Comparative analysis of Streptococcus suis genomes identifies novel candidate virulence-associated genes in North American isolates

Abstract

Streptococcus suis is a significant economic and welfare concern in the swine industry. Pan-genome analysis provides an in-silico approach for the discovery of genes involved in pathogenesis in bacterial pathogens. In this study, we performed pan-genome analysis of 208 S. suis isolates classified into the pathogenic, possibly opportunistic, and commensal pathotypes to identify novel candidate virulence-associated genes (VAGs) of S. suis. Using chi-square tests and LASSO regression models, three accessory pan-genes corresponding to S. suis strain P1/7 markers SSU_RS09525, SSU_RS09155, and SSU_RS03100 (>95% identity) were identified as having a significant association with the pathogenic pathotype. The proposed novel SSU_RS09525 + /SSU_RS09155 + /SSU_RS03100 + genotype identified 96% of the pathogenic pathotype strains, suggesting a novel genotyping scheme for predicting the pathogenicity of S. suis isolates in North America. In addition, mobile genetic elements carrying antimicrobial resistance genes (ARGs) and VAGs were identified but did not appear to play a major role in the spread of ARGs and VAGs.

Keywords: Streptococcus suis; comparative genomics; pathotype; virulence-associated genes (VAGs).

Figure 1

Illustration of the pan-genome approach for identifying candidate virulence-associated genes (VAGs). A The first data set analyzed consisted of all 208 isolates and B the second data set consisted of a subset of 161 isolates. Genes with a significant association (p-value < 0.05) with pathotype, and present in more than 50% (69/139) of isolates within the pathogenic pathotype and present in less than 50% (11/22) of isolates within the commensal pathotype, were analyzed using the LASSO shrinkage regression model. Genes identified in both LASSO analyses were selected as the ‘best’ predictors of pathotype.

Figure 2

Summary statistics for the pan-genome analysis of 208 S. suis isolates. A Number of conserved genes, B unique genes, and C total number of genes plotted against the number of genomes included in the analysis.

Figure 3

Predicted Cluster of Orthologous Groups (COG) functional classes. COG functional classes were determined for the 995 pan-gene clusters that met the criteria (psiblast, coverage ≥ 70%, percent identity ≥ 70%, and e-value ≤ 10–5). A The total frequencies of pan-gene clusters classified into each COG functional class (single-letter code) were plotted and color-coded. B The percentages of accessory and core pan-gene clusters classified into each COG functional class were plotted. Dark and light shading represents core and accessory pan-gene clusters, respectively. C The percentages of pan-gene clusters (from each COG class) present in each pathotype. Dark, medium, and light shading represents pan-gene clusters present in the pathogenic, possibly opportunistic, and commensal pathotypes, respectively.

Figure 4

Antimicrobial resistance genes (ARGs) and drug classes determined in S. suis isolates (n = 208). A Percentage of isolates possessing each of the 15 ARGs identified. ARGs were subdivided by pathotype (stacked bar sections; pathogenic, possibly opportunistic, and commensal). B Percentage of each pathotype possessing ARGs conferring resistance to each of the five drug classes aminoglycosides, lincosamides, macrolides, nucleosides, and tetracyclines.

Figure 5

Mobile genetic elements carrying ARGs and VAGs. Percentage of each pathotype (pathogenic, possibly opportunistic, and commensal) possessing A various MGE types, B ARG-associated MGEs, and C VAG-associated MGEs. S. suis strain 05ZYH33 89 K PAI sequences CH1/CH2 (5′ flanking region), CH3/CH4 (716 bp internal fragment), CH5/CH6 (3′ flanking region). Total percentage and number of isolates possessing each type of MGE are listed under its respective name.