DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes

Abstract

While serotyping and phage typing have been used widely to characterize Salmonella isolates, sensitive subtyping methods that allow for evolutionary analyses are essential for examining Salmonella transmission, ecology, and evolution. A set of 25 Salmonella enterica isolates, representing five clinically relevant serotypes (serotypes Agona, Heidelberg, Schwarzengrund, Typhimurium, and Typhimurium var. Copenhagen) was initially used to develop a multilocus sequence typing (MLST) scheme for Salmonella targeting seven housekeeping and virulence genes (panB, fimA, aceK, mdh, icdA, manB, and spaN). A total of eight MLST types were found among the 25 isolates sequenced. A good correlation between MLST types and Salmonella serotypes was observed; only one serotype Typhimurium var. Copenhagen isolate displayed an MLST type otherwise typical for serotype Typhimurium isolates. Since manB, fimA, and mdh allowed for the highest subtype discrimination among the initial 25 isolates, we chose these three genes to perform DNA sequencing of an additional 41 Salmonella isolates representing a larger diversity of serotypes. This "three-gene sequence typing scheme" allowed discrimination of 25 sequence types (STs) among a total of 66 isolates; STs correlated well with serotypes and allowed within-serotype differentiation for 9 of the 12 serotypes characterized. Phylogenetic analyses showed that serotypes Kentucky and Newport could each be separated into two distinct, statistically well supported evolutionary lineages. Our results show that a three-gene sequence typing scheme allows for accurate serotype prediction and for limited subtype discrimination among clinically relevant serotypes of Salmonella. Three-gene sequence typing also supports the notion that Salmonella serotypes represent both monophyletic and polyphyletic lineages.

FIG. 1.

Phylogenetic analysis of manB alleles. The phylogenetic tree was built on the maximum-likelihood framework using an alignment of manB sequences representing each manB allelic type found among the 66 Salmonella isolates sequenced, with the exception of the manB1 and manB2 sequences, which include those for all three Salmonella serotype Montevideo isolates that showed an manB gene duplication. Bootstrap values of >50.0 are given at the nodes above the branches. The scale bar indicates relative sequence distance. Allele assignments, followed by the number of isolates within each allelic type (in parentheses), and the serotype associated with each allelic type are also shown.

FIG. 2.

Compatibility matrix for all seven MLST genes. The matrix was constructed using RETICULATE and a concatenated alignment of the seven genes (shown on the plot) for 25 Salmonella isolates. The figure is a plot of all pairwise comparisons of 152 informative sites that are phylogenetically compatible (white squares) or incompatible (black squares). Intragenic comparisons are marked within the triangles, and the number of informative sites for each gene is given in parentheses after the gene name.

FIG. 3.

Compatibility matrix for fimA, manB, and mdh. The matrix was constructed using RETICULATE and a concatenated alignment of fimA, manB, and mdh obtained for 66 Salmonella isolates. The figure is a plot of all pairwise comparisons of 69 informative sites that are phylogenetically compatible (white squares) or incompatible (black squares). Intragenic comparisons are marked within triangles, while intergenic comparisons are marked in rectangles. The number of informative sites for each gene is given in parentheses after the gene name.

FIG. 4.

Phylogenetic tree of 25 Salmonella isolates based on a concatenated alignment of the seven MLST genes (fimA, mdh, manB, spaN, icdA, panB, aceK) sequenced. The phylogenetic tree was built using the maximum-likelihood method. The branch length of the outgroup was collapsed so as to best view the topology of the tree. Bootstrap values of >50.0 are given at the nodes above the branches. Posterior probabilities of >0.50 are given at the nodes below the branches. The scale bar indicates relative sequence distance. Sequence type assignments, followed by the serotypes for the isolates within each sequence type, are given for all isolates. No sequence data for icdA and panB were available for the five Salmonella serotype Schwarzengrund isolates (since PCR primers did not yield amplification products), and appropriate gaps were introduced into the alignment for the Salmonella Schwarzengrund isolates.

FIG. 5.

Phylogenetic tree for all sequence types found among 66 Salmonella isolates based on sequencing of three genes. The phylogenetic tree was built on the Bayesian framework using a concatenated alignment of fimA, manB, and mdh sequences representing all 25 sequence types found among the 66 isolates. The branch length of the outgroup was collapsed so as to best view the topology of the tree. Posterior probabilities of >0.50 are given at the nodes above the branches. Bootstrap values of >50.0 are given in parentheses below the branches. The scale bar indicates relative sequence distance. Sequence type assignments, followed by the number of isolates within each sequence type and their serotypes, are given for all isolates. Serogroups are given in parentheses after the serotype designation.