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Carbapenemases: how to detect them ?

Laurent Poirel

Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, Department of Medicine, University of Fribourg, Fribourg, Switzerland

French INSERM European Unit, University of Fribourg (LEA-IAME), Switzerland

National Reference Center for Emerging Antibiotic Resistance (Switzerland)© ESCMID eLibrary by a

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Carbapenem resistance

Carbapenemases

Combined mechanisms of resistance

© ESCMID eLibrary by a

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Enterobacteriaceae (++Enterobacter spp.)

Resistance to 3GC

• Overproduced

cephalosporinase

• Plasmid-mediated

cephalosporinase

• Plasmid-mediated

ESBL

Decreased OM permeability

Resistance to carbapenems

Frequent mechanism of resistance to carbapenems in Enterobacteriaceae: combined mechanisms of resistance

IPM

FEPMOX

IPM

FEP

MOX


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Emergence of carbapenemasesin Enterobacteriaceae

2004

FRANCE

(TURKEY)

Poirel, Heritier,

Tolün,

Nordmann

AAC 2004 : 48:

15-22

2009

SWEDEN

(INDIA)

Yong, Toleman,

Giske, Cho,

Sundman, Lee,

Walsh

AAC 2009 : 53:

5046-5054

NmcA

Enterobacter

cloacae

IMP-1

Serratia

marcescens

KPC

Klebsiella

pneumoniae

OXA-48

Klebsiella

pneumoniae

NDM-1

Klebsiella

pneumoniae


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Carbapenemases in Enterobacteriaceae

Penicillins 1G, 2Gcephalosporins

3G, 4Gcephalosporins

ß-lactam / clavulanic acid

CarbapenemsENZYME

Amblerclass

A

B

D

KPC, IMI, GES …

Metallo-ß-lactamases : VIM, IMP, NDM-1

Oxacillinases : OXA-48, OXA-181, OXA-204© ESCMID eLibrary b

y author

Class A carbapenemases

• Are inhibited by clavulanic acid and tazobactam (not that

well for KPC)

• Are inhibited in-vitro by boronic acid

• Hydrolyse penicillins, broad-spectrum cephalosporins,

carbapenems, and monobactams, but NOT cephamycins

(except the GES-14 variant)© ESCMID eLibrary by a

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Metallo-ß-lactamases (class B)

• Requires zinc ions to be functional

• Not inhibited by clavulanic acid nor tazobactam

• Inhibited in vitro by EDTA and dipicolinic acid

• Hydrolyse penicillins, broad-spectrum

cephalosporins, cephamycins, carbapenems, but

NOT monobactams

• Hydrolyse carbapenems at a high level© ESCMID eLibrary b

y author

The acquired class B ß-lactamases

• VIM (P. aeruginosa + Enterobacteriaceae)

• IMP (P. aeruginosa + Enterobacteriaceae)

• SPM, GIM (P. aeruginosa)

• NDM: Enterobacteriaceae ++++

A. baumannii ++

P. aeruginosa +© ESCMID eLibrary b

y author

Metallo-ß-lactamase; E. coli (NDM-1)


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Chromosome

OmpC deficience Multiresistance

OmpF deficience Mutliresistance

AmpC ß-lactam R

cephalosporinase

GyrA, ParC Fluoroquinolone R

PlasmidsNDM-1, CTX-M-15 Broad-spectrum ß-lactam R

TEM-1, OXA-1,OXA-9, OXA-10, Narrow-spectrum ß-lactam R

ArmA, RmtB, AAC6’ Broad-spectrum aminoglycoside R

AphA, AAC3’

Acetylase Chloramphenicol R

Ribosylase Rifampin R

QepA Quinolone R

ErmB, mel, mphB Macrolide R

BleMBL Bleomycin R

SulI Sulfamide R

Dhfr1, Dhfr 12 Trimethoprim R

QacE Quaternary ammonium R

merATPADE Heavy metals R

160 kb130 kb

36 kb 110 kb


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Class D carbapenemases

. OXA-23 A. baumannii (+ Proteus mirabilis)

. OXA-40 A. baumannii + P. aeruginosa

. OXA-58 Acinetobacter sp.

. OXA-143 A. baumannii

. OXA-48 (and derivatives)

Enterobacteriaceae© ESCMID eLibrary b

y author


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Weak impact of OXA-48 productionin a wild-type E. coli background

• OXA-48 hydrolyzes carbapenems, but spare ceftazidime and cefotaxime


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- OXA-48

- OXA-47

- TEM-1

- SHV-2a

- OmpK36-

Sept. 2001, Istanbul, Turkey …

K. pneumoniae 11978UTI, previous meropenem treatment

MIC (mg/L)

the first OXA-type carbapenemase in enterics© ESCMID eLibrary b

y author

K. pneumoniae


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Reduced susceptibility to carbapenems is the clue

CASFM EUCAST CLSI

Imipenem

Meropenem

Ertapenem

Doripenem

8 2 8 2 8 4

8 2 8 2 8 4

1 0.5 1 0.5 4 2

2 1 2 1 ND ND

SR≤>

SR≤>

SR≤>

Current debate on breakpoints !!!


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From 48 h Pasteur’s Microbiology to

Rapid Diagnostic testing (30 min)

+ 24 h

+ 24 h

Antibiotic susceptibility testing

Clinical sample

In-vitro culture (E. coli)© ESCMID eLibrary by a

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Infections

D 2

D 0

Blood culturesUrineOther samples

D 1+

+

Antibiogramm

Antibiogramm


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E. coli

K. pneumoniaeK. pneumoniaeK. pneumoniae

E. coliE. coli

Question : where are the carbapenemase producers here ?

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM


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Susceptibility testing : imipenem, ertapenem, meropenem: CLSI, EUCAST guidelines

Phenotypic detection

- Hodge test; modified Hodge test, CIM method- Inhibition; EDTA, clavulanic acid, boronic acid…

Carbapenem hydrolysis (UV spectrophotometry, mass spectro)

Molecular biology

- Specific PCR , multiplex PCR +/- sequencing- Real time PCR- DNA Microarray

Currently available diagnostic tests for carbapenemase producers

Which is the situation ?

In addition

A rapid, specific, sensitive, cheap test for detection of carbapenemases in Enterobacteriaceae worldwide


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Carbapenemase detection tests (1)


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Carbapenemase detection tests (2)


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Molecular detection of

carbapenemase genes


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• Real-Time PCR :

- Check-MDR Real-Time PCR- Detect the presence of the carbapenemase gene- 4-5 h- Cost +++

• Specific PCR +/- sequencing :

- OXA-48-like / KPC / VIM / IMP / NDM- 3 to 5 h- Expertise ++- Cost +

Molecular biology : PCR Techniques


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Protocol

DNA chip with DNA fragments on a solid support

0 Extraction of DNA

1 Hybridization + Ligation

2 Amplification

3 Loading on the chip : hybridization

4 Revelation and analysis

Advantages

Detection of carbapenemase genes but also ESBL or AmpC genes

Disadvantages

Cost, duration of the technique

Detect only known genes (variants ???)

Micro-array hybridization


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The principle of the Carba NP test

N

O

R

COOH

S-R

Carbapenems

ImipenemMeropenemErtapenemDoripenem

H2N

R

COOH

S-RO

HO

Acid production

Carbapenemase

pH

Colorimetric detection


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Interpretation of the Carba NP test

+-Imipenem

No inoculation

Non-carbapenemase producer

Carbapenemase producer


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Results. Check your plate at 15 min, 30 min, 1 h anda maximum 2 h of incubation

E. coli VIM-1

K. pneumoniae CTX-M15 + impermeability

K. pneumoniae OXA-48

K. pneumoniae KPC-2

E. coli NDM-1

E. coli IMP-1

Carbapenem- +

Yellow = carbapenem hydrolysis


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The Carba NP test

Nordmann, Poirel, Dortet, Emerg Infect Dis, 2012, 18:1503-1507

Sensitivity = 100%

Specificity = 100%


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Ambler

classCarbapenemase type

Mean time for

positivity

A KPC 15 min- 1h

A GES-2, -5 1h-1h30

B NDM 20-50 min

B VIM 20-50 min

B IMP 5-30 min

D OXA-48 30-40 min

The Carba NP test


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Excellent results also with Pseudomonas sp.

119 : Controls20 : SPM-121 : DIM-122-23 : KPC24-30 : VIM31-35 : IMP36-37 : NDM38 : GIM39 : AIM40 : BIC41-42 : GES

100% negative

100% positive

Negative (2 strains)

Specificity = 100%, sensitivity = 94.4%

36 carbapenemase producers72 control strains

Dortet L, Poirel L, Nordmann P, J Clin Microbiol. 2013


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Question ; any carbapenemase here ?

E. coli

K. pneumoniae K. pneumoniae K. pneumoniae

E. coliE. coli

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM

ETP

IMP

MEM


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Question : any carbapenemase here ?

E. coli VIM-1

K. pneumoniaeCTX-M15 + impermeability K. pneumoniae OXA-48 K. pneumoniae KPC-2

E. coli NDM-1E. coli IMP-1

(+) 30 min(-) 2 hours (+) 5 min

(+)25 min(+) 30 min (+) 20 min


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Infections

D 2

D 0

Blood culturesUrineOther samples

Carba NP test

D 1+

Carba NP test

+

Carbapenem hydrolysis (mass spectrometry)

Carbapenem hydrolysis (mass spectrometry)

D 3

Carba NP test

Confirmatory phenotypical tests (Modified Hodge test, inhibition tests)

Molecular identification of carbapenems (sequencing, hydridization)

Hydrolysis of carbapenems (UV Spectrophotometry)

Detection using molecular biology (PCR, RT-PCR)


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The Carba NP test

① Rapid; less than 2 h

② Sensitive; 100%

③ Specific: 100%

④ Detection of any type carbapenemase activity

⑤ Cheap : 0.5 euro

⑥ Easy-to-handle

⑦ Implementable worldwide

Nordmann P, Poirel L, Dortet L, Emerg Infect Dis 2012


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This test is now commercially available


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Principal

- KPC inhibited by boronic acid or clavulanic acid

- MBL inhibited by EDTA or dipicolinic acid

Tests available

- Combined Test (ROSCO) : meropenem +/- cloxacillin or ac. dicolinic acide or boronic ac.

- E-test MBL

- Inhibition by EDTA (« home-made technique »)

Imipenem + EDTA Imipenem alone

Inhibition tests


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Apart from molecular biology…


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Protocol :

1) Broth culture with the strain to be tested + carbapenem : 3-6h2) Mass spectrometry

Mass spectrometry : MALDI-TOF

CarbapenemCarbapenem

hydrolysis product

3) if carbapenemase + : hydrolysis of the carbapenem molecule leading to a degradation product

Advantages :

NDM-1

IMP-1

Specific / sensitiveFastness +Cheap if you dispose from the machine !

DisadvantagesMaterialExpertise

Hrabák et al. JCM. 2011Burckhardt et al. JCM. 2011Hrabák et al. JCM. 2012


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D0: Overnight culture in broth D1: centrifugation

pellet sonication: lysis of bacteriacentrifugationsurpernatant = enzymes

Mesurement of the OD: carbapenem + crude enzyme extract

Advantage : Low cost

Disadvantage : Expertise ++

Hydrolysis of carbapenems measured by UV spectrophotometry

Protocol :

∆ OD > 0.0005 carbapenems

Imipenem only

Imipenem + carbapenemase


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High-throughput sequencing

Dune et al., Eur J Clin Microbiol Infect Dis 2012;31:1719-26

Theoritically, will allow :- accurate identification- obtention of a « virtual » antibiogram- typing (criteria to be defined)

Preparation of the sample is critical Gene expression levelCost


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- Rapid detection of carbapenemase producers in Enterobacteriaceae is now possible. To be implemented worldwide

- Carba NP test interesting for antibiotic stewardship and outbreak control

- CIM, inhibition techniques….. Laborious, delay for obtaining the results….

- Molecular techniques costly and not implementable worldwide, but useful for epidemiological purposes

Conclusion


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Thank you

[email protected]


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