Salmonella Typhimurium and its monophasic variant S . 4,[5],12:i:- are the dominant serotypes associated with pigs in many countries. We investigated their population structure on nine farms using whole genome sequencing, and their genotypic and phenotypic variation. The population structure revealed the presence of phylogenetically distinct clades consisting of closely related clones of S . Typhimurium or S . 4,[5],12:i:- on each pig farm, that persisted between production cycles. All the S . 4,[5],12:i:- strains carried the Salmonella genomic island-4 (SGI-4), which confers resistance to heavy metals, and half of the strains contained the mTmV prophage, harbouring the sopE virulence gene. Most clonal groups were highly drug resistant due to the presence of multiple antimicrobial resistance (AMR) genes, and two clades exhibited evidence of recent on-farm plasmid-mediated acquisition of additional AMR genes, including an IncHI2 plasmid. Biofilm formation was highly variable but had a strong phylogenetic signature. Strains capable of forming biofilm with the greatest biomass were from the S . 4,[5],12:i:- and S . Typhimurium DT104 clades, the two dominant pandemic clones found over the last 25 years. On-farm microevolution resulted in enhanced biofilm formation in subsequent production cycle.

Epidemic and pandemic clones of bacterial pathogens with distinct characteristics continually emerge, replacing those previously dominant through mechanisms that remain poorly characterized. Here, whole-genome-sequencing-powered epidemiology linked horizontal transfer of a virulence gene, sopE, to the emergence and clonal expansion of a new epidemic Salmonella enterica serovar Typhimurium (S. Typhimurium) clone. The sopE gene is sporadically distributed within the genus Salmonella and rare in S . enterica Typhimurium lineages, but was acquired multiple times during clonal expansion of the currently dominant pandemic monophasic S. Typhimurium sequence type (ST) 34 clone. Ancestral state reconstruction and time-scaled phylogenetic analysis indicated that sopE was not present in the common ancestor of the epidemic clade, but later acquisition resulted in increased clonal expansion of sopE-containing clones that was temporally associated with emergence of the epidemic, consistent with increased fitness. The sopE gene was mainly associated with a temperate bacteriophage mTmV, but recombination with other bacteriophage and apparent horizontal gene transfer of the sopE gene cassette resulted in distribution among at least four mobile genetic elements within the monophasic S . enterica Typhimurium ST34 epidemic clade. The mTmV prophage lysogenic transfer to other S. enterica serovars in vitro was limited, but included the common pig-associated S . enterica Derby (S. Derby). This may explain mTmV in S. Derby co-circulating on farms with monophasic S. Typhimurium ST34, highlighting the potential for further transfer of the sopE virulence gene in nature. We conclude that whole-genome epidemiology pinpoints potential drivers of evolutionary and epidemiological dynamics during pathogen emergence, and identifies targets for subsequent research in epidemiology and bacterial pathogenesis.

Salmonella enterica serovar Typhimurium (S. Typhimurium) comprises a group of closely related human and animal pathogens that account for a large proportion of all Salmonella infections globally. The epidemiological record of S. Typhimurium in Europe is characterized by successive waves of dominant clones, each prevailing for approximately 10–15 years before replacement. Succession of epidemic clones may represent a moving target for interventions aimed at controlling the spread and impact of this pathogen on human and animal health. Here, we investigate the relationship of phage sensitivity and population structure of S. Typhimurium using data from the Anderson phage typing scheme. We observed greater resistance to phage predation of epidemic clones circulating in livestock over the past decades compared to variants with a restricted host range implicating increased resistance to phage in the emergence of epidemic clones of particular importance to human health. Emergence of monophasic S. Typhimurium ST34, the most recent dominant multidrug-resistant clone, was accompanied by increased resistance to phage predation during clonal expansion, in part by the acquisition of the mTmII prophage that may have contributed to the fitness of the strains that replaced ancestors lacking this prophage.

Salmonella Typhimurium is the second most common cause of foodborne salmonellosis. Phage-mediated horizontal gene transfer contributes to the virulence of S. Typhimurium. An example of a phage-encoded virulence gene is sopE, a T3SS effector, found rarely in Typhimurium and associated with epidemics. The current pandemic and multi-drug resistant monophasic variant of S. Typhimurium (S. 4,[5],12:i:-) acquired sopE in multiple events following the lysogeny of a previously undescribed bacteriophage, mTmV. The current study aimed to further investigate the association of the virulence gene with S. 4,[5],12:i:- and assess its epidemiological impact. To this end, a large collection of clinical S. Typhimurium isolates from the UK have been analysed for the sopE gene and mTmV presence using a phylogenomic approach. While a large proportion of S. 4,[5],12:i:- (41 %) carried the sopE gene, few isolates outside the epidemic clade harboured it. Notably, the mTmV bacteriophage was identified only in S. 4,[5],12:i:-, although laboratory experiments demonstrated that the phage host range is not restricted to it. Nonetheless, we identified the phage in other S. enterica serovars circulating in the same ecological niche of S. 4,[5],12:i:-. In addition, a genomic characterisation of mTmV was performed revealing an unexpected level of phage variation. Finally, we identified a novel phage-like element harbouring the gene. The study revealed the large dissemination and selection of the virulence gene in the current epidemic, which is mobilised by multiple and distinct mobile genetic elements.

IncHI plasmids account for multiple antimicrobial resistance in Salmonella and other enterobacterial genera. These plasmids are generally very stable in their bacterial hosts. R27 is the archetype of IncHI1 plasmids. A high percentage of the R27-encoded open reading frames (ORFs) (66.7%) do not show similarity to any known ORFs. We performed a deletion analysis of all non-essential R27 DNA sequences to search for hitherto non-identified plasmid functions that might be required for plasmid stability. We report the identification of a short DNA sequence (incC) that is essential for R27 stability. That region contains several repeats (incC repeats), belongs to one of the three-plasmid replicons (R27 FIA-like) and is targeted by the R27 E protein. Deletion of the incC sequence drastically reduces R27 stability both in Escherichia coli and in Salmonella, the effect being more pronounced in this latter species. Interfering with incC–E protein interaction must lead to a reduced IncHI1 plasmid stability, and may represent a new approach to combat antimicrobial resistance.