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Genomic characterization of two Salmonella entericasubsp. enterica var. Issatschenko strains possessing selective rodenticidal properties

Although Salmonella enterica subsp. enterica is mostly known as a causative agent of severe gastrointestinal diseases in humans, its actual host range spans vast outside the human domain. In the current study, we performed whole‐genome sequencing and genome assembly for two strains, namely 29‐1 and 32‐3 attributed to the Issatschenko pathovar. Having been originally isolated from bird feces, these strains demonstrated exclusive rodenticidal properties. Genomes of both strains were sequenced with both long (Oxford Nanopore MinION) and short (Illumina HiSeq) reads, with assembly first performed with Raven v1.7.0. Long‐reads‐first assemblies were then subjected to long‐read polishing by four consecutive runs of Minimap2 v2.22 and Racon v1.4.3 followed by short‐read polishing with bowtie2 v2.4.2 and Pilon v1.23.0. Genome annotation was performed with Prokka v1.14.5. The strains’ attribution to serovar Enteritidis was confirmed by using SISTR standalone suite v1.1.1. The resulting assemblies were both 4.75 Mb in size with N50 values of 4.65 Mb. Both assemblies comprised of three replicons, including 92.9 Kb virulence plasmids and 4.6 Kb extrachromosomal selfish element, both identical between the two strains. The major plasmid harbored 109 genes, of which 15 correspond to known virulence factors deposited in the Virulence Factor Database, including Pef fimbrial operon members, adhesion protein EaeH, and type III secretion system components. In addition, the chromosomes of both strains bore an apparently duplicated fragment harboring 39 protein‐coding genes including 5 virulence factors’ homologs. Notably, some loci represented host adaptation determinants, namely, the adhesion‐mediating zirTS system, zink transporter ZntB ensuring cations transport into Salmonella‐containing vacuoles, and metabolic components allowing intracellular survival such as antioxidant peroxiredoxin and stress‐related protein UspE. Lesser replicons bore three genes and shared more than 99% similarity with the previously reported salmonellan plasmid pN18S1943‐2. We then compared our strains with the known diversity of the closest serovar, S. Enteritidis. 2263 raw sequencing projects from the NCBI SRA (Sequence Read Archive) database were downloaded to reveal Issatschenko‐specific variants. Variants were called using snippy v4.6.0 against assembled genomes, and the obtained VCF files were merged with bcftools v1.13‐25. We then selected 9 polymorphic sites identified in more than 2000 strains. Of these, 5 synonymous and 4 non‐synonymous mutations were detected. Interestingly, three mutations affected genes encoding for primary metabolism enzymes, e.g., ribonucleoside‐diphosphate reductase and formate dehydrogenase, whereas one mutation was identified in the locus coding for type III secretion system translocon protein SseC suggesting probable involvement of these proteins in the control of host‐pathogen selectivity.