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Evaluation of methods for detection of

carbapenemase producing

E. coli and Salmonella in meat samples

- a progress report

M. San José, J. Fischer, S. Schmoger, R. Helmuth, Y. Agerso, H. Hasman, B. Guerra

et al.

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 2

Carbapenemase-producers in animals and their environment

Smet et al. JAC 2012.

Belgium 2012

Pomba et al. AAC 2014,

Portugal 2009

Acinetobacter OXA-23

Horse, CatE. coli and Klebsiella OXA-48

Dogs

Stolle et al. JAC 2013.

Germany 2013

E. coli NDM-1

Dogs and cats

Shaheen. AAC 2013.

USA 2013

Poirel et al. EID 2012.

France 2011

Acinetobacter

OXA-23

Cattle

Kempf et al. PloS one 2012.

Senegal 2012

Acinetobacter

OXA-23

Human lice

Wang et al. PloS one 2012.

Zhang et al. JAC 2013.

China 2012, 2013

Acinetobacter

NDM-1

Broiler and swine

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 3

RESET-2 IP-1: BfR-Resi

Molecular epidemiology of extended-spectrum β-lactamases (ESBL), AmpC-β-lactamases and carbapenemases,

and its relation with plasmid encoded quinolone resistance determinants in Enterobacteriaceae from healthy

food-producing animals, their environment and their resulting food-products

Task 3 WP 1a: Definition and validation of methods to detect carbapenemase-encoding

isolates from animal and food samples. Evaluation of different methods (commercial and

non-commercial) using spiked samples with known quantity of bacteria.

RESET CPE positive farms RESET-1 (IP-1 BfR-Resi, IP-3 FU)

S1: Salmonella (outside the farm, mice faeces)

S2: Salmonella (pig faeces),

E.coli (pig faeces, manure, flies)

S4: Salmonella (pig faeces)

G1: Salmonella (dust sample poultry farm)

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 4

Members of the Working Group on Carbapenem resistance in food animal ecosystems: Patrick Butaye, Teresa Coque, Beatriz Guerra Román, Henrik Hasman, Annemarie Kasbohrer, Anna Pelagia Magiorakos, Vivi Miriagou, Laurent Poirel, John Threlfall and EFSA staff: Ernesto Liebana and Pietro Stella.

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 5

Questions to be answered:

- Pre-enrichment: selective vs. non selective?

- In house vs. commercial media (Chromogenic)?

- E. coli vs. E. coli, Salmonella and other producers (Enterobacteria and non-fermenters)

- Meat vs. faeces /caecal samples

www.efsa.europa.eu/efsajournal

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 6

Bacterial strainsSalmonella R3 (VIM-1, ACC-1)

E. coli R178 (VIM-1, ACC-1)

Salmonella β59 (NDM-1, CMY-16)

E. coli β38 (NDM-1, TEM, CTX-M)

E. coli β61 (KPC-2, MOX)

Salmonella Ken26 (OXA-48)

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

ATCC 25922 (susceptibility quality control)

Sample

Fresh bovine minced meat

Validation of detection methods for carbapenemase producing

E. coli and Salmonella in meat samples

Sept- 1013- on.

Collaboration with the DTU (spiking protocols, standardization of methods,

materials and isolates, results interpretation, improvements, feasibility study).

RESET German project and NRL-AR

Objective: To find a method with high sensitivity and specificity, easy to be

performed by the sampling laboratories

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 7

Phenotypic beta-lactam susceptibility test

E. coli R178 (swine)

DD cut-offS I R S R (WT ≥ Y mm)

Imipenem ≥ 23 20-22 ≤ 19 ≥ 22 mm < 16 mm ≥ 24 mmErtapenem ≥ 23 20-22 ≤ 19 ≥ 25 mm < 22 mm ≥ 29 mmMeropenem ≥ 23 20-22 ≤ 19 ≥ 22 mm < 16 mm ≥ 26 mm

CLSI (2011) EUCAST (2011) breakpoints breakpoints

Salmonella R3 (poultry)

IMP: 20 mmETP: 24 mm

E. coli ATCC 25922

IMP 30 mm ETP 32 mmIMP 22 mm ETP 24 mm

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 8

Carbapenem non-susceptible (CNS)

vs. Carbapenem resistant

MHK cut-offS I R S R (WT ≤ X mg/l)

Imipenem ≤ 1 mg/l 2 ≥ 4 mg/l ≤ 2 mg/l > 8 mg/l ≤ 0.5 mg/lErtapenem ≤ 0.25 mg/l 0.5 ≥ 1 mg/l ≤ 0.5 mg/l > 1 mg/l ≤ 0.064 mg/lMeropenem ≤ 1 mg/l 2 ≥ 4 mg/l ≤ 2 mg/l > 8 mg/l ≤ 0,125 mg/l

CLSI (2011) EUCAST (2011)breakpointsbreakpoints

E. coli ββββ1ACC-1MIC test

E. coli ATCC 25922

ETP[µg/ml]

0,5 1 2 4 8 16 0,5 1 2 4 8 16

IMP[µg/ml]

IMP/ ETP[µg/ml]

0,5 1 0,5 1

E. coli “R178”VIM-1+ACC-1

IMP[µg/ml]

0,5 1 2 4 8 16

Salmonella “R3”VIM-1+ACC-1

IMP[µg/ml]

0,5 1 2 4 8 16

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 9

Evaluation of detection methods for carbapenemase producing

Enterobacteria in bovine fresh meat samples:

Preliminary tests

To learn about:

- Which isolate do grow where?

- Where do they grow better (selective media, temperature)

- How do the isolates look like on the different media?

- Can we find them after spiking? Easily? Quality of the meat?

- How to spike the meat?

- Which bacterial dose?

- Not all MacConkey are MacConkey: variation on broths and agars

We did preliminary tests with:

- Single isolates, combinations of isolates without meat/ with meat

- Doses: first onc. (109); then 108-10, finally 103-1 (0 control)

- Chromogenic and non-chromogenic. 37°C and 44°C

- Spiking each meat aliquot vs. infecting all meat, and then make the aliquots

- For pre- enrichment: pepton water; MacConkey purple broth (OXOID), MacConkey

broth(Merck)

- MacConkey 1-3 (Oxoid); MacConkey Agar Merck

- Duplicates of all; only duplicates of pre-enrichment (A, B)

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 10

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 11

Evaluation of methods for detection of carbapenemase producing

Enterobacteriaceae in bovine minced meat

Per isolate10 x 25g + 10 x 5g

Beatriz Guerra Román, EURL-AR Workshop Lingby 08.04.2014 slide 12

Evaluation of methods for detection of carbapenemase producing

Enterobacteriaceae in bovine minced meat

Preliminar experiments (onc infected meat)