SUPPLEMENTAL TESTING

Tan Thean Yen

To Supplement
Definition: add as a supplement to what seems insufficient

"supplement your diet"

Why supplement?
Current methods dont work well Additional information provided by supplemental testing Alternative approaches to current methods More rapid testing results

Why NOT supplement?

Current breakpoints are adequate More work Confusing Slower testing results

Three areas to cover

1. inducible resistance v.s. de-repressed resistance 2. Beta-lactamases & beta-lactamase inhibitors

3. Heterogenous populations

Part One

INDUCIBLE V.S. DE-REPRESSED

Inducible resistance

I N D U C T I O N

Inducible resistance
I N D U C T I O N

De-repressed or resistant

May be

Always

OR

Inducible resistance

STAPHYLOCOCCI

Clindamycin & Erythromycin

Macrolide
Erythromycin Clarithromycin Azithromycin

Lincosamide
Clindamycin Lincomycin

Streptogramin
QuinupristinDalfopristin Pristinamycin

E R Y T H R O M Y C I N
& C L I N D A M Y C I N

Erythromycin S R

Clindamycin Interpretation S R Organism susceptible to both Organism resistant to both May have inducible resistance

E R Y T H R O M Y C I N
& C L I N D A M Y C I N

E R Y T H R O M Y C I N
& C L I N D A M Y C I N

Inducible resistance

E R Y T H R O M Y C I N
& C L I N D A M Y C I N

Recommendations for MLSb

Clindamycin & erythromycin discs
Staphylococci: 15-26mm apart Streptococci: 12 mm apart look for flattening of zone of inhibition (D-zone)

applicable to staphylococci, beta-haemolytic streptococci, S. pneumoniae, oral streptococci

Leclercq R. Mechanisms of Resistance to Macrolides and Lincosamides: Nature of the Resistance Elements and Their Clinical Implications. Clin Infect Dis 2002;34:48292.

E R Y T H R O M Y C I N
& C L I N D A M Y C I N

Recommendations for MLSb

broth dilution:
use combination of erythromycin 4 g/ml and clindamycin 0.5 g/ml in a single well growth = inducible resistance

OR use disc testing methods, on purity plate.

E R Y T H R O M Y C I N
& C L I N D A M Y C I N

Other methods
Agar dilution Sensitivity = 91%, specificity = 97%

Vitek card Sensitivity = 91%, specificity = 100%

Lavallee, C., et al. (2010). Performance of an Agar Dilution Method and a Vitek 2 Card for Detection of Inducible Clindamycin Resistance in Staphylococcus spp.. J. Clin. Microbiol. 48: 1354-1357

Part Two

BETA-LACTAMASES

Beta-lactamases
CLASS A: extended-spectrum beta-lactamases CLASS C: ampC beta-lactamases

CLASS B: metallo-beta-lactamases

Plasmid (acquired)
G E N E
A C Q U I S I T I O N

Chromosomal
(born with it)

beta-lactamases

ampC

ampC
Chromosomal
A M P C
E N Z Y M E S

Plasmid-mediated Klebsiella spp. Salmonella spp. Proteus mirabilis

Enterobacter spp. & most other Enterobacteriaceae Pseudomonas aeruginosa

(E. coli)

ampC
inducible
A M P C
E N Z Y M E S

chromosomal
de-repressed (always on)

inducible
plasmid genes de-repressed (always on)

CAZ
A M P C
E N Z Y M E S

IMP

CAZ
A M P C
E N Z Y M E S

IMP

A M P C
E N Z Y M E S

Inducible ampC resistance

ampC inducers
Antibiotic
A M P C
E N Z Y M E S

Inducer Strong Strong Moderate Weak

Hydrolysed Effect Yes No Yes Resistant Susceptible Resistant Susceptible

Cefoxitin Carbapenem Clavulanic acid Aztreonam

ampC inhibitors
A M P C
E N Z Y M E S

Inhibitors cloxacillin oxacillin boronic acid

No inhibitory effect clavulanate tazobactam sulbactam

beta-lactamases

Extended spectrum betalactamases (ESBL)

Extended spectrum betalactamases

actually a family of related beta-lactamases
E S B L

SHV
TEM

three main groups:

SHV TEM CTX

CTX

usually plasmid-borne

Beta lactam

sulbactam

Beta lactam inhibitor

E S B L

clavulanic acid

tazobactam

ESBL & beta-lactamase inhibitors

E S B L

ESBL enzyme breaks down cephalosporin antibiotic

ESBL & beta-lactamase inhibitors

Inhibitor binds to ESBL enzyme. Prevents ESBL from breaking down antibiotic.

E S B L

antibiotic

ESBL & beta-lactamase inhibitors

Beta-lactamase inhibitors

E S B L

compete
with the beta-lactamase enzyme

ESBL & beta-lactamase inhibitors

Inhibitor > ESBL = susceptible
ESBL > Inhibitor = resistant

E S B L

Double disk approximation

E S B L Clavulanic acid

cephalosporin

Cefotaxime
E S B L

Cefotaxime & clavulanate

Ceftazidime + Clavulanate MIC 0.25

E S B L

Ceftazidime MIC > 32

ampC and ESBL

Supplementary methods to detect beta-lactamases

2 main methods
Antibiotic interactions Effect of inhibitors

Beta-lactamases & inhibitors

Beta-lactamase ampC Inhibitor cloxacillin boronic acid EDTA mercaptopropionic acid (MPA) other chelating agents boronic acid

MBL

KPC

Same principle
Substrate Enzyme Inhibitor

ampC
Substrate
imipenem Strong inducer.. but generally not broken down by ampC

Enzyme

Inhibitor

ampC
Substrate
imipenem

Enzyme

Inhibitor

strong inducer of ampC AND broken down by ampC

cefoxitin

ampC

cloxacillin

ampC
cefoxitin (by itself) = small zone (disc) high MIC (dilution)

cefoxitin & cloxacillin

larger zone (disc) lower MIC (dilution)

Ceftazidime

Ceftazidime 21 mm

Meropenem 17 mm

Antibiotic 1
21 mm Ceftazidime & clavulanate

Ceftazidime & Meropenem & clavulanate EDTA 16 mm 28 mm

Antibiotic 2
27 mm

Interpretation

Part Three

HETEROGENEOUS POPULATION

Heterogeneous

mostly susceptible

small number of resistant strains

Methicillin resistance
mediated by the mecA gene
M E T H I C I L L I N

(mostly)

Methicillin testing (disc)

S. aureus & S. lugdunensis coagulase negative staph
M E T H I C I L L I N

Oxacillin (MIC testing only) Cefoxitin (disc testing) Cefoxitin (disc testing)

Thymidine-dependent Small Colonial Variant S. aureus

Kahl B C et al. J. Clin. Microbiol. 2003;41:410-413

MRSA and SCVs

slow-growing, atypical phenotype
M E T H I C I L L I N

often seen in: cystic fibrosis, foreign-body infections &osteomyelitis susceptibility may be difficult to test best to use pbp2a or mecA detection
Frank Kipp, Karsten Becker, Georg Peters, and Christof von Eiff. Evaluation of Different Methods To Detect Methicillin Resistance in Small-Colony Variants of Staphylococcus aureus. J Clin Microbiol. 2004 March; 42(3): 12771279

other methods
detection of pbp2a
M E T H I C I L L I N

latex agglutination kits immunochromatographic kits (Binax)

detection of mecA gene by hybridisation

Evigene

detection of mecA gene by PCR

BD Diagnostics, Cepheid, Roche Molecular Diagnostics

Other resistant S. aureus

M E T H I C I L L I N

mecC gene confers resistance to oxacillin and beta-lactams

reliably detected by phenotypic methods* not detected by genotypic methods for mecA may show cefoxitin (R), Oxacillin (S) phenotype in Vitek

Skov R, et al. Phenotypic detection of mecC-MRSA: cefoxitin is more reliable than oxacillin. J Antimicrob Chemother. Sep 2013. Cartwright EJP, et al. Use of Vitek 2 antimicrobial susceptibility profile to identify mecC in methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2013;51:27324.

Moderately resistant S. aureus

M E T H I C I L L I N

dont have the mecA gene altered pbp (penicillin binding proteins)

cefoxitin (S), oxacillin (R)

rare phenotype should respond to drugs like augmentin, cephalexin
Massidda, Orietta, et al. Borderline methicillin-susceptible Staphylococcus aureus strains have more in common than reduced susceptibility to penicillinase-resistant penicillins. Antimicrobial Agents and Chemotherapy 40.12 (1996): 2769-2774.

Vancomycin
V A N C O M Y C I N

large molecule, diffuses slowly in agar no disc diffusion criteria for S. aureus

resistance to vancomycin: low-level (intermediate-resistance) high-level (vanA mediated) heterogenous (spectrum)

hVISA:
Laboratory
V A N C O M Y C I N

Heterogenous resistance to vancomycin

Clinical exposure to vancomycin (prolonged) high bacterial load

S. aureus isolate with susceptible vancomycin MIC but with proportion of cells with reduced vancomycin susceptibility

Howden, BP., et al. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications. Clinical Microbiology Reviews 23.1 (2010): 99-139.

hVISA
V A N C O M Y C I N

hVISA
slow growing
V A N C O M Y C I N

phenotypic variation MIC = S unstable phenotype

Howden, BP., et al. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications. Clinical Microbiology Reviews 23.1 (2010): 99-139.

hVISA
GRD strip
V A N C O M Y C I N

Screening plates with Va

varying sensitivity and specificity ? gold standard

Etest macro method

Population analysis

hVISA
Susceptibility
VSSA VSSA with increased MIC

Definition
MIC 2 (!)

Clinical

Laboratory

Potential clinical failure

hVISA

MIC 2 Screening (+) PAP 0.9

MIC 4

Potential clinical failure

Avoid vancomycin

Perform screening if risk factors present

VISA / VRSA

Holmes NE, et al. Relationship between vancomycin-resistant Staphylococcus aureus, vancomycin-intermediate S. aureus, high vancomycin MIC, and outcome in serious S. aureus infections. J Clin Microbiol 2012;50:254852.

No Through Road

SUPPLEMENTAL TESTING OR MIC?

Traditional way
Tested result Piperacillin -tazobactam Cefotaxime Ceftazidime Cefepime Reported result Piperacillin -tazobactam Cefotaxime Ceftazidime Cefepime ESBL present

S R S S

? R R R

key-hole effect present

Its the MIC way

Tested result Piperacillin -tazobactam Cefotaxime Ceftazidime Cefepime Reported result Piperacillin -tazobactam Cefotaxime Ceftazidime Cefepime

S R S S

S R S S

key-hole effect present

Why?

For the gene

The presence of a resistance gene makes a difference to whether or not a particular antibiotic will work.
Its not just the MIC.

For the gene

Look for particular resistance enzymes or phenotype: ampC ESBL MBL Modify the susceptibility according to the presence of resistance enzymes.

For the M.I.C.

The breakpoint for each antibiotic determines whether patient will respond to that antibiotic.
It doesnt matter what the resistance gene is.

FOR Life is much simpler!

AGAINST Is it **really** true?

Simple = more consistent lab results

Is it true for **every** enzyme?

Opinion!
Change susceptibility if present inducible clindamycin resistance (blood & bone) Enterobacter spp. and cephalosporin susceptible (blood) Dont know plasmid ampC ESBL KPC MBL (probably dont change tested result)

Conclusions
Standard susceptibility methods work for most organism / antibiotic combinations.

Some resistance phenotypes may need supplemental methods to detect.

Conclusions
Supplement or MIC alone? (need more clinical evidence)

Its complicated.

Tan Thean Yen