All acid-fast organisms in smear-positive clinical 

specimens were identified directly by sequencing the 

SecA1 gene. 

• This approach was used in the last 5 years to identify 

mycobacteria and nocardia in clinical specimens from 

more than patients. 

• 17 species of mycobacteria (including M. tuberculosis, 

M. bovis, M. leprae) and 3 species of Nocardia were 

identified in many different specimens (e.g., respiratory, 

CSF, synovial fluid, duodenal fluid, skin lesions, sinus 

aspirates, urine, and tissues).

Susan Harrington, PhD 

• PhD, Department of Microbiology, 

University of Maryland School of 

Medicine 



• Former Associate Director, 

Microbiology Laboratory, Albany 

Medical Center, New York 

• Current position: Cleveland Clinic - 

Associate Medical Director, 

Mycobacteriology & Specimen 

Processing

Genotypic Analysis of Invasive S. pneumoniae 

Strains from Mali Africa 

• Previous work demonstrated that 78% of the S. pneumoniae 

strains responsible for IPD in Mali were not in PCV7. 

• Used PFGE and repPCR to determine if clonal dissemination 

of predominant serotypes had occurred. 

• The two methods were equally discriminatory within a 

specific pneumococcal serotype. 

• Using both methods, isolates within serotypes 2, 5 and 7 

(the most common serotypes) formed 3 large clusters 

containing 1 genotype each, which is consistent with clonal 

dissemination of these serotypes.

• Born Venezuela 

Clemente Montero, PhD 

• PhD, Department of Chemical 

and Biomolecular Engineering, 

North Carolina State University 

• NIH Microbiology Fellow (2006- 

09) 


• Current position: Research 

Associate, Weill Medical College 

of Cornell University

Evaluation of Pyrosequencing for Yeast Identification

Evaluation of Pyrosequencing for Yeast Identification 

• 133 isolates of clinically relevant yeasts, representing 43 

species, were analyzed by gene sequencing. 

• Sanger cycle sequencing of the ITS1-5.8S-ITS2 region 

identified 79% of the isolates at the species level. 

• Pyrosequencing of the hyper-variable ITS region identified 

69% of the isolates at the species level. 

• Although most species of Candida could be identified 

accurately by pyrosequencing, Trichosporon species and 

some Cryptococcus species could not be differentiated.

Lindsay Stevenson, PhD 

• PhD, Department of Microbiology 

and Molecular Genetics, Emory 

University 

• NIH Microbiology Fellow (2007- 

09) 


• Current position: CPT, US Army 

Assistant Chief, Infectious Disease 

Laboratory, Walter Reed Army 

Medical Center and Bethesda 

Naval Hospital, Washington, DC

Microbial Identification by Matrix Assisted Laser 

Desorption/Ionization Time of Flight (MALDI-TOF) 

Mass Spectrometry 

Intens. 

[a.u.] 

500 

0 

400 

0 

300 

0 

200 

0 

100 

0 

4364 

5096 

5380 

6254 

6315 

6410 

7157 

7273 

0 

4000 5000 6000 7000 8000 

7870 

8368 

m/z

MALDI-TOF Mass Spectrometry 

Technical Considerations 

• Most biomarkers detected in MALDI spectra are intracellular 

basic proteins (2000 to 25,000 Da). 

– Bacteria – ribosomal proteins 

– Fungi – ribosomal and mitochondrial proteins 

• Spectral profiles are influenced by culture conditions (e.g., 

media, age of colonies), pre-analytic processing, and assay 

conditions. 

• Although reproducibility of spectra can be poor, unique 

profiles of conserved, discriminatory peaks are obtained and 

are sufficient to identify a wide spectrum of organisms.


Yeast Identification 

• 194 clinical isolates were tested (6 genera, 23 species) 

• 192 (99.0%) were identified correctly; 2 isolates (Candida 

rugosa and Cryptococcus neoformans) had spectral scores 


Identification of Isolates in Positive Blood Cultures 

• 212 positive blood cultures were analyzed: 

– 20% with no identification 

– 95% of the remaining organisms were identified 

correctly at the species level 

– 8 organisms were misidentified (all Streptococcus mitis 

isolates were misidentified as S. pneumoniae) 

• The most common organisms with no ID were 

Propionibacterium and coagulase-negative 

staphylococci

• PhD, Yale University School of 

Medicine 

Lilia Mijares, PhD 

• NIH Fellow in Clinical Microbiology 

Laboratory and National Human 

Genome Institute, Julie Segre lab 

(2008-present)


Nocardia and Mycobacteria Identification 

• Techniques used to bacteria and yeasts failed to produce 

acceptable spectra for nocardia and mycobacteria. 

• A modified protein extraction procedure was developed 

using heat inactivation and mechanical steps to disperse 

bacteria and fragment their cell walls. 

• Databases were developed for 37 species of mycobacteria 

and 43 species of nocardia, and then challenged with >300 

strains of mycobacteria and nocardia. 

• M. tuberculosis complex strains were identified accurately 

at the complex level but could not be identified at the 

species level. All other strains were identified accurately at 

the species level.


• Routinely used in the NIH lab for the 

identification: 

– Aerobic and anaerobic bacteria 

– Yeasts and limited selection of molds 

– Bacteria and yeasts recovered in blood culture broths 

– Nocardia and mycobacteria 

• Virtually all organisms identified at the species 

level unless: 

– Inoculum insufficient 

– Organism not adequately represented in database

Reflections 

• My career is rooted in mentors and colleagues who taught me 

the clinical significance of this discipline, the skills to critically 

analyze data and write my thoughts coherently, and the 

intricacies of managing a clinical lab; they gave me the 

opportunities and support to function at a national level; 

• I have benefitted most recently by being surrounded by 

fellows and colleagues who are willing to explore new 

approaches for improving diagnostic testing. 

• I think the future of clinical microbiology is bright because we 

have a new generation of microbiologists who have the 

training and intellectual skills to discover novel solutions to 

clinical problems.

ABMM/ABMLI Professional Recognition Award

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