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΄)