Using CLSI M45-A2 for
Antimicrobial Susceptibility Testing
of Infrequently-isolated Organisms
Erik Munson
Clinical Microbiology
Wheaton Franciscan Laboratory
Wauwatosa, Wisconsin
The presenter states no conflict of interest and has no financial relationship
to disclose relevant to the content of this presentation.
OUTLINE
I.
M45-A2 guideline
A.
B.
II.
Appropriate format
Microbiology review
Caveats and other intelligence for your life
A.
B.
C.
Clinical presentation
Taxonomy
Antibiogram
BRIEF HISTORY
M45-A2 Methods for Antimicrobial Dilution
and Disk Susceptibility Testing of
Infrequently Isolated or Fastidious
Bacteria; Approved Guideline-Second Edition (2010)
M45-A
Ibid (2006)
M45-P
Ibid (2005)
You Make the Call
QUESTION ONE
Can the microbiology laboratory perform
antimicrobial 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
Medium
Cation-adjusted Mueller-Hinton
broth with 2.5-5.0% (v/v) lysed
horse blood plus 1 mg/mL
pyridoxal hydrochloride
Inoculum
20-24 h direct colony suspension
equivalent to 0.5 McFarland
Incubation
35°C ambient air
20-24 hours
Agents to consider
for primary testing
Penicillin
Cefotaxime or ceftriaxone
Vancomycin
Disk Diffusion
NOTES
Former nutritionally-deficient,
-variant streptococci; requires
cysteine or pyridoxal for growth
Koneman, 5th ed.
Testing of isolates from normally-sterile sources
may be warranted, especially in immunodeficient
patients
Diminished susceptibility to penicillin
Interpretive criteria derived from Streptococcus
spp. breakpoints (CLSI M100 series)
CLSI M45-A2
LIMITED DATA
46.7% susceptibility to macrolide agents
J. Clin. Microbiol. 42: 4323-4326; 2004
Penicillin
Amoxicillin
Ceftriaxone
Granulicatella
55
81
63
Abiotrophia
8
92
83
Meropenem
96
100
Diagn. Microbiol. Infect. Dis. 38: 189-191; 2000
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 antibiotics
Pain worsened overnight; came back to ER
WBC 24,200/mL (89.0% segmented neutrophils)
Sutures removed to reveal purulent discharge
Septic pre-patellar bursitis; surgical debridement
QUESTION TWO
What is the most likely infectious etiology
for 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. shigelloides
Format
Broth microdilution
Disk Diffusion
Medium
Cation-adjusted
Mueller-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 air
16-20 hours
35°C ambient air
16-18 hours
Agents to consider
for primary testing
Amoxicillin-clavulanic acid
3° and 4° cephems
Fluoroquinolones
Trimethoprim-sulfamethoxazole
Amoxicillin-clavulanic acid
3° and 4° cephems
Fluoroquinolones
Trimethoprim-sulfamethoxazole
NOTES
Aeromonas caviae complex
Aeromonas hydrophila complex
Aeromonas veronii complex (incl. biovar sobria)
Plesiomonas shigelloides
Often limited to isolates from extraintestinal sites
Aeromonas spp. resistant to ampicillin
Ampicillin resistance in P. shigelloides unclear
Interpretive criteria derived from Enterobacteriaceae
breakpoints (CLSI M100 series)
CLSI M45-A2
Bacillus spp.
Format
Broth microdilution
Medium
Cation-adjusted
Mueller-Hinton broth
Inoculum
Direct colony suspension
equivalent to 0.5 McFarland
Incubation
35°C ambient air
16-20 hours
Agents to consider
for primary testing
Vancomycin
Fluoroquinolones
Clindamycin
Disk Diffusion
NOTES
Not Bacillus anthracis
Testing of isolates from normally-sterile sources
may be warranted, especially in immunodeficient
patients
Generally resistant to penicillins and cephems;
b-lactamase testing is unreliable
Interpretive criteria derived from Staphylococcus
spp. breakpoints (CLSI M100 series)
CLSI M45-A2
LIMITED DATA
Penicillin
Cefotaxime
Imipenem
Erythromycin
Clindamycin
Tetracycline
Vancomycin
Ciprofloxacin
Trimethoprim-sulfamethoxazole
B. cereus
0
Non-B. cereus
40
0
100
94
71
97
97
72
59
100
98
40
71
100
100
0
97
Antimicrob. Agents Chemother. 32: 642-645; 1988
Campylobacter jejuni/coli
Format
Broth microdilution
Disk Diffusion
Medium
Cation-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
Direct colony suspension
equivalent to 0.5 McFarland
equivalent to 0.5 McFarland
Incubation
36-37°C maerophilic; 48 hours
42°C maerophilic; 24 hours
36-37°C maerophilic; 48 hrs
42°C maerophilic; 24 hours
Agents to consider
for primary testing
Erythromycin
Ciprofloxacin
Erythromycin
Ciprofloxacin
NOTES
Microaerophilic (10% CO2, 5% O2, 85% N2)
Compressed gas incubator
Microaerophilic gas-generating sachets
Sealed plastic bags/pouches not reproducible
Testing may be useful for epidemiology or for
patients with protracted or severe symptoms
Ciprofloxacin, tetracycline interpretive criteria
derived 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 perform
antimicrobial 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.; CORYNEFORMS
Format
Broth microdilution
Medium
Cation-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 air
24-48 hours
Agents to consider
for primary testing
Penicillin
Vancomycin
Gentamicin
Erythromycin
Disk Diffusion
NOTES
All Corynebacterium spp.
Arcanobacterium spp.
Brevibacterium spp.
Cellulomonas spp.
Dermabacter spp.
Leifsonia spp.
Microbacterium spp.
Oerskovia spp.
Rothia spp.
Turicella spp.
Testing of isolates from normally-sterile sources
may be warranted, especially in immunodeficient
patients
Some Corynebacterium spp. resistant to multiple
drug classes
CLSI M45-A2
MORE NOTES
Interpretations of “resistant” may be reported at
24 hours; isolates appearing “susceptible” to blactam 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 distributions
Rest from Staphylococcus spp.
CLSI M45-A2
Erysipelothrix rhusiopathiae
Format
Broth microdilution
Medium
Cation-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 air
20-24 hours
Agents to consider
for primary testing
Penicillin or ampicillin
Disk Diffusion
NOTES
Looks like a-hemolytic streptococci
H2S-positive
gefor.4t.com
Testing not necessarily required
ID may be more important in context of potentially
fulminant endocarditis (d/c empiric therapy
covering Gram-positives)
Erythromycin and clindamycin testing may be
warranted in patients with penicillin allergy
CLSI M45-A2
MORE NOTES
Intrinsic resistance to vancomycin (PELL)
Derivations of interpretive criteria (CLSI M100)
Ciprofloxacin from Staphylococcus spp.
Rest from Streptococcus spp.
CLSI M45-A2
Hs & As
Haemophilus aphrophilus
Haemophilus paraphrophilus
Haemophilus segnis
A. actinomycetemcomitans
Aggregatibacter spp.
(HA)CEK GROUP
Format
Broth microdilution
Medium
Cation-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 enrichment
24-48 hours
Agents to consider
for primary testing
Ampicillin
Amoxicillin-clavulanic acid
Ceftriaxone or cefotaxime
Imipenem
Ciprofloxacin or levofloxacin
Trimethoprim-sulfamethoxazole
Disk Diffusion
NOTES
Aggregatibacter spp.
Cardiobacterium spp.
Eikenella corrodens
Kingella spp.
Testing of isolates from normally-sterile sources
may be warranted, especially in immunodeficient
patients or those unable to tolerate PO b-lactams
Testing of E. corrodens isolates from bite wounds
may not be necessary due to high probability of
susceptibility to amoxicillin-clavulanic acid
CLSI M45-A2
MORE NOTES
b-lactamase production predicts (most) ampicillin
resistance (exception: Aggregatibacter spp.)
Watch growth control wells; could be problematic
Derivations of interpretive criteria (CLSI M100)
Chloramphenicol from Streptococcus spp.
Penicillin from population distributions
Rest from Haemophilus influenzae
CLSI M45-A2
Helicobacter pylori
Format
Broth mdilution
AGAR DILUTION
Medium
Mueller-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 maerophilic
72 hours
Agents to consider
for primary testing
Clarithromycin main driver
of a regimen that includes
proton-pump inhibitor and
possibly additional
antimicrobials
Disk Diffusion
Lactobacillus spp.
Format
Broth microdilution
Medium
Cation-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 enrichment
24-48 hours
Agents to consider
for primary testing
Penicillin or ampicillin
Gentamicin (for combined Rx)
Disk Diffusion
NOTES
Testing of isolates from normally-sterile sources
may be warranted
Intrinsic resistance to vancomycin (PELL),
particularly those that grow well in ambient air
Derivation of interpretive criteria (CLSI M100)
Gentamicin, linezolid, vancomycin from
Staphylococcus spp.
Clindamycin, imipenem from population distributions
Rest from Enterococcus spp.
CLSI M45-A2
LIMITED DATA
Penicillin
Ceftriaxone
Imipenem
Erythromycin
Gentamicin
Tetracycline
Chloramphenicol
Ciprofloxacin
Trimethoprim-sulfamethoxazole
Lactobacillus
0
31
100
100
100
15
100
38
0
Antimicrob. Agents Chemother. 34: 543-549; 1990
Leuconostoc spp.
Format
Broth microdilution
Medium
Cation-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 air
20-24 hours
Agents to consider
for primary testing
Penicillin or ampicillin
Gentamicin (for combined Rx)
Disk Diffusion
NOTES
Testing of isolates from normally-sterile sources
may be warranted (e.g., endocarditis)
Interpretive criteria only provided for penicillin,
ampicillin, gentamicin, chloramphenicol,
minocycline
Intrinsic resistance to vancomycin (PELL)
Interpretive of interpretive criteria (CLSI M100)
Gentamicin from Staphylococcus spp.
Rest from Enterococcus spp.
CLSI M45-A2
LIMITED DATA
Penicillin
Leuconostoc
6
Ampicillin
Ceftriaxone
Imipenem
Erythromycin
2
57
81
100
Gentamicin
Tetracycline
Chloramphenicol
100
77
98
Ciprofloxacin
Trimethoprim-sulfamethoxazole
24
70
Antimicrob. Agents Chemother. 34: 543-549; 1990
Listeria monocytogenes
Format
Broth microdilution
Medium
Cation-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 air
20-24 hours
Agents to consider
for primary testing
Penicillin or ampicillin
Trimethoprim-sulfamethoxazole
Disk Diffusion
NOTES
Testing of isolates may be limited to suspected
treatment failures or patients with penicillin allergy
Intrinsically resistant to cephems
Ampicillin and penicillin data previously published
(CLSI M100-S15; 2005)
Trimethoprim-sulfamethoxazole interpretive criteria
derived from Streptococcus spp. breakpoints
(CLSI M100 series)
CLSI M45-A2
Moraxella catarrhalis
Format
Broth microdilution
Disk Diffusion
Medium
Cation-adjusted
Mueller-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 air
20-24 hours
35°C with 5% CO2
20-24 hours
Agents to consider
for primary testing
Amoxicillin-clavulanic acid
Cefaclor or cefuroxime
Trimethoprim-sulfamethoxazole
Amoxicillin-clavulanic acid
Trimethoprim-sulfamethoxazole
NOTES
b-lactamase production predicts ampicillin and
amoxicillin resistance
Testing may be useful for epidemiology or for
patients with protracted or severe infections
Derivation of interpretive criteria (CLSI M100)
Macrolides from population distributions
Rest from Haemophilus spp.
CLSI M45-A2
DATA
Penicillin
Amoxicillin-clavulanic acid
Ceftriaxone
Cefuroxime
Azithromycin
Levofloxacin
Tetracycline
Trimethoprim-sulfamethoxazole
M. catarrhalis
5
100
100
99
100
100
99
93
J. Clin. Microbiol. 44: 3775-3777; 2006
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 presentation
Slightly tachycardic and tachypneic
WBC 12,100/mL (77.8% segmented neutrophils)
Empiric ampicillin-sulbactam
Gram-negative bacillus; no growth MacConkey
Susceptible to penicillin
Discharge on hospital day 3
QUESTION FOUR
What is the most likely infectious etiology
for 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
Medium
Cation-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 20-24 h direct colony suspension
equivalent to 0.5 McFarland
equivalent to 0.5 McFarland
Incubation
35°C ambient air
20-24 hours
35°C ambient air
16-18 hours
Agents to consider
for primary testing
Penicillins
Amoxicillin-clavulanic acid
Ceftriaxone
Fluoroquinolones
Tetracyclines
Macrolides
Trimethoprim-sulfamethoxazole
Penicillins
Amoxicillin-clavulanic acid
Ceftriaxone
Fluoroquinolones
Tetracyclines
Macrolides
Trimethoprim-sulfamethoxazole
NOTES
Routine testing usually not necessary on isolates
from bite wounds; empiric therapy for polymicrobial
infection
Testing of isolates from normally-sterile sources
may be warranted, especially in immunodeficient
patients
b-lactamase testing can have value; rare isolates
with penicillin, amoxicillin MIC > 0.5 mg/mL
Interpretive criteria derived from population
distributions
CLSI M45-A2
Pediococcus spp.
Format
Broth microdilution
Medium
Cation-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 air
20-24 hours
Agents to consider
for primary testing
Penicillin or ampicillin
Gentamicin (for combined Rx)
Disk Diffusion
NOTES
Testing of isolates from normally-sterile sources
may be warranted, especially in immunodeficient
patients
Interpretive criteria only provided for penicillin,
ampicillin, gentamicin, chloramphenicol,
imipenem
Intrinsic resistance to vancomycin (PELL)
Derivations of interpretive criteria (CLSI M100)
Gentamicin from Staphylococcus spp.
Rest from Enterococcus spp.
CLSI M45-A2
LIMITED DATA
Penicillin
Pediococcus
0
Ampicillin
Ceftriaxone
Imipenem
Erythromycin
0
38
100
92
Gentamicin
Tetracycline
Chloramphenicol
100
4
100
Ciprofloxacin
Trimethoprim-sulfamethoxazole
0
54
Antimicrob. Agents Chemother. 34: 543-549; 1990
Vibrio spp.
Format
Broth microdilution
Disk Diffusion
Medium
Cation-adjusted
Mueller-Hinton broth
Mueller-Hinton agar
Inoculum
20-24 h direct colony suspension 20-24 h direct colony suspension
equivalent to 0.5 McFarland
equivalent to 0.5 McFarland
Incubation
33-37°C ambient air
16-20 hours
33-37°C ambient air
16-18 hours
Agents to consider
for primary testing
Cefotaxime
Ceftazidime
Tetracycline
Fluoroquinolones
Cefotaxime
Ceftazidime
Tetracycline
Fluoroquinolones
Vibrio cholerae
Format
Broth microdilution
Disk Diffusion
Medium
Cation-adjusted
Mueller-Hinton broth
Mueller-Hinton agar
Inoculum
20-24 h direct colony suspension 20-24 h direct colony suspension
equivalent to 0.5 McFarland
equivalent to 0.5 McFarland
Incubation
33-37°C ambient air
16-20 hours
33-37°C ambient air
16-18 hours
Agents to consider
for primary testing
Azithromycin
Doxycycline
Sulfonamides
Chloramphenicol
Ampicillin
Tetracycline
Trimethoprim-sulfamethoxazole
Azithromycin
Doxycycline
Sulfonamides
Chloramphenicol
Ampicillin
Tetracycline
Trimethoprim-sulfamethoxazole
NOTES
Direct colony suspension in 0.85% saline; obviates
need to supplement Mueller-Hinton-based medium
Testing most often limited to isolates from
extraintestinal sites
Halophilic Vibrio spp. generally resistant to
penicillin, sulfonamides, some cephems
Derivations of interpretive criteria (CLSI M100)
Azithromycin from Staphylococcus spp.
Rest from Enterobacteriaceae
CLSI M45-A2
You Make the Call
QUESTION FIVE
What does all of this mean to you (i.e., how will
your 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
Organisms that previously lacked defined methods
Coryneform bacteria
Abiotrophia
Bacillus (not anthracis)
Granulicatella
Gram-positive organisms with intrinsic
glycopeptide resistance
Fastidious Gram-negative organisms
(HA)CEKs
Pasteurella
More-detailed guidance
Campylobacter
M. catarrhalis
Listeria monocytogenes
Aeromonas/Plesiomonas
SUMMARY
Abiotrophia, 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)