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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)
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