Prevalence, virulence determinants, and genetic diversity in Yersinia enterocolitica isolated from slaughtered pigs and pig carcasses

Abstract

Yersinia enterocolitica is an important zoonotic foodborne pathogen that could be transferred from infected pigs to their carcasses at slaughter, with subsequent introduction of the pathogen into the food chain. The aim of the present study was to study the prevalence, virulence characteristics, and genetic diversity of Y. enterocolitica isolates present in slaughtered pig tonsils and carcasses by using the WGS approach. A total of 200 slaughtered pig tonsils from 11 pig farms were collected in 2020-2021 at six slaughterhouses located in Latvia. Out of these samples, n = 190 were obtained from slaughtered pigs raised on Latvian farms while n = 10 were of Lithuanian origin, with the number of farms sampled being 10 and 1, respectively. Additionally, 30 pig carcasses were sampled at five slaughterhouses from pigs originating from five farms in 2021. Samples were investigated microbiologically, Y. enterocolitica isolates were biotyped and serotyped. Y. enterocolitica 4/O:3 was screened for antimicrobial resistance with the EUVSEC test panels. Whole genome sequence analysis (WGS) was performed in order to detect virulence genes and to assess the genetic diversity of Y. enterocolitica isolates. A total of 139 isolates, including one to three isolates from 84 Y. enterocolitica positive slaughtered pig tonsils and 13 pig carcass samples, were subjected to WGS analysis. The prevalence of Y. enterocolitica 4/O:3 in slaughtered pig tonsils and carcasses was 35% (70/200) and 13% (4/30), respectively. Antimicrobial resistance to ampicillin and tetracycline was detected in 97% (72/74) and 1% (1/74) of Y. enterocolitica 4/O:3 isolates. Y. enterocolitica 4/O:3 was represented only by ST18, while Y. enterocolitica 1A by ST3, ST147, ST304, ST307, and ST473. The ST18 isolates harbored the same main chromosomal (ail, inv, myfA, ystA) and majority shared plasmid-borne virulence genes (virF, yadA, yop virulon). The main virulence genes were not identified within the STs of Y. enterocolitica 1A and only minor differences were found between ST3, ST147, ST304, ST307, and ST473. Among ST18 isolates, cgMLST analysis revealed 43 cgMLST genotypes while 16 cgMLST genotypes were found among Y. enterocolitica 1A STs. The present study has shown the distribution of genetically distant cgMLST genotypes in slaughtered pigs from pig farms located in different geographical regions of Latvia, with one to 11 cgMLSTs identified within each sampled farm. The presence of undistinguishable cgMLST genotypes in slaughtered pig tonsils and the respective carcasses supported the link between the slaughter of Y. enterocolitica - positive pigs and carcass contamination with Y. enterocolitica 4/O:3.

Keywords: Antimicrobial resistance; Core genome multilocus sequence typing (cgMLST); Latvia; Sequence type (ST); Virulence.