-Colistin Resistance Genes Mobilized by IncX4, IncHI2, and IncI2 Plasmids in of Pigs and White Stork in Spain

Colistin has become the last-line antimicrobial for the treatment of multidrug resistant (MDR) in human medicine. To date, several colistin resistance genes have been described. Of them -1 is disseminated worldwide in of human and animal origin. The aim of this study was to characterize -mediated resistance plasmids from of animal origin in Spain. From our strain collection, 70 of pig origin collected between 2005 and 2014 (10 per year, except for years 2009-2010-2013) were randomly selected and screened for the presence of -genes. Additionally, 20 isolated in 2011 from white storks () from the same urban household waste landfill associated colony were also included. Whole genome sequencing of -positive isolates was carried out on a MiSeq (Illumina). Hybrid whole genome sequencing strategy combining nanopore and Illumina technologies were performed in a selection of isolates to close the genomes and plasmids and identify the presence of antimicrobial resistance genes. Minimum inhibitory concentration (MIC) was used to assess the susceptibility to colistin. Mating experiments were carried out to evaluate transferability of the -genes. A total of 19 -1 and one -4 positive isolates were detected, 15 from pigs distributed during the study period, and five from storks collected in 2011. No other -variants were found. The MICs for colistin ranged between 4 and >4 mg/L. High diversity of STs were detected among the positive isolates, with only ST-10 shared between pigs and white storks. Except for one isolate, all were genotypic and phenotypically MDR, and five of them also harbored cephalosporin resistance genes ( , , and three ). -1 genes were mobilizable by conjugation, associated with IncX4, IncHI2, and IncI2 plasmids. In our study, -1 genes have been circulating in pig farms since 2005 harbored by a variety of clones. Its persistence may be driven by co-selection since plasmids containing -1 also exhibit resistance to multiple drugs used in veterinary medicine. Furthermore, this is the first report of the presence of -1 gene in isolates from white storks in Spain. This finding highlights the potential importance of wildlife that forage at urban household waste landfills in the transmission and spread of colistin resistance genes.