Study on molecular determinants for colistin resistance in Avian Pathogenic Escherichia coli (APEC) from commercial poultry in Italy.

Since its recent discovery in November 2015, the mcr-1 gene has been detected in a variety of Enterobacteriaceae from humans, animals and the environment. In Italy, only a few reports describing the presence of mcr-1 gene in pathogenic and commensal Escherichia coli isolates from humans and poultry have been published to date. Mcr-1 is a plasmid-borne gene encoding for the phosphoethanolamine transferase conferring resistance to colistin, which is considered a life-saving antibiotic for humans. In poultry, colistin is widely used for treating or preventing bacterial infections. The aim of this study was to estimate the presence of the mcr-1 gene in Avian Pathogenic Escherichia coli (APEC) isolated from intensive poultry farms. The screening was carried out on a collection of non-repetitive APEC isolated from organs and tissues collected from commercial poultry, showing lesions suggestive of colibacillosis. A total of 229 APEC strains, collected between 2008 and 2012, were screened to detect the presence of the mcr-1 gene. In detail, strains were isolated from turkeys (n=109), broilers (n=98), and layer hens (n=22). To detect the presence of the mcr-1 gene, a PCR to amplify an internal fragment of 308 bp was performed. Afterwards, the whole mcr-1 gene was sequenced using a different set of primers amplifying a fragment of 1,600 bp. PCR protocols were carried out according to the publication of Liu and colleagues (2016). Finally, strains with a confirmed presence of the mcr-1 gene were analysed by Minimum Inhibitory Concentration (MIC), according to the European Committee on Antimicrobial Susceptibility Testing guidelines (EUCAST, 2016). Out of 229 strains, 29 (12,6%) were positive to the mcr-1 gene. Sequence analysis of all strains showed a sequence identity of 100% compared to the original sequence (NCBI accession number: NG_050417). Most strains (n = 20) had MIC values of ≥ 2 mg/L, which is the epidemiological cut-off value (ECOFF) for colistin, according to EUCAST. The remaining strains (n=9) had MIC values of < 2 mg/L, which correspond to the wild type phenotype. Until now, most of the studies investigating on the dissemination of mcr-1 gene in poultry or its products have been carried out on commensal E. coli, even in Italy. In this study, we focused on the occurrence of colistin resistance in APEC strains, which can cause infections with serious economic consequences for the poultry industry. Our results demonstrate a high prevalence of the mcr-1 gene in the studied APEC population. Interestingly, the MIC values of some of the analysed mcr-1 carrying isolates are consistent with intermediate resistance or even susceptibility to colistin. Of note, this phenomenon might lead to a silent dissemination of the mcr-1 gene among bacterial populations with unknown implications. Even more, this finding suggests that colistin resistance assessed only by phenotypic susceptibility assays could be misleading when determining the prevalence of the mcr-1 gene. Further investigation is crucial because colistin is an essential antimicrobial drug for the poultry sector as well as for public health. We are currently investigating also on the presence of the mcr-2 gene in order to obtain a full spectrum of the plasmid-borne resistance determinants for colistin in APEC isolates.