Phenotypic and genetic characterization of antimicrobial resistance in Salmonella enterica serovar Choleraesuis isolates from humans and animals in Spain from 2006 to 2021

Abstract

Objectives: While an increase in the levels of MDR in Salmonella enterica sevorar Choleraesuis has been reported in Europe, little is known about the situation in Spain. Therefore, we first aimed to assess the phenotypic resistance profile and to determine the presence of genetic determinants of resistance of S. Choleraesuis isolates collected in animal and human. Our second objective was to identify and characterize clusters of highly related isolates.

Methods: We analysed 50 human and 45 animal isolates retrieved from 2006 to 2021 using the disc diffusion method and performed WGS followed by analyses of genetic determinants and phylogenetic analysis.

Results: All isolates were of ST145 and corresponded to the variant Kunzendorf. Swine isolates harboured a significantly higher number of antimicrobial resistance genes than human isolates, and often carried plasmid replicons of the IncHI2/IncHI2A type (42% of all animal isolates). In addition, we identified several MDR S. Choleraesuis strains circulating in humans and swine between 2006 and 2021. The phylogenetic analyses identified four clades associated with specific patterns of resistance genes and plasmid replicons. The clades also included isolates that differed in terms of year and region of isolation as well as host of origin.

Conclusions: This One Health approach highlights that reducing human MDR S. Choleraesuis infections may require the adoption of strategies that not only seek to prevent cases in humans but also to characterize and reduce the infection burden in swine.

Figure 1.

Heatmap of antimicrobial susceptibility of all the isolates included in the study. Black is used for resistance, dark grey for intermediate and light grey for susceptibility. Missing data are represented in white. Susceptibility to antimicrobial agents was determined using the Kirby–Bauer disc diffusion method and interpreted according to the clinical breakpoints of the EUCAST. Isolates are presented in the order of Figure 3.

Figure 2.

Mapping of isolates with (a) Choleraesuis specific plasmid (pSCV50), (b) S. enterica subsp enterica plasmid (pCFSA629) and (c) E. coli plasmid (pEGY1) performed by PlasmidID. Annotations for resistance genes and plasmid replicons are indicated. From the outside track to the inner track: (i) reference plasmid size indicators, (ii) histogram with the number of reads mapped in each position of the reference plasmid, (iii) two layers including contig and plasmid database annotation results from Prokka software. Each purple and grey box indicated a predicted named gene or protein coding sequence with its identity tag placed over the sequence, for contigs and plasmid database respectively. Genes over the line and darker are located in + strand, the rest in—strand, (iv) the contig track represents local alignments of contigs sequences that match plasmid region and (v) the complete contig track represent the full length of contigs that are homologous to the reference plasmid.

Figure 2.

Mapping of isolates with (a) Choleraesuis specific plasmid (pSCV50), (b) S. enterica subsp enterica plasmid (pCFSA629) and (c) E. coli plasmid (pEGY1) performed by PlasmidID. Annotations for resistance genes and plasmid replicons are indicated. From the outside track to the inner track: (i) reference plasmid size indicators, (ii) histogram with the number of reads mapped in each position of the reference plasmid, (iii) two layers including contig and plasmid database annotation results from Prokka software. Each purple and grey box indicated a predicted named gene or protein coding sequence with its identity tag placed over the sequence, for contigs and plasmid database respectively. Genes over the line and darker are located in + strand, the rest in—strand, (iv) the contig track represents local alignments of contigs sequences that match plasmid region and (v) the complete contig track represent the full length of contigs that are homologous to the reference plasmid.

Figure 3.

Maximum-likelihood phylogenetic tree of 94 S. Choleraesuis ST145 isolates. Sequences were aligned to genome CP075026. The coloured labels indicate the four identified clades [groups identified with BAPS (level 1)]. The following information is presented to the left of the isolate IDs: host of origin, region of collection, presence/absence of ARGs and plasmid replicons. Branches with a bootstrap values >90 support are shown in red in the tree. Isolates in red were selected for the PlasmidID analyses. Details on the possible events of transmission and the identification of prophages can be found in the Supplementary Materials.