Download - Antimicrobial susceptibility testing 2013
Antimicrobial Susceptibility Testing
Lourens Robberts, PhD, D(ABMM), FCCM. 25 September’13
Sir Alexander Fleming
Minimum Inhibitory Concentration
• MIC – The MIC is the lowest concentration of
antimicrobial agent that completely inhibits growth of the organism in the tube as detected by the unaided eye
• MBC – Minimum Bactericidal Concentration is the
lowest concentration of an antibiotic killing the majority (99.9%) of a bacterial inoculum
• MIC Methods – Broth macrodilution – Broth microdilution – Agar dilution
• Diffusion methods – Kirby-Bauer disc diffusion assay
• E-test
Methods of determining the MIC
• Reference method • Defined media • Mueller-Hinton agar
base (CLSI)
5 ml 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml
Medium
5 ml
5 ml
512 256 128 64 32 16 8 4 µg/ml
4 8 16 32 64 128 256 512
Broth Dilution Method
Microdilution Method
• Series of agar plates containing Mueller-Hinton agar and discrete concentrations of an antimicrobial agent
512 µg/ml 256 µg/ml 128 µg/ml 64 µg/ml
Agar Dilution Method
Agar Dilution: Replica Plating
Growth control
Ampicillin 16 µg/ml Ampicillin 8 µg/ml
• Predefined gradient of antimicrobial concentration
• Immobilized
• Calibrated plastic strips
• DOES NOT REQUIRE DIFFUSION OF AGENT TO CREATE CONCENTRATION GRADIENT
Elipsometer (E-test®), Strip
E-test® (AB Biodisk), Daptomycin
Disk Diffusion (Kirby-Bauer)
P
Disk Diffusion (Kirby-Bauer)
Distance from disc (mm)
Con
cent
ratio
n (m
g/m
l)
Disk Diffusion (Kirby-Bauer)
Small
0
Large
P
• Requirement for diffusion of drug through the agar medium
• Zone edges not always clear
• Colonies within zone
Vancomycin Amikacin
Distance from disc (mm)
Con
cent
ratio
n (m
g/m
l)
Disk Diffusion (Kirby-Bauer)
Small
0
Large
P
P
Distance from disc (mm)
Con
cent
ratio
n (m
g/m
l)
0
Bacterial grow
th
Scatterplot of cefditoren MICs by agar dilution versus disk diffusion zone diameters. Each symbol indicates one isolate. Dashed line shows regression line. Disk diffusion breakpoints for cefditoren at MIC breakpoints of 1, 2, and 4 µg/ml are shown and are 20, 17 to 19, and 16 mm, respectively.
Kelly, L. et al. J Clin Microbiol 1999
MIC distribution of wild type (normal) isolates
The MIC per se is not useful clinically unless it can be compared to the concentration of the antibiotic achieved at the site of infection. Thus, we need INTERPRETIVE BREAKPOINTS
Breakpoints (S, I, R)
MIC distribution of resistant isolates
Time course after antibiotic administration
• Susceptible – Isolates are inhibited by the usually
achievable concentrations of antimicrobial agent when the recommended dosage is used for the site of infection
Breakpoints (S, I, R)
• Intermediate – Isolates with MICs that approach usually attainable
blood and tissue levels and for which response rates may be lower than for susceptible isolates.
– Efficacy in body sites where the drugs are concentrated (quinolones and β-lactams).
Breakpoints (S, I, R)
• Resistant – Isolates are not inhibited by the usually
achievable concentrations of the agent with normal dosage schedules.
Breakpoints (S, I, R)
CLSI MIC Interpretive Standards
CLSI MIC Interpretive Standards
• Clinically significant isolates – All sterile site isolates – Significant isolates as determined by
laboratory reporting standards • Surveillance isolates
Which Isolates to Test?
• Consecutive isolates obtained from the same body site should be tested to detect resistance development (after 3 – 4 days)
• Examples of why: – Enterobacter, Citrobacter, Serratia with 3rd
generation cephs – P. aeruginosa – all drugs – Staphylococci – quinolones – VSSA (vanco Susc S. aureus) may become
VISA (intermediate) during vancomycin Tx
Repeat Isolates Resting?
• Decided in concert with: • Clinical laboratory • ID practitioners • Pharmacy and therapeutics committee • Infection control
Which Drugs to Test?
• Factors to consider: – Efficacy – Prevalence of resistance – Minimizing emergence of resistance – Cost – Clinical indications – Current consensus recommendations for first-
choice and alternative drugs
Which Drugs to Test?
CLSI M100-S17
• Selective reporting improves clinical relevance of test reports
• Minimize selection of multi-resistant nosocomial strains by overuse of broad-spectrum agents
• Unexpected resistance should be reported (i.e. resistant to secondary agent but susceptible to primary agent)
How to Report?
Clusters:
Interpretive results and clinical efficacy is similar
Cross-resistance and cross-susceptibility is nearly complete
(Predictive)
CLSI M100-S17
Agents reported should be the agents tested
*One exception
• Specific therapeutic concerns – “Enterococcal endocarditis requires
combination therapy with high-dose penicillin/ -ampicillin/vancomycin/teicoplanin PLUS gentamicin or streptomycin for bacterial eradication.”
– “Enterobacter, Citrobacter and Serratia harbor inducible AmpC beta-lactamases. Treatment with second or third generation cephalosporins may select derepressed mutants, and is therefore not recommended.”
Inclusion of Comments
• Accurate species identification • Acceptable QC
– Test medium (Mueller-Hinton broth, agar etc) – Drug activity (expiration dates, storage,
handling) – Incubation – Reading – Recommended QC organism strains for which
acceptable values have been established by CLSI
Quality Control
WARNING - TRICKERY-
May appear susceptible in vitro but are not effective clinically and should not be reported as “S”
ESBL-producing K. pneumoniae, K. oxytoca, E. coli, P. mirabilis
Penicillins, cephalosporins, aztreonam
Salmonella spp., Shigella spp. 1st – and 2nd –generation cephalosporins, cephamycins, aminoglycosides
Oxacillin-resistant Staphylococci Penicillins, β-lactam/ β-lactamase inhibitor combinations, cephems, carbapenems
Enterococci Aminoglycosides (except high concentrations), cephems, clindamycin, TMP-SMX
• Many bacterial species harbor resistance determinants that are not readily detected by standard MIC, E-test or disk diffusion techniques
• Many of these resistance characteristics are produced by the cells in response to specific environmental stimuli, these need to be induced in the laboratory for phenotypic detection
AST “Cryptography”
• Staphylococcus and Streptococcus spp. • Requires MIC determination in presence
of inducer (clindamycin + erythromycin) • msrA gene – active efflux of macrolides
– Constitutive • erm genes – target site methylation (50S
ribosomal subunit) – Inducible (to constitutive)
Inducible Macrolide Resistance
S
None
D
ermD
Inducible
HD
ermA or ermC or ermA + msrA
Constitutive
R
ermA or ermC or ermB
+/- msrA
Constitutive
Staphylococcus aureus Macrolide Resistance Based on findings by Steward et al. J Clin Microbiol 2005
D+
ermC +/- ermA
Inducible
Negative
msrA
Constitutive
Group A
TEM & SHV CTX-M
KPC
Pen Cef ’s
Penems
Inhibitor Sensitive
Group C
AmpC
Cef ’s Oxa
Inhibitor Resistant
Group D
OXA
Pen esp Oxa
Inhibitor
S / R
Group B
IMP & VIM
Penems
Inhibitor Resistant
Active site Serine Active site Zn
(metallo)
β-lactamase classification
• Beta-lactamases are expressed in response to the presence of an inducer (e.g. beta-lactam)
• Appears susceptible in absence of inducer • Single mutation can transform an inducible
phenotype into a derepressed hyperproducing phenotype
• Enterobacter, Citrobacter freundii, Serratia, Morganella morganii, Providencia stuartii, P. rettgeri
Inducible Beta-lactamase enzymes Chromosomal AmpC enzymes
Inducing power St
abili
ty
Ampicillin Narrow spectrum cephalosporins
Xtended Ceph Ureido,
Carboxypens
Carbapenems Temocillin Cefepime Cefpirome
-
- +
S S
SR
Inducible phenotype Derepressed phenotype
Enterobacter spp.
0
20
40
60
80
100
≤ 1 µg/ml 2 µg/ml 4 µg/ml 8 µg/ml
Success
Failure
MIC
Clinical outcome of patients with ESBL-producing
K. pneumoniae and E. coli bacteraemia treated with cephalosporin monotherapy
n = 42
• Current recommendations call for detection of ESBLs:
• E. coli, K. pneumonaie, K. oxytoca, P. mirabilis
• Screening with CPD, CAZ, CTX, ATM
• If positive screen, proceed to confirmation (clavulanate)
• Ambler class A beta-lactamases are inhibited by clavulanate
• Clavulanate acts as “suicide” inhibitor – thereby protecting the accompanying beta-lactam (clavulanic acid, sulbactam, tazobactam)
Extended Spectrum Beta-lactamases ESBLs
Kirby-Bauer disc diffusion synergy
E-test
OR MIC
≥ 4-fold increase
Kirby-Bauer disc augmentation
∆ ≥ 5 mm
ESBL Confirmatory Methods
Carbapenemases, KPC
E. coli
ATCC 21922
Positive control Negative control
E. coli
AMP
AMC
PTZ
CEF
FOX
CXM
CAZ
CTX
FEP
ATM
IMP
Interpretation Edit
K. oxytoca R R R R S R S S S R S K1 (HIGH) CXM, CAZ?
R R R R R R R R S r S AmpC acquired
E. coli
R R R R R R R r S r S AmpC (HIGH)
R R S r R r r r S ESBL Confirm. + 2/3/4 gen R
R R S R R R S R S ESBL Confirm. + 2/3/4 gen R
Interpretive reading
Some examples
PCG AMP OXA CEPH IMP/MEM INTERPRETATION EDIT
Staphylococci ANY ANY R ANY ANY MRSA ALL beta-lactams to R
(PBP2’) E. faecalis
R R R R R Probably E. faecium
Check speciation (PBP-5)
Interpretive reading
Some examples
Interpretive reading
Some examples GEN NET TOB AMK KAN NEO INTERPRETATION EDIT
Enterobacteriaceae
R R R S R S AAC(3) II
S/r R R R R R AAC(6΄) GEN best avoided
P. aeruginosa
R S R S R R AAC(3) II
R S R S R R ANT(2΄)
R R R R R R IMPERMEABILITY
Interpretive reading
Some examples GEN NET TOB AMK KAN NEO INTERPRETATION EDIT
Staphylococci
S S R R R S ANT(4΄)
S S S S R R APH(3΄)
E. faecium
R R R R R R AAC(6΄) Intrinsic chromosomal
R R HR HR HR R ANT(4΄)