Validating a selection method for. H. Hasman, Y. Agersø, C. Svendsen, H. Nielsen, N.S. Jensen, B. Guerra, Aarestrup, FM

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1 Validating a selection method for detecting ESBL/AmpC/CPE H. Hasman, Y. Agersø, C. Svendsen, H. Nielsen, N.S. Jensen, B. Guerra, Aarestrup, FM

2 Validating a selection method for detecting ESBL/AmpC/CPE in meat 4. Specific monitoring of ESBL- or AmpC- or carbapenemase-producing Salmonella and E. coli 4.1. Method for detection of ESBL- or AmpC- or carbapenemase-producing E. coli in broilers, fattening turkeys, fattening pigs, bovines under one year of age and fresh meat of broilers, pig meat and bovine meat For the purpose of estimating the proportion of samples containing ESBL- or AmpC- or carbapenemase-producing E. coli amongst the caecal samples collected from broilers, fattening turkeys, fattening pigs, bovines under one year of age, fresh meat of broilers, pig meat and bovine meat in accordance with point 1(d) of this Part, the following method shall apply.

3 Method For the detection of ESBL- or AmpC-producing E. coli the method shall start by a pre-enrichment step, followed by inoculation on McConkey agar containing a third generation cephalosporin in a selective concentration according to the most recent version of the detailed protocol for standardisation of the European Union Reference Laboratory for Antimicrobial Resistance ( 3 ). The microbial species E. coli shall be identified using an appropriated method.

4 E. coli strains ESBLs - -M-1 - SHV-12 - TEM-52 AmpC - CMY-2 CPE* - VIM-1 - NDM-1 - KPC-2 - OXA-48 Note: Not a cephalosporinase! Control - ATCC25922 * Carbapenemase producing Enterobacteriae

5 5*190 g meat 5*10 g for premix with meat in different concentrations 180 g = h 37C McConkey Plates (44C) 0 CFU/g 10 3 CFU/g 0.1 CFU/g 10 2 CFU/g 10 1 CFU/g 1 CFU/g * 25 g 2 * 25 g 2 * 25 g 3*5 g b ml 225 ml + Loop 10 µl 225 ml h 44C 5 6 % AB 45 ml McConkey 3*5 g Loop 10 µl McConkey Plates (44C)

6 Spiking the meat 6*190 g meat 6*10 g for premix with bacteria in different concentrations 10 3 CFU/g 10 2 CFU/g 10 1 CFU/g 1 CFU/g 0 CFU/g 0.1 CFU/g

7 Meat sample Preenrichment: buffer method vs MacConkey method AB selective enrichment: Ceftriaxone () vs Cefotaxime ()

8 6 different methods #1 The peptone buffer method 25 gram of meat 225 ml peptone media No AB selective enrichment Incubation o.n. at 37 C MacConkey + (1µg/mL) Plating on MacConkey + (1µg/mL) (44 C)

9 5*190 g meat 5*10 g for premix with meat in different concentrations 10 3 CFU/g 10 2 CFU/g 10 1 CFU/g 1 CFU/g 0 CFU/g 0.1 CFU/g 2 * 25 g h 37 C ml McConkey Plates 44 C

10 6 different methods #2 The peptone buffer method + (1µg/ml) 25 gram of meat 225 ml peptone media Selective enrichment using ceftriaxone (1µg/mL) Incubation o.n. at 37 C Plating on MacConkey + (1µg/mL) (44 C)

11 6 different methods #2 The peptone buffer method + (1µg/ml) 25 gram of meat 225 ml peptone media Selective enrichment using cefotaxime (1µg/mL) Incubation o.n. at 37 C Plating on MacConkey + (1µg/mL) (44 C)

12 5*190 g meat 5*10 g for premix with meat in different concentrations 10 3 CFU/g 10 2 CFU/g 10 1 CFU/g 1 CFU/g 0 CFU/g 0.1 CFU/g 2 * 25 g 2 * 25 g 2 * 25 g h 37 C ml ml ml + Loop 10 µl McConkey Plates 44 C

13 6 different methods #4 The MacConkey buffer method 5 gram of meat 45 ml MacConkey media No AB selective enrichment Incubation o.n. at 44 C MacConkey + (1µg/mL) Plating on MacConkey + (1µg/mL) (44 C)

14 6 different methods #5 The MacConkey method + (1µg/ml) 5 gram of meat 45 ml MacConkey media Selective enrichment using ceftriaxone (1µg/mL) Incubation o.n. at 44 C Plating on MacConkey + (1µg/mL) (44 C)

15 6 different methods #6 The MacConkey method + (1µg/ml) 5 gram of meat 45 ml MacConkey media Selective enrichment using cefotaxime (1µg/mL) Incubation o.n. at 44 C Plating on MacConkey + (1µg/mL) (44 C)

16 5*190 g meat 5*10 g for premix with meat in different concentrations 10 3 CFU/g 10 2 CFU/g 10 1 CFU/g 1 CFU/g 0 CFU/g 0.1 CFU/g 2 * 25 g h 37 C McConkey Plates 44 C 1 2 * 25 g 2 * 25 g 225 ml ml + Loop 10 µl 225 ml h 44 C 5 6 3*5 g 4 % AB 45 ml McConkey 3*5 g Loop 10 µl McConkey Plates 44 C

17 Scoring and verification colonies for PCR 2 MacConkey + / 1 mg/l 3

18 -M1 Ceftriaxone () Cefotaxime () No antibiotics MacConkey

19 TEM-52 Ceftriaxone () Cefotaxime () No antibiotics MacConkey

20 CMY-2 Ceftriaxone () Cefotaxime () No antibiotics MacConkey

21 VIM-1 (+ACC-1) Ceftriaxone () Cefotaxime () No antibiotics MacConkey

22 KPC-1 Ceftriaxone () Cefotaxime () No antibiotics MacConkey

23 OXA-48 Ceftriaxone () Cefotaxime () No antibiotics MacConkey

24 ATCC25922 (negative control) Ceftriaxone () Cefotaxime () No antibiotics MacConkey

25 Conclusions - Food All of the methods detected the spiked bacteria (except the OXA-48 producing E. coli). Detection limits is <1 CFU/gram. The peptone buffer methods had a tendency to generated more background growth of accompanying flora compared to the MacConkey method.

26 Suggestion for method to detect ESBL/AmpC producing E. coli in meat #1 The peptone buffer method 25 gram of meat 225 ml media No AB selective enrichment Incubation o.n. at 37 C Plating on MacConkey + (1µg/mL) (44 C)

27 -M1 5*6 g faeces Dilluted spiking samples 6 g = 10 3 CFU/g 10 2 CFU/g 10 1 CFU/g 1 CFU/g h 37C MacConkey Plates 44 C 2 * 1 g c 9 ml h 44C e 2*1 g f % AB 0 CFU/g 9 ml McConkey 2*1 g MacConkey plates

28 -M1 Ceftriaxone () Cefotaxime () No antibiotics MacConkey

29 Detecting carbapenemases? To be continued.

30 5*190 g meat 5*10 g for premix with meat in different concentrations 180 g = h 37C CARBA OXA McConkey Plates (44C) 0 CFU/g 10 3 CFU/g 0.1 CFU/g 10 2 CFU/g 10 1 CFU/g 1 CFU/g * 25 g 2 * 25 g 2 * 25 g 3*5 g b ml 225 ml + Loop 10 µl 225 ml h 44C 5 6 % AB 45 ml McConkey 3*5 g Loop 10 µl McConkey Plates (44C) CARBA OXA

31 Suggestion for method to detect ESBL/AmpC nd Carbapenemase producing E. coli in meat #1 The peptone buffer method 25 gram of meat 225 ml media No AB selective enrichment Incubation o.n. at 37 C MacConkey + (1µg/mL) (44 C) Plating on MacConkey + AXO (1µg/mL) ChromID CARBA (BioMerieux)(37 /44 C) CARBA OXA ChromID CARBA SMART

32 -M1 5*6 g faeces Dilluted spiking samples 6 g = 10 3 CFU/g 10 2 CFU/g 10 1 CFU/g 1 CFU/g h 37C MacConkey Plates 44 C BioMerieux 37 C 2 * 1 g c 9 ml h 44C CARBA OXA e 2*1 g f % AB 0 CFU/g 9 ml McConkey 2*1 g MacConkey plates CARBA OXA CARBA OXA

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