How can the Laboratory Detect Carbapenemases

You first

• Do you screen for carbapenemases Rectal screening

AST results only

• Do you use Chromogenic media for carbapenemase screens

• How many use the Modified Hodge Test?

• Do you perform any molecular confirmations on possible carbapenemases?

Outline

• Carbapenemases Brief description

Will not discuss epidemiology

• Detection methods Development of phenotypic methods

(Molecular methods)

(Novel techniques)

Carbapenems

• Broad spectrum of activity

• Stable to hydrolysis by most ‐lactamases

• Drugs of choice for treatment of infections caused by penicillin‐ or cephalosporin‐resistant GNB

• Resistance more common in nonfermenters Pseudomonas aeruginosa

Acinetobacter spp.

Carbapenem Resistance

Enterobacteriaceae

• Lack of drug penetration porin mutations

efflux pumps

• Acquisition of carbapenemase genes that encode enzymes capable of degrading carbapenems

Ambler class B metallo‐‐lactamases; NDM, VIM, IMP

Ambler class A serine enzymes; KPC

Ambler class D serine enzymes; OXA‐48 types

CarbapenemResistant

(R+I)

2012

Escherichia coli

Pseudomonas aeruginosa

http://www.ecdc.europa.eu/

Klebsiella pneumoniae

2010EARS-Net

MeropenemMIC Distributions

K. pneumoniaeE. coli

P. aeruginosa

E. cloacae

Carbapenem screening criteria

Screening criteria Clinical breakpoints

Organism MIC (mg/L) ZD (mm) ECOFF (mg/L) a EUCAST CLSI

Meropenem E. coli, Klebsiella spp., Enterobacter spp., Salmonella spp. > 0.25 b < 25 0.125 ≤ 2 ≤ 1

Citrobacter spp., Serratia spp., Proteus spp., M. morganii, Providencia spp.

> 0.25 < 25 0.25 ≤ 2 ≤ 1

Pseudomonas spp., Acinetobacter spp. > 4 ≤ 26 2 ≤ 2 ≤ 2

The recommended screening criteria is meropenem MIC >0.125 mg/L; Vitek 2 and Phoenix card range is 0.25-16 mg/L. As such >0.25 mg/L is used.

Meropenem has the best balance of sensitivity and specificity

EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance. December 2013

CLSI M100 S24

Ambler class Enzyme

Hydrolysis profile

Inhibitor2GC 3GC AZT CAR

A

SME‐1 to ‐3 ‐ + + ++Clavulanate,tazobactam, sulbactam, NXL‐104

NmcA ‐ + ‐ ++

IMI‐2 ‐ + ‐ ++

GES‐4, ‐5, ‐6 + + ‐ +

KPC‐2 to ‐12 ‐ ++ + ++Clavulanate, tazobactam, boronicacid, sulbactam

BIMP, VIM ++ ++ ‐ ++ EDTA, thiol agents 

(SMA/MPA)NDM ++ ++ ‐ +

D OXA‐48 like +/‐ +/‐ ‐ + NaCl

Nordman et al, (2012) Trends in Mol Med 18:263SMA = mercaptoacetic acidMPA = mercaptopropionic acid

Carbapenemase Detection Methods

• Imipenem hydrolysis (UV spectrophometric)• Use of various enzyme inhibitors

Combined Disc Tests Double Disc Synergy Tests

• Modified Hodge Test • MIC strips • Colorimetric Detection • Chromogenic Agars• Microdilution Tests • Detection of genes using molecular methods• Immunochromatography Assay• Imipenem hydrolysis (MALDI‐TOF mass spectrometry)

Algorithm for Phenotypic Detection of Carbapenemase

Synergy with BA AND cloxacillin

Synergy with DPA/EDTA only No synergy

KPC (or class A carbapenemase)

AmpC (chromosomal or plasmid-acquired)

plus porin loss

Metallo-β-lactamase (MBL)

ESBL + porin loss AND OXA-48

Meropenem10 µg ZD <=25 mmMIC > 0.12 mg/L

Synergy with BA

Interpretation of Phenotypic confirmation tests (carbapenemases in bold type)

β-lactamase

Synergya with meropenem(10 µg) disc/tablet and Temocillin

MIC > 32mg/L ZD <11 mm

DPA/ EDTA

APBA/ PBA

DPA+APBA CLOX

MBL (class B) Yes No No No Variable b

KPC (class A) No Yes No No variableMBL + KPC c v v Yes No variable

OXA-48-like (class D) No No No No YesAmpC + porin loss No (Yes) no (Yes) variable

ESBL + porin loss No No No No No

Abbreviations: DPA = dipicolinic acid; APBA = aminophenyl boronic acid; PBA = phenyl boronic acid; CLOX = cloxacillin

Tsakris A et al. JAC. 2010;65:1664

AmpC/ESBL

Three combined‐disc testβ‐lactamasedetected

PBA (400 µg) 

EDTA(292 µg)

PBA+EDTA

Sensitivity Specificity

KPC + ‐ + 100% 98.5%

MBL ‐ + + 100% 100%

MBL/KPC ‐ ‐ + 96.8% 100%

KPC/VIM/ESBL KPC/ESBL VIM

Increase ≥ 5 mm

Carbapenemase Detection KitsMASTDISCS™ ID• Carbapenemase(Enterobacteriaceae) detection disc set (D70C) A Carbapenem 10 µg B Carbapenem 10 µg + MBL inhibitor

C Carbapenem 10 µg + KPC inhibitor

D Carbapenem 10 µg + AmpC inhibitor

[Temocillin 30 µg]

Rosco Diagnostica CE• KPC/MBL and OXA‐48 Confirm Kit (98015)  MRP10 meropenem 10 µg MRPBO meropenem 10 µg + phenylboronic acid (AmpC/KPC inhibitor)

MRPCX meropenem 10 µg + (AmpC inhibitor)

MRPDP meropenem 10 µg + dipicolinic acid (MBL inhibitor)

Temocillin 30 µg

Escherichia coli (NDM)MASTID™ID

D70CROSCO

98015

In-house

ROSCO Diagnostica CE

Total Metallo‐β‐lactamase Confirm Kit (98016)

McConkey Agar

The kit contains 5 tablets:• MRP10 Meropenem 10 µg

• DPA  Dipicolinic acid

• IMI10  Imipenem 10 µg

• IM+DP Imipenem 10 µg + DPA

• IM10E  Imipenem 10 µg + EDTA

MHA

MAC

ROSCO Diagnostica CE

KPC/MBL in P. aeruginosa/Acinetobacter Kit• uses two substrates to enhance the detection

The kit contains 5 tablets:• MRP10 meropenem (10 ug)• MRPBO meropenem (10 ug) + 

phenylboronic acid (KPC and AmpCinhibitor)

• MRCXH meropenem (10 ug) + cloxacillin high (AmpC inhibitor)

• IMP10 imipenem (10 ug)• DPA dipicolinic acid

McConkey Agar

Disc Methods

• Availability of commercial discs/tests have helped to standardise methods

• Isolates with multiple resistance mechanisms may make interpretation difficult 

• Optimum distance between discs for detection of synergy may vary for different countries/regions

• Changes to standard guidelines

• Stability of discs and inhibitors one manufacturer recommends 3 day shelf life for meropenem discs once cartridge opened!

“Convenient” DDST Test

• Imipenem based test with TE + MPA (1:320) most sensitive (100%) and specific (100%)

• TE alone did not detect 2 IMP‐1 A. baumanniiand 1 SPM‐1 P. aeruginosa

• MPA alone failed to detect 1 VIM‐2‐like P. aeruginosa

Kim et al. JCM (2007) 45: 2798

Convenient DDST Test – for MBLs

SMA 3 µl TE + MPA (1:320)

Tris-EDTAimipenem 10 µg10 cm edge to edge

Kim et al JCM 2007; 45: 2798

Modified Hodge Test

• CLSI‐recommended confirmatory test for carbapenemase production

• Lacks specificity

• Long turnaround time

• Poor specificity for MBL detection

Positive

negative

Positive

Enhanced growth of E. coli ATCC 25922

Detecting class B enzymes:MBL Etests

• imipenem (I) vs. imipenem + EDTA (IPI)• ratio 8 consistent with MBL production• zone distortion consistent with MBL production• sensitivity - good ; specificity - poor

MIC Strip Tests

• Etest® KPC MP/MPB Meropenem (0.25 μg/ml – 16 μg/ml) / Meropenem (0.064 μg/ml – 4 μg/ml) + boronic acid derivative (constant level)

• Liofilchem® MIC Test strip MRP/MBO  Meropenem (0.25 μg/ml – 16 μg/ml) / Meropenem+Boronic acid (0.032 μg/ml –2 μg/ml)

• Liofilchem® MIC Test strip ETP/EBO Ertapenem (0.25 μg/ml – 16 μg/ml) / Ertapenem+Boronicacid (0.062 μg/ml –4 μg/ml)

• Several methodology changes since 1st

published Whole bacterial cells, rather then supernatant after lysis

Eppendorf tubes rather than micotitre tray

Increased concentration of imipenem

Dortet et al, JCM 2012; 50:3773-3776

Nordman et al, EID 2012; 18:1503-1507 Dortet et al, AAC 2012; 56:6437-6440

Dortet et al, JMM 2014; 63:772

Carba NP (Nordmann‐Poirel) Test

SOLUTIONS• Solution A: 0.05% phenol 

red + 0.1 mM ZnSO4

• Solution A + imipenem (6 mg/mL)

Carba NP (Nordmann‐Poirel) TestREQUIREMENTS• 1.5 ml eppendorf tubes• 0.5% (w/v) phenol red solution• Imipenem‐cilastatin (MSD 

Primaxin® 500)• 10 mM ZnSO4• 20 mM Tris‐HCl lysis buffer 

B‐PER II  or SoluLyse(ThermoFisher Scientific)

• Negative (wild‐type E. coli) and positive (K. pneumoniae OXA‐48 or K. pneumoniae KPC‐2) controls.

reconstitute imipenem in solution A each day of test

stable at RT for 1 week -20°C several months

Dortet et al, JMM 2014; 63:772

Carba NP (Nordmann‐Poirel) Test

PROTOCOL• Each isolate tested in paired tubes• Add 100 μl of 20 mM Tris‐HCl lysis buffer (B‐PERII) in each of 

two 1.5 ml eppendorf tubes• Resuspend a 1/4 to 1/3 calibrated loop (10 μl) of bacterial 

colonies in each tube 

Can be taken directly from around carbapenem disc on MHA DO NOT use colonies from Drigalski or McConkey plates

• Vortex suspensions to ensure colonies well suspended• Add 100 μl of Solution A in the first tube and 100 μl Solution A 

+ imipenem 6 mg/ml in the second tube, mix well• Incubate at 37°C for a maximum of 2 hours

1 ul loopful

Carba NP (Nordmann‐Poirel) Test

No antibiotic

Imipenem

Carbapenemase

Non producerRed Red

Carbapenemaseproducer

RedOrange/

Yellow

interpretable Yellow Yellow

Modifications/Adaptations• Blood cultures

2ml BHI + 70 mg/ml ZnSO4, no imipeneminoculated with 5 drops of positive blood culture into 2 ependorf tubes

Incubate at 37oC (agitated) for 3h

Centriguge at 10,000 g for 5 min

Perform Carba NP test on pellet

• CarbAcineto NP Test Acinetobacter spp. OXA‐types

Uses 5 M NaCl instead for PER bufferDortet et al, JCM 2014; 23 April

Blue‐Carba

• Carba NP variant

• Bromothymol blue as the indicator (pH 6.0 to 7.6)

Optimal pH range for most β‐lactamases

• No lysis buffer

• Direct colony approach

Pires et al, JCM 2013; 51:4281-4283

NDM

OXA-23

OXA-48

ATCC 25922

Blue‐Carba Test

• Test solution: 0.04% bromothymol blue (pH 6.0)

+ 0.1mmol/L ZnSO4

+ 3 mg/ml imipenem

Final pH 7.0

• Negative control solution• 0.04% bromothymol blue (pH 7.0)

• Colonies suspended directly into 100 ul test solution

Pires et al, JCM 2013; 51:4281-4283

NDM

OXA-23

OXA-48

ATCC 25922

OXA-181+ VIMK. pneumoniae

IMP-4P. mirabilis

NDME. coli

PER BTB NaCl CARB

OXA-181K. pneumoniae

negativeK. pneumoniae

OXA-48K. pneumoniae

PER BTB NaCl CARB

VIMP. mirabilis

VIM-18P. aeruginosa

PER BTB NaCl CARB

Colorimetric Tests: Summary• 2 hour test, often within 15 minutes• High sensitivity (100%) and specificity often > 99%• CarbaNP: colour changes slower for VIM‐positive isolates and 

isolates with low carbapenemMICs• Commercially available 

Rapid CARB Screen Kit; Rosco Diagnostica 98021 Some reported difficulties in reading 

• Modified procedure available for blood cultures• Modified procedure for Acinetobacter spp  ‐ CarbAcineto NP

Uses 5M NaCl rather than PER buffer• Blue Carba

easier to perform and interpret; cheaper alternative to Carba NP Able to detect OXA‐48‐like enzymes

Vasoo et al, JCM 2013; 51:3097-3101 Hunag et al, JCM 2014; online 21 May 2014

ChromID ESBL

chromID Carba Brilliance CRE

chromID® CARBA SMART

OXA-48

Other CPE

CHROMagar KPC

Chromatic CRELiofilchem®

Chromogenic Media

MicroorganismCarbapenem‐resistant Klebsiella pneumoniaeCarbapenem‐resistant Escherichia coliCarbapenem‐resistant Enterobacter spp.Carbapenem‐resistant Citrobacter spp.Carbapenem‐resistant Non EnterobacteriaceaeOther microorganisms

Typical appearance of the coloniesBlue‐violetRedBlue‐greenBlue with red haloWhite to natural pigmentedInhibited

• Carbapenemase negative P. aeruginosa and Enterobacteriaceae isolates with reduced susceptibility to carbapenems (eg. AmpC with porinloss) can grow with direct screening,

• Enterococcus spp. and Candida spp. may also grow

Colours may difficult to discriminate

Published Evaluations

UK Protocols | P 8 | Issue no: 1.1 | Issue date: 08.05.14 | Page: 19 of 25 UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

General comments

• Predominance of a single type of carbapenemase

• different media may show different performances in different geographical locations

• It is likely that most methods have been optimised for KPC carbapenemases, as these predominate in several of the larger markets

• Enrichment broths supplemented with carbapenemshave also been advocated.  inferior performance to commercially available chromogenic agars 

extra day is required to obtain colonies for further testing

Molecular Methods

• Confirm the presence of carbapenemase genes

• Can be used for screening

• Mostly confined to research laboratories

• Only detect genes that are recognized by the repertoire of available probes Can miss detection of new enzymes

Mendes et al (2007) JCM 45;544

Multiplex Real‐Time PCR

blaIMP‐typeblaVIM‐typeblaGIM‐1blaSPM‐1blaSIM‐1

Genes detected

Metallo‐β‐lactamases

Molecular Methods

MCA using SYBR green

OXA-48-like and KPC multiplex

OXA-181OXA-48

KPC

IMP-4

IMP-14 VIM

IMP-7

IMP/VIM types multiplex

MicroArrays

• Detect over 100 specific DNA markers in one test 

• Only ligated probes are amplified

• A small camera in the tube reader takes a picture of the array

• Analyzed using software

Check‐Direct CPE

• Real‐time PCR

• Directly from rectal or perianal swabs

• Results in 2 hours

• KPC, OXA‐48, or VIM/NDM

• Check‐MDR CT103XL OXA‐23, ‐OXA‐24, OXA‐58, GES, SPM, GIM and ESBLs (GES, PER, VEB, BEL)

Immunochromatography (IC) Assay

• Quick Chaser® IMP

• Detects all known IMP‐subtypes (IMP‐1 to ‐44)

• Enterobacteriaceae and NF‐GNB

Notake et al, JCM 2013; 51:1762-1768Kitao et al, JMM 2011; 87:330-337

Interpretation ~ 20 min

¥1,000 per test¥1,000 per test

Other Approaches

• Whole genomic sequence data

• MALDI‐TOF Mass Spectrometry

• Line probe assay AID, Germany; (ESBL and KPC)

Stoesser et al JAC May 2013

Van Belkum et al JCM (2013) 51:2018-2022

Bloemberg et al JAC Sep 2013

Summary• Need for prompt detection to limit spread

Essential for implementation of infection control procedures

May provide guidance for individual patient management 

• Numerous detection methods now available. Chromogenicagars have improved and Mass Spectrometry is now available in many institutions

• Many organism have multiple enzymes and resistance mechanisms

• Be aware of changes to standard guidelines

• Although not mandatory, all laboratories are advised by the DoH to screen for carbapenemases

• The Blue Carba or CarbaNP can easily be incorporated into the routine workflow