Serotype and Genotype (Multilocus Sequence Type) of Streptococcus suis Isolates from the United States Serve as Predictors of Pathotype

Abstract

Streptococcus suis is a significant cause of mortality in piglets and growing pigs worldwide. The species contains pathogenic and commensal strains, with pathogenic strains causing meningitis, arthritis, endocarditis, polyserositis, and septicemia. Serotyping and multilocus sequence typing (MLST) are primary methods to differentiate strains, but the information is limited for strains found in the United States. The objective of this study was to characterize the diversity of 208 S. suis isolates collected between 2014 and 2017 across North America (mainly the United States) by serotyping and MLST and to investigate associations between subtype and pathotype classifications (pathogenic, possibly opportunistic, and commensal), based on clinical information and site of isolation. Twenty serotypes were identified, and the predominant serotypes were 1/2 and 7. Fifty-eight sequence types (STs) were identified, and the predominant ST was ST28. Associations among serotypes, STs, and pathotypes were investigated using odds ratio and clustering analyses. Evaluation of serotype and ST with pathotype identified a majority of isolates of serotypes 1, 1/2, 2, 7, 14, and 23 and ST1, ST13, ST25, ST28, ST29, ST94, ST108, ST117, ST225, ST373, ST961, and ST977 as associated with the pathogenic pathotype. Serotypes 21 and 31, ST750, and ST821 were associated with the commensal pathotype, which is composed of isolates from farms with no known history of S. suis-associated disease. Our study demonstrates the use of serotyping and MLST to differentiate pathogenic from commensal isolates and establish links between pathotype and subtype, thus increasing the knowledge about S. suis strains circulating in the United States.

Keywords: MLST; Streptococcus suis; multilocus sequence typing; pathogenic; pathotype; porcine; serotyping.

FIG 1

The number and percentage of S. suis isolates characterized from each state. States are colored according to the number of isolates characterized from each state. States without isolates are gray. Isolates from Canada (n = 4) and Mexico (n = 1) are not shown.

FIG 2

Distribution of S. suis STs by serotype. The stacked histogram illustrates the serotypes identified in this study, which were subdivided by STs. The x axis represents each serotype while the y axis represents the frequency of each serotype. Bar sections are labeled with their respective STs. The category 1or14 and (nontypeable (NT) represents isolates with serotypes that could not be differentiated by coagglutination, PCR, or WGS.

FIG 3

Distribution of S. suis pathotypes by serotype. The stacked histogram illustrates the serotypes identified in this study, which were subdivided by pathotype (pathogenic, possibly opportunistic, and commensal). The x axis represents each serotype while the y axis represents the frequency of each pathotype. Bar sections are labeled with their respective pathotypes. The category 1or14 and NT (nontypeable) represents isolates with serotypes that could not be differentiated by coagglutination, PCR, or WGS.

FIG 4

Distribution of S. suis pathotypes by ST. The stacked histogram illustrates the STs identified in this study, which were subdivided by pathotype (pathogenic, possibly opportunistic, and commensal). The x axis represents each ST while the y axis represents the frequency of each pathotype. Bar sections are labeled with their respective pathotypes. Not found (NF) indicates ST could not be determined because one housekeeping gene could not be identified for MLST classification.

FIG 5

Ternary and OR plots summarizing the associations between S. suis pathotype and serotype. Only serotypes containing more than a single isolate are illustrated in the ternary plot, and only pathotype-serotype combinations containing more than a single isolate are illustrated in the OR plot. (A) The proportions of isolates classified as the pathogenic, possibly opportunistic, and commensal pathotype in each of the 16 serotypes (and the NT category) were plotted. The gray lines and color shading denote pathotype boundaries. (B) OR plot for 16 serotypes (and the NT category) versus pathotype. The dotted lines illustrate the minimum lower limit (OR, 0.3) and typical threshold (OR, 1) for identifying significant ORs. Error bars represent the 95% confidence intervals. Inf, Infinite. Nontypeable (NT) represents isolates which could not be serotyped using coagglutination, PCR, or WGS.

FIG 6

Ternary and OR plots summarizing the associations between S. suis pathotype and ST. Only STs containing more than a single isolate are illustrated in the ternary plot, and only pathotype-ST combinations containing more than a single isolate are illustrated in the OR plot. (A) The proportions of isolates classified as the pathogenic, possibly opportunistic, and commensal pathotype in each of the 17 STs (and the NF category) were plotted. The gray lines and color shading denote pathotype boundaries. (B) OR plot for 16 STs (and the NF category) versus pathotype. ST119 contained a single isolate of each pathogenetic and possibly opportunistic pathotype failing to meet the criteria for the plot. The dotted lines illustrate the minimum lower limit (OR, 0.3) and typical threshold (OR, 1) for identifying significant ORs. Error bars represent the 95% confidence intervals. Infinite (Inf) is represented by the value 10,000 for visualization purposes. Not found (NF) indicates ST could not be determined because one housekeeping gene could not be identified for MLST classification.

FIG 7

Three-way MCA analyzing the associations among pathotypes, serotypes, and STs. The ellipses represent 95% of isolates in each pathotype. Dots represent isolates colored by their respective pathotype, and hollow diamonds represent the three active variables (pathotype, serotype, and ST). Nontypeable (NT) represents isolates which could not be serotyped using coagglutination, PCR, or WGS. Not found (NF) indicates ST could not be determined because one housekeeping gene could not be identified for MLST classification.

FIG 8

eBURST illustration of the global S. suis population. Primary founders (blue) are positioned at the center of the cluster and subgroup founders are shown in yellow. Clusters of linked STs correspond to CCs. Magenta dots mark the STs identified in our study and arrows mark the CCs relevant to this study. Individual CCs have been expanded to illustrate relationships among STs.

FIG 9

Ternary and OR plots summarizing the associations between pathotype and CC. (A) The proportions of isolates classified as the pathogenic, possibly opportunistic, and commensal pathotype in each of the five CCs (and the noCC category) were plotted. The gray lines and color shading denote pathotype boundaries. (B) OR plot for all five CCs (and the noCC category) versus pathotype. The dotted lines illustrate the minimum lower limit (OR, 0.3) and typical threshold (OR, 1) for identifying significant ORs. Error bars represent the 95% confidence intervals. Infinite (Inf) is represented by the value 10,000 for visualization purposes. noCC represents the group of isolates lacking a CC.

FIG 10

MLST sequence identity heatmap of S. suis. Isolates are annotated (colored rectangles) by pathotype (1), serotype (2), and ST (3). The five CCs are indicated by black brackets, with the number of isolates in the CC. Blue brackets represent clusters of isolates without a CC. Nontypeable (NT) represents isolates which could not be serotyped using coagglutination, PCR, or WGS. #, group of isolates lacking a CC; +, ST13 not within a CC but closest to CC1; ∼, ST979 not within a CC but closest to CC94.