Virulence and antimicrobial resistance determinants of verotoxigenic Escherichia coli (VTEC) and of ESBL-producing multidrug resistant E. coli from foods of animal origin illegally imported to Europe

Microbial risk due to illegal food import has not been investigated so far. Here we aimed to reveal frequency, phenotype and genotype of verotoxigenic E. coli (VTEC) and ESBL-producing multidrug resistant (MDR) E. coli isolated from foods of animal origin confiscated at the EU airport borders. Of the 1500 food samples confiscated at the airports of Austria, Germany and Slovenia, the most frequent were cheese and meat products primarily from Turkey and from Balkan countries. The VTEC bacteria were isolated using ISO 16654:2001 for O157 and Ridascreen® ELISA based PCR testing of stx genes or ISO/ TS13136 for non-O157 VTEC, resulting in 15 isolates of VTEC (1%). In addition 600 samples from the Vienna airport were also tested for ESBL-producing MDR E. coli, using cefotaxime-McConkey agar. We identified 14 E. coli strains as ESBL/MDR E. coli. (0,9%) for phenotyping for antimicrobial resistance and for genotypiing by microarray (Identibac®,AMR05). The 15 VTEC isolates were phenotyped as Stx toxin producing non-O157 strain. Only one isolate, from Turkish cheese, proved to be EHEC (O26:H46). The remaining 14 strains represent uncommon VTEC serotypes with stx1 and/or stx2 genes. Microarray analysis (Identibac®, Ec03) revealed a wide range of other non-LEE encoding virulence genes. Pulsed field electrophoresis (PFGE) showed high genetic diversity of the strains. Multilocus sequence typing (MLST) established three new ST types (ST4505, 4506 and 4507) in the MLST database, and indicated the existence of 5 small clusters with no relation to origin or serotype/genotype of the strains, but representing several human-related ST types. All VTEC isolates were sensitive to 18 antimicrobials relevant to human and/or animal health, and did not contain resistance genes. ESB/MDR E. coli were resistant to at least 3 classes of antimicrobials. Microarray analysis detected TEM-1 in all but one strain and a variety of genes encoding resistances to other ESBLs (CTXM-1, OXA-1), trimethromprim, tetracycline, aminoglycosides and class1/class2 integrons (8/14 isolates). E.coli virulence microarray detected 2-6 virulence genes in all but one MDR E. coli, and one of the strains qualified as an atypical EPEC . Even though the frequency and attributes of isolated VTEC and ESBL/MDR E. coli did not represent an immediate major risk through illegal food import for the countries involved, it is suggested that the unusual serovars of VTEC as well as the virulence and antimicrobial resistance determinants of ESBL/MDR E. coli detected here, may indicate a future emerging threat by strains in illegally imported foods. Acknowledgement is due to: EU FP7 PROMISE (Grant No: 265877), to Dr. Mária Herpay, National Institute for Epidemiology, Budapest

Virulence and antimicrobial resistance determinants of verotoxigenic Escherichia coli (VTEC) and of multidrug-resistant E. coli from foods of animal origin illegally imported to the EU by flight passengers

The aim of this study was to reveal phenotype/genotype characteristics of verotoxigenic Escherichia coli (VTEC) and multidrug resistant E. coli in food products of animal origin confiscated as illegal import at Austrian, German and Slovenian airports. VTEC isolates were obtained by using ISO guidelines 16654:2001 for O157 VTEC or ISO/ TS13136:2012 for non-O157 VTEC, with additional use of the RIDASCREEN® Verotoxin immunoassay. The testing of 1526 samples resulted in 15 VTEC isolates (1.0%) primarily isolated from hard cheese from Turkey and Balkan countries. Genotyping for virulence by using a miniaturized microarray identified a wide range of virulence determinants. One VTEC isolate (O26:H46) possessing intimin (eae) and all other essential genes of Locus of Enterocyte Effacement (LEE) was designated as enterohemorrhagic E. coli (EHEC). None of the other VTEC strains belonged to serogroups O157, O145, O111, O104 or O103. VTEC strains harbored either stx1 (variants stx1a or stx1c) or stx2 (variants stx2a, stx2b, stx2a/d or stx2c/d) genes. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) demonstrated high genetic diversity and identified three new sequence types (STs): 4505, 4506 and 4507. Food samples collected from the Vienna airport were also tested for E. coli quantities using the ISO 16649:2001, and for detection of multidrug resistant phenotypes and genotypes. The resulting 113 commensal E. coli isolates were first tested in a pre-screening against 6 selected antimicrobials to demonstrate multidrug resistance. The resulting 14 multidrug resistant (MDR) E. coli isolates, representing 0.9% of the samples, were subjected to further resistance phenotyping and to microarray analyses targeting genetic markers of antimicrobial resistance and virulence. Genotyping revealed various combinations of resistance determinants as well as the presence of class 1, class 2 integrons. The isolates harbored 6 to 11 antibiotic resistance genes as well as 1 to 14 virulence genes. In this panel of 14 MDR E. coli two strains proved to carry CTX-M type ESBLs, and one single isolate was identified as enteropathogenic E. coli (EPEC). In general, isolates carrying a high number of resistance determinants had lower number of virulence genes and vice versa. In conclusion, this first pilot study on the prevalence of VTEC and of MDR/ESBL E. coli in illegally imported food products of animal origin suggests that these strains could represent reservoirs for dissemination of potentially new types of pathogenic and MDR E. coli in Europe