Genomic, Phenotypic, and Virulence Analysis of Streptococcus sanguinis Oral and Infective-Endocarditis Isolates

Abstract

Streptococcus sanguinis, an abundant and benign inhabitant of the oral cavity, is an important etiologic agent of infective endocarditis (IE), particularly in people with predisposing cardiac valvular damage. Although commonly isolated from patients with IE, little is known about the factors that make any particular S. sanguinis isolate more virulent than another or, indeed, whether significant differences in virulence exist among isolates. In this study, we compared the genomes of a collection of S. sanguinis strains comprised of both oral isolates and bloodstream isolates from patients diagnosed with IE. Oral and IE isolates could not be distinguished by phylogenetic analyses, and we did not succeed in identifying virulence genes unique to the IE strains. We then investigated the virulence of these strains in a rabbit model of IE using a variation of the Bar-seq (barcode sequencing) method wherein we pooled the strains and used Illumina sequencing to count unique barcodes that had been inserted into each isolate at a conserved intergenic region. After we determined that several of the genome sequences were misidentified in GenBank, our virulence results were used to inform our bioinformatic analyses, identifying genes that may explain the heterogeneity in virulence. We further characterized these strains by assaying for phenotypes potentially contributing to virulence. Neither strain competition via bacteriocin production nor biofilm formation showed any apparent relationship with virulence. Increased cell-associated manganese was, however, correlated with blood isolates. These results, combined with additional phenotypic assays, suggest that S. sanguinis virulence is highly variable and results from multiple genetic factors.

Keywords: Streptococcus; biofilms; genomics; infective endocarditis; manganese; viridans; virulence.

FIG 1

Cluster analysis of 19 genomes by PGAP in GF mode. Upper curve, pangenome calculation; lower curve, core genome calculation.

FIG 2

Phylograms computed by PGAP using the neighbor-joining method of clustering. Clades are labeled and accentuated by darker branches emanating from the clade’s most basal node. Red indicates blood isolates, and blue denotes oral isolates. (A) Pangenome-based phylogram created using a gene distance matrix approach built upon gene presence or absence in each strain. (B) SNP-based phylogram created from analysis of core genes lacking paralogs.

FIG 3

Selection of a conserved intergenic region (CIR) for insertion of exogenous DNA into S. sanguinis strains. The spectinomycin resistance cassette is denoted aad9. The stem and loop structure represents the terminator sequence.

FIG 4

Examination of SK36 derivatives in a rabbit model of IE. Rabbits were coinoculated with the strains indicated. Like shapes indicate values obtained from the same rabbit in a single experiment. Geometric means are indicated by horizontal bars. Significance was determined by repeated-measures ANOVA with Tukey-Kramer post hoc test. **, P < 0.001.

FIG 5

Examination of SK36 and SK405 derivatives in a rabbit model of IE. Rabbits were coinoculated with the strains indicated. Like shapes indicate values obtained from the same rabbit in a single experiment. Geometric means are indicated by horizontal bars. Significance was determined by repeated-measures ANOVA with Tukey-Kramer post hoc test. **, P < 0.01; ***, P < 0.001.

FIG 6

Use of a conserved intergenic region for barcode insertion. Stem and loop structure indicates terminator sequence. An asterisk indicates the last bp of stop codon in SSA_1217. Downward arrow indicates barcode (BC) insertion site. Right arrow indicates start of terminator sequence. P1 and P2 designate primers used to amplify DNA from inoculum cells and from harvested rabbit vegetations for Illumina sequencing.

FIG 7

Misidentified GenBank WGS sequences as determined by our analysis.

FIG 8

Examination of marked strains in a rabbit model of IE. Six rabbits were coinoculated with 17 barcoded strains in one of two duplicate pools. Blue and red indicate oral and blood isolates, respectively. % Exp., percent expected. (A) The % Exp. Inoculum for each strain was determined by dividing the BC counts for each strain by the total BC counts for that inoculum and then multiplying by 17. (B) The % Exp. Recovered was determined similarly by the equation (% each strain in the total recovered counts for each rabbit)/(% each strain in the total inoculum counts). The dashed line represents 1/17 of total bacterial burden in each inoculum (A) and percent abundance in each rabbit (B) relative to its abundance in the corresponding inoculum. There was no significant association between strain source (blood of IE patient versus oral cavity) and virulence (P = 0.1234 by unpaired t test).

FIG 9

S. sanguinis biofilm production. Strains were grown in biofilm medium supplemented with either 1% sucrose (A) or 1% glucose (B) and grown under anaerobic or aerobic conditions, respectively. Biofilm formation was determined by absorbance at 560 nm. Blue indicates oral isolates, while red indicates blood isolates. Results are from four experiments. There was no significant correlation between biofilm formation and virulence, as determined by Pearson correlation, for results shown in panel A (P = 0.2392) or B (P = 0.5686). There was no significant relationship between biofilm formation and strain origin, as determined by unpaired t test, for results shown in panel A (P = 0.6493) or B (P = 0.3029).

FIG 10

Cell-associated manganese content of S. sanguinis strains. Strains were grown in 12% O₂ and analyzed by ICP-OES, displayed in order of decreasing virulence (A) or decreasing Mn content (B). Means and standard deviations from four independent experiments are shown. (A) There was no significant correlation between virulence and Mn content, as determined by Pearson correlation (P = 0.9350). (B) There was significant correlation between strain origin and Mn content, as determined by unpaired t test (P = 0.0124).