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Escherichia coli with extended-spectrum beta-lactamases or transferable AmpC beta-lactamases and Salmonella on meat imported into Sweden
International Journal of Food Microbiology 171 (2014) 8–14
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short communication
Escherichia coli with extended-spectrum beta-lactamases or transferable AmpC beta-lactamases and Salmonella on meat imported into Sweden Maria Egervärn a,⁎, Stefan Börjesson b, Sara Byfors a, Maria Finn b, Caroline Kaipe a, Stina Englund b, Mats Lindblad a a b
National Food Agency, Box 622, SE-75126 Uppsala, Sweden National Veterinary Institute (SVA), SE-75189 Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 30 June 2013 Received in revised form 21 October 2013 Accepted 5 November 2013 Available online 14 November 2013 Keywords: Antimicrobial resistance Cephalosporin ESBL-producing E. coli pAmpC-producing E. coli Salmonella Meat
a b s t r a c t The presence of Enterobacteriaceae producing extended spectrum beta-lactamases (ESBL) or transferable AmpC beta-lactamases (pAmpC) is increasingly being reported in humans and animals world-wide. Their occurrence in food-producing animals suggests that meat is a possible link between the two populations. This study investigated the occurrence and characteristics of Salmonella and ESBL- or pAmpC-producing E. coli in 430 samples of beef, pork and broiler meat imported into Sweden, in order to provide data required for assessing the potential public health risk of these bacteria in food. Depending on region of origin, ESBL/pAmpC-producing E. coli were found in 0–8% of beef samples, 2–13% of pork samples and 15–95% of broiler meat samples. The highest prevalence was in South American broiler meat (95%), followed by broiler meat from Europe (excluding Denmark) (61%) and from Denmark (15%). Isolates from meat outside Scandinavia were generally defined as multiresistant. A majority of the ESBL/pAmpC genes were transferable by conjugation. BlaCTX-M-2 and blaCTX-M-8 were the dominant genes in E. coli from South American broiler meat, whereas blaCMY-2 and blaCTX-M-1 dominated in European meat. The majority of blaCMY-2 and blaCTX-M-1 were situated on plasmids of replicon type incK and incI1, respectively. The same combinations of ESBL/pAmpC genes and plasmids have been described previously in clinical human isolates. Salmonella was found in five samples tested, from European pork and broiler meat. No Salmonella isolate was resistant to third-generation cephalosporins. In conclusion, meat imported into Sweden, broiler meat in particular, is a potential source of human exposure to ESBL- and pAmpC-producing E. coli. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The presence of Enterobacteriaceae producing extended spectrum beta-lactamases (ESBL) or transferable AmpC beta-lactamases (pAmpC) is a rapidly emerging public health problem (Pitout and Laupland, 2008). Furthermore, the presence of ESBL- and pAmpC-producing E. coli and Salmonella is increasingly being reported in food-producing animals and in meat worldwide (EFSA, 2011), with 44–93% prevalence of ESBL- and pAmpC-producing E. coli in broiler meat from countries such as Germany (Kola et al., 2012), the Netherlands (Overdevest et al., 2011) and Spain (Egea et al., 2012). In beef and pork, the occurrence of E. coli with ESBL/pAmpC appears to be several-fold lower (EFSA, 2011). Recent Dutch studies have shown that the predominant ESBL genes and plasmids in E. coli obtained from broiler meat are also present in clinical isolates from humans (Leverstein-van Hall et al., 2011; Overdevest et al., 2011), indicating potential for transmission to humans via food (EFSA, 2011; Smet et al., 2010b). However, the extent to which food ⁎ Corresponding author at: National Food Agency, Risk and Benefit Assessment Department, Box 622, SE-751 26 Uppsala, Sweden. Tel.: +46 18 17 53 15; fax: +46 18 10 58 48. E-mail address: [email protected] (M. Egervärn). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.11.005
serves as a dissemination route for these bacteria to humans and the impact on human health have not yet been established (EFSA, 2011; Smet et al., 2010b). In Sweden, the occurrence of ESBL- and pAmpC-producing E. coli on domestic meat is regularly investigated as part of the Swedish Veterinary Antimicrobial Resistance Monitoring (SVARM) programme. Meat imported into Sweden is currently not included in the programme. Considering that 42% of the red meat and 38% of the poultry meat sold on the Swedish market are imported (estimates based on Anonymous, 2012), studies mapping the presence of ESBL- and pAmpC-producing E. coli in imported meat are needed. Such data are also required for assessing the potential public health risk of these bacteria in food. The aim of the present study was to investigate the occurrence and characteristics of Salmonella and ESBL/pAmpC-producing E. coli in meat imported into Sweden from Europe and South America. 2. Material and methods 2.1. Sampling A stratified sampling strategy was applied, with the intention of allowing comparison of the proportions of ESBL-positive samples from
M. Egervärn et al. / International Journal of Food Microbiology 171 (2014) 8–14
the most common countries or regions exporting beef, pork and broiler meat to Sweden. The division of countries or regions into strata was based on statistics from the Swedish Board of Agriculture (2009) on the import value of meat in 2008. The beef strata were: ‘Other European countries’, Ireland and South America, representing 57, 35 and 7% of imports, respectively. The pork strata were: Denmark, Germany and ‘other European countries’, representing 49, 26 and 25% of imports, respectively. The broiler meat strata were: Denmark, ‘other European countries’ and South America, representing 71, 29 and b1% of imports. South America was included due to recent findings of ESBL-producing E. coli in South American broiler meat (Dhanji et al., 2010; Warren et al., 2008). The target was to collect about 40–50 samples from each stratum except beef from ‘other European countries’, for which the target was ~ 100 samples. In total, samples of imported broiler meat (n = 133), beef (n = 178) and pork (n = 119) were collected fresh or frozen at retail stores and outlets from January 2010 to June 2011. Each sample represented a unique batch or single production date. Samples were collected based on their availability in retail outlets, by official inspectors. 2.2. Isolation and antibiotic susceptibility testing of ESBL- or pAmpC-producing E. coli Approximately 25 g meat were homogenised by stomaching in 225 mL buffered peptone water. A 100 mL portion of the suspension was supplemented with 1 μg/mL cefotaxime (Sigma Aldrich) and incubated at 37 °C for 18–24 h. In order to capture both ESBL- and pAmpCproducing bacteria from the sample, aliquots of the enrichment culture were then plated in parallel onto two selective media, CHROMagar™ ESBL and CHROMagar™ Orientation (EMM Life Science AB), containing 1 μg/mL cefotaxime. After 18–24 h incubation at 37 °C, a tentative, single E. coli colony was primarily selected from CHROMagar™ ESBL. Species identity was confirmed using a biochemical API20E test (bioMérieux Sweden). Beta-lactamase production was verified by Etest®ESBL and Etest®AmpC (bioMérieux, Sweden). Susceptibility to 14 antibiotics was assessed by broth microdilution according to CLSI (2008) using VetMIC™ GN-mo panels (SVA Sweden). Susceptibility of ESBL- or pAmpC-producing E. coli to carbapenems was tested by Etest®Imipenem. 2.3. Identification of ESBL/pAmpC genes and plasmids To determine the gene group responsible for the ESBL and AmpC phenotypes, all isolates were subjected to multiplex-PCRs detecting the following gene groups: blaCTX-M (Woodford et al., 2006), pAmpC (Perez-Perez and Hanson, 2002) and blaSHV, blaTEM and blaOXA-1 (Fang et al., 2008). The specific gene variants were determined by sequencing as previously described by Börjesson et al. (2013a), with additional primers for blaCTX-M-2 (Hopkins et al., 2006), blaCTX-M-8 (this study; 5’-CTT TTT GTG CTG ACT GTG AA-3’ and 5’-GCA GCM GCG AGY ACG TCA-3’), blaCTX-M-9 (McGettigan et al., 2009), blaCTX-M-25 (Chmelnitsky et al., 2005) and blaSHV (Ryoo et al., 2005). Isolates testing positive for blaCTX-M-15 were subjected to multilocus sequence typing (MLST) according to http://mlst.ucc.ie/. Transferability of ESBL/pAmpC genes from the isolates was tested by conjugation to E. coli HMS174 (E. coli GSC6576). The donor and recipient strains were inoculated in NY-broth and incubated at 37 °C overnight. After incubation, the donor and recipient were mixed (1:4) and centrifuged. The pellets obtained were suspended in saline buffer, spotted on LA and incubated at 37 °C for 6 h. Bacterial growth was collected and resuspended in saline buffer. Serial dilutions were spread on LA containing cefotaxime (2 mg/L) and rifampin (50 mg/L) and incubated at 37 °C overnight. Transformation was performed on isolates unable to transfer ESBL/pAmpC phenotypes by conjugation and transconjugants testing PCR-positive for multiple plasmid replicon types. Plasmid DNA was extracted according to the miniprep protocol described by
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Sambrook and Russell (2001) with minor modification. After the NaC2H3O2 step, the mix was centrifuged and the supernatant was collected, mixed with 5 M LiCl (1:1), incubated at −20 °C and centrifuged. Plasmid DNA was collected from the supernatant by ethanol precipitation and transformed (~60 ng μL−1) with a voltage of 1250 V using ElectroMax™ DH10B™ (Gibco Invitrogen) according to the manufacturer's instruction. Transconjugants and transformants were subjected to PCR-based plasmid replicon typing (Carattoli et al., 2005) and multiplex-PCRs for genes encoding ESBL/pAmpC (Fang et al., 2008; Perez-Perez and Hanson, 2002; Woodford et al., 2006). 2.4. Isolation and antibiotic susceptibility testing of Salmonella Salmonella was isolated in accordance with the method described by the Nordic Committee on Food Analysis, NMKL 71. Isolates were identified to genus level using API20E (bioMérieux Sweden), with subsequent serotyping according to the standard Kaufmann–White procedure. Susceptibility to 12 antibiotics was assessed using VetMIC™ panels for Salmonella bacteria according to CLSI standards for microdilution (CLSI, 2008). 3. Results 3.1. Occurrence and characteristics of ESBL/pAmpC-producing E. coli 3.1.1. Broiler meat ESBL- and pAmpC-producing E. coli were isolated from broiler meat samples originating from all geographical regions included (Table 1a). The highest prevalence was in meat imported from South America, foremost Brazil (95%), followed by meat from Europe excluding Denmark (61%) and Denmark (15%). The most prevalent ESBL/pAmpC genes in broiler meat imported from South America were blaCTX-M-2 and blaCTX-M-8 (Table 2a), while the most prevalent genes in broiler meat imported from ‘other EU countries’ were blaCMY-2 and blaCTX-M-1. The blaCTX-M-8 gene was identified in isolates from South American meat only, whereas blaCTX-M-1 was identified in isolates from European meat only. The single E. coli isolated from French broiler meat contained the only blaCTX-M-25 found. One isolate from German broiler meat contained both blaCMY-2 and blaSHV-12. In Danish meat, blaCMY-2 was the only ESBL/pAmpC gene identified. In total, 86% of the isolates from Danish meat and 85% of the isolates from meat obtained from ‘other EU countries’ were able to transfer the ESBL/pAmpC genes by conjugation, whereas 76% of the isolates from South American meat were able to transfer the gene. After applying transformation on the isolates unable to transfer the gene by conjugation, nine genes were still non-transferable. Isolates containing blaCTX-M-1, blaCTX-M-8, blaSHV-12 or blaTEM frequently (90%) carried those genes on plasmids belonging to replicon type incI1. Similarly, 74% of the blaCMY-2-positive isolates carried the pAmpC gene on incK plasmids. The specific location of blaCTX-M-2 was in most cases not possible to decide by transferability experiments and replicon typing. When it was transferred, the frequency was low overall and most plasmids were PCR-positive for both incP and incHI2. Non-ESBL blaTEM were not co-transferred with ESBL genes. Three plasmids carrying ESBL or pAmpC genes that were transferred proved to be untypeable by replicon-typing PCR (Table 2a). In total, 81% of the 75 broiler meat isolates were multiresistant, i.e. resistant to three or more antibiotic classes (Table 2a). Overall, isolates from South American meat samples showed resistance to a wider range of antibiotics than isolates from European meat samples. Resistance to quinolones, aminoglycosides and sulfamethoxazole was the most common trait. Resistance to chloramphenicol (5 isolates) and florfenicol (1 isolate) was exclusively found among the isolates from South American meat. One blaCMY-2-positive isolate from Chilean meat was susceptible to colistin only. Two isolates from German meat samples and all but one isolate from Danish and Finnish meat samples
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Table 1 Prevalence of ESBL/pAmpC-producing E. coli in (A) broiler meat, (B) beef and (C) pork available on the Swedish market 2010–2011. A) Region of origin
Samples, n
ESBL- E. coli, n
Denmark 46 0 Germany 28 14c Finland 9 0 Othera 7 3 Europed, total 44 17 Brazil 40 38 Otherb 3 1 S. America, total 43 39 a Less than five samples tested per country: Croatia, Estonia, France, Lithuania, Netherlands, Poland b Less than five samples tested per country: Argentina, Chile c One isolate was also pAmpC-producing d Denmark excluded
pAmpC- E. coli, n
ESBL/pAmpC- E. coli, n (%)
7 9 1 0 10 0 2 2
7 (15) 23 (82) 1 (11) 3 (43) 27 (61) 38 (95) 3 (100) 41 (95)
B) Region of origin
Samples, n
ESBL- E. coli, n
Ireland 40 0 Germany 35 1 The Netherlands 25 5 Othera 36 2 Europeb, total 96 8 Brazil 18 0 Uruguay 18 0 Argentina 6 0 S. America, total 42 0 a Less than five samples tested per country: Denmark, Estonia, Italy, Lithuania, Poland, UK, Austria b Ireland excluded
pAmpC- E. coli, n
ESBL/pAmpC- E. coli, n (%)
0 0 0 0 0 0 0 0 0
0 (0) 1 (3) 5 (20) 2 (6) 8 (8) 0 (0) 0 (0) 0 (0) 0 (0)
C) Region of origin
Samples, n
ESBL- E. coli, n
Denmark 44 1 Germany 44 3 Italy 20 3 Othera 11 0 Europeb, total 31 3 a Less than five samples tested per country: Finland, Netherlands, Poland, Spain b Denmark and Germany excluded
were resistant to beta-lactams only. None of the isolates was resistant to imipenem. 3.1.2. Beef ESBL-producing E. coli was found in 8 out of 96 samples of beef imported into Sweden from ‘other EU countries’, excluding Ireland (Table 1b). Five of the positive samples originated from Dutch beef. ESBL-producing E. coli was not found in Irish or South American samples and pAmpC-producing E. coli was not found in sample from any geographical region included (Table 1b). ESBL production was encoded by three gene variants: blaCTX-M-1 (n = 5), blaCTX-M-15 (n = 2) and blaTEM-52 (n = 1; Table 2b). All isolates were able to transfer genes encoding ESBL by conjugation. Two E. coli of MLST type ST10 (data not shown) from Dutch and Austrian minced meat contained blaCTX-M-15 that was identified on incI1 plasmids. The blaCTX-M-1 genes were identified on plasmids belonging to different replicon types and the plasmid carrying blaTEM-52 was untypeable by replicon-typing PCR. All but one ESBL-producing E. coli isolates were multiresistant. None of the isolates showed reduced susceptibility to imipenem.
pAmpC- E. coli, n
ESBL/pAmpC- E. coli, n (%)
0 0 0 1 1
1 (2) 3 (7) 3 (15) 1 (9) 4 (13)
ESBL production was encoded by three gene variants: blaCTX-M-1 (n = 5) on incI1 plasmids or an incN plasmid, blaCTX-M-14 (n = 1) on an incF plasmid and blaTEM-52 (n = 1) on an incI1 plasmid (Table 2c). The single pAmpC enzyme identified was encoded by blaCMY-2 on an incA/C plasmid. All isolates were multiresistant except one from Italian pork. None of the isolates showed reduced susceptibility to imipenem. 3.2. Occurrence of antibiotic resistant Salmonella Salmonella was detected in five of the 430 samples tested; three and two samples of meat from broilers and pigs, respectively (Table 3). All isolates were isolated from German meat except one isolated from Italian pork. Serotypes identified were Salmonella Typhimurium, S. Infantis and S. Paratyphi B. variant Java. One isolate from German broiler meat was susceptible to all antibiotics tested, whereas the remaining isolates were multiresistant. Resistance to ampicillin, tetracycline and sulfamethoxazole was most common. Two of the isolates from German broiler meat were resistant to quinolones. None of the isolates was resistant to third-generation cephalosporins (Table 3). 4. Discussion
3.1.3. Pork ESBL-producing E. coli was found in 1 out of 44 Danish pork samples, 3 out of 44 German pork samples and 3 out of 31 pork samples from other EU countries (Table 1c). The latter positive samples originated from Italian pork. pAmpC-producing E. coli was found in one sample tested, from Polish pork. All isolates were able to transfer genes encoding ESBL or pAmpC by conjugation.
ESBL- and pAmpC-producing E. coli were frequently found in samples of imported beef, pork and broiler meat available on the Swedish market, with the highest prevalence in broiler meat. The occurrence in various meats was of the same order of magnitude as in a similar study on imported and domestic meat in Denmark, performed during the same period (DANMAP, 2010). The results of the present study are
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Table 2 Characteristics of ESBL/pAmpC-producing E. coli in (A) broiler meat, (B) beef and (C) pork available on the Swedish market 2010–2011. Ciprofloxacin (Ci), nalidixic acid (Nal), chloramphenicol (CM), florfenicol (Ff), colistin (Cs), gentamicin (Gm), kanamycin (Km), streptomycin (Sm), tetracycline (Tc), sulfamethoxazole (Su), trimethoprim (Tm). Epidemiological cut-off values for resistance according to EUCAST (www.eucast.org). A) Region of origin Denmark
Isolates, n (country of origina)
Beta-lactam gene variant/s/
3 (DK) blaCMY-2 2 (DK) blaCMY-2 + blaTEM-1 1 (DK) blaCMY-2 1 (DK) blaCMY-2 Europe 2 (DE, FI) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 + blaTEM-1 1 (DE) blaCMY-2 + blaSHV-12 1 (DE) blaCTX-M-1 1 (PL) blaCTX-M-1 1 (DE) blaCTX-M-1 1 (DE) blaCTX-M-1 1 (DE) blaCTX-M-1 + blaTEM-1 1 (DE) blaCTX-M-1 + blaTEM-1 1 (DE) blaCTX-M-1 + blaTEM-135 1 (DE) blaCTX-M-2 1 (NL) blaCTX-M-2 1 (FR) blaCTX-M-25 2 (DE) blaSHV-12 1 (DE) blaSHV-12 + blaTEM-1 1 (DE) blaTEM-19 1 (DE) blaTEM-52 1 (DE) blaTEM-52 S. America 1 (AR) blaCMY-2 1 (CL) blaCMY-2 1 (BR) blaCTX-M-2 2 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (AR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 2 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 + blaTEM-1 1 (BR) blaCTX-M-2 + blaTEM-1 1 (BR) blaCTX-M-2 + blaTEM-1 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 2 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 2 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 2 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 + blaTEM-1 1 (BR) blaCTX-M-8 + blaTEM-1 1 (BR) blaCTX-M-8 + blaTEM-1 a DK = Denmark, DE = Germany, FI = Finland, NL = the Netherlands, PL = Poland, FR = France, AR = b All isolates resistant to ampicillin, cefotaxime and ceftazidime c ? = Non-typeable using PCR-based plasmid replicon typing d Transformant positive for blaCMY-2 e NT = Non-transferable with transconjugation or transformation
Pattern of non-beta-lactam resistanceb – – Km – – Ci, Nal Ci, Nal, Su Ci Nal, Su Ci, Nal, Cs, Km, Sm, Su, Tm, Tc Gm, Km, Sm, Su Gm, Sm, Su Gm, Km, Sm, Su, Tm Ci, Nal, Su,Tm, Tc Ci, Nal, Cm, Ff, Su, Tc Ci, Nal, Tc Ci, Nal, Sm, Su, Tm Sm, Su, Tc Su, Tc – Ci, Nal, Sm, Su, Tm, Tc Sm, Su, Tm Ci, Nal, Gm, Km, Sm, Su, Tc Cm, Km, Sm, Su, Tc Ci, Nal, Km, Su, Tc Cm, Sm, Su, Tc Cm, Ci, Nal, Su, Tm, Tc Ci, Nal, Su Ci, Nal, Tc Km, Sm, Su, Tm Ci, Nal, Km, Sm, Su, Tm Ci, Nal, Cm, Ff, Gm, Km, Sm, Su, Tm, Tc Ci, Nal, Gm, Km, Sm, Su, Tm, Tc Ci, Nal, Sm, Su, Tm, Tc Ci, Nal, Km, Sm, Su, Tm, Tc Ci, Nal, Km, Sm, Su, Tm, Tc Ci, Nal, Gm, Km, Sm, Su, Tm Ci, Nal, Gm, Km, Sm, Su, Tc Ci, Nal, Gm, Km, Sm, Su, Tc Ci, Nal, Gm, Km, Sm, Su, Tc Ci, Nal, Gm, Km, Sm, Tc Ci, Nal, Gm, Km, Su, Tc Ci, Nal, Cm, Gm, Su, Tc Ci, Nal, Cm, Sm, Su, Tc Gm, Km, Sm, Su, Tc Cm, Km, Sm, Su, Tc Gm, Km, Sm, Su, Tc, Tm Ci, Nal, Cm, Gm, Su, Tm, Tc Ci, Nal, Cs, Su, Sm, Tm Ci, Nal, Gm, Km, Sm, Su, Tm, Tc Ci, Nal, Gm, Sm, Su, Tm, Tc Ci, Nal, Sm, Su, Tm, Tc Ci, Nal, Gm, Sm, Su, Tm Ci, Nal, Km, Sm, Su, Tc Ci, Nal, Sm, Tm Ci, Nal, Sm, Su, Tm Nal, Sm, Su, Tm Ci, Sm, Su, Tm Sm, Su, Tm, Tc Km, Sm, Tm, Tc Sm Ci, Nal Km Ci, Nal, Gm, Km, Sm, Su, Tm, Tc Sm, Tc Ci, Nal, Tc Argentine, BR = Brazil, CL = Chile
ESBL /pAmpC location incK incK incK incI1 incK ?c incK incI1 incK incK incI1 incK incI1 incKd incP/incO incI1 incI1 incI1 ? incI1 incI1 NTe NT incA/C incI1 incI1 incI1 incI1 incI1 incI1 incP incP/incHI2 incHI2 NT incO ? NT incI1 incP/incHI2 incP/incHI2 incP/incHI2 NT NT incP/incHI2 NT incP/incHI2 NT NT incI1 incI1 incI1 incI1 NT incI1 incI1 incI1 incI1 incI1 incI1 incI1 incI1 incI1 incI1 incI1 incI1
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Table 2 (continued) B) Region of origin
Isolates, n (country of origina)
Pattern of non-beta-lactam resistanceb
ESBL /pAmpC location
– Ci, Nal, Gm, Sm, Su, Tm, Tc Gm, Su, Tm, Tc Sm, Su, Tm, Tc Ci, Nal, Sm Cm, Km, Sm, Su, Tc Ci, Nal, Su, Tm, Tc
incI1 incF incFIIA incHI2 incI1 incI1 ?c
Beta-lactam gene variant/s/
Pattern of non-beta-lactam resistanceb
ESBL /pAmpC location
blaCTX-M-1 + blaCTX-M-1 + blaCTX-M-1 + blaCTX-M-14 blaCMY-2 blaCTX-M-1 blaCTX-M-1 + blaTEM-52
Nal, Km, Sm, Su, Tm, Tc Sm, Su, Tm, Tc Km, Sm, Su, Tm Km, Su, Tm, Tc Cm, Ff, Sm, Su, Tc Su, Tm, Tc Ci, Nal, Cm, Sm, Su, Tm, Tc –
incN incI1 incI1 incF incA/C incI1 incI1 incI1
Beta-lactam gene variant/s/
Europe
2 (DE, NL) blaCTX-M-1 1 (NL) blaCTX-M-1 1 (NL) blaCTX-M-1 + blaTEM-1 1 (NL) blaCTX-M-1 + bladTEM 1 (NL) blaCTX-M-15 + blaTEM-1 1 (AU) blaCTX-M-15 + blaTEM-1 1 (ES) blaTEM-52 a DK = Denmark, DE = Germany, NL = the Netherlands, AU = Austria, ES = Estonia b All isolates resistant to ampicillin, cefotaxime and ceftazidime c ? = Non-typeable using PCR-based plasmid replicon typing d Negative by sequencing of the tem gene fragment C) Region of origin
Isolates, n (country of origina)
Denmark Germany
1 (DK) 1 (DE) 1 (DE) 1 (DE) Europe 1 (PL) 1 (IT) 1 (IT) 1 (IT) a DK = Denmark, DE = Germany, PL = Poland, IT = Italy b All isolates resistant to ampicillin, cefotaxime and ceftazidime
blaTEM-1 blaTEM-1 blaTEM-1
blaTEM-1
also in agreement with data on ESBL/pAmpC prevalence in meat previously summarised by the European Food Safety Authority (2011). In contrast to many earlier European studies, CMY-2-producing E. coli was the most common finding on the meat samples investigated here. However, it should be noted that most previous studies did not actively investigate the occurrence of pAmpC-producing E. coli in meat. Considering that resistance due to production of transferable AmpC enzymes is also deemed a significant public health problem (EFSA, 2011), prevalence studies such as this should include screening that captures both ESBL- and pAmpC-producing E. coli from a sample. However, the proportion of E. coli with pAmpC phenotype in the samples may have been underestimated also in the present study, because tentative colonies were primarily selected from selective ESBL plates inhibiting the growth of AmpC producers. Moreover, the results are in strong contrast to a recently published Swedish study, which showed that none of the samples tested of Mediterranean foods imported into Sweden contained ESBL-producing Enterobacteriaceae (Tham et al., 2012). The main reason for the difference is probably that Tham et al. (2012) used a non-selective culture method. This underlines the importance of using a selective culture approach in targeted surveys that include samples where the levels of ESBL- and pAmpC-producing E. coli can be low.
The high occurrence of ESBL- and/or pAmpC-producing E. coli on European broiler meat reported here has previously been described in several countries within the EU (Egea et al., 2012; Kola et al., 2012; Overdevest et al., 2011). This includes Sweden, with 44% prevalence in Swedish broiler meat (Börjesson et al., 2013a). A high proportion of ESBL/pAmpC-producing bacteria on broiler meat is also increasingly being reported in various countries outside Europe, such as the USA (Doi et al., 2010) and Japan (Ahmed et al., 2009). In the present study, ESBL-producing E. coli were frequently found in samples from South American broiler meat, confirming findings in two previous British studies (Dhanji et al., 2010; Warren et al., 2008). As in the present study, the dominant ESBL genes in those studies, blaCTX-M-2 and blaCTX-M-8, in E. coli from South American meat differed from the ESBL gene dominating in European meat, blaCTX-M-1. This supports the theory of different CTX-M type lineages dominating in different geographical regions (Canton and Coque, 2006; Smet et al., 2010a). The combination of blaCMY-2 and incK frequently identified in E. coli from European broiler meat has recently also been identified in clinical isolates from Swedish patients (Börjesson et al., 2013b) and has disseminated among E. coli isolates in intensive care units in Canada (Baudry et al., 2009). In addition, the majority of blaCTX-M-1 detected in the present study was situated on plasmids of replicon type incI1, i.e. the
Table 3 Serotypes and antibiotic resistance pattern of salmonella detected in samples collected in 2010–2011 from meat imported into Sweden. Epidemiological cut-off values for resistance according to EUCAST (www.eucast.org). Sample type
Country of origin
Serotype
Resistance patterna
Pork fillet Loin of pork Whole chicken Whole chicken Chicken fillet
Italy Germany Germany Germany Germany
S. subsp. I S. Typhimurium S. subsp. I S. paratyphi B.b S. Infantis
Am, Sm, Tc, Su Am, Tc, Su, Tm – Am, Ci, Nal, Cm, Sm, Tc, Su, Tm Am, Ci, Nal, Su
a Ampicillin (Am), cefotaxime (Ctx), ciprofloxacin (Ci), nalidixic acid (Nal), chloramphenicol (Cm), florfenicol (Ff), gentamicin (Gm), kanamycin (Km), streptomycin (Sm), tetracycline (Tc), sulfamethoxazole (Su), trimethoprim (Tm) b variant Java
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same combination of ESBL gene and plasmid previously described in Salmonella isolates from poultry and humans in France (Cloeckaert et al., 2010). In contrast, the dominant ESBL/pAmpC gene among clinical isolates in Sweden, blaCTX-M-15, was found in only 2% of the 91 E. coli isolates from meat samples examined in the present study. Furthermore, blaCTX-M-15 was identified on incI1 plasmids in E. coli typed as ST10 (data not shown) and thus was not associated to the most prevalent plasmid type and clonal lineage among clinical E. coli, i.e. incFII and ST131 (Naseer and Sundsfjord, 2011). This is in agreement with similar British and Swiss studies, which report a predominance of blaCTX-M-1 and blaCTX-M-15 in E. coli isolates from animals/meat and humans, respectively (Geser et al., 2012a; Geser et al., 2012b; Randall et al., 2011). In contrast, recent Dutch studies have described identical ESBL genes, plasmids and genotypes of ESBL-producing E. coli in isolates from broiler meat and from Dutch patients, suggesting that broiler meat is a source of ESBL-producing bacteria in the Netherlands (Leverstein-van Hall et al., 2011; Overdevest et al., 2011). The E. coli isolates from meat outside Scandinavia were frequently found to be multiresistant, as is often the case with ESBL/pAmpC producers and may reflect the usage of antimicrobials in production of food-producing animals (EMA, 2012). Four of the five Salmonella isolated from European pork and broiler meat in the present study were also multiresistant, with similar resistance patterns for Salmonella in these types of meat as stated in the EU summary report on antimicrobial resistance (EFSA, 2012). Data in the present study revealed no cephalosporin resistance, probably due to the limited number of Salmonella found in the samples, whereas EFSA (2012) reported 3% cefotaxime resistance in Salmonella isolated from German broiler meat after non-selective culture. In conclusion, samples from beef, pork and broiler meat imported into Sweden frequently contained ESBL- and pAmpC-producing E. coli, with the highest prevalence in broiler meat. The combinations of ESBL/pAmpC genes and plasmids identified in E. coli isolated from meat were partly similar to those previously found in clinical human isolates. On-going work at our department is focusing on genetic comparisons of ESBL/pAmpC genes, plasmids and E. coli isolates from meat, patients and healthy carriers in Sweden, in order to investigate the extent to which meat available on the Swedish market serves as a source of human exposure to ESBL- and pAmpC-producing E. coli. Funding This work was carried out with financial support from the Swedish Civil Contingencies Agency. References Ahmed, A.M., Shimabukuro, H., Shimamoto, T., 2009. Isolation and molecular characterization of multidrug-resistant strains of Escherichia coli and Salmonella from retail chicken meat in Japan. J. Food Sci. 74, M405–M410. Anonymous, 2009. Sweden's imports and exports of agrofood products 2006–2008. Swedish Board of Agriculture (SJV). Report 2009:18 (abstract in English). Anonymous, 2012. Yearbook of agricultural statistics 2012 including food statistics. Swedish Board of Agriculture and Statistics of Sweden. Baudry, P.J., Mataseje, L., Zhanel, G.G., Hoban, D.J., Mulvey, M.R., 2009. Characterization of plasmids encoding CMY-2 AmpC beta-lactamases from Escherichia coli in Canadian intensive care units. Diagn. Microbiol. Infect. Dis. 65, 379–383. Börjesson, S., Egervärn, M., Lindblad, M., Englund, S., 2013a. Frequent occurrence of extended-spectrum beta-lactamase- and transferable ampc beta-lactamaseproducing Escherichia coli on domestic chicken meat in Sweden. Appl. Environ. Microbiol. 79, 2463–2466. Börjesson, S., Jernberg, C., Brolund, A., Edquist, P., Finn, M., Landen, A., Olsson-Liljequist, B., Tegmark Wisell, K., Bengtsson, B., Englund, S., 2013b. Characterization of plasmidmediated AmpC-producing E. coli from Swedish broilers and association with human clinical isolates. Clin. Microbiol. Infect. 19 (7), E309–11. Canton, R., Coque, T.M., 2006. The CTX-M beta-lactamase pandemic. Curr. Opin. Microbiol. 9, 466–475. Carattoli, A., Bertini, A., Villa, L., Falbo, V., Hopkins, K.L., Threlfall, E.J., 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63, 219–228. Chmelnitsky, I., Carmeli, Y., Leavitt, A., Schwaber, M.J., Navon-Venezia, S., 2005. CTX-M-2 and a new CTX-M-39 enzyme are the major extended-spectrum beta-lactamases in
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multiple Escherichia coli clones isolated in Tel Aviv, Israel. Antimicrob. Agents Chemother. 49, 4745–4750. Cloeckaert, A., Praud, K., Lefevre, M., Doublet, B., Pardos, M., Granier, S.A., Brisabois, A., Weill, F.X., 2010. IncI1 plasmid carrying extended-spectrum-beta-lactamase gene blaCTX-M-1 in Salmonella enterica isolates from poultry and humans in France, 2003 to 2008. Antimicrob. Agents Chemother. 54, 4484–4486. CLSI, 2008. Performance Standards for Antimicrobial Disc and Dilution Susceptibility Test for Bacteria Isolated from Animals; Approved Standard, third edition. Clinical and Laboratory Standard Institute. DANMAP, 2010. Use of antimicrobial agents and occurence of antimicrobial resistance in bacteria from food animals, foods and humans in Denmark in 2010. Dhanji, H., Murphy, N.M., Doumith, M., Durmus, S., Lee, S.S., Hope, R., Woodford, N., Livermore, D.M., 2010. Cephalosporin resistance mechanisms in Escherichia coli isolated from raw chicken imported into the UK. J. Antimicrob. Chemother. 65, 2534–2537. Doi, Y., Paterson, D.L., Egea, P., Pascual, A., Lopez-Cerero, L., Navarro, M.D., Adams-Haduch, J.M., Qureshi, Z.A., Sidjabat, H.E., Rodriguez-Bano, J., 2010. Extended-spectrum and CMY-type beta-lactamase-producing Escherichia coli in clinical samples and retail meat from Pittsburgh, USA and Seville, Spain. Clin. Microbiol. Infect. 16, 33–38. EFSA, 2011. Scientific opinion on the public health risks of bacterial strains produicng extended-spectrum betalactamases and/or AmpC betalactamases in food and foodproducing animals. EFSA Panel on Biological hazards. EFSA J. 9, 2322. EFSA, 2012. The European Union summary report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in the European Union in 2010. EFSA and ECDC. EFSA J. 10, 2598. Egea, P., Lopez-Cerero, L., Torres, E., Gomez-Sanchez Mdel, C., Serrano, L., Navarro Sanchez-Ortiz, M.D., Rodriguez-Bano, J., Pascual, A., 2012. Increased raw poultry meat colonization by extended spectrum beta-lactamase-producing Escherichia coli in the south of Spain. Int. J. Food Microbiol. 159, 69–73. EMA, 2012. European Medicines Agency, 2012. Sales of veterinary antimicrobial agents in 19 EU/EEA countries in 2010 (EMA/88728/2012). Second ESVAC report. Fang, H., Ataker, F., Hedin, G., Dornbusch, K., 2008. Molecular epidemiology of extendedspectrum beta-lactamases among Escherichia coli isolates collected in a Swedish hospital and its associated health care facilities from 2001 to 2006. J. Clin. Microbiol. 46, 707–712. Geser, N., Stephan, R., Hachler, H., 2012a. Occurrence and characteristics of extendedspectrum beta-lactamase (ESBL) producing Enterobacteriaceae in food producing animals, minced meat and raw milk. BMC Vet. Res. 8, 21. Geser, N., Stephan, R., Korczak, B.M., Beutin, L., Hachler, H., 2012b. Molecular identification of extended-spectrum-beta-lactamase genes from Enterobacteriaceae isolated from healthy human carriers in Switzerland. Antimicrob. Agents Chemother. 56, 1609–1612. Hopkins, K.L., Batchelor, M.J., Liebana, E., Deheer-Graham, A.P., Threlfall, E.J., 2006. Characterisation of CTX-M and AmpC genes in human isolates of Escherichia coli identified between 1995 and 2003 in England and Wales. Int. J. Antimicrob. Agents 28, 180–192. Kola, A., Kohler, C., Pfeifer, Y., Schwab, F., Kuhn, K., Schulz, K., Balau, V., Breitbach, K., Bast, A., Witte, W., Gastmeier, P., Steinmetz, I., 2012. High prevalence of extendedspectrum-beta-lactamase-producing Enterobacteriaceae in organic and conventional retail chicken meat, Germany. J. Antimicrob. Chemother. 67, 2631–2634. Leverstein-van Hall, M.A., Dierikx, C.M., Cohen Stuart, J., Voets, G.M., van den Munckhof, M.P., van Essen-Zandbergen, A., Platteel, T., Fluit, A.C., van de Sande-Bruinsma, N., Scharinga, J., Bonten, M.J., Mevius, D.J., 2011. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin. Microbiol. Infect. 17, 873–880. McGettigan, S.E., Hu, B., Andreacchio, K., Nachamkin, I., Edelstein, P.H., 2009. Prevalence of CTX-M beta-lactamases in Philadelphia, Pennsylvania. J. Clin. Microbiol. 47, 2970–2974. Naseer, U., Sundsfjord, A., 2011. The CTX-M conundrum: dissemination of plasmids and Escherichia coli clones. Microb. Drug Resist. 17, 83–97. Overdevest, I., Willemsen, I., Rijnsburger, M., Eustace, A., Xu, L., Hawkey, P., Heck, M., Savelkoul, P., Vandenbroucke-Grauls, C., van der Zwaluw, K., Huijsdens, X., Kluytmans, J., 2011. Extended-spectrum beta-lactamase genes of Escherichia coli in chicken meat and humans, The Netherlands. Emerg. Infect. Dis. 17, 1216–1222. Perez-Perez, F.J., Hanson, N.D., 2002. Detection of plasmid-mediated AmpC betalactamase genes in clinical isolates by using multiplex PCR. J. Clin. Microbiol. 40, 2153–2162. Pitout, J.D., Laupland, K.B., 2008. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect. Dis. 8, 159–166. Randall, L.P., Clouting, C., Horton, R.A., Coldham, N.G., Wu, G., Clifton-Hadley, F.A., Davies, R.H., Teale, C.J., 2011. Prevalence of Escherichia coli carrying extended-spectrum betalactamases (CTX-M and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009. J. Antimicrob. Chemother. 66, 86–95. Ryoo, N.H., Kim, E.C., Hong, S.G., Park, Y.J., Lee, K., Bae, I.K., Song, E.H., Jeong, S.H., 2005. Dissemination of SHV-12 and CTX-M-type extended-spectrum beta-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae and emergence of GES3 in Korea. J. Antimicrob. Chemother. 56, 698–702. Sambrook, J., Russell, D., 2001. Molecular Cloning: A Laboratory Manual, third edition. Cold Spring Harbor Laboratory Press. Smet, A., Martel, A., Persoons, D., Dewulf, J., Heyndrickx, M., Herman, L., Haesebrouck, F., Butaye, P., 2010a. Broad-spectrum beta-lactamases among Enterobacteriaceae of animal origin: molecular aspects, mobility and impact on public health. FEMS Microbiol. Rev. 34, 295–316. Smet, A., Martel, A., Persoons, D., Dewulf, J., Heyndrickx, M., Herman, L., Haesebrouck, F., Butaye, P., 2010b. Broad-spectrum beta-lactamases among Enterobacteriaceae of animal origin: molecular aspects, mobility and impact on public health. FEMS Microbiol. Rev. 34, 295–316.
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Tham, J., Walder, M., Melander, E., Odenholt, I., 2012. Prevalence of extendedspectrum beta-lactamase-producing bacteria in food. Infect. Drug Resist. 5, 143–147. Warren, R.E., Ensor, V.M., O'Neill, P., Butler, V., Taylor, J., Nye, K., Harvey, M., Livermore, D.M., Woodford, N., Hawkey, P.M., 2008. Imported chicken meat as a potential source
of quinolone-resistant Escherichia coli producing extended-spectrum beta-lactamases in the UK. J. Antimicrob. Chemother. 61, 504–508. Woodford, N., Fagan, E.J., Ellington, M.J., 2006. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum (beta)-lactamases. J. Antimicrob. Chemother. 57, 154–155.
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short communication
Escherichia coli with extended-spectrum beta-lactamases or transferable AmpC beta-lactamases and Salmonella on meat imported into Sweden Maria Egervärn a,⁎, Stefan Börjesson b, Sara Byfors a, Maria Finn b, Caroline Kaipe a, Stina Englund b, Mats Lindblad a a b
National Food Agency, Box 622, SE-75126 Uppsala, Sweden National Veterinary Institute (SVA), SE-75189 Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 30 June 2013 Received in revised form 21 October 2013 Accepted 5 November 2013 Available online 14 November 2013 Keywords: Antimicrobial resistance Cephalosporin ESBL-producing E. coli pAmpC-producing E. coli Salmonella Meat
a b s t r a c t The presence of Enterobacteriaceae producing extended spectrum beta-lactamases (ESBL) or transferable AmpC beta-lactamases (pAmpC) is increasingly being reported in humans and animals world-wide. Their occurrence in food-producing animals suggests that meat is a possible link between the two populations. This study investigated the occurrence and characteristics of Salmonella and ESBL- or pAmpC-producing E. coli in 430 samples of beef, pork and broiler meat imported into Sweden, in order to provide data required for assessing the potential public health risk of these bacteria in food. Depending on region of origin, ESBL/pAmpC-producing E. coli were found in 0–8% of beef samples, 2–13% of pork samples and 15–95% of broiler meat samples. The highest prevalence was in South American broiler meat (95%), followed by broiler meat from Europe (excluding Denmark) (61%) and from Denmark (15%). Isolates from meat outside Scandinavia were generally defined as multiresistant. A majority of the ESBL/pAmpC genes were transferable by conjugation. BlaCTX-M-2 and blaCTX-M-8 were the dominant genes in E. coli from South American broiler meat, whereas blaCMY-2 and blaCTX-M-1 dominated in European meat. The majority of blaCMY-2 and blaCTX-M-1 were situated on plasmids of replicon type incK and incI1, respectively. The same combinations of ESBL/pAmpC genes and plasmids have been described previously in clinical human isolates. Salmonella was found in five samples tested, from European pork and broiler meat. No Salmonella isolate was resistant to third-generation cephalosporins. In conclusion, meat imported into Sweden, broiler meat in particular, is a potential source of human exposure to ESBL- and pAmpC-producing E. coli. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The presence of Enterobacteriaceae producing extended spectrum beta-lactamases (ESBL) or transferable AmpC beta-lactamases (pAmpC) is a rapidly emerging public health problem (Pitout and Laupland, 2008). Furthermore, the presence of ESBL- and pAmpC-producing E. coli and Salmonella is increasingly being reported in food-producing animals and in meat worldwide (EFSA, 2011), with 44–93% prevalence of ESBL- and pAmpC-producing E. coli in broiler meat from countries such as Germany (Kola et al., 2012), the Netherlands (Overdevest et al., 2011) and Spain (Egea et al., 2012). In beef and pork, the occurrence of E. coli with ESBL/pAmpC appears to be several-fold lower (EFSA, 2011). Recent Dutch studies have shown that the predominant ESBL genes and plasmids in E. coli obtained from broiler meat are also present in clinical isolates from humans (Leverstein-van Hall et al., 2011; Overdevest et al., 2011), indicating potential for transmission to humans via food (EFSA, 2011; Smet et al., 2010b). However, the extent to which food ⁎ Corresponding author at: National Food Agency, Risk and Benefit Assessment Department, Box 622, SE-751 26 Uppsala, Sweden. Tel.: +46 18 17 53 15; fax: +46 18 10 58 48. E-mail address: [email protected] (M. Egervärn). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.11.005
serves as a dissemination route for these bacteria to humans and the impact on human health have not yet been established (EFSA, 2011; Smet et al., 2010b). In Sweden, the occurrence of ESBL- and pAmpC-producing E. coli on domestic meat is regularly investigated as part of the Swedish Veterinary Antimicrobial Resistance Monitoring (SVARM) programme. Meat imported into Sweden is currently not included in the programme. Considering that 42% of the red meat and 38% of the poultry meat sold on the Swedish market are imported (estimates based on Anonymous, 2012), studies mapping the presence of ESBL- and pAmpC-producing E. coli in imported meat are needed. Such data are also required for assessing the potential public health risk of these bacteria in food. The aim of the present study was to investigate the occurrence and characteristics of Salmonella and ESBL/pAmpC-producing E. coli in meat imported into Sweden from Europe and South America. 2. Material and methods 2.1. Sampling A stratified sampling strategy was applied, with the intention of allowing comparison of the proportions of ESBL-positive samples from
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the most common countries or regions exporting beef, pork and broiler meat to Sweden. The division of countries or regions into strata was based on statistics from the Swedish Board of Agriculture (2009) on the import value of meat in 2008. The beef strata were: ‘Other European countries’, Ireland and South America, representing 57, 35 and 7% of imports, respectively. The pork strata were: Denmark, Germany and ‘other European countries’, representing 49, 26 and 25% of imports, respectively. The broiler meat strata were: Denmark, ‘other European countries’ and South America, representing 71, 29 and b1% of imports. South America was included due to recent findings of ESBL-producing E. coli in South American broiler meat (Dhanji et al., 2010; Warren et al., 2008). The target was to collect about 40–50 samples from each stratum except beef from ‘other European countries’, for which the target was ~ 100 samples. In total, samples of imported broiler meat (n = 133), beef (n = 178) and pork (n = 119) were collected fresh or frozen at retail stores and outlets from January 2010 to June 2011. Each sample represented a unique batch or single production date. Samples were collected based on their availability in retail outlets, by official inspectors. 2.2. Isolation and antibiotic susceptibility testing of ESBL- or pAmpC-producing E. coli Approximately 25 g meat were homogenised by stomaching in 225 mL buffered peptone water. A 100 mL portion of the suspension was supplemented with 1 μg/mL cefotaxime (Sigma Aldrich) and incubated at 37 °C for 18–24 h. In order to capture both ESBL- and pAmpCproducing bacteria from the sample, aliquots of the enrichment culture were then plated in parallel onto two selective media, CHROMagar™ ESBL and CHROMagar™ Orientation (EMM Life Science AB), containing 1 μg/mL cefotaxime. After 18–24 h incubation at 37 °C, a tentative, single E. coli colony was primarily selected from CHROMagar™ ESBL. Species identity was confirmed using a biochemical API20E test (bioMérieux Sweden). Beta-lactamase production was verified by Etest®ESBL and Etest®AmpC (bioMérieux, Sweden). Susceptibility to 14 antibiotics was assessed by broth microdilution according to CLSI (2008) using VetMIC™ GN-mo panels (SVA Sweden). Susceptibility of ESBL- or pAmpC-producing E. coli to carbapenems was tested by Etest®Imipenem. 2.3. Identification of ESBL/pAmpC genes and plasmids To determine the gene group responsible for the ESBL and AmpC phenotypes, all isolates were subjected to multiplex-PCRs detecting the following gene groups: blaCTX-M (Woodford et al., 2006), pAmpC (Perez-Perez and Hanson, 2002) and blaSHV, blaTEM and blaOXA-1 (Fang et al., 2008). The specific gene variants were determined by sequencing as previously described by Börjesson et al. (2013a), with additional primers for blaCTX-M-2 (Hopkins et al., 2006), blaCTX-M-8 (this study; 5’-CTT TTT GTG CTG ACT GTG AA-3’ and 5’-GCA GCM GCG AGY ACG TCA-3’), blaCTX-M-9 (McGettigan et al., 2009), blaCTX-M-25 (Chmelnitsky et al., 2005) and blaSHV (Ryoo et al., 2005). Isolates testing positive for blaCTX-M-15 were subjected to multilocus sequence typing (MLST) according to http://mlst.ucc.ie/. Transferability of ESBL/pAmpC genes from the isolates was tested by conjugation to E. coli HMS174 (E. coli GSC6576). The donor and recipient strains were inoculated in NY-broth and incubated at 37 °C overnight. After incubation, the donor and recipient were mixed (1:4) and centrifuged. The pellets obtained were suspended in saline buffer, spotted on LA and incubated at 37 °C for 6 h. Bacterial growth was collected and resuspended in saline buffer. Serial dilutions were spread on LA containing cefotaxime (2 mg/L) and rifampin (50 mg/L) and incubated at 37 °C overnight. Transformation was performed on isolates unable to transfer ESBL/pAmpC phenotypes by conjugation and transconjugants testing PCR-positive for multiple plasmid replicon types. Plasmid DNA was extracted according to the miniprep protocol described by
9
Sambrook and Russell (2001) with minor modification. After the NaC2H3O2 step, the mix was centrifuged and the supernatant was collected, mixed with 5 M LiCl (1:1), incubated at −20 °C and centrifuged. Plasmid DNA was collected from the supernatant by ethanol precipitation and transformed (~60 ng μL−1) with a voltage of 1250 V using ElectroMax™ DH10B™ (Gibco Invitrogen) according to the manufacturer's instruction. Transconjugants and transformants were subjected to PCR-based plasmid replicon typing (Carattoli et al., 2005) and multiplex-PCRs for genes encoding ESBL/pAmpC (Fang et al., 2008; Perez-Perez and Hanson, 2002; Woodford et al., 2006). 2.4. Isolation and antibiotic susceptibility testing of Salmonella Salmonella was isolated in accordance with the method described by the Nordic Committee on Food Analysis, NMKL 71. Isolates were identified to genus level using API20E (bioMérieux Sweden), with subsequent serotyping according to the standard Kaufmann–White procedure. Susceptibility to 12 antibiotics was assessed using VetMIC™ panels for Salmonella bacteria according to CLSI standards for microdilution (CLSI, 2008). 3. Results 3.1. Occurrence and characteristics of ESBL/pAmpC-producing E. coli 3.1.1. Broiler meat ESBL- and pAmpC-producing E. coli were isolated from broiler meat samples originating from all geographical regions included (Table 1a). The highest prevalence was in meat imported from South America, foremost Brazil (95%), followed by meat from Europe excluding Denmark (61%) and Denmark (15%). The most prevalent ESBL/pAmpC genes in broiler meat imported from South America were blaCTX-M-2 and blaCTX-M-8 (Table 2a), while the most prevalent genes in broiler meat imported from ‘other EU countries’ were blaCMY-2 and blaCTX-M-1. The blaCTX-M-8 gene was identified in isolates from South American meat only, whereas blaCTX-M-1 was identified in isolates from European meat only. The single E. coli isolated from French broiler meat contained the only blaCTX-M-25 found. One isolate from German broiler meat contained both blaCMY-2 and blaSHV-12. In Danish meat, blaCMY-2 was the only ESBL/pAmpC gene identified. In total, 86% of the isolates from Danish meat and 85% of the isolates from meat obtained from ‘other EU countries’ were able to transfer the ESBL/pAmpC genes by conjugation, whereas 76% of the isolates from South American meat were able to transfer the gene. After applying transformation on the isolates unable to transfer the gene by conjugation, nine genes were still non-transferable. Isolates containing blaCTX-M-1, blaCTX-M-8, blaSHV-12 or blaTEM frequently (90%) carried those genes on plasmids belonging to replicon type incI1. Similarly, 74% of the blaCMY-2-positive isolates carried the pAmpC gene on incK plasmids. The specific location of blaCTX-M-2 was in most cases not possible to decide by transferability experiments and replicon typing. When it was transferred, the frequency was low overall and most plasmids were PCR-positive for both incP and incHI2. Non-ESBL blaTEM were not co-transferred with ESBL genes. Three plasmids carrying ESBL or pAmpC genes that were transferred proved to be untypeable by replicon-typing PCR (Table 2a). In total, 81% of the 75 broiler meat isolates were multiresistant, i.e. resistant to three or more antibiotic classes (Table 2a). Overall, isolates from South American meat samples showed resistance to a wider range of antibiotics than isolates from European meat samples. Resistance to quinolones, aminoglycosides and sulfamethoxazole was the most common trait. Resistance to chloramphenicol (5 isolates) and florfenicol (1 isolate) was exclusively found among the isolates from South American meat. One blaCMY-2-positive isolate from Chilean meat was susceptible to colistin only. Two isolates from German meat samples and all but one isolate from Danish and Finnish meat samples
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Table 1 Prevalence of ESBL/pAmpC-producing E. coli in (A) broiler meat, (B) beef and (C) pork available on the Swedish market 2010–2011. A) Region of origin
Samples, n
ESBL- E. coli, n
Denmark 46 0 Germany 28 14c Finland 9 0 Othera 7 3 Europed, total 44 17 Brazil 40 38 Otherb 3 1 S. America, total 43 39 a Less than five samples tested per country: Croatia, Estonia, France, Lithuania, Netherlands, Poland b Less than five samples tested per country: Argentina, Chile c One isolate was also pAmpC-producing d Denmark excluded
pAmpC- E. coli, n
ESBL/pAmpC- E. coli, n (%)
7 9 1 0 10 0 2 2
7 (15) 23 (82) 1 (11) 3 (43) 27 (61) 38 (95) 3 (100) 41 (95)
B) Region of origin
Samples, n
ESBL- E. coli, n
Ireland 40 0 Germany 35 1 The Netherlands 25 5 Othera 36 2 Europeb, total 96 8 Brazil 18 0 Uruguay 18 0 Argentina 6 0 S. America, total 42 0 a Less than five samples tested per country: Denmark, Estonia, Italy, Lithuania, Poland, UK, Austria b Ireland excluded
pAmpC- E. coli, n
ESBL/pAmpC- E. coli, n (%)
0 0 0 0 0 0 0 0 0
0 (0) 1 (3) 5 (20) 2 (6) 8 (8) 0 (0) 0 (0) 0 (0) 0 (0)
C) Region of origin
Samples, n
ESBL- E. coli, n
Denmark 44 1 Germany 44 3 Italy 20 3 Othera 11 0 Europeb, total 31 3 a Less than five samples tested per country: Finland, Netherlands, Poland, Spain b Denmark and Germany excluded
were resistant to beta-lactams only. None of the isolates was resistant to imipenem. 3.1.2. Beef ESBL-producing E. coli was found in 8 out of 96 samples of beef imported into Sweden from ‘other EU countries’, excluding Ireland (Table 1b). Five of the positive samples originated from Dutch beef. ESBL-producing E. coli was not found in Irish or South American samples and pAmpC-producing E. coli was not found in sample from any geographical region included (Table 1b). ESBL production was encoded by three gene variants: blaCTX-M-1 (n = 5), blaCTX-M-15 (n = 2) and blaTEM-52 (n = 1; Table 2b). All isolates were able to transfer genes encoding ESBL by conjugation. Two E. coli of MLST type ST10 (data not shown) from Dutch and Austrian minced meat contained blaCTX-M-15 that was identified on incI1 plasmids. The blaCTX-M-1 genes were identified on plasmids belonging to different replicon types and the plasmid carrying blaTEM-52 was untypeable by replicon-typing PCR. All but one ESBL-producing E. coli isolates were multiresistant. None of the isolates showed reduced susceptibility to imipenem.
pAmpC- E. coli, n
ESBL/pAmpC- E. coli, n (%)
0 0 0 1 1
1 (2) 3 (7) 3 (15) 1 (9) 4 (13)
ESBL production was encoded by three gene variants: blaCTX-M-1 (n = 5) on incI1 plasmids or an incN plasmid, blaCTX-M-14 (n = 1) on an incF plasmid and blaTEM-52 (n = 1) on an incI1 plasmid (Table 2c). The single pAmpC enzyme identified was encoded by blaCMY-2 on an incA/C plasmid. All isolates were multiresistant except one from Italian pork. None of the isolates showed reduced susceptibility to imipenem. 3.2. Occurrence of antibiotic resistant Salmonella Salmonella was detected in five of the 430 samples tested; three and two samples of meat from broilers and pigs, respectively (Table 3). All isolates were isolated from German meat except one isolated from Italian pork. Serotypes identified were Salmonella Typhimurium, S. Infantis and S. Paratyphi B. variant Java. One isolate from German broiler meat was susceptible to all antibiotics tested, whereas the remaining isolates were multiresistant. Resistance to ampicillin, tetracycline and sulfamethoxazole was most common. Two of the isolates from German broiler meat were resistant to quinolones. None of the isolates was resistant to third-generation cephalosporins (Table 3). 4. Discussion
3.1.3. Pork ESBL-producing E. coli was found in 1 out of 44 Danish pork samples, 3 out of 44 German pork samples and 3 out of 31 pork samples from other EU countries (Table 1c). The latter positive samples originated from Italian pork. pAmpC-producing E. coli was found in one sample tested, from Polish pork. All isolates were able to transfer genes encoding ESBL or pAmpC by conjugation.
ESBL- and pAmpC-producing E. coli were frequently found in samples of imported beef, pork and broiler meat available on the Swedish market, with the highest prevalence in broiler meat. The occurrence in various meats was of the same order of magnitude as in a similar study on imported and domestic meat in Denmark, performed during the same period (DANMAP, 2010). The results of the present study are
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Table 2 Characteristics of ESBL/pAmpC-producing E. coli in (A) broiler meat, (B) beef and (C) pork available on the Swedish market 2010–2011. Ciprofloxacin (Ci), nalidixic acid (Nal), chloramphenicol (CM), florfenicol (Ff), colistin (Cs), gentamicin (Gm), kanamycin (Km), streptomycin (Sm), tetracycline (Tc), sulfamethoxazole (Su), trimethoprim (Tm). Epidemiological cut-off values for resistance according to EUCAST (www.eucast.org). A) Region of origin Denmark
Isolates, n (country of origina)
Beta-lactam gene variant/s/
3 (DK) blaCMY-2 2 (DK) blaCMY-2 + blaTEM-1 1 (DK) blaCMY-2 1 (DK) blaCMY-2 Europe 2 (DE, FI) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 1 (DE) blaCMY-2 + blaTEM-1 1 (DE) blaCMY-2 + blaSHV-12 1 (DE) blaCTX-M-1 1 (PL) blaCTX-M-1 1 (DE) blaCTX-M-1 1 (DE) blaCTX-M-1 1 (DE) blaCTX-M-1 + blaTEM-1 1 (DE) blaCTX-M-1 + blaTEM-1 1 (DE) blaCTX-M-1 + blaTEM-135 1 (DE) blaCTX-M-2 1 (NL) blaCTX-M-2 1 (FR) blaCTX-M-25 2 (DE) blaSHV-12 1 (DE) blaSHV-12 + blaTEM-1 1 (DE) blaTEM-19 1 (DE) blaTEM-52 1 (DE) blaTEM-52 S. America 1 (AR) blaCMY-2 1 (CL) blaCMY-2 1 (BR) blaCTX-M-2 2 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (AR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 2 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 1 (BR) blaCTX-M-2 + blaTEM-1 1 (BR) blaCTX-M-2 + blaTEM-1 1 (BR) blaCTX-M-2 + blaTEM-1 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 2 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 2 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 2 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 1 (BR) blaCTX-M-8 + blaTEM-1 1 (BR) blaCTX-M-8 + blaTEM-1 1 (BR) blaCTX-M-8 + blaTEM-1 a DK = Denmark, DE = Germany, FI = Finland, NL = the Netherlands, PL = Poland, FR = France, AR = b All isolates resistant to ampicillin, cefotaxime and ceftazidime c ? = Non-typeable using PCR-based plasmid replicon typing d Transformant positive for blaCMY-2 e NT = Non-transferable with transconjugation or transformation
Pattern of non-beta-lactam resistanceb – – Km – – Ci, Nal Ci, Nal, Su Ci Nal, Su Ci, Nal, Cs, Km, Sm, Su, Tm, Tc Gm, Km, Sm, Su Gm, Sm, Su Gm, Km, Sm, Su, Tm Ci, Nal, Su,Tm, Tc Ci, Nal, Cm, Ff, Su, Tc Ci, Nal, Tc Ci, Nal, Sm, Su, Tm Sm, Su, Tc Su, Tc – Ci, Nal, Sm, Su, Tm, Tc Sm, Su, Tm Ci, Nal, Gm, Km, Sm, Su, Tc Cm, Km, Sm, Su, Tc Ci, Nal, Km, Su, Tc Cm, Sm, Su, Tc Cm, Ci, Nal, Su, Tm, Tc Ci, Nal, Su Ci, Nal, Tc Km, Sm, Su, Tm Ci, Nal, Km, Sm, Su, Tm Ci, Nal, Cm, Ff, Gm, Km, Sm, Su, Tm, Tc Ci, Nal, Gm, Km, Sm, Su, Tm, Tc Ci, Nal, Sm, Su, Tm, Tc Ci, Nal, Km, Sm, Su, Tm, Tc Ci, Nal, Km, Sm, Su, Tm, Tc Ci, Nal, Gm, Km, Sm, Su, Tm Ci, Nal, Gm, Km, Sm, Su, Tc Ci, Nal, Gm, Km, Sm, Su, Tc Ci, Nal, Gm, Km, Sm, Su, Tc Ci, Nal, Gm, Km, Sm, Tc Ci, Nal, Gm, Km, Su, Tc Ci, Nal, Cm, Gm, Su, Tc Ci, Nal, Cm, Sm, Su, Tc Gm, Km, Sm, Su, Tc Cm, Km, Sm, Su, Tc Gm, Km, Sm, Su, Tc, Tm Ci, Nal, Cm, Gm, Su, Tm, Tc Ci, Nal, Cs, Su, Sm, Tm Ci, Nal, Gm, Km, Sm, Su, Tm, Tc Ci, Nal, Gm, Sm, Su, Tm, Tc Ci, Nal, Sm, Su, Tm, Tc Ci, Nal, Gm, Sm, Su, Tm Ci, Nal, Km, Sm, Su, Tc Ci, Nal, Sm, Tm Ci, Nal, Sm, Su, Tm Nal, Sm, Su, Tm Ci, Sm, Su, Tm Sm, Su, Tm, Tc Km, Sm, Tm, Tc Sm Ci, Nal Km Ci, Nal, Gm, Km, Sm, Su, Tm, Tc Sm, Tc Ci, Nal, Tc Argentine, BR = Brazil, CL = Chile
ESBL /pAmpC location incK incK incK incI1 incK ?c incK incI1 incK incK incI1 incK incI1 incKd incP/incO incI1 incI1 incI1 ? incI1 incI1 NTe NT incA/C incI1 incI1 incI1 incI1 incI1 incI1 incP incP/incHI2 incHI2 NT incO ? NT incI1 incP/incHI2 incP/incHI2 incP/incHI2 NT NT incP/incHI2 NT incP/incHI2 NT NT incI1 incI1 incI1 incI1 NT incI1 incI1 incI1 incI1 incI1 incI1 incI1 incI1 incI1 incI1 incI1 incI1
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Table 2 (continued) B) Region of origin
Isolates, n (country of origina)
Pattern of non-beta-lactam resistanceb
ESBL /pAmpC location
– Ci, Nal, Gm, Sm, Su, Tm, Tc Gm, Su, Tm, Tc Sm, Su, Tm, Tc Ci, Nal, Sm Cm, Km, Sm, Su, Tc Ci, Nal, Su, Tm, Tc
incI1 incF incFIIA incHI2 incI1 incI1 ?c
Beta-lactam gene variant/s/
Pattern of non-beta-lactam resistanceb
ESBL /pAmpC location
blaCTX-M-1 + blaCTX-M-1 + blaCTX-M-1 + blaCTX-M-14 blaCMY-2 blaCTX-M-1 blaCTX-M-1 + blaTEM-52
Nal, Km, Sm, Su, Tm, Tc Sm, Su, Tm, Tc Km, Sm, Su, Tm Km, Su, Tm, Tc Cm, Ff, Sm, Su, Tc Su, Tm, Tc Ci, Nal, Cm, Sm, Su, Tm, Tc –
incN incI1 incI1 incF incA/C incI1 incI1 incI1
Beta-lactam gene variant/s/
Europe
2 (DE, NL) blaCTX-M-1 1 (NL) blaCTX-M-1 1 (NL) blaCTX-M-1 + blaTEM-1 1 (NL) blaCTX-M-1 + bladTEM 1 (NL) blaCTX-M-15 + blaTEM-1 1 (AU) blaCTX-M-15 + blaTEM-1 1 (ES) blaTEM-52 a DK = Denmark, DE = Germany, NL = the Netherlands, AU = Austria, ES = Estonia b All isolates resistant to ampicillin, cefotaxime and ceftazidime c ? = Non-typeable using PCR-based plasmid replicon typing d Negative by sequencing of the tem gene fragment C) Region of origin
Isolates, n (country of origina)
Denmark Germany
1 (DK) 1 (DE) 1 (DE) 1 (DE) Europe 1 (PL) 1 (IT) 1 (IT) 1 (IT) a DK = Denmark, DE = Germany, PL = Poland, IT = Italy b All isolates resistant to ampicillin, cefotaxime and ceftazidime
blaTEM-1 blaTEM-1 blaTEM-1
blaTEM-1
also in agreement with data on ESBL/pAmpC prevalence in meat previously summarised by the European Food Safety Authority (2011). In contrast to many earlier European studies, CMY-2-producing E. coli was the most common finding on the meat samples investigated here. However, it should be noted that most previous studies did not actively investigate the occurrence of pAmpC-producing E. coli in meat. Considering that resistance due to production of transferable AmpC enzymes is also deemed a significant public health problem (EFSA, 2011), prevalence studies such as this should include screening that captures both ESBL- and pAmpC-producing E. coli from a sample. However, the proportion of E. coli with pAmpC phenotype in the samples may have been underestimated also in the present study, because tentative colonies were primarily selected from selective ESBL plates inhibiting the growth of AmpC producers. Moreover, the results are in strong contrast to a recently published Swedish study, which showed that none of the samples tested of Mediterranean foods imported into Sweden contained ESBL-producing Enterobacteriaceae (Tham et al., 2012). The main reason for the difference is probably that Tham et al. (2012) used a non-selective culture method. This underlines the importance of using a selective culture approach in targeted surveys that include samples where the levels of ESBL- and pAmpC-producing E. coli can be low.
The high occurrence of ESBL- and/or pAmpC-producing E. coli on European broiler meat reported here has previously been described in several countries within the EU (Egea et al., 2012; Kola et al., 2012; Overdevest et al., 2011). This includes Sweden, with 44% prevalence in Swedish broiler meat (Börjesson et al., 2013a). A high proportion of ESBL/pAmpC-producing bacteria on broiler meat is also increasingly being reported in various countries outside Europe, such as the USA (Doi et al., 2010) and Japan (Ahmed et al., 2009). In the present study, ESBL-producing E. coli were frequently found in samples from South American broiler meat, confirming findings in two previous British studies (Dhanji et al., 2010; Warren et al., 2008). As in the present study, the dominant ESBL genes in those studies, blaCTX-M-2 and blaCTX-M-8, in E. coli from South American meat differed from the ESBL gene dominating in European meat, blaCTX-M-1. This supports the theory of different CTX-M type lineages dominating in different geographical regions (Canton and Coque, 2006; Smet et al., 2010a). The combination of blaCMY-2 and incK frequently identified in E. coli from European broiler meat has recently also been identified in clinical isolates from Swedish patients (Börjesson et al., 2013b) and has disseminated among E. coli isolates in intensive care units in Canada (Baudry et al., 2009). In addition, the majority of blaCTX-M-1 detected in the present study was situated on plasmids of replicon type incI1, i.e. the
Table 3 Serotypes and antibiotic resistance pattern of salmonella detected in samples collected in 2010–2011 from meat imported into Sweden. Epidemiological cut-off values for resistance according to EUCAST (www.eucast.org). Sample type
Country of origin
Serotype
Resistance patterna
Pork fillet Loin of pork Whole chicken Whole chicken Chicken fillet
Italy Germany Germany Germany Germany
S. subsp. I S. Typhimurium S. subsp. I S. paratyphi B.b S. Infantis
Am, Sm, Tc, Su Am, Tc, Su, Tm – Am, Ci, Nal, Cm, Sm, Tc, Su, Tm Am, Ci, Nal, Su
a Ampicillin (Am), cefotaxime (Ctx), ciprofloxacin (Ci), nalidixic acid (Nal), chloramphenicol (Cm), florfenicol (Ff), gentamicin (Gm), kanamycin (Km), streptomycin (Sm), tetracycline (Tc), sulfamethoxazole (Su), trimethoprim (Tm) b variant Java
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same combination of ESBL gene and plasmid previously described in Salmonella isolates from poultry and humans in France (Cloeckaert et al., 2010). In contrast, the dominant ESBL/pAmpC gene among clinical isolates in Sweden, blaCTX-M-15, was found in only 2% of the 91 E. coli isolates from meat samples examined in the present study. Furthermore, blaCTX-M-15 was identified on incI1 plasmids in E. coli typed as ST10 (data not shown) and thus was not associated to the most prevalent plasmid type and clonal lineage among clinical E. coli, i.e. incFII and ST131 (Naseer and Sundsfjord, 2011). This is in agreement with similar British and Swiss studies, which report a predominance of blaCTX-M-1 and blaCTX-M-15 in E. coli isolates from animals/meat and humans, respectively (Geser et al., 2012a; Geser et al., 2012b; Randall et al., 2011). In contrast, recent Dutch studies have described identical ESBL genes, plasmids and genotypes of ESBL-producing E. coli in isolates from broiler meat and from Dutch patients, suggesting that broiler meat is a source of ESBL-producing bacteria in the Netherlands (Leverstein-van Hall et al., 2011; Overdevest et al., 2011). The E. coli isolates from meat outside Scandinavia were frequently found to be multiresistant, as is often the case with ESBL/pAmpC producers and may reflect the usage of antimicrobials in production of food-producing animals (EMA, 2012). Four of the five Salmonella isolated from European pork and broiler meat in the present study were also multiresistant, with similar resistance patterns for Salmonella in these types of meat as stated in the EU summary report on antimicrobial resistance (EFSA, 2012). Data in the present study revealed no cephalosporin resistance, probably due to the limited number of Salmonella found in the samples, whereas EFSA (2012) reported 3% cefotaxime resistance in Salmonella isolated from German broiler meat after non-selective culture. In conclusion, samples from beef, pork and broiler meat imported into Sweden frequently contained ESBL- and pAmpC-producing E. coli, with the highest prevalence in broiler meat. The combinations of ESBL/pAmpC genes and plasmids identified in E. coli isolated from meat were partly similar to those previously found in clinical human isolates. On-going work at our department is focusing on genetic comparisons of ESBL/pAmpC genes, plasmids and E. coli isolates from meat, patients and healthy carriers in Sweden, in order to investigate the extent to which meat available on the Swedish market serves as a source of human exposure to ESBL- and pAmpC-producing E. coli. Funding This work was carried out with financial support from the Swedish Civil Contingencies Agency. References Ahmed, A.M., Shimabukuro, H., Shimamoto, T., 2009. Isolation and molecular characterization of multidrug-resistant strains of Escherichia coli and Salmonella from retail chicken meat in Japan. J. Food Sci. 74, M405–M410. Anonymous, 2009. Sweden's imports and exports of agrofood products 2006–2008. Swedish Board of Agriculture (SJV). Report 2009:18 (abstract in English). Anonymous, 2012. Yearbook of agricultural statistics 2012 including food statistics. Swedish Board of Agriculture and Statistics of Sweden. Baudry, P.J., Mataseje, L., Zhanel, G.G., Hoban, D.J., Mulvey, M.R., 2009. Characterization of plasmids encoding CMY-2 AmpC beta-lactamases from Escherichia coli in Canadian intensive care units. Diagn. Microbiol. Infect. Dis. 65, 379–383. Börjesson, S., Egervärn, M., Lindblad, M., Englund, S., 2013a. Frequent occurrence of extended-spectrum beta-lactamase- and transferable ampc beta-lactamaseproducing Escherichia coli on domestic chicken meat in Sweden. Appl. Environ. Microbiol. 79, 2463–2466. Börjesson, S., Jernberg, C., Brolund, A., Edquist, P., Finn, M., Landen, A., Olsson-Liljequist, B., Tegmark Wisell, K., Bengtsson, B., Englund, S., 2013b. Characterization of plasmidmediated AmpC-producing E. coli from Swedish broilers and association with human clinical isolates. Clin. Microbiol. Infect. 19 (7), E309–11. Canton, R., Coque, T.M., 2006. The CTX-M beta-lactamase pandemic. Curr. Opin. Microbiol. 9, 466–475. Carattoli, A., Bertini, A., Villa, L., Falbo, V., Hopkins, K.L., Threlfall, E.J., 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63, 219–228. Chmelnitsky, I., Carmeli, Y., Leavitt, A., Schwaber, M.J., Navon-Venezia, S., 2005. CTX-M-2 and a new CTX-M-39 enzyme are the major extended-spectrum beta-lactamases in
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