Detecting Reporting Resistance in Fastidious Bacteria - SWACM

Detecting / Reporting 

Resistance in Fastidious 

Bacteria 

Janet A. Hindler, MCLS MT(ASCP)

CLSI M45-A2 Guideline 

“Methods for Antimicrobial 

Dilution and Disk Susceptibility 

Testing of Infrequently Isolated 

or Fastidious Bacteria” 

M45-A2 August 2010 

M45-A May 2006 

M45-P Oct. 2005

CLSI M45-A2 Guideline 

Abiotrophia / Granulicatella Lactobacillus 

*Aeromonas / Plesiomonas Leuconostoc 

Bacillus spp. (not anthrax) 

Listeria monocytogenes 

*Campylobacter jejuni / coli *Moraxella catarrhalis 

Corynebacterium 

Erysipelothrix 

HACEK Group 

Helicobacter pylori 

*Pasteurella 

Pediococcus 

*Vibrio spp. (incl. cholera) 

Potential Bacterial Agents 

of Bioterrorism 

* disk diffusion method described in addition 

to reference MIC method

When should we test M45 organisms? 

“Testing should ONLY be undertaken 

in consultation with infectious disease 

or other expert clinicians that can 

assist in determining if susceptibility 

testing is needed in the management 

of a specific patient and in the 

interpretation of any results 

generated.” 

CLSI M45-A2 

Section 5

Organisms Included in 

CLSI M45-A2 

May not require treatment 

– Some often “normal flora” (e.g., 

Corynebacterium spp., Lactobacillus spp.) 

– Some environmental organisms (e.g., Bacillus 

spp.) 

Generally, test isolates from sterile 

sites only 

– But even for isolates from sterile sites, testing 

may not be needed as infections often treated 

empirically…respond to drugs of choice

Empiric Therapy Recommendations 

Order ($$) at: medicalletter.org 

2007 

2010 

Order ($$) at: sanfordguide.com

Table 17. Summary of Testing Conditions and 

QC Recommendations for Infrequently 

Isolated or Fastidious Bacteria 

CLSI M45-A2 

Table 17

Recommended Media M45-A2 

(Broth microdilution MIC) 

Plain Mueller Hinton broth 

– Aeromonas, Plesiomonas, Vibrio, Bacillus, M. catarrhalis, 

some BT agents 

Mueller Hinton + 2.5-5% lysed horse blood – 

same as recommendation for streptococci 

– Abiotrophia/Granulicatella (+0.001% pyridoxal), 

Campylobacter, Coryneforms and Corynebacterium spp., 

Erysipelothrix, HACEK, Lactobacillus, Leuconostoc, 

Listeria, Pasteurella, Pediococcus 

(some need CO 2 or other special incubation atmosphere 

and/or extended incubation) 

Other 

– Helicobacter pylori, some BT agents

Can we test M45-A2 organisms with a 

commercial AST system? 

Disk diffusion - OK 

– Number of drug/bugs for which disk diffusion method 

is described is limited 

MICs limited 

– Few tests FDA cleared for M45-A2 organisms; low 

range of drug concentrations may not be available 

If NOT FDA cleared 

– Confirm reliability by performing in house verification / 

validation 

– Qualify results 

Remember: if you cannot confidently test in your 

laboratory, SEND to a reliable reference 

laboratory

What commercial AST systems 

might we use? 

Etest or commercial broth microdilution MIC 

system 

– Perform test according to manufacturer’s 

recommendations; use manufacturer’s QC ranges 

For Etest - check technical information and 

references 

– Etest lists some breakpoints for M45 organisms, some 

of which differ from those of CLSI 

Note: the extent to which commercial systems have been 

evaluated for most M45-A2 organisms is very limited 

CLSI does not endorse commercial test systems

Qualifying Results 

Optional Comments 

“Testing performed per Dr. Jones 

request” 

“Infectious Disease consult suggested” 

“Testing performed using a method that 

is not FDA cleared for testing Abiotrophia 

spp. but was validated in this laboratory” 

Consider for All M45 organism AST reports ….following 

discussion with medical staff and review of request / 

organism / drugs / results / method 

Note: Will not include on all examples in this talk due to space.

Specimen: Blood 

Diagnosis: Endocarditis 

Abiotrophia spp. 

Should we perform AST? 

What method? 

What drugs?

Abiotrophia spp. / Granulicatella spp. (1) 

Nutritionally variant streptococci 

Requires 0.001% (1 µg/ml) pyridoxal hydrochloride 

– Add 5 µl of 20 µg/ml solution to 100 µl of drug in 

microdilution well (CAMHB + 2.5-5% LHB) 

May show decreased penicillin susceptibility… can 

cause difficulties in treating endocarditis 

– Combination therapy with penicillin or vancomycin plus 

aminoglycoside often prescribed…or 3 rd or 4 th generation 

cephalosporin 

– May require surgical intervention 

Many labs may not separate two genera 

– Genus identification not critical to guide therapy

Baddour et al. 2005. Circulation. 111:e394-434.

CLSI M45-A2 Table 1 

Abiotrophia / Granulicatella

Drugs to Test / Report 

“Agents to Consider for Primary 

Testing” 

Other agents listed that might be 

useful for a specific patient 

“In consultation with the clinician 

caring for the patient, a priority list of 

critical drugs for a specific patient’s 

isolate can be developed” 

CLSI M45-A2 

Section 6.1


Abiotrophia / Granulicatella 

Derivation of interpretive criteria (breakpoints)….

How were interpretive criteria 

(breakpoints) established? 

Searched literature 

Surveyed experience of working group 

members 

Performed limited studies 

Adapted breakpoints from similar bugs in 

M100, when appropriate 

Did NOT use extensive microbiological, 

clinical, and pharmacodynamic databases 

that are typically used to set breakpoints 

(described in CLSI M23 document)

“S” only breakpoint 

Abiotrophia spp. – Vancomycin 

MIC (µg/ml) 

Susc Int Res 

vancomycin 1.0 - - 

investigate any NS isolate 

..Repeat ID and AST 

..Save isolate 

..Send to reference lab (test by CLSI reference MIC method) 

CLSI M45-A2 

Table 1




Test performed with 

CLSI Reference Method 

Report Example 

MIC (µg/ml) 

ceftriaxone 

penicillin 

vancomycin 

2.0 I 

1.0 I 

0.5 S 

“Testing performed per Dr. Jones request.” 

Infectious Disease consult suggested.




ceftriaxone 


vancomycin 

Test performed with Etest 

Report Example 

MIC (µg/ml) 

2.0 I 

1.0 I 

0.5 S 

“Testing performed per Dr. Jones request. Testing 

performed by a method that is not FDA cleared for testing 

Abiotrophia spp. but was validated in this laboratory. 

Infectious Disease consult suggested.”

Specimen: Bone marrow 

Diagnosis: Leukemia 

Corynebacterium spp. 


What method? 

What drugs?

Coryneforms and 


Includes: 

– Corynebacterium spp. 

– Coryneforms - Arcanobacterium, Arthrobacter, 

Brevibacterium, Cellulomonas, Cellulosimicrobium, 

Corynebacterium, Dermabacter, Leifsonia, 

Microbacterium, Oerskovia, Rothia, Turicella, and 

aerotolerant Actinomyces such as A. naeslundii, A 

odontolyticus, A neuii 

Resistance 

– C. jekeium, C. urealyticum most “R” Corynebacterium 

spp. 

– Variety of results for other species 

-lactam “S” results should not be reported until 

48 h




MIC (µg/ml) 

erythromycin > 2 R 

gentamicin 1 S 

penicillin - 

vancomycin 1 S 

24 h preliminary 

results 

(pen ≤ 0.5 µg/ml) 

“Penicillin results pending”




MIC (µg/ml) 

erythromycin > 2 R 

gentamicin 1 S 

penicillin 0.5 S 

vancomycin 1 S 

48 h final 

results

Specimen: Dog bite 

Diagnosis: Localized abscess 

Pasteurella multocida 


What method? 

What drugs?


Pasteurella spp. (1)


Pasteurella spp. (2) 

Additional information including test for -lactamase…..

Why should we do -lactamase test on P. 

multocida from “normally sterile and 

respiratory sources” (vs. wounds)? 

Physicians might use a single agent to 

treat infections at “normally sterile and 

respiratory sites” 

In contrast, wounds are usually treated 

with agent that would cover for - 

lactamase producing organisms

Bite Wound Infections 

Common therapy: -lactam / -lactamase 

inhibitor (e.g., amoxicillin-clavulanic acid) 

– Covers -lactamase producing organisms 

Organisms commonly present that are 

sometimes -lactamase positive 

– Pasteurella multocida 

– Eikenella corrodens 

Organisms commonly present that are 

usually -lactamase positive 

– Staphylococcus aureus

-Lactamase Testing of M45 Bugs 

Recommended for P. multocida, HACEK 

Use chromogenic cephalosporin (e.g., 

nitrocefin) 

Positive reaction within 5 minutes 

(Lion, C. personal communication) 

+ _

Specimen: Cerebrospinal fluid 

Diagnosis: Ventricular shunt infection 

Preliminary report: 

alpha hemolytic streptococcus-like colonies 

(patient initially treated with vancomycin 

without significant improvement) 

Final report: 

Leuconostoc spp. 

(patient switched to penicillin)

Specimen: Cerebrospinal fluid 

Diagnosis: Ventricular shunt infection 

Leuconostoc spp. 


MIC (µg/ml) 

2 S 

“Leuconostoc spp. are intrinsically resistant 

to vancomycin. Infectious Disease consult 

suggested.”

Vancomycin-R Gram Positives 

(colonies may sometimes look like streptococci) 

Organism 

Vancomycin 

Vancomycin 

breakpoints? 

Erysipelothrix All “R” No 

Lactobacillus 

Some species “S” 

(e.g. L. acidophilus grp) 

Yes 

Leuconostoc All “R” No 

Pediococcus All “R” No

Procedure used in some labs to expedite 

identification of vancomycin-R grampositive 

cocci… 

Gram stain: GPC chains 

Drop vancomycin disk on BAP subculture 

– Note: Not standardized procedure 

No zone and colony morphology 

consistent with vancomycin-R species – 

report “presumptive” vancomycin 

resistance 

– Vancomycin often used empirically for suspected 

gram-positive organism infections

Some additional notes on the 

other CLSI M45-A2 organisms…

HACEK Group 

Haemophilus aphrophilus, H. paraphrophilus, H. 

segnis, Aggregatibacter (formerly Actinobacillus) 

actinomycetemcomitans, Cardiobacterium spp., 

Eikenella corrodens, Kingella spp. 

Often associated with endocarditis 

– Previous treatment recommendation was ampicillin + 

gentamicin 

– Increasing numbers of -lactamase producers 

– Therapy now often recommend is cefotaxime / ceftriaxone 

Perform -lactamase test 

Some Haemophilus spp. and Actinobacillus spp. 

may be “R” to ampicillin by mechanisms other than 

-lactamase

MIC Testing of HACEK 

CAMHB plus 2.5-5% lysed horse blood 

Some isolates may not grow well using 

recommended testing conditions 

Ensure adequate growth in growth control 

well 

Sometimes growth enhanced with fresh 

and maximum amount (5%) of lysed horse 

blood



Cardiobacterium spp. 

“Unable to obtain sufficient growth 

for antimicrobial susceptibility 

testing.”

Specimen: CSF 

Diagnosis: meningitis 

Listeria monocytogenes 

MIC ( g/ml) 

ampicillin 

trimeth-sulfa 

1 S 

0.5/9.5 S 

“Infectious Disease consult suggested. 

Cephalosporins are NOT effective against Listeria 

monocytogenes”

Moraxella catarrhalis 

“If desired, -lactamase testing can be performed, using 

chromogenic cephalosporin methods such as 

nitrocefin.” (>90% -lactamase positive) 

Respiratory infections due to M. catarrhalis respond to 

drugs commonly prescribed (e.g., amoxicillin/clavulanic 

acid, macrolide) for respiratory tract infections 

“Testing may be useful for epidemiological purposes or 

for management of patients with prolonged or severe 

infections.” 

In addition to MIC method, disk diffusion can now be 

used for: amoxicillin-clavulanic acid, azithromycin, 

clarithromycin, erythromycin, tetracycline, and TMP- 

SMX 

CLSI M45-A2 Table 12

Antibiotic therapy 

Vibrio cholerae 

– An adjunct to fluid replacement 

– For severe cases (otherwise, mortality up to 60%) 

– Used to reduce duration of diarrhea and excretion 

of V. cholerae to control spread 

Drugs of choice for V. cholerae 

– Doxycycline 

– Fluoroquinolone 

– Trimeth-sulfa (strain 0139 is resistant) 

Sanford Guide, 2011

Outcomes of Single Dose Azithromycin vs. 

Ciprofloxacin for Cholera in Adults 

Outcome 

Azithromycin 

(97 patients) 

Ciprofloxacin 

(98 patients) 

P value 

Clinical successful 71(73)* 26 (27)

What about quality control?…

Table 17. Summary of Testing Conditions and 

QC Recommendations for Infrequently 

Isolated or Fastidious Bacteria 

CLSI M45-A2 

Table 17

Quality Control 

Adapted from M02, M07, M100 as much as possible 

Follow M02, M07 testing/frequency 

recommendations 

QC ranges in M45-A2 same as in M100 

If already performing specific drug-organism QC as 

part of M100 testing, no need to redo for M45-A2 

drug-organism 

– Example: 

S. pneumoniae ATCC 49619 is used to QC CAMHB-LHB 

(2.5-5% v/v) panel and weekly QC program in place 

Panel is used to test penicillin and P. multocida 

No supplemental QC needed for penicillin

Minimum Laboratory Requirements for 

Testing Infrequently Isolated or 

Fastidious Bacteria 

Performs disk diffusion AST at least once/week if 

testing Aeromonas spp., Plesiomonas, spp. Vibrio 

spp., M. catarrhalis, Pasteurella spp., or 

Campylobacter by disk diffusion 

Performs broth microdilution MICs (visually read) 

at least once/week 

Possesses current CLSI M02, M07, and M100 

Laboratory coordinated by director-level clinical 

microbiologist, ID MD or pathologist with expertise 

in antimicrobial susceptibility testing 

CLSI M45-A2 

Section 9.1

Summary 

M45-A2 describes reference methods for testing 

several types of infrequently isolated or fastidious 

bacteria 

M45-A2 organisms generally do not require AST 

because…. 

– They may not be causing an infection 

– Infections caused by them are often treated empirically 

AST of M45-A2 organisms should only be performed 

– Following consultation with an Infectious Disease MD 

– In a laboratory that meets the qualifications suggested in M45-A 

Currently, there are no FDA cleared systems for 

testing most of the organisms listed in M45-A2

Detecting Reporting Resistance in Fastidious Bacteria - SWACM

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