Occurrence and characteristics of extended- spectrum b-lactamase (ESBL) producing Enterobacteriaceae in food producing animals, minced meat and raw milk

Background: The impact of food animals as a possible reservoir for extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, and the dissemination of such strains into the food production chain need to be assessed. In this study 334 fecal samples from pigs, cattle, chicken and sheep were investigated at slaughter. Additionally, 100 raw milk samples, representing bulk tank milk of 100 different dairy farms, 104 minced meat (pork and beef) samples and 67 E. coli isolates from cattle E. coli mastitis were analyzed. Results: As many as 15.3% of the porcine, 13.7% of the bovine, 8.6% of the sheep and 63.4% of the chicken fecal samples yielded ESBL producers after an enrichment step. In contrast, none of the minced meat, none of the bulk tank milk samples and only one of the mastitis milk samples contained ESBL producing strains. Of the total of 91 isolates, 89 were E. coli, one was Citrobacter youngae and one was Enterobacter cloacae. PCR analysis revealed that 78 isolates (85.7%) produced CTX-M group 1 ESBLs while six isolates (6.6%) produced CTX-M group 9 enzymes. Five detected ESBLs (5.5%) belonged to the SHV group and 2 isolates (2.2%) contained a TEM-type enzyme. A total of 27 CTX-M producers were additionally PCR-positive for TEM-beta-lactamase. The ESBL-encoding genes of 53 isolates were sequenced of which 34 produced CTX-M-1, 6 produced CTX-M-14, 5 produced CTX-M-15 and also 5 produced SHV-12. Two isolates produced TEM-52 and one isolate expressed a novel CTX-M group 1 ESBL, CTX-M117. One isolate–aside from a CTX-M ESBL– contained an additional novel TEM-type broad-spectrum betalactamase, TEM-186. Conclusions: The relatively high rates of ESBL producers in food animals and the high genetic diversity among these isolates are worrisome and indicate an established reservoir in farm animals. Background Antimicrobial resistance in bacteria has emerged as a problem in both human and veterinary medicine. One of the currently most important resistance mechanisms in Enterobacteriaceae, which reduces the efficacy even of modern expanded-spectrum cephalosporins (except cephamycins and carbapenems) and monobactams is based on plasmid-mediated production of enzymes that inactivate these compounds by hydrolyzing their b-lactam ring. Such resistance is encoded by an increasing number of different point-mutational variants of classical broad-spectrum b-lactamases (BSBL). These variants are called extended spectrum b-lactamases (ESBL): most are derivates of TEM and SHV b-lactamase families, whereas other groups, such as CTX-M, PER and KPC b-lactamases have been described more recently [1]. The phenotypical difference between BSBLs and ESBLs is that the latter efficiently hydrolyze 3rdand 4th-generation cephalosporins, additionally to penicillins and lower generation cephalosporins as the BSBLs are capable of. BSBLs and ESBLs are inhibited by clavulanic acid, sulbactam and tazobactam [2], a feature that is used (i) as a criterion for classification of b-lactamases and (ii) for diagnostic ESBL detection purposes. Until * Correspondence: [email protected] Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, CH-8057 Zurich, Switzerland Geser et al. BMC Veterinary Research 2012, 8:21 http://www.biomedcentral.com/1746-6148/8/21 © 2012 Geser et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. now more than 600 ESBL variants are known http:// www.lahey.org/Studies/ (last accessed January 2012). Among them, the over 100 CTX-M enzymes so far reported may be grouped into five main subgroups. Each of them is characterized by a group-representative single structure according to their amino acid sequence (group 1: CTX-M-1, group 2: CTX-M-2, group 8: CTXM-8, group 9: CTX-M-9, and group 25: CTX-M-25) [3]. As a matter of growing concern, resistance caused by ESBLs is often associated with resistance to other classes of antibiotics like fluoroquinolones, aminoglycosides and trimethoprim-sulfmethoxazole [1,4]. Since the first description of ESBL producing Enterobacteriaceae isolated from hospitalized humans [5], many nosocomial outbreaks have been reported. However, since a few years, there is an increase in the detection of ESBL producing strains in the community [6]. More recently, reports have also raised concern about the dissemination of ESBL producing E. coli in healthy food producing animals in several countries in Europe [7-9] and Asia [10,11] or in food products like meat, fish and raw milk [12-14]. Recently, Wittum et al. [15] and Doi et al. [16] described for the first time ESBL producers in healthy dairy cattle and retail meat in the USA. Therefore, the impact of healthy farm animals as a possible reservoir for ESBL producing Enterobacteriaceae on the food processing chain has to be assessed. The aim of the present study was to screen for the occurrence of ESBL producing Enterobacteriaceae in healthy swine, cattle, sheep and chicken at slaughter as well as in milk and meat in Switzerland and to further characterize isolates. Results After an enrichment step ESBL producers were isolated from 90 (26.9%) of the investigated 334 fecal samples, and one ESBL producer (1.5%) was found in 67 E. coli mastitis milk isolates, but none was isolated from either minced meat (pork and beef) or bulk tank milk samples. The ESBL prevalence among cattle was 13.7%, 25.3% among calves (animals under 6 months), 8.6% among sheep, and 15.3% among pigs. For chickens (herd level) a very high prevalence of 63.4% was determined (Table 1). All suspected isolates were phenotypically confirmed, in that they showed a synergy effect with at least 1 of 3 strips when tested with Etest-ESBL strips containing cefepime, cefotaxime or ceftazidime, and they yielded factors > 8 when ratios of MIC (cephalosporin)/MIC (cephalosporin plus clavulanic acid) were calculated. Almost all isolated ESBL producers were E. coli (89 out of 91), the exceptions being one Enterobacter cloacae isolated from a sheep, and one Citrobacter youngae isolated from a calf (Table 2). The ESBL-encoding genes of all isolates were further characterized by PCR. A total of 78 isolates (85.7%) produced CTX-M group 1 ESBLs while six isolates (6.6%) produced CTX-M group 9 enzymes. Five isolates (5.5%) were detected as producers of the SHV-ESBLs and 2 isolates (2.2%) exclusively produced TEM-type enzymes. Twenty-seven CTX-M carriers were additionally PCRpositive for blaTEM genes. Of the 91 ESBL producing isolates, 53 were selected for sequencing of the involved bla genes (Figure 1). Thirty-four isolates were CTX-M-1 producers, eight expressed additional TEM-1 and one isolate–from a pig– additionally expressed a TEM-type enzyme, TEM-186 http://www.lahey.org/Studies/, never found before (nucleotide sequence accession number JN227084). Six isolates carried CTX-M-14 with TEM-1 and five isolates specified CTX-M-15, one of which producing additional TEM-1. One isolate from a calf produced TEM-1 in combination with CTX-M-117 http:// Table 1 Occurrence of ESBL producers in food-producing animals at slaughter as well as in minced meat, bulk tank milk and isolates from bovine mastitis in Switzerland Origin n Number of samples with ESBL producers (percentage) Cattle, fecal samples 124 17 (13.7%; [95% CI, 8.1; 21.0]) calves 63 16 (25.3%; [95% CI, 15.3; 37.9]) others 61 1 (1.6%; [95% CI, 0.4; 8.7]) Pig, fecal samples 59 9 (15.3%; [95% CI, 7.2; 26.9]) Chicken, fecal samples from crates of different flocks 93 59 (63.4%; [95% CI, 52.8; 73.2]) Sheep, fecal samples 58 5 (8.6%; [95% CI, 2.9; 18.9]) lambs 40 2 (5.0%; [95% CI, 0.6; 16.9]) others 18 3 (16.7%; [95% CI, 3.5; 41.4]) Mined meat (pork, beef) 104 0 (0.0%; [95% CI, 0.0; 3.4]) Bulk tank milk 100 0 (0.0%; [95% CI, 0.0; 3.6]) E. coli isolates from mastitis milk 67 1 (1.5%; [95% CI, 0.3; 8.0]) n: number of samples tested CI: confidence interval Geser et al. 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