Using CLSI M45-A2 for Antimicrobial Susceptibility Testing of Infrequently-isolated Organisms Erik...
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Transcript of Using CLSI M45-A2 for Antimicrobial Susceptibility Testing of Infrequently-isolated Organisms Erik...
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
Incubation 35°C ambient air16-20 hours
35°C ambient air16-18 hours
Agents to consider for primary testing
Amoxicillin-clavulanic acid3° and 4° cephemsFluoroquinolones
Trimethoprim-sulfamethoxazole
Amoxicillin-clavulanic acid3° and 4° cephemsFluoroquinolones
Trimethoprim-sulfamethoxazole
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
Medium Cation-adjustedMueller-Hinton broth
Inoculum Direct colony suspension equivalent to 0.5 McFarland
Incubation 35°C ambient air16-20 hours
Agents to consider for primary testing
VancomycinFluoroquinolones
Clindamycin
NOTES
Not Bacillus anthracis
Interpretive criteria derived from Staphylococcusspp. breakpoints (CLSI M100 series)
Testing of isolates from normally-sterile sourcesmay be warranted, especially in immunodeficientpatients
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
MediumCation-adjusted Mueller-Hinton broth with 2.5-5.0% (v/v) lysed
horse blood
Mueller-Hinton agar with 5% sheep blood
Inoculum 24-48 h direct colony suspension equivalent to 0.5
McFarland
Direct colony suspension equivalent to 0.5 McFarland
Incubation 36-37°C aerophilic; 48 hours42°C aerophilic; 24 hours
36-37°C aerophilic; 48 hrs42°C aerophilic; 24 hours
Agents to consider for primary testing
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
MediumCation-adjusted Mueller-Hinton broth with 2.5-5.0% (v/v) lysed
horse blood; daptomycin caveat
Inoculum 20-24 h direct colony suspension equivalent to 0.5
McFarland
Incubation 35°C ambient air24-48 hours
Agents to consider for primary testing
PenicillinVancomycinGentamicin
Erythromycin
NOTESAll Corynebacterium spp. Leifsonia spp.Arcanobacterium spp. Microbacterium spp.Brevibacterium spp. Oerskovia spp.Cellulomonas spp. Rothia spp.Dermabacter spp. Turicella spp.
Testing of isolates from normally-sterile sourcesmay be warranted, especially in immunodeficientpatients
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
MediumCation-adjusted Mueller-Hinton broth with 2.5-5.0% (v/v) lysed
horse blood
Inoculum 1-3 d direct colony suspension equivalent to 0.5 McFarland
Incubation 35°C ambient air20-24 hours
Agents to consider for primary testing
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
Derivations of interpretive criteria (CLSI M100)
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
MediumCation-adjusted Mueller-Hinton broth with 2.5-5.0% (v/v) lysed
horse blood
Inoculum 24-48 h direct colony suspension equivalent to 0.5
McFarland
Incubation 35°C with 5% CO2 enrichment24-48 hours
Agents to consider for primary testing
AmpicillinAmoxicillin-clavulanic acidCeftriaxone or cefotaxime
ImipenemCiprofloxacin or levofloxacin
Trimethoprim-sulfamethoxazole
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
Derivations of interpretive criteria (CLSI M100)
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
Agents to consider for primary testing
Clarithromycin main driver of a regimen that includes proton-pump inhibitor and
possibly additional antimicrobials
Lactobacillus spp.Format Broth microdilution Disk Diffusion
MediumCation-adjusted Mueller-Hinton broth with 2.5-5.0% (v/v) lysed
horse blood
Inoculum 24-48 h direct colony suspension equivalent to 0.5
McFarland
Incubation 35°C with 5% CO2 enrichment24-48 hours
Agents to consider for primary testing
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
Antimicrob. Agents Chemother. 34: 543-549; 1990
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
MediumCation-adjusted Mueller-Hinton broth with 2.5-5.0% (v/v) lysed
horse blood
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
Penicillin or ampicillinGentamicin (for combined Rx)
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
Intrinsic resistance to vancomycin (PELL)
Interpretive of interpretive criteria (CLSI M100)
Gentamicin from Staphylococcus spp.Rest from Enterococcus spp.
LIMITED DATA
Antimicrob. Agents Chemother. 34: 543-549; 1990
Leuconostoc
Penicillin 6
Ampicillin 2
Ceftriaxone 57
Imipenem 81
Erythromycin 100
Gentamicin 100
Tetracycline 77
Chloramphenicol 98
Ciprofloxacin 24
Trimethoprim-sulfamethoxazole 70
Listeria monocytogenesFormat Broth microdilution Disk Diffusion
MediumCation-adjusted Mueller-Hinton broth with 2.5-5.0% (v/v) lysed
horse blood
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
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
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
Incubation 35°C ambient air20-24 hours
35°C with 5% CO2 20-24 hours
Agents to consider for primary testing
Amoxicillin-clavulanic acidCefaclor or cefuroxime
Trimethoprim-sulfamethoxazole
Amoxicillin-clavulanic acid
Trimethoprim-sulfamethoxazole
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
Trimethoprim-sulfamethoxazole 93
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
MediumCation-adjusted Mueller-Hinton broth with 2.5-5.0% (v/v) lysed
horse blood
Mueller-Hinton agar with 5% sheep blood
Inoculum 20-24 h direct colony suspension equivalent to 0.5
McFarland
20-24 h direct colony suspension equivalent to 0.5 McFarland
Incubation 35°C ambient air20-24 hours
35°C ambient air16-18 hours
Agents to consider for primary testing
PenicillinsAmoxicillin-clavulanic acid
CeftriaxoneFluoroquinolones
TetracyclinesMacrolides
Trimethoprim-sulfamethoxazole
PenicillinsAmoxicillin-clavulanic acid
CeftriaxoneFluoroquinolones
TetracyclinesMacrolides
Trimethoprim-sulfamethoxazole
NOTESRoutine testing usually not necessary on isolatesfrom bite wounds; empiric therapy for polymicrobialinfection
Testing of isolates from normally-sterile sourcesmay be warranted, especially in immunodeficientpatients
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
MediumCation-adjusted Mueller-Hinton broth with 2.5-5.0% (v/v) lysed
horse blood
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
Penicillin or ampicillinGentamicin (for combined Rx)
NOTES
Derivations of interpretive criteria (CLSI M100)
Gentamicin from Staphylococcus spp.Rest from Enterococcus spp.
Interpretive criteria only provided for penicillin,ampicillin, gentamicin, chloramphenicol,imipenem
CLSI M45-A2
Testing of isolates from normally-sterile sourcesmay be warranted, especially in immunodeficientpatients
Intrinsic resistance to vancomycin (PELL)
LIMITED DATA
Antimicrob. Agents Chemother. 34: 543-549; 1990
Pediococcus
Penicillin 0
Ampicillin 0
Ceftriaxone 38
Imipenem 100
Erythromycin 92
Gentamicin 100
Tetracycline 4
Chloramphenicol 100
Ciprofloxacin 0
Trimethoprim-sulfamethoxazole 54
Vibrio spp.Format Broth microdilution Disk Diffusion
Medium Cation-adjustedMueller-Hinton broth
Mueller-Hinton agar
Inoculum 20-24 h direct colony suspension equivalent to 0.5
McFarland
20-24 h direct colony suspension equivalent to 0.5 McFarland
Incubation 33-37°C ambient air16-20 hours
33-37°C ambient air16-18 hours
Agents to consider for primary testing
CefotaximeCeftazidimeTetracycline
Fluoroquinolones
CefotaximeCeftazidimeTetracycline
Fluoroquinolones
Vibrio choleraeFormat Broth microdilution Disk Diffusion
Medium Cation-adjustedMueller-Hinton broth
Mueller-Hinton agar
Inoculum 20-24 h direct colony suspension equivalent to 0.5
McFarland
20-24 h direct colony suspension equivalent to 0.5 McFarland
Incubation 33-37°C ambient air16-20 hours
33-37°C ambient air16-18 hours
Agents to consider for primary testing
AzithromycinDoxycycline
SulfonamidesChloramphenicol
AmpicillinTetracycline
Trimethoprim-sulfamethoxazole
AzithromycinDoxycycline
SulfonamidesChloramphenicol
AmpicillinTetracycline
Trimethoprim-sulfamethoxazole
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
Derivations of interpretive criteria (CLSI M100)
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)