the best method for detection of carbapenemaseproducing e ... · protocols kindly provided by l....

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 20

The best method for detection of carbapenemase producing

E. coli in bovine minced meat

Sensitivity:

•1 cfu/g

Specificity:

•Limited

accompanying

flora

Non selective preenrichment in

peptone water (37°C) and use of

ChromIDTM CARBA (Biomerieux) (44°C

or 37°C)

10 x 25g + 10 x 5g



Enterobacteriaceae (general screening) in bovine minced meat

37ºC

Sensitivity:

•1 cfu/g

Specificity:

•Limited

accompanying

flora

Non selective preenrichment in

peptone water (37°C) and use of

ChromIDTM CARBA (Biomerieux) (37°C) 10 x 25g + 10 x 5g



Enterobacteriaceae in bovine minced meat

→ Selective preenrichment with meropenem is not recomended.

→ The use of BrillianceTM CRE Agar plates could give false negatives

10 x 25g + 10 x 5g


Isolation of CNP Enterobacteriaceae from Food and Faecal samples

Faeces/Caecal samples

•1 g Material zu 9 ml Pepton water (1:10)

Stomacher/Vortex

•Swabs

Chrom-

ID

CARBA*

37 °C, 24 h

37 °C, 18-22 h

37 °C/44 °C, 24 h

* Chrom ID CARBA SMART ???

Direct

Meat

25 g Material and 225 ml Peptonwater (1:10)

Stomacher/Vortex

10 µl

Species Identification

3 Colonies

100 µl

Chrom-

ID

CARBA*

Sensitivity: 1 cfu/g;Specificity: Limited accompanying flora

Antimicrobial susceptibility test

MIC (EUVEC + EUVEC2)

PW

* Chrom ID OXA-48


Evaluation of methods for detection of carbapenemase producing

E. coli and Salmonella in meat and faeces samples:

To be continued…

Meat:

ATCC 25922 (susceptibility quality control)

Salmonella Ken26 (OXA-48)

E. coli OXA-48 ?

Klebsiella β39 (OXA-48, SHV, TEM, CTX)

E. coli β61 (KPC-2, MOX)

Faeces:

At least E. coli R178 (VIM-1, ACC), Salmonella R3 (VIM-1, ACC-1), Salmonella β59

(NDM-1, CMY-16) and ATCC 25922.

Only pre-selected methods.

In colaboration with FU Berlin.


www.efsa.europa.eu/efsajournal

Isolates:

•Species characterization

•MIC

•Confirmation of carbapenemase production


Antimicrobial susceptibility test for CNP Enterobacteriaceae from

Food and Faecal samples (according to 2013/652/EU)

EUVEC2

http://eur-lex.europa.eu


Phenotypic detection of carbapenemase producing strains

Carba NP II test (Nordmann et al., 2012)

Nordmann et al., 2012. Emerg Infect Dis. 18: 1503-1507

Based on in vitro hydrolysis of imipenem by a bacterial lysate

Lysis solution

(B-PERII)

Solution A(pH indicator)

+

IMP

+ IMP + IMP+ TAZ + EDTAIMP: 6 mg/ml

TAZ: 40 mg/ml

EDTA: 0.03 M

Protocols kindly provided by L. Poirel


Phenotypic detection of carbapenemase producing strains:

Carba NP II Test (Nordmann et al., 2012)

Results

VIM

(class B)

NDM

(class B)

KPC

(class A)

oxa-48

(class D)

ATCC 25922

Protocols kindly provided by L. Poirel


Phenotypic detection of carbapenemase producing strains

Blue-CARBA test (Pires et al., 2013)

Modified test for the direct detection from bacterial cultures.

Pires et al., 2013. JCM. 51:4281-3

Test

solution

Control

solution

Bromothymol blue 0.04%

pH indicator

+ IMP - IMPpH 7 pH 7

IMP: TienamTM 500 (3 mg/ml)

Visual reaction

Protocols kindly provided by L. Peixe


Phenotypic detection of carbapenemase producing strains:

Blue-CARBA test (Pires et al., 2013)

Pires et al., 2013. JCM 51: 4281-3

Results

VIM

(class B)

NDM

(class B)

KPC

(class A)

oxa-48

(class D)

ATCC 25922

Not very clear in some cases

- Cheaper and faster than Carba NP II

- Resuls; yes/no, but not the class

Protocols kindly provided by L. Peixe


Molecular detection of carbapenemase producing strains

Dallene et al., JAC 2010:

IMP-VIM-KPC-OXA-48

Dallene et al., 2010. J Antimicrob Chemother. 65: 490-495.

Poirel et al. 2011. Diagnost. Microbiol.Infect. Dis. 70:119-123

Poirel et al. DMID 2011:

IMP, VIM, NDM, SPM, AIM, DIM, GIM, SIM, KPC,

BIC, and OXA-48

Enterobacteriaceae

Acinetobacter baumanii

Woodford et al., 2006:

oxa51-oxa23-oxa24-oxa58

Woodford et al., 2006. International Journal of Antimicrobial Agents 27: 351-353

There are several approaches…

some Multiplex PCR-amplification based methods:


Evaluation of methods for detection of carbapenemase producing

E. coli and Salmonella in meat and faeces samples:

To be continued…

Meat:


Salmonella Ken26 (OXA-48)

E. coli OXA-48

Klebsiella β39 (OXA-48, SHV, TEM, CTX)


Faeces:

At least E. coli R178 (VIM-1, ACC), Salmonella R3 (VIM-1, ACC-1), Salmonella β59

(NDM-1, CMY-16) and ATCC 25922.


In colaboration with EURL-AR and FU Berlin.


Recomended steps for the detection of carbapenemase-producing Enterobacteriaceae:

Summary

1. Non selective pre-enrichment in PW

2. Selective isolation on a chromogen medium

3. Selection of colonies

4. Species identification

5. Antimicrobial susceptibility test

Recomended:

6. Biochemical/phenotypic or molecular confirmation


Evaluation of methods for detection of CP E. coli and Salmonella in

meat and caecal-faeces samples:

To be continued…

Meat:


Salmonella Ken26 (OXA-48), E. coli OXA-48, Klebsiella β39 (OXA-48, SHV, TEM, CTX),


Faeces/Caecal samples:

E. coli R178 (VIM-1, ACC), Salmonella R3 (VIM-1, ACC-1), Salmonella β59 (NDM-1,

CMY-16) and ATCC 25922.


In colaboration with EURL-AR and FU Berlin.

The EURL-AR will provide the compulsatory method for isolation

of CP E. coli


Thanks to all et al.

B. Baumann

S. Jahn

K. Thomas

B. Lauzat

E. Guiral

J. Beutlich

Y. Pfeifer (RKI, Wernigerode, Germany)

U. Roesler and A. Friese (FU, Berlin, Germany)

L. Poirel (Friburg University)

V. Miriagu (Pasteur Insitute, Athens, Greece)

L. Peixe (University of Porto, Portugal)

the best method for detection of carbapenemaseproducing e ... · protocols kindly provided by l....

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