Characterization of Resistance Mechanisms to Critically Important Antibiotics in Multidrug Escherichia coli Isolated from Food-Producing Animals

Abstract

Bacteria resistant to critically important antibiotics are a public and animal health problem. Extended-spectrum β-lactamase (ESBL)/plasmid-mediated AmpC β-lactamase (PMAβ) has been increasing during the last years among Escherichia coli isolated from food-producing animals and animal-derived foods. The aim of this study was to determine the prevalence of resistance to critically important antibiotics among ESBL/PMAβ-producing E. coli isolated from bovine and swine cecal contents, and to characterize the main acquired antibiotic resistance genes. In this study, a total of 266 E. coli isolates obtained in 2017, under the scope of the national surveillance programme for the detection of ESBL and PMAβ-producing bacteria in food-producing animals, were used. The minimum inhibitory concentration values, obtained through broth microdilution technique, were used to determine the antimicrobial susceptibility profile of bovine (n=104) and swine (n=164) isolates, with reduced susceptibility to third generation cephalosporins and or cephamycines. Molecular characterization of plasmid-mediated genes encoding β-lactamases, quinolone and polymyxin resistance-encoding genes, as well as integrase genes, was achieved by multiplex PCR on 96 selected multidrug resistant (MDR) isolates. Sanger sequencing was performed to determine bla gene variants, and to identify point mutations and amino acid substitutions in the chromosomal AmpC β-lactamase, as well as in gyrase and topoisomerase IV encoding genes. Three MDR isolates were selected for mating experiments to investigate horizontal gene transfer. Whole genome sequencing of eleven isolates was performed using Illumina NextSeq platform, and genomic data were analyzed using bioinformatic tools available on the Center for Genomic Epidemiology (CGE). The results showed high frequency of antibiotic resistance in ESBL/PMAβ E. coli isolates, being sulfamethoxazole, tetracycline and β-lactams, the antibiotics with the highest resistance frequencies. Among the critically important antibiotics, the prevalence of resistance to ciprofloxacin was higher than to colistin. Resistance levels to sulfamethoxazole, trimethoprim, nalidixic acid, colistin and cefoxitin in isolates from swine were significantly different from bovine isolates. High prevalence of MDR was found in isolates form swine origin (91%, 147/162) and bovine origin (63%, 65/104). Furthermore, 17.9% of MDR isolates exhibited a resistance profile including resistances to third generation cephalosporins, sulfonamides, aminoglycosides, tetracyclines, trimethoprim and fluoroquinolones. ESBL presumptive profile was more frequently found than PMAβ, either in isolates from bovine (99% and 3.85%) or swine (87.0% and 22.8%). High diversity of β-lactamase encoding genes was found in ESBL-producing E. coli isolates, being blaCTX-M the most prevalent genes. The blaCTX-M-1 gene was the most frequent variant, followed by blaCTX-M-32, blaCTX-M-15, blaCTX-M-55, blaCTX-M-14, blaCTX-M-27, blaCTX-M-8, blaCTX-M-2, blaSHV-12,. In PMAβ-producing isolates, the blaCMY-2 gene was the only variant identified. Moreover, one ESAC-producing E. coli was found among swine isolates. Carbapenem resistance genes were not identified among the isolates. All colistin resistant isolates carried the mcr-1 gene, with higher frequency being found in swine isolates. In 50% of bovine and 40% of swine isolates resistant to ciprofloxacin harbored PMQR genes, namely qnrB, qnrS, and aac(6')Ib-cr, with an overall higher frequency of qnrS. Nevertheless, chromosomal mutations were also observed in gyrase and topoisomerase IV-encoding genes. Genetic elements like integrons class 1 and class 2 were found in 79% of isolates, with a higher prevalence of class 1. Horizontal gene transfer of blaCTX-M-8, blaSHV-12, qnrB and class 1 integron was achieved through conjugation assays. Genomic analysis identified several plasmids encoding resistant determinants, namely IncF, IncH, IncI, InX and Col156. A great diversity of plasmids was found to be involved in the dissemination of resistance traits, and a variety of additional resistance genes other than those involved in resistance to critically important antibiotics were identified, as well as several virulence genes. Additionally several sequence types were found among isolates, with concern for ST-410 and ST-10 known to be found in human isolates. Our findings highlight the spread of blaESBL, blaPMAβ, PMQR and PMCR genes within food-producing animals, with a particularly high prevalence of co-resistance to critically important antibiotics, as they are generally associated to horizontal gene transfer through plasmids. Higher frequency of polymyxin resistance is found in swine isolates. The high prevalence of integrons in most MDR E. coli isolates highlights their high potential to be transmissible through horizontal gene transfer. Our results confirm the horizontal transfer of bla and qnr genes, as well as integrons, corroborating with the rapid dissemination of these resistance determinants. In summary, this work provides evidence for the existence of resistance determinants reservoirs in the intestinal tract of bovine and swine, where numerous bacteria coexist, allowing the dissemination of resistance genes mediated by plasmids. To our knowledge, this was the first report of CTX-M-55 and CTX-M-2 producing E. coli isolated from food-producing animals, in Portugal. This study reinforces the need for continuous surveillance and research, in order to preserve antibiotic effectiveness and contradict the global trend foreseen by the World Health Organization, of 10 million deaths per year in 2050. Additionally, it emphasized the benefits of using genomic approaches, allowing a more complete understanding of all the factors involved in antibiotic resistance and its horizontal transfer.