using clsi m45-a2 for antimicrobial susceptibility testing of infrequently-isolated organisms

Using CLSI M45-A2 forAntimicrobial Susceptibility Testingof Infrequently-isolated Organisms

Erik MunsonClinical Microbiology

Wheaton Franciscan LaboratoryWauwatosa, Wisconsin

The presenter states no conflict of interest and has no financial relationshipto disclose relevant to the content of this presentation.

OUTLINE

I. M45-A2 guideline

A. Appropriate formatB. Microbiology review

II. Caveats and other intelligence for your life

A. Clinical presentationB. TaxonomyC. Antibiogram

M45-A2 Methods for Antimicrobial Dilutionand Disk Susceptibility Testing ofInfrequently Isolated or FastidiousBacteria; Approved Guideline--Second Edition (2010)

BRIEF HISTORY

M45-A Ibid (2006)

M45-P Ibid (2005)

You Make the Call

QUESTION ONE

Can the microbiology laboratory performantimicrobial susceptibility testing on Abiotrophia spp.?

A. No

B. Possibly

C. Yes; John Tesh advised us how

D. Yes; the microbiology laboratory is omniscient

E. What *#$% is Abiotrophia spp.

Abiotrophia spp.; Granulicatella spp.Format Broth microdilution Disk Diffusion

MediumCation-adjusted Mueller-Hinton broth with 2.5-5.0% (v/v) lysed

horse blood plus 1 g/mL pyridoxal hydrochloride

Inoculum 20-24 h direct colony suspension equivalent to 0.5

McFarland

Incubation 35°C ambient air20-24 hours

Agents to consider for primary testing

PenicillinCefotaxime or ceftriaxone

Vancomycin

NOTESFormer nutritionally-deficient,-variant streptococci; requirescysteine or pyridoxal for growth

Testing of isolates from normally-sterile sourcesmay be warranted, especially in immunodeficientpatients

CLSI M45-A2

Interpretive criteria derived from Streptococcusspp. breakpoints (CLSI M100 series)

Diminished susceptibility to penicillin

Koneman, 5th ed.

LIMITED DATA

Diagn. Microbiol. Infect. Dis. 38: 189-191; 2000

Granulicatella Abiotrophia

Penicillin 55 8

Amoxicillin 81 92

Ceftriaxone 63 83

Meropenem 96 100

46.7% susceptibility to macrolide agents

J. Clin. Microbiol. 42: 4323-4326; 2004

You Make the Call

CASE PRESENTATION

29-year-old female wading in Milwaukee River;fell and scraped knee on rock

Visited ER for cleaning & suturing; no antibioticsPain worsened overnight; came back to ER

Septic pre-patellar bursitis; surgical debridement

WBC 24,200/L (89.0% segmented neutrophils)Sutures removed to reveal purulent discharge

QUESTION TWO

What is the most likely infectious etiologyfor this clinical presentation?

A. Methicillin-susceptible Staphylococcus aureus

B. Aeromonas species

C. Non-tuberculous Mycobacterium species

D. Pseudomonas aeruginosa

E. A 24,000 white count doesn’t really alarm me.

A. hydrophila complex; P. shigelloidesFormat Broth microdilution Disk Diffusion

Medium Cation-adjustedMueller-Hinton broth

Mueller-Hinton agar

Inoculum Direct colony suspension equivalent to 0.5 McFarland

Direct colony suspension equivalent to 0.5 McFarland


35°C ambient air16-18 hours


Amoxicillin-clavulanic acid3° and 4° cephemsFluoroquinolones

Trimethoprim-sulfamethoxazole

Amoxicillin-clavulanic acid3° and 4° cephemsFluoroquinolones


NOTESAeromonas caviae complexAeromonas hydrophila complexAeromonas veronii complex (incl. biovar sobria)Plesiomonas shigelloides

Often limited to isolates from extraintestinal sites

CLSI M45-A2

Aeromonas spp. resistant to ampicillinAmpicillin resistance in P. shigelloides unclear

Interpretive criteria derived from Enterobacteriaceaebreakpoints (CLSI M100 series)

Bacillus spp.Format Broth microdilution Disk Diffusion





VancomycinFluoroquinolones

Clindamycin

NOTES

Not Bacillus anthracis

Interpretive criteria derived from Staphylococcusspp. breakpoints (CLSI M100 series)


CLSI M45-A2

Generally resistant to penicillins and cephems;-lactamase testing is unreliable

LIMITED DATA

Antimicrob. Agents Chemother. 32: 642-645; 1988

B. cereus Non-B. cereus

Penicillin 0 40

Cefotaxime 0 71

Imipenem 100 97

Erythromycin 94 97

Clindamycin 72 40

Tetracycline 59 71

Vancomycin 100 100

Ciprofloxacin 98 100

Trimethoprim-sulfamethoxazole 0 97

Campylobacter jejuni/coliFormat Broth microdilution Disk Diffusion


horse blood

Mueller-Hinton agar with 5% sheep blood


McFarland


Incubation 36-37°C aerophilic; 48 hours42°C aerophilic; 24 hours

36-37°C aerophilic; 48 hrs42°C aerophilic; 24 hours


ErythromycinCiprofloxacin

ErythromycinCiprofloxacin

NOTESMicroaerophilic (10% CO2, 5% O2, 85% N2)

Compressed gas incubatorMicroaerophilic gas-generating sachetsSealed plastic bags/pouches not reproducible

Testing may be useful for epidemiology or forpatients with protracted or severe symptoms

Ciprofloxacin, tetracycline interpretive criteriaderived from Enterobacteriaceae breakpoints(CLSI M100 series); population distributions

CLSI M45-A2

DATA

Emerg. Infect. Dis. 8: 1501-1503; 2002

You Make the Call

QUESTION THREE

Can the microbiology laboratory performantimicrobial susceptibility testing on diphtheroids?

A. No

B. Possibly

C. Yes; John Tesh advised us how

D. Yes; the microbiology laboratory is omniscient

E. Do we really want to advertise this?

Corynebacterium spp.; CORYNEFORMSFormat Broth microdilution Disk Diffusion


horse blood; daptomycin caveat


McFarland



PenicillinVancomycinGentamicin

Erythromycin

NOTESAll Corynebacterium spp. Leifsonia spp.Arcanobacterium spp. Microbacterium spp.Brevibacterium spp. Oerskovia spp.Cellulomonas spp. Rothia spp.Dermabacter spp. Turicella spp.


Some Corynebacterium spp. resistant to multipledrug classes

CLSI M45-A2

MORE NOTES

Interpretations of “resistant” may be reported at24 hours; isolates appearing “susceptible” to -lactam agents are re-incubated to be read @ 48h

Derivations of interpretive criteria (CLSI M100)

Cephems from Streptococcus spp.Linezolid from Enterococcus spp.Penicillin, erythromycin from population distributionsRest from Staphylococcus spp.

CLSI M45-A2

Erysipelothrix rhusiopathiaeFormat Broth microdilution Disk Diffusion


horse blood

Inoculum 1-3 d direct colony suspension equivalent to 0.5 McFarland



Penicillin or ampicillin

NOTES

Looks like -hemolytic streptococciH2S-positive

CLSI M45-A2

Testing not necessarily required

ID may be more important in context of potentiallyfulminant endocarditis (d/c empiric therapycovering Gram-positives)

Erythromycin and clindamycin testing may bewarranted in patients with penicillin allergy

gefor.4t.com

MORE NOTES


Ciprofloxacin from Staphylococcus spp.Rest from Streptococcus spp.

Intrinsic resistance to vancomycin (PELL)

CLSI M45-A2

Haemophilus aphrophilusHaemophilus paraphrophilusHaemophilus segnisA. actinomycetemcomitans

Aggregatibacter spp.

Hs & As

(HA)CEK GROUPFormat Broth microdilution Disk Diffusion


horse blood


McFarland

Incubation 35°C with 5% CO2 enrichment24-48 hours


AmpicillinAmoxicillin-clavulanic acidCeftriaxone or cefotaxime

ImipenemCiprofloxacin or levofloxacin


NOTES

Aggregatibacter spp. Eikenella corrodensCardiobacterium spp. Kingella spp.

Testing of isolates from normally-sterile sourcesmay be warranted, especially in immunodeficientpatients or those unable to tolerate PO -lactams

Testing of E. corrodens isolates from bite woundsmay not be necessary due to high probability ofsusceptibility to amoxicillin-clavulanic acid

CLSI M45-A2

MORE NOTES

-lactamase production predicts (most) ampicillinresistance (exception: Aggregatibacter spp.)

Watch growth control wells; could be problematic

CLSI M45-A2


Chloramphenicol from Streptococcus spp.Penicillin from population distributionsRest from Haemophilus influenzae

Helicobacter pyloriFormat Broth dilution AGAR DILUTION Disk Diffusion

MediumMueller-Hinton agar with

aged (≥ 2-week-old) sheep blood (5% v/v)

Inoculum 72 h saline suspension equivalent to 2.0 McFarland

Incubation 33-37°C aerophilic72 hours


Clarithromycin main driver of a regimen that includes proton-pump inhibitor and

possibly additional antimicrobials

Lactobacillus spp.Format Broth microdilution Disk Diffusion


horse blood


McFarland

Incubation 35°C with 5% CO2 enrichment24-48 hours


Penicillin or ampicillinGentamicin (for combined Rx)

NOTESTesting of isolates from normally-sterile sourcesmay be warranted

CLSI M45-A2

Derivation of interpretive criteria (CLSI M100)

Gentamicin, linezolid, vancomycin fromStaphylococcus spp.

Clindamycin, imipenem from population distributions

Rest from Enterococcus spp.

Intrinsic resistance to vancomycin (PELL),particularly those that grow well in ambient air

LIMITED DATA


Lactobacillus

Penicillin 0

Ceftriaxone 31

Imipenem 100

Erythromycin 100

Gentamicin 100

Tetracycline 15

Chloramphenicol 100

Ciprofloxacin 38

Trimethoprim-sulfamethoxazole 0

Leuconostoc spp.Format Broth microdilution Disk Diffusion


horse blood


McFarland




NOTES

Interpretive criteria only provided for penicillin,ampicillin, gentamicin, chloramphenicol,minocycline

Testing of isolates from normally-sterile sourcesmay be warranted (e.g., endocarditis)

CLSI M45-A2


Interpretive of interpretive criteria (CLSI M100)

Gentamicin from Staphylococcus spp.Rest from Enterococcus spp.

LIMITED DATA


Leuconostoc

Penicillin 6

Ampicillin 2

Ceftriaxone 57

Imipenem 81

Erythromycin 100

Gentamicin 100

Tetracycline 77

Chloramphenicol 98

Ciprofloxacin 24


Listeria monocytogenesFormat Broth microdilution Disk Diffusion


horse blood


McFarland



Penicillin or ampicillinTrimethoprim-sulfamethoxazole

NOTES

Ampicillin and penicillin data previously published(CLSI M100-S15; 2005)

Trimethoprim-sulfamethoxazole interpretive criteriaderived from Streptococcus spp. breakpoints(CLSI M100 series)

Testing of isolates may be limited to suspectedtreatment failures or patients with penicillin allergy

CLSI M45-A2

Intrinsically resistant to cephems

Moraxella catarrhalisFormat Broth microdilution Disk Diffusion


Mueller-Hinton agar




35°C with 5% CO2 20-24 hours


Amoxicillin-clavulanic acidCefaclor or cefuroxime


Amoxicillin-clavulanic acid


NOTES

-lactamase production predicts ampicillin andamoxicillin resistance

Testing may be useful for epidemiology or forpatients with protracted or severe infections

Derivation of interpretive criteria (CLSI M100)

Macrolides from population distributionsRest from Haemophilus spp.

CLSI M45-A2

DATA

J. Clin. Microbiol. 44: 3775-3777; 2006

M. catarrhalis

Penicillin 5

Amoxicillin-clavulanic acid 100

Ceftriaxone 100

Cefuroxime 99

Azithromycin 100

Levofloxacin 100

Tetracycline 99


You Make the Call

CASE PRESENTATION

55-year-old female bitten by family cat on R palm;progressive erythema over next 12-18 hours

Afebrile upon presentationSlightly tachycardic and tachypneicWBC 12,100/L (77.8% segmented neutrophils)Empiric ampicillin-sulbactam

Gram-negative bacillus; no growth MacConkeySusceptible to penicillin

Discharge on hospital day 3

QUESTION FOUR

What is the most likely infectious etiologyfor this clinical presentation?

A. Viridans group Streptococcus

B. CDC group EF-4a

C. Pasteurella species

D. A strictly veterinary pathogen

E. I’m doomed. Help me, John Tesh!!!

Pasteurella spp.Format Broth microdilution Disk Diffusion


horse blood

Mueller-Hinton agar with 5% sheep blood


McFarland

20-24 h direct colony suspension equivalent to 0.5 McFarland


35°C ambient air16-18 hours


PenicillinsAmoxicillin-clavulanic acid

CeftriaxoneFluoroquinolones

TetracyclinesMacrolides


PenicillinsAmoxicillin-clavulanic acid

CeftriaxoneFluoroquinolones

TetracyclinesMacrolides


NOTESRoutine testing usually not necessary on isolatesfrom bite wounds; empiric therapy for polymicrobialinfection


Interpretive criteria derived from populationdistributions

CLSI M45-A2

-lactamase testing can have value; rare isolateswith penicillin, amoxicillin MIC > 0.5 g/mL

Pediococcus spp.Format Broth microdilution Disk Diffusion


horse blood


McFarland




NOTES


Gentamicin from Staphylococcus spp.Rest from Enterococcus spp.

Interpretive criteria only provided for penicillin,ampicillin, gentamicin, chloramphenicol,imipenem

CLSI M45-A2



LIMITED DATA


Pediococcus

Penicillin 0

Ampicillin 0

Ceftriaxone 38

Imipenem 100

Erythromycin 92

Gentamicin 100

Tetracycline 4

Chloramphenicol 100

Ciprofloxacin 0


Vibrio spp.Format Broth microdilution Disk Diffusion


Mueller-Hinton agar


McFarland


Incubation 33-37°C ambient air16-20 hours

33-37°C ambient air16-18 hours


CefotaximeCeftazidimeTetracycline

Fluoroquinolones

CefotaximeCeftazidimeTetracycline

Fluoroquinolones

Vibrio choleraeFormat Broth microdilution Disk Diffusion


Mueller-Hinton agar


McFarland


Incubation 33-37°C ambient air16-20 hours

33-37°C ambient air16-18 hours


AzithromycinDoxycycline

SulfonamidesChloramphenicol

AmpicillinTetracycline


AzithromycinDoxycycline

SulfonamidesChloramphenicol

AmpicillinTetracycline


NOTESDirect colony suspension in 0.85% saline; obviatesneed to supplement Mueller-Hinton-based medium

Testing most often limited to isolates fromextraintestinal sites

CLSI M45-A2


Azithromycin from Staphylococcus spp.Rest from Enterobacteriaceae

Halophilic Vibrio spp. generally resistant to penicillin, sulfonamides, some cephems

You Make the Call

QUESTION FIVE

What does all of this mean to you (i.e., how willyour knowledge of M45-A2 impact your workplace)?

A. We’re all good.

B. Dude, we could perform AST on more isolates!!

C. We’re going to stop “incorrect” practices.

D. We’re going to change to correct practices.

E. Sorry--I’m just waking up from my food coma; I need another cookie.

SUMMARY

Gram-positive organisms with intrinsicglycopeptide resistance

Organisms that previously lacked defined methods

Coryneform bacteria Bacillus (not anthracis)Abiotrophia Granulicatella

Fastidious Gram-negative organisms

(HA)CEKs Pasteurella

More-detailed guidance

Campylobacter Listeria monocytogenesM. catarrhalis Aeromonas/Plesiomonas

SUMMARYAbiotrophia, Granulicatella

Aeromonas, Plesiomonas

Bacillus (not anthracis)

Campylobacter jejuni/coli

Corynebacterium

Erysipelothrix rhusiopathiae

(HA)CEK group

Helicobacter pylori

Lactobacillus

Leuconostoc

Listeria monocytogenes

Moraxella catarrhalis

Pasteurella

Pediococcus

Vibrio

THE END

Stuff we missed

Bioterrorism agents (formerly in CLSI M100)

AFB, Nocardia, other actinomycetes (CLSI M11-A8)Anaerobes (CLSI M11-A8)Yeast and moulds (CLSI M27-A3, M44-A2, M38-A2, M51-A)

Yes, we can (sort of)

using clsi m45-a2 for antimicrobial susceptibility testing of infrequently-isolated organisms

Documents

hours35c ambient air16

hours42c maerophilic

hrs42c maerophilic

microbiology reviewcaveats

question onecan

granulicatella spp

question threecan

clinical presentationb