Complex Population Structure and Virulence Differences among Serotype 2 Streptococcus suis Strains Belonging to Sequence Type 28

Abstract

Streptococcus suis is a major swine pathogen and a zoonotic agent. Serotype 2 strains are the most frequently associated with disease. However, not all serotype 2 lineages are considered virulent. Indeed, sequence type (ST) 28 serotype 2 S. suis strains have been described as a homogeneous group of low virulence. However, ST28 strains are often isolated from diseased swine in some countries, and at least four human ST28 cases have been reported. Here, we used whole-genome sequencing and animal infection models to test the hypothesis that the ST28 lineage comprises strains of different genetic backgrounds and different virulence. We used 50 S. suis ST28 strains isolated in Canada, the United States and Japan from diseased pigs, and one ST28 strain from a human case isolated in Thailand. We report a complex population structure among the 51 ST28 strains. Diversity resulted from variable gene content, recombination events and numerous genome-wide polymorphisms not attributable to recombination. Phylogenetic analysis using core genome single-nucleotide polymorphisms revealed four discrete clades with strong geographic structure, and a fifth clade formed by US, Thai and Japanese strains. When tested in experimental animal models, strains from this latter clade were significantly more virulent than a Canadian ST28 reference strain, and a closely related Canadian strain. Our results highlight the limitations of MLST for both phylogenetic analysis and virulence prediction and raise concerns about the possible emergence of ST28 strains in human clinical cases.

Fig 1

A) Genome atlas of Canadian S. suis ST28 strain NSUI002. Depicted data from innermost to outermost circles represent genome size in Mbp (circle 1); percent G+C content (circle 2); GC skew (circle 3), (G-C)/(G+C) averaged over a moving window of 10,000 bp, with excess G and excess C shown in green and purple, respectively; annotated coding sequences (CDSs) encoded on the forward/direct (circle 4, red), and reverse/complementary (circle 5, blue) chromosomal strands; TBLASTN comparisons of the CDSs predicted in ST28 strains NSUI002 and 05HAS68 (circle 6, percent identity defined in the Fig), TBLASTN comparisons of ST28 strain NSUI002 and ST1 strain P1/7 (circle 7, percent identity defined in the Fig); reference genome landmarks (circle 8): ribosomal RNAs are labeled in green; mobile genetic elements are labeled in black, genes used in the S. suis MLST scheme are labeled in light blue; genes encoding resistance to antimicrobial agents are labeled in red; other genes are labeled in black. B) Venn diagram depicting unique and shared CDSs in each of the S. suis strains as identified by ortholog analysis. Each strain is represented by one color, and the number of CDSs are displayed in the same color. Numbers in the intersectional regions indicate CDSs shared by two or three strains. Since there may be more than one CDS in the same ortholog cluster, number of CDSs in the intersections are slightly different between strains C) Collinearity of the genomes of S. suis ST28 strains NSUI002 and 05HAS68. The genomes of the strains were aligned using progressiveMauve. Sequence alignments that are free of rearrangements are shown as colored local collinear blocks (LCBs). Sequence inversions are denoted by differential positioning of the LCBs relative to a reference axis. Several genome rearrangements between NSUI002 and 05HAS68 are noticeable.

Fig 2

A) Results of Bayesian analysis of recombination for the 51 ST28 S. suis strains. The names and countries of isolation of the strains are shown on the right. The colored bars denote the recombination events in the strains along the core genome. The coloring of the bars at a specific genomic location reflects the clustering of the recombination events into groups, and is unrelated to other bars at distant genomic locations. CAN: Canada, USA: United States of America; JAP: Japan; Tha: Thailand. B) Neighbor-joining phylogenetic tree depicting the relationships between the 51 ST28 S. suis strains. The tree was constructed using 1,421 SNPs identified against the core genome (see text for details). Two singletons and five distinct clades (I to V) were identified.

Fig 3. Venn diagram depicting unique and shared ortholog gene clusters in each of the five clades defined among the S. suis strains.

Numbers shown in the different sections indicate the numbers of ortholog groups. The two ST28 singletons were not included in this analysis.

Fig 4. Results of animal experimental infections.

A) Survival of mice inoculated with the different S. suis ST28 strains. All the mice in the NSUI002 and NSUI062 groups survived, while approx. 75% of the animals in the NSUI036 group died from septicemia or meningitis. Animals that received strains NSUI010 or NSUI081 showed reduced mortality compared to NSUI036. Significant differences in survival (LogRank test) are depicted in the Fig Bacteremia at 24 h (B) and 48 h (C) post-infection (pi). NSUI002 and NSUI062 were isolated at lower titers than the other three strains following inoculation. The different symbols represent values from individual mice. The horizontal lines indicate the geometrical mean for each group. Significant differences in isolation from blood were noted at 24 h between NSUI036 and NSUI002 and NSUI062 and 48 h pi between NSUI002 and NSUI010, NSUI081 and NSUI036 and between NSUI062 and NSUI036 only (ANOVA on ranks, P < 0.05).

Fig 5. Genetic organization and predicted open reading frames of NSUI002 (clade I) and NSUI036 (clade V) regions containing a fucose utilization operon and zmpC genes.

The region spans from position 1,054,531 to 1,219,953 in strain NSUI002, and contains 152 CDSs. In NSUI036, the region is notably smaller (83,658 bp). Differences in size are mainly due to the absence in the genome of strain NSUI036 of an approx. 83 kbp mobile genetic element (MGE), highlighted in light blue, that contains gene tetO. Other differences include a small MGE (highlighted in light blue) in NUI036 that contains gene ermB. In the conserved area, we observed a high degree of gene content conservation, with the exception of a zmpC gene also known as iga, encoding an IgA protease involved in S. suis virulence [59, 60], which is intact in NSUI036 but disrupted by a transposon insertion in strain NSUI002.