Phylogeographic Clustering Suggests that Distinct Clades of Salmonella enterica Serovar Mississippi Are Endemic in Australia, the United Kingdom, and the United States.

Salmonella enterica serovar Mississippi is the 2nd and 14th leading cause of human clinical salmonellosis in the Australian island state of Tasmania and the United States, respectively. Despite its public health relevance, relatively little is known about this serovar. Comparison of whole-genome sequence (WGS) data of S. Mississippi isolates with WGS data for 317 additional S. enterica serovars placed one clade of S. Mississippi within S. enterica clade B ("clade B Mississippi") and the other within section Typhi in S. enterica clade A ("clade A Mississippi"), suggesting that these clades evolved from different ancestors. Phylogenetic analysis of 364 S. Mississippi isolates from Australia, the United Kingdom, and the United States suggested that the isolates cluster geographically, with U.S. and Australian isolates representing different subclades (Ai and Aii, respectively) within clade A Mississippi and clade B isolates representing the predominant S. Mississippi isolates in the United Kingdom. Intraclade comparisons suggested that different mobile elements, some of which encode virulence factors, are responsible for the observed differences in gene content among isolates within these clades. Specifically, genetic differences among clade A isolates reflect differences in prophage contents, while differences among clade B isolates are due to the acquisition of a 47.1-kb integrative conjugative element (ICE). Phylogenies inferred from antigenic components (fliC, fljB, and O-antigen-processing genes) support that clade A and B Mississippi isolates acquired these loci from different ancestral serovars. Overall, these data support that different S. Mississippi phylogenetic clades are endemic in Australia, the United Kingdom, and the United States. IMPORTANCE The number of known so-called "polyphyletic" serovars (i.e., phylogenetically distinct clades with the same O and H antigenic formulas) continues to increase as additional Salmonella isolates are sequenced. While serotyping remains a valuable tool for reporting and monitoring Salmonella, more discriminatory analyses for classifying polyphyletic serovars may improve surveillance efforts for these serovars, as we found that for S. Mississippi, distinct genotypes predominate at different geographic locations. Our results suggest that the acquisition of genes encoding O and H antigens from different ancestors led to the emergence of two Mississippi clades. Furthermore, our results suggest that different mobile elements contribute to the microevolution and diversification of isolates within these two clades, which has implications for the acquisition of novel adaptations, such as virulence factors.

The Majority of Typhoid Toxin-Positive Salmonella Serovars Encode ArtB, an Alternate Binding Subunit.

Salmonella enterica encodes a wide array of virulence factors. One novel virulence factor, an A2B5 toxin known as the typhoid toxin (TT), was recently identified among a variety of S. enterica serovars. While past studies have shown that some serovars encode both the TT (active subunits CdtB and PltA and binding subunit PltB) and a second binding subunit (ArtB), these serovars were thought to be the exception. Here, we show that genes encoding the TT are detected in more than 100 serovars representing distinct phylogenetic lineages of S. enterica subsp. enterica, although clade B and section Typhi are significantly more likely to encode TT genes than serovars from other clades. Furthermore, we show that 81% of these TT-positive serovars also encode artB, suggesting that the cooccurrence of both toxin binding subunits is considerably more common than previously thought. A combination of in silico modeling, bacterial two-hybrid system screening, and tandem affinity purification (TAP) of toxin subunits suggests that ArtB and PltB interact in vitro, at least under some growth conditions. While different growth conditions yielded slightly higher transcript abundances of artB and pltB, both genes had their highest relative transcript abundances when Salmonella was grown under low-Mg2+ conditions, suggesting that ArtB and PltB may compete for inclusion in the TT. Together, our results suggest that ArtB likely plays an important and previously underappreciated role in the biology of the TT produced by typhoidal and nontyphoidal SalmonellaIMPORTANCE While previous reports had suggested that the typhoid toxin (TT) could potentially use ArtB as an alternate binding subunit, this was thought to play a minor role in the evolution and biology of the toxin. In this study, we establish that both TT genes and artB are widespread among Salmonella enterica subsp. enterica, suggesting that TT likely plays a broader role in Salmonella virulence that extends beyond its proposed role in typhoid fever. Furthermore, our data suggest the selective maintenance of both toxin binding subunits, which may compete for inclusion in the holotoxin. Last, our data support the importance of characterizing diverse nontyphoidal Salmonella (NTS) serovars, as the presence of classically defined typhoidal virulence factors among NTS serovars continues to challenge the typhoid-nontyphoid Salmonella paradigm.