Prevalence of broad-spectrum cephalosporin-resistant Escherichia coli isolates in food samples in Tunisia, and characterization of integrons and antimicrobial resistance mechanisms implicated

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Abstract

The presence of broad-spectrum-cephalosporin-resistant Escherichia coli isolates and the implicated mechanisms of resistance were investigated in 79 food samples of animal origin obtained in different supermarkets and local butcheries in Tunisia. Ten of these samples (12.6%) harbored extended-spectrum beta-lactamase (ESBL) producing E. coli isolates and 13 ESBL-positive isolates were recovered (one or two/sample), which exhibited nine different Pulsed-Field-Gel-Electrophoresis (PFGE) patterns. ESBLs detected were the following: CTX-M-1 (10 strains), CTX-M-1 + TEM-1b (2 strains) and CTX-M-1 + TEM-20 (1 strain). The orf477 sequence was identified downstream of blaCTX-M-1 gene in all 13 strains and ISEcp1 upstream in 9 strains. All ESBL-positive strains were included into phylogenetic group A or B1 (4 and 9 strains, respectively). Three of the 79 food samples (3.8%) contained broad-spectrum-cephalosporin-resistant and ESBL-negative E. coli isolates with AmpC phenotype. One isolate per sample was studied, and they showed unrelated PFGE patterns. The CMY-2 type beta-lactamase was identified in one of these 3 strains and specific point mutations in the promoter/attenuator region of ampC gene (at positions 42, − 18, − 1 and + 58) were detected in the remaining two strains. Twelve ESBL-positive and one ESBL-negative E. coli strains contained class 1 integrons with the following gene cassette arrangements: dfrA1+aadA (6 strains) and dfrA17+aadA5 (7 strains). E. coli strains from food samples could represent a reservoir of ESBL-encoding genes and integrons that could be transmitted to humans through the food chain.

Introduction

Escherichia coli is a normal inhabitant of the intestinal tract of most animals and humans (Tannock 1995), and it can easily contaminate food products during animal evisceration at slaughter or during food manipulation. This microorganism can also be implicated in human infections and broad-spectrum cephalosporins are important agents used for the treatment of serious human E. coli infections. Previous studies have reported that E. coli isolates from animals and from food products can harbor resistance determinants to many classes of antimicrobial agents, constituting an important reservoir for transmissible resistance genes (Sáenz et al., 2004a, Smet et al., 2008). The inclusion of different antimicrobial resistance genes on mobile elements such as plasmids, transposons, and integrons facilitates the rapid dissemination of these genes among bacteria. E. coli strains are highly capable of acquiring and transferring antimicrobial resistance genes to other microorganisms (Sunde and Norström 2006).

In the past few years, there has been an important concern in the scientific community about the emergence and dissemination of E. coli isolates producing extended-spectrum β-lactamases (ESBLs) in human medicine, especially of the CTX-M class, very frequently associated to community infections (Pitout et al., 2005, Livermore et al., 2007). ESBLs hydrolyze broad-spectrum cephalosporins and aztreonam, but not cephamicins (as cefoxitin), and these enzymes are inhibited by clavulanic acid. CTX-M-class beta-lactamases hydrolyze cefotaxime very efficiently, and six different groups of enzymes have been described, being those of the CTX-M-1 and CTX-M-9 groups the most prevalent ones in human infections (Livermore et al., 2007). In the last few years, different reports have alerted about the dissemination of ESBL-positive E. coli isolates among the intestinal microbiota of healthy humans (Vinué et al., 2009), as well as of food-producing animals and also in food products (Briñas et al., 2003, Briñas et al., 2005, Blanc et al., 2006, Girlich et al., 2007, Jouini et al., 2007, Liu et al., 2007, Smet et al., 2008, Escudero et al., 2009). These resistant bacteria could enter the food chain, representing a problem for food safety because they can transfer resistance genes to pathogenic bacteria.

Plasmidic class C beta-lactamases constitute an important mechanism of resistance that inactivates broad-spectrum cephalosporins as well as cephamycins (cefoxitin), and these enzymes are not inhibited by clavulanic acid. Among them, those of the CMY class are acquiring great relevance in the last years, due to the problems they cause for therapy in E. coli infections (Haldorsen et al., 2008). Few data do exist in the literature about the dissemination of this type of enzymes among E. coli isolates of food origin.

It is known that all E. coli isolates harbor a chromosomal ampC beta-lactamase gene, and due to its low natural expression level, it is not associated to beta-lactam resistance. Nevertheless, specific nucleotidic point mutations in the promoter/attenuator region of this gene (specifically at the − 42 or − 32 positions) with respect to the one of E. coli K12 are associated with the hyperproduction of the chromosomal AmpC beta-lactamase (Caroff et al., 2000, Haldorsen et al., 2008). Hyperproduction of this AmpC beta-lactamase is associated with resistance to some beta-lactam antimicrobials as ampicillin, amoxicillin–clavulanic acid, and cefoxitin, and also confer low level resistance to broad-spectrum cephalosporins. The prevalence of this mechanism of resistance among food E. coli isolates has been scarcely analyzed (Briñas et al., 2002).

In a previous study carried out by our group (Jouini et al., 2007), the prevalence of ESBL-containing E. coli isolates was analyzed in a small number of food samples of animal origin obtained in 2006, but the presence of plasmidic class C beta-lactamases or the hyperproduction of chromosomal ampC beta-lactamase were not tested. The aim of the present study was to characterize the mechanisms of broad-spectrum cephalosporin resistance in E. coli isolates recovered from food samples of animal origin in Tunisia during 2007, and to characterize the type of ESBL genes implicated in resistance, their genetic environments and the associated antimicrobial resistance genes and integrons. It was also our interest to determine if the ESBL-producing E. coli isolates detected in this study harbored the shiga-toxin genes or belonged to the O157 serotype, due to the importance of these characteristics in food safety. The present study will provides more information about the real problem of ESBL and of other mechanisms of beta-lactam resistance in food E. coli isolates, and will represent a valuable help to control this emerging problem and to track its future evolution.

Section snippets

Samples and E. coli isolation

Seventy-nine food samples of animal origin (26 of poultry, 28 of sheep, 14 of beef, 10 of fish, and 1 of horse) were obtained between February and November 2007. They were recovered in 41 butcheries, 7 local markets and one sheep farm from 12 different cities all around Tunisia. Refrigerated samples were transported to the laboratory, and all of them were tested within 24 h of collection. A 30 g portion of each sample was vigorously homogenized with 270 ml of buffered peptone water and incubated

E. coli recovery from food samples

E. coli isolates with a phenotype of resistance or reduced susceptibility to broad-spectrum-cephalosporins (specifically cefotaxime) were recovered in 13 of the 79 food samples analyzed (16.4%), and 20 isolates were further characterized. Seventeen of the 20 cefotaxime-resistant isolates (obtained from 10 of the 13 positive food samples) exhibited a positive ESBL-screening test (Table 1). The remaining 3 cefotaxime-resistant E. coli isolates (obtained from 3 of the 13 positive food samples)

Acknowledgements

This study was performed through the financial support of the AECID from the Ministerio de Asuntos Exteriores of Spain and from the Tunisian Ministry of Higher Education, Scientific Research and Technology.

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