Molecular epidemiology of Salmonella Infantis in Europe: insights into the success of the bacterial host and its parasitic pESI-like megaplasmid

Abstract

Salmonella Infantis is one of the five serovars most frequently causing human salmonellosis in Europe, mainly associated with poultry. A clone harbouring a conjugative plasmid of emerging S. Infantis (pESI)-like megaplasmid, carrying multidrug resistant (MDR) and extended-spectrum beta-lactamases (ESBL) genes, has spread in the Italian broiler chicken industry also causing human illness. This work is aimed at elucidating the molecular epidemiology of S. Infantis and pESI-like in Europe using whole-genome sequencing and bioinformatics analysis, and to investigate the genetic relatedness of S. Infantis clones and pESI-like from animals, meat, feed and humans provided by institutions of nine European countries. Two genotyping approaches were used: chromosome or plasmid SNP-based analysis and the minimum spanning tree (MST) algorithm based on core-genome multilocus sequence typing (cgMLST). The European S. Infantis population appeared heterogeneous, with different genetic clusters defined at core-genome level. However, pESI-like variants present in 64.1 % of the isolates were more genetically homogeneous and capable of infecting different clonal lineages in most of the countries. Two different pESI-like with ESBL genes (n=82) were observed: bla_CTX-M-1-positive in European isolates and bla_CTX-M-65-positive in American isolates (study outgroup). Both variants had toxin-antitoxin systems, resistance genes towards tetracyclines, trimethoprim, sulphonamides and aminoglycosides, heavy metals (merA) and disinfectants (qacEΔ). Worryingly, 66 % of the total isolates studied presented different gyrA chromosomal point mutations associated with (fluoro)quinolone resistance (MIC range 0.125-0.5 mg/L), while 18 % displayed transferable macrolide resistance mediated by mph, mef and erm(B) genes. Proper intervention strategies are needed to prevent further dissemination/transmission of MDR S. Infantis and pESI-like along the food chain in Europe.

Keywords: ESBL (Extended Spectrum Beta-Lactamases); Salmonella Infantis; megaplasmids; multidrug resistance; pESI-like; whole genome sequencing.

Fig. 1.

Maximum-likelihood SNP-based phylogeny of 382 S. Infantis genomes using as reference S. Infantis SINFA (LN649235) and based on a total of 8444 informative SNPs. (a) Circular representation of the phylogeny, obtained using iTOL (http://itol.embl.de/) ignoring branch length. Dotted lines mark the subcluster containing isolates from human clinical cases, harbouring mostly bla _CTX-M-65 (study outgroup). Colours of the isolate ID indicate the different country of origin. Colour squares indicate, from the innermost to the outermost: sample type, pESI determinants, ESBL/AmpC resistance genes, bla_TEM gene type 1A, 1B or 216 (blue square). Year of isolation and the presence of gyrA chromosomal point mutations are also indicated for each genome. (b) Circular representation of the phylogeny showing the branch lengths.

Fig. 2.

Representation as MSTs of the cgMLST (a) and the SNP tree (b), coloured according to the country of origin. Representation as MSTs of the cgMLST(a1,b1) and the SNP tree (a2,b2), coloured according respectively to the sample type (a1,a2) and the presence of pESI-like (b1,b2).

Fig. 3.

Comparison of chromosome and plasmid-reference-based trees built with S. Infantis pESI-like positive isolates. Blotted lines of the same colour link the same isolate IDs included in the two trees. Two small well-differentiated clusters were identified as clusters A_p and B_p. Cluster A_p was the only one whose group correlates with the chromosome reference-based phylogenetic tree (blue dotted line). The other three groups were chosen, as an example, to evince the genetic relatedness of isolates belonging to different clusters in the two trees.

Fig. 4.

Graphical representation of the antimicrobial resistance genotypes (accessory resistance genes) of the 382 S. Infantis isolates clustered according to the chromosome SNP-based phylogeny, (linear representation). Coloured squares indicate the presence of resistance genes for the following antimicrobial classes: green squares: trimethoprim; black squares: tetracyclines; light blue squares: sulphonamides; orange squares: phenicols; pink squares: macrolides; yellow squares: polymyxins; red squares: ESBL/AmpC genes; blue squares: aminoglycosides.