PANGENOME ANALYSIS AND ANTIMICROBIAL RESISTOME OF Salmonella enterica SEROVARS TYPHIMURIUM AND 1,4,[5],12:I:- SEQUENCE TYPE 34

Salmonella enterica serovar 1,4,[5],12:i:- has recently emerged in food-borne epidemics of multi-drug resistance (MDR) strains associated to several outbreaks in Europe (EU) as well as in other continents. The existence of a European clone, associated to Multi-Locus Sequence Types ST34 and to R-Type ASSuT, has been described. The aim of the present study was to investigate the geographical structure and antimicrobial resistance gene patterns of a set of Salmonella serovars Typhimurium and 1,4,[5],12:i:- genomes belonging to ST34. A core genome gene-by-gene approach was performed on 1,254 publicly available S. 1,4,[5],12:i:- and S. Typhimurium genomes belonging to ST34 and including 148 newly sequenced genomes of Italian S. 1,4,[5],12:i:- isolates collected from humans, swine and pork meat between 2012 and 2014. Strains gathered in several clusters irrespectively of the year and of the source of isolation. Three subclades, STY-, WE- and NA-clade, accounting for roughly the 60% of the genomes were identified as significantly associated to Italian, United Kingdom and North American origin, respectively (Fisher’s exact test; P<0.0001). The AMR patterns of all genomes were predicted by in silico identification of antimicrobial resistance associated genes (ARGs) from ResFinder database. Almost all genomes (96.5%) were positive for at least one ARG, whereas a limited number (45) did not show any positive match for ARGs in Resfinder database. The 63% of all ST34 isolates and the 66% of Italian ones were predicted to harbour the R-Type ASSuT. Within the 1,254 ST34 tested genomes, 55 different ARGs of the ResFinder database were found. The number of fluoroquinolone detected ARGs was 67 with qnrB gene as the most prevalent one (46). Aminoglycosides ARGs were detected 2,352 times with aph(3'')-Ib and aph(6)-Id genes as the most reported (925 and 963 respectively). Beta-lactam ARGs were rarely detected with the exception of blaTEM-1B (960). Sulfonamides ARGs were frequently detected (1,090) with sul2 (1,006) as the most identified. The frequency of detection of tetracycline ARGs was high (1,226) with tetB (1,137) as the most represented. In relation to the country of origin, Italian genomes and non-Italian genomes showed similar percentages of the most abundant ARGs (around 70- 80% for aph(3'')-Ib, aph(6)-Id and blaTEM-1B genes and around 80-90% for tetB gene). However, in relation to the other ARGs, a lower percentage of ARGs was observed in Italian genomes with the exception of floR2 (7% Italian vs 5 % non-Italian genomes) and mcr genes (5 Italian genomes vs 0 non-Italian genomes). Regarding the 5 Italian S. 1,4,[5],12:i:- genomes found positive for mcr genes, three isolates and one isolate harboured mcr-1 and mcr-5 genes respectively and were collected from pigs between 2012 and 2014; one isolate carried mcr-4 and was collected from human in 2007. In conclusion, the identification of most prevalent ARGs might be useful to identify key genetic biomarkers of antimicrobial resistance in S. Typhimurium and S. 1,4,[5],12:i:-. Moreover, the identification of geographically segregated clades in combination with their antimimicrobial resistome gave first insights to uncover the evolution and potential routes of spread of antimicrobial resistance genes from country to country