Supplemental Testing (4)

7/29/2019 Supplemental Testing (4)

1/18

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 bysupplemental testing

Alternative approaches to current methods

More rapid testing results

Why NOT supplement?

Current breakpoints are adequate

More work

Confusing

Slower testing results


2/18

Three areas to cover

1. inducible resistance

v.s. de-repressed resistance

2. Beta-lactamases& beta-lactamase inhibitors

3. Heterogenous populations INDUCIBLE V.S. DE-REPRESSED

Part One

IND

UC

TIO

N

Inducible resistance


May be

OR

De-repressed or resistant

Always

IND

UC

TI

O

N


3/18

STAPHYLOCOCCI


Clindamycin & Erythromycin

Macrolide

ErythromycinClarithromycin

Azithromycin

Lincosamide

ClindamycinLincomycin

Streptogramin

Quinupristin-Dalfopristin

Pristinamycin

Erythromycin Clindamycin Interpretation

S S Organism susceptibleto both

R ROrganism resistant to

both

R SMay have inducible

resistance

ER

YT

HRO

MY

CIN

&

CL

IN

DAM

YC

IN

ER

YT

HRO

MY

CI

N

&

CL

IN

DAM

YC

IN


4/18


ER

YT

HRO

MY

CIN

&

CL

IN

DAM

YC

IN

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

ER

YT

HRO

MY

CI

N

&

CL

IN

DAM

YC

IN

Lec lerc q R. Mechanisms of Resistance to Macrolides and Lincosami des: Nature of the Resistance Elements and Their Clinical

Implicat ions. Cli n Infect Dis 2002;34:48292.

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.

ER

YT

HRO

MY

CIN

&

CL

IN

DAM

YC

IN

Other methods

Agar dilution

Sensitivity = 91%, specificity = 97%

Vitek card

Sensitivity = 91%, specificity = 100%

Lava lle e, C., et al. (2010). Performance of an Agar Diluti on Method and a Vitek 2 Card for Detection of Inducible Clindamycin

Resistance i n Staphylococcus spp..J. Clin. Microbiol. 48: 1354-1357

ER

YT

HRO

MY

CI

N

&

CL

IN

DAM

YC

IN


5/18

BETA-LACTAMASES

Part Two

Beta-lactamases

CLASS A:

extended-spectrum beta-lactamases

CLASS C:

ampC beta-lactamases

CLASS B:

metallo-beta-lactamases

Chromosomal

(born with it)

GE

NE

AC

QUI

SI

TIO

N

Plasmid

(acquired)

ampC

beta-lactamases


6/18

Plasmid-mediated

Klebsiella spp.

Salmonella spp.

Proteus mirabilis

(E. coli)

Chromosomal

Enterobacterspp. & most

other Enterobacteriaceae

Pseudomonas aeruginosa

AM

PC

EN

ZYM

ES

ampC ampC

AM

PC

EN

ZYM

ES

chromosomal

inducible

de -repressed (always on)

plasmid genes

inducible

de -repressed (always on)

AM

PC

EN

ZYM

ES

IMP

CAZ

AM

PC

EN

ZYM

ES

IMP

CAZ


7/18

AM

PC

EN

ZYM

ES

Inducible

ampC resistance

ampC inducers

Antibiotic Inducer Hydrolysed Effect

Cefoxitin Strong Yes Resistant

Carbapenem Strong No Susceptible

Clavulanic

acidModerate - Resistant

Aztreonam Weak Yes Susceptible

AM

PC

EN

ZYM

ES

ampC inhibitors

Inhibitors

cloxacillin

oxacillin

boronic acid

No inhibitory effect

clavulanate

tazobactam

sulbactam

AM

PC

EN

ZYM

ES Extended spectrum beta-

lactamases (ESBL)

beta-lactamases


8/18

Extended spectrum beta-

lactamasesactually a family of

related beta-lactamases

three main groups:

SHV

TEM

CTX

usually plasmid-borne

CTX

SHVTEM

E

S

B

L

sulbactam

clavulanic acid

tazobactam

E

S

B

L

Beta lactam

inhibitor

Beta lactam

ESBL & beta-lactamase inhibitors

ESBL enzyme breaks

down cephalosporinantibiotic

E

S

B

L


Inhibitor binds to

ESBL enzyme.

Prevents ESBL from

breaking down

antibiotic.

antibiotic

E

S

B

L


9/18


Beta-lactamase inhibitors

compete

with the

beta-lactamase enzyme

E

S

B

L


ESBL > Inhibitor

=resistant

Inhibitor > ESBL

=susceptibleE

S

B

L

Double disk approximation

Clavulanicacid cephalosporin

E

S

B

L

Cefotaxime

Cefotaxime

& clavulanate

E

S

B

L


10/18

Ceftazidime + Clavulanate

MIC 0.25

CeftazidimeMIC > 32

E

S

B

L

Supplementary methods todetect beta-lactamases

ampC and ESBL

2 main methods

Antibiotic interactions Effect of inhibitors

Beta-lactamases & inhibitors

Beta-lactamase

ampC

MBL

KPC

Inhibitor

cloxacillinboronic acid

EDTA

mercaptopropionic acid (MPA)

other chelating agents

boronic acid


11/18

Same principle

Substrate Enzyme Inhibitor

ampC


imipenem

Strong inducer..

but generally

not broken

down by ampC

ampC


imipenem

cefoxitin ampC cloxacillin

strong inducer of

ampC

AND

broken down by ampC

ampC

cefoxitin = small zone (disc)

(by itself) high MIC (dilution)

cefoxitin = larger zone (disc)

& cloxacillin lower MIC (dilution)


12/18

Antibiotic 1

Ce fta zidime Ce fta zidime Me rope nem

21 mm 21 mm 17 mm

Antibiotic 2

Ceftazidime &

clavulanate

Ceftazidime &

clavulanate

Meropenem &

EDTA

27 mm 16 mm 28 mm

Interpret-

ation

HETEROGENEOUS POPULATION

Part Three

Heterogeneous mostly susceptible

small number of resistant

strains

Methicillin resistance

mediated by the mecA geneME

THI

CI

LLI

N

(mostly)


13/18

Methicillin testing (disc)

S. aureus & S. lugdunensis

Oxacillin

(MIC testing only)

Cefoxitin

(disc testing)

coagulase negative staph

Cefoxitin

(disc testing)

ME

THI

CI

LLI

N

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

Thymidine-dependent Small

Colonial Variant S. aureus

MRSA and SCVs

slow-growing, atypical phenotype

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 Becke r, Georg Peters, and Christof von Eiff. Evaluation of Different Methods To Detec t Methicilli n

Resistanc e in Smal l-Colony Variants ofStaphylococcus aureus.J Clin Microbiol. 2004 March; 42(3): 12771279

ME

THI

CI

LLI

N

other methods

detection of pbp2a

latex agglutination kits

immunochromatographic kits (Binax)

detection ofmecA gene by hybridisation

Evigene

detection ofmecA gene by PCR

BD Diagnostics, Cepheid, Roche MolecularDiagnostics

ME

THI

CI

LLI

N


14/18

Other resistant S. aureus

mecCgene

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

ME

THI

CI

LLI

N

Skov R, et al. Phenotypic detec tion of mecC-MRSA: cefoxitin is more reli able than oxacillin.

J Anti microb Chemother. Sep 2013.

Cartwright E JP, 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

dont have the mecA gene

altered pbp (penicillin binding proteins)

cefoxitin (S), oxacillin (R)

rare phenotype

should respond to drugs like augmentin,

cephalexin

ME

THI

CI

LLI

N

Massidda, Oriet ta, et al. Borderline methicillin-susceptible Staphylococcus aureus strains have more in common than reduced susceptibility to

penic il linase-resistant penici llins. Antimic robial Agents and Chemotherapy 40.12 (1996): 2769-2774.

Vancomycin

large molecule, diffuses s lowly in agar

no disc diffusion criteria for S. aureus

resistance to vancomycin:

low-level (intermediate-resistance)

high-level (vanA mediated)

heterogenous (spectrum)

VA

NCO

MY

CIN

hVISA: Heterogenous resistance to vancomycin

Laboratory

S. aureus isolate with

susceptible vancomycin

MIC but with proportion

of cells with reduced

vancomycin susceptibility

Clinical

exposure to vancomycin

(prolonged)

high bacterial load

Howden, BP., et al. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomyci n-intermediate and heterogeneous

vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications.

Clinical Microbiology Reviews 23.1 (2010): 99-139.

VA

NCO

MY

CI

N


15/18

hVISA

VA

NCO

MY

CIN

hVISA

slow growing

phenotypic variation

MIC = S

unstable phenotype

VA

NCO

MY

CI

N

Howden, BP., et al. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomyci n-intermediate and heterogeneous

vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications.

Clinical Microbiology Reviews 23.1 (2010): 99-139.

hVISA

GRD strip

Screening plates with Va

Etest macro method

Population analysis

varying sensitivity and

specificity

? gold standard

VA

NCO

MY

CIN

hVISA

Susceptibility Definition Clinical Laboratory

VSSA MIC 2

VSSA with

increased MIC

(!) Potential clinical

failure

hVISA MIC 2

Screening (+)

PAP 0.9

Potential clinical

failure

Perform screening

if risk factors

present

VISA / VRSA MIC 4 Avoid vancomycin

Holmes NE, et al. Relati onship between vancomycin-resistant Staphylococcus aureus, vancomycin-intermediate S. aureus, high vancomycin MIC,

and outcome in serious S. aureus infections. J Clin Microbiol2012;50:254852.


16/18

SUPPLEMENTAL TESTINGOR MIC?

No Through Road

Traditional way

Tested result

Piperacillin

-tazobactam S

Cefotaxime R

Ceftazidime S

Cefepime S

key-hole effect present

Reported result

Piperacillin

-tazobactam ?

Cefotaxime R

Ceftazidime R

Cefepime R

ESBL present

Its the MIC way

Tested result

Piperacillin

-tazobactam S

Cefotaxime R

Ceftazidime S

Cefepime S

key-hole effect present

Reported result

Piperacillin

-tazobactam S

Cefotaxime R

Ceftazidime S

Cefepime S

Why?


17/18

For the gene

The presence of a resistance genemakes 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 determineswhether patient will

respond to that antibiotic.

It doesnt matter what the

resistance gene is.

FOR

Life is much simpler!

Simple = more consistent

lab results

AGAINST

Is it **really** true?

Is it true for **every**

enzyme?


18/18

Opinion!

inducible clindamycin

resistance

(blood & bone)

Enterobacterspp. and

cephalosporin susceptible

(blood)

plasmid ampC

ESBL

KPC

MBL

(probably dont change

tested result)

Change susceptibility if

present

Dont know

Conclusions

Standard susceptibility methods work for mostorganism / 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

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Supplemental Testing (4)