Pilus distribution among lineages of group b streptococcus: an evolutionary and clinical perspective

Abstract

Background: Group B Streptococcus (GBS) is an opportunistic pathogen in both humans and bovines. Epidemiological and phylogenetic analyses have found strains belonging to certain phylogenetic lineages to be more frequently associated with invasive newborn disease, asymptomatic maternal colonization, and subclinical bovine mastitis. Pilus structures in GBS facilitate colonization and invasion of host tissues and play a role in biofilm formation, though few large-scale studies have estimated the frequency and diversity of the three pilus islands (PIs) across diverse genotypes. Here, we examined the distribution of pilus islands (PI) 1, 2a and 2b among 295 GBS strains representing 73 multilocus sequence types (STs) belonging to eight clonal complexes. PCR-based RFLP was also used to evaluate variation in the genes encoding pilus backbone proteins of PI-2a and PI-2b.

Results: All 295 strains harbored one of the PI-2 variants and most human-derived strains contained PI-1. Bovine-derived strains lacked PI-1 and possessed a unique PI-2b backbone protein allele. Neonatal strains more frequently had PI-1 and a PI-2 variant than maternal colonizing strains, and most CC-17 strains had PI-1 and PI-2b with a distinct backbone protein allele. Furthermore, we present evidence for the frequent gain and loss of genes encoding certain pilus types.

Conclusions: These data suggest that pilus combinations impact host specificity and disease presentation and that diversification often involves the loss or acquisition of PIs. Such findings have implications for the development of GBS vaccines that target the three pilus islands.

Figure 1

Evolutionary relationships and pilus island (PI) profiles. The Neighbor-Joining method was used to infer the evolutionary history among 73 sequence types (STs) representing 3,456 nucleotides, or seven genes. Evolutionary distances were calculated using the p-distance method that represents the number of base differences per site. Numbers at the ends of each branch indicate the STs; grey shading represents human-derived strains from patients with invasive disease while STs shown in red are bovine-derived. Four STs (1, 2, 19, and 23) comprised strains from both humans with and without disease as well as bovines and are indicated in red. The seven clonal complexes (CCs) contained STs that clustered together with significant bootstrap support or that were identified in prior studies. Bootstrap values are indicated at the nodes. Pilus profiles for each ST are shown as colored circles: PI-1 (blue), PI-2a (red), and PI-2b (yellow). Black circles represent those STs containing strains that lacked the PI-1 but possessed an occupied PI-1 integration site.

Figure 2

Recombination among GBS genotypes. The Neighbor-net analysis highlighted a complex network with evidence of recombination, which is represented as parallelograms, among the 73 multilocus sequence types (STs). Clonal complexes (CCs) are presented in different colors. Closely related STs were collapsed into a single point to improve the clarity of the figure.

Figure 3

Frequency of pilus island (PI) types by clonal complexes (CCs). All 295 stains were screened for the presence of PI-1, PI-2a, and PI-2b using multiplex PCR. The frequency of each PI is illustrated across CCs, which are listed in tree order as determined using the Neighbor-Joining method (Figure 1). Strains representing STs that did not belong to one of the seven CCs were combined into a group of singletons.

Figure 4

Frequency of pilus island (PI) backbone protein genes by clonal complex (CC). The distribution of A) six gbs59 alleles specific for PI-2a is illustrated in 161 group B streptococcal strains and B) three san1519 alleles specific for PI-2b in 113 strains belonging to the seven CCs. In each figure, the CCs are listed in tree order based on the Neighbor-Joining phylogeny (Figure 1). Singletons (n = 21) were excluded from this analysis.

Figure 5

Gain and loss of pilus islands among GBS sequence types (STs). eBURST analysis was conducted on the MLST allele profiles for all 295 strains. The founding genotype was assigned to the ST that varies from the largest number of STs at a single locus. STs grouped into three main groups bovine strains indicated by red print. The PI profile distribution is indicated by the color of the circle representing each ST. Double locus variants are connected via dashed lines and STs with multiple pilus profiles are connected with orange lines. In cases where the founding genotype had strains with multiple PI profiles, the ST was listed twice. The predicted founding and co-founding genotypes were used to predict acquisition and loss of PIs, as indicated by the grey arrows.