Serotypes, Virulence-Associated Factors, and Antimicrobial Resistance of Streptococcus suis Isolates Recovered From Sick and Healthy Pigs Determined by Whole-Genome Sequencing

Abstract

Streptococcus suis is ubiquitous in swine, and yet, only a small percentage of pigs become clinically ill. The objective of this study was to describe the distribution of serotypes, virulence-associated factor (VAF), and antimicrobial resistance (AMR) genes in S. suis isolates recovered from systemic (blood, meninges, spleen, and lymph node) and non-systemic (tonsil, nasal cavities, ileum, and rectum) sites of sick and healthy pigs using whole-genome sequencing. In total, 273 S. suis isolates recovered from 112 pigs (47 isolates from systemic and 136 from non-systemic sites of 65 sick pigs; 90 isolates from non-systemic sites of 47 healthy pigs) on 17 Ontario farms were subjected to whole-genome sequencing. Using in silico typing, 21 serotypes were identified with serotypes 9 (13.9%) and 2 (8.4%) as the most frequent serotypes, whereas 53 (19.4%) isolates remained untypable. The relative frequency of VAF genes in isolates from systemic (Kruskal-Wallis, p < 0.001) and non-systemic (Kruskal-Wallis, p < 0.001) sites in sick pigs was higher compared with isolates from non-systemic sites in healthy pigs. Although many VAF genes were abundant in all isolates, three genes, including dltA [Fisher's test (FT), p < 0.001], luxS (FT, p = 0.01), and troA (FT, p = 0.02), were more prevalent in isolates recovered from systemic sites compared with non-systemic sites of pigs. Among the isolates, 98% had at least one AMR gene, and 79% had genes associated with at least four drug classes. The most frequently detected AMR genes were tetO conferring resistance to tetracycline and ermB conferring resistance to macrolide, lincosamide, and streptogramin. The wide distribution of VAFs genes in S. suis isolates in this study suggests that other host and environmental factors may contribute to S. suis disease development.

Keywords: Streptococcus suis; antimicrobial resistance; in silico serotyping; swine; virulence factors; whole genome sequencing.

Figure 1

In silico serotype of 273 S. suis isolates in four classified groups based on disease status (confirmed, probable infection, and healthy) and isolation source. Systemic sites included blood, meninges, spleen, and lymph node. Non-systemic sites included tonsil, nasal cavities, ileum, and rectal sample.

Figure 2

Distribution of virulence-associated factor genes and other genes carried by 273 S. suis isolates. Density plots of relative frequencies of known/putative VAF genes (red) against all other genes (gray) carried by each isolate. A relative frequency > 1 denotes a higher prevalence of gene within isolate group; (A) isolates recovered from systemic sites of pigs with confirmed infections (SC), (B) isolates recovered from non-systemic sites of pigs with confirmed infections (NSC), (C) isolates recovered from probable infections (NSP), (D) isolates recovered from non-systemic sites of healthy pigs (NSH).

Figure 3

Virulence-associated factor gene distribution among isolates in SC, NSC, NSP, and NSH groups. Darker shades show a higher proportion of isolates of a serotype carrying a particular gene. White indicates an absence of gene in that serotype. (A) Isolates recovered from systemic sites of pigs with confirmed infections (SC), (B) isolates recovered from non-systemic sites of pigs with confirmed infections (NSC), (C) isolates recovered from probable cases (NSP), and (D) isolates recovered from non-systemic sites of healthy pigs (NSH).

Figure 4

NMDS clustering based on virulence-associated factor gene presence and absence profiles of S. suis isolates. Ordination was based on a Jaccard distance matrix generated using VAF gene presence and absence data. Ellipses represent clusters within a 95% confidence level for each isolate group. Serotypes with prevalence below 5% were grouped together as “other”.

Figure 5

Antimicrobial resistance genes distribution in 267 S. suis isolates from SC and NSC and NSP and NSH groups. Darker shades show a higher proportion of isolates of serotypes carrying a particular gene. White indicates an absence of gene in that serotype.

Figure 6

Association plot for presence of AMR genes in 273 S. suis isolates belonging to different serotypes. Generated using Pearson's chi-squared test of independence of multi-way contingency table. Shade represents a degree of departure from expected values; blue for higher and red for lower.