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

Abstract

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.

Keywords: Salmonella; phylogeography; polyphyly; prophage; whole-genome sequencing.

FIG 1

S. Mississippi isolates cluster within S. enterica subsp. enterica clades A and B. (A) Phylogeny inferred from maximum likelihood analysis of 10,905 core SNPs compared across 318 unique serovars, including representative isolates of each S. Mississippi clade (denoted with red [clade A, section Typhi] and blue [clade B] stars). A total of 100 bootstrap repetitions were performed. Branches are color-coded to reflect the phylogenetic clade of the serovar. The tree is rooted by S. enterica subsp. arizonae (GenBank assembly accession number GCA_000018625), which has been used previously as an outgroup for S. enterica (22). (B and C) Core SNPs for serovars that clustered with clade B Mississippi (29 serovars; 57,090 core SNPs) (B) and clade A Mississippi (29 serovars; 61,144 core SNPs) (C) were identified, and phylogenetic trees were inferred. The bootstrap values listed represent the averages from 1,000 bootstrap repetitions. Colored strips on the right show the serogroup (rightmost strip) and phase 1 (H1) and phase 2 (H2) flagellar antigens (middle and leftmost strips, respectively) reported for a given serovar. Some serovars do not encode a phase 2 flagellar antigen, and therefore, these serovars lack a colored square to signify that they are monophasic. Outgroups were selected based on the phylogenetic analyses in panel A and were S. Wagenia and S. Bovismorbificans for clades A and B, respectively.

FIG 2

Phylogeographic clustering of S. Mississippi clades suggests that different S. Mississippi isolates are endemic in Australia, the United Kingdom, and the United States. A phylogeny was inferred from maximum likelihood analysis of 54,880 core SNPs among 364 S. Mississippi isolates and representative isolates of serovars that share an ancestor with clade A and B Mississippi as determined from Fig. 1B and C. A total of 500 bootstrap repetitions were performed, and the tree was rooted with the clade C serovar S. Maricopa as the outgroup. The colored squares shown external to the tree are colored to reflect the country of isolation.

FIG 3

Diversification of clade Ai and Aii isolates is mediated by the acquisition and loss of prophages. (A) Comparison of core genes (present in at least 99% of isolates in the comparison) among clade Ai and Aii isolates as well as genes shared by isolates in both clades. (B) Categories of genes that were present in at least 90% of isolates in one clade but that were absent from all isolates in the other clade. Genes were categorized manually into each group based on annotation suggested by InterPro and/or Prokka. Genes in the phage-associated category were annotated as encoding phage components (such as tail fibers and capsid proteins, etc.) or integration-related machinery necessary for prophage insertion (such as integrase and recombinase, etc.). Hypothetical proteins represent genes that did not have any annotation suggested by InterPro, while genes in the “other” category represent genes with annotations suggesting that they were associated with nonvirulence and nonphage functions. A full list of all genes and their annotations can be found in Data Set S3 and Table S1 in the supplemental material. CDS, coding DNA sequences. (C) Box plot summaries of the nucleotide lengths of prophages Entero_mEp460, Gifsy-2, Salmon_118970_sal3, and Salmon_vB_SosS_Oslo for all isolates in clade Ai (n = 99 isolates) and clade Aii (n = 124 isolates). Nucleotide lengths were summed from hits for each local BLAST alignment for each prophage.

FIG 4

Differences in gene contents of clade Bi and Bii Mississippi isolates are mediated by the acquisition of a 47.1-kb integrative conjugation element by clade Bii Mississippi. (A) Comparison of core genes (present in at least 99% of isolates in the comparison) among clade Bi and Bii isolates as well as genes shared by isolates in both clades. (B) Categories of genes that were present in at least 90% of isolates in one clade but that were absent from all isolates in the other clade. Genes were categorized manually into each group based on annotation suggested by InterPro and/or Prokka. Genes in the integrative conjugative element category were annotated as genes associated with integration or conjugative transfer (such as integrases and Tra proteins, etc.). Hypothetical proteins represent genes that did not have any annotation suggested by InterPro, while genes in the “other” category represent genes with annotation suggesting that they were associated with nonvirulence and nonphage functions. See Data Set S3 and Table S3 in the supplemental material for a full list of all genes and their annotations. (C) Organization and annotation of genes identified in panel B that were located within the 47.1-kb integrative conjugative element found in all 7 clade Bii Mississippi isolates. (D) Results of a discontiguous BLAST search for the clade Bii ICE in other Salmonella isolates and other bacteria. Only hits with >70% query coverage are shown. E. albertii, Escherichia albertii; K. variicola, Klebsiella variicola.

FIG 5

Phylogenies inferred for fliC and fljB support the acquisition of these genes from different ancestors for clade A and B Mississippi. (A and B) Maximum likelihood inference of fliC before (A) and after (B) a recombination breakpoint at nt 356 to 357 detected by GARD for representative isolates of clade A and B Mississippi and 33 additional serovars. (C to E) Maximum likelihood inference for fljB segments representing nucleotides 1 to 512 (C), 513 to 935 (D), and 936 to 1521 (E) for representative isolates of clade A and B Mississippi and 80 additional serovars. Branches are color-coded to reflect the phylogenetic clade of the serovar. Stars represent the branches corresponding to clade A (red stars) and clade B (blue stars) S. Mississippi fljB sequences. Phylogenies were inferred with IQ-TREE with 1,000 ultrafast bootstrap repetitions. Values shown on branches represent ultrafast bootstrap approximations; values are shown only for branches with >95% bootstrap support.

FIG 6

Comparison of Mississippi A and B serogroup O13 (G) O antigen gene clusters suggests acquisition from different ancestors. (A and B) O-antigen gene clusters established previously (4) were mapped onto the phylogenetic trees shown in Fig. 1B and C for clade A (A) and clade B (B) S. Mississippi. Genes are color-coded based on their predicted function as described previously by Liu and colleagues (4). Branches represent O antigens that are initiated with either galactose (Gal initiated) (yellow) or N-acetylglucosamine/N-acetylgalactosamine (GlcNAc/GalNAc initiated) (teal). (C) O-antigen gene cluster for serogroup O13(G) as described previously by Liu and colleagues (4). Only wzx, wzy, wcmC, and wfbI were compared here as gne and wfbG were missing from most of the serovars that also react with O22 antisera, and gmd, fcl, gmm, manC, and manB are also present in the colanic acid synthesis operon; therefore, sequences for these genes could not be reliably extracted for all isolates or serovars examined. (D) Concatenated sequences for wzx, wzy, wcmC, and wfbI were used to infer a phylogeny for 24 serovars, including representative strains of clade A and B S. Mississippi. Branches are colored based on the phylogenetic clade of the serovar determined in Fig. 1 (red, clade A; blue, clade B). Bootstrap values of >95% are listed at the nodes (1,000 ultrafast bootstrap repetitions were performed). Colored bars represent the O type (O22 or O23) listed in the serovars’ antigenic formulas. The tree is midpoint rooted.