Streptococcus anginosus is a prevalent member of the human flora. While it has been found in the microbiota of \"healthy\" asymptomatic individuals, it has also been associated with genitourinary tract infections and bacteremia. Based upon multilocus sequence analysis, two subspecies and two genomosubspecies have been characterized for the species. We previously conducted whole-genome sequencing of 85 S. anginosus isolates from the urinary tract. Here, we present genomic analysis of this species, including isolates from the urinary tract as well as gut and fecal, vaginal, oral, respiratory, and blood and heart samples. Average nucleotide identity and core genome analysis revealed that these strains form two distinct groups. Group 1 is comprised of the S. anginosus type strain and other previously identified S. anginosus subspecies and genomosubspecies, including isolates from throughout the human body. In contrast, group 2 consists of predominantly urinary streptococci (n = 77; 85.6%). Both of these S. anginosus groups are distinct from other members of the Streptococcus anginosus group (SAG) species S. intermedius and S. constellatus. Genes conserved among all strains of one group but not in any strains in the other group were next identified. Group 1 strains included genes found in S. intermedius and S. constellatus, suggesting that they were lost within the ancestor of the group 2 strains. In contrast, genes unique to the group 2 strains were homologous to more distant streptococci, indicative of acquisition via horizontal gene transfer. These genes are ideal candidates for use as marker genes to distinguish between the two groups in the human microbiota. IMPORTANCE Whole-genome analysis of S. anginosus strains provides greater insight into the diversity of this species than from marker genes alone. Our investigation of 166 publicly available S. anginosus genomes via average nucleotide identity and core genome analysis revealed two phylogenomically distinct groups of this species, with one group almost exclusively consisting of isolates from the urinary tract. In contrast, only 8 urinary strains were identified within the other group, which contained the S. anginosus type strain, as well as all identified subspecies and genomosubspecies. While genomic analysis suggested that this urinary group of S. anginosus is genomically different from the previously characterized S. anginosus subspecies, phenotypic characterization is still needed. Given prior reports of the prevalence of S. anginosus in the urinary tract of both continent and incontinent females, future studies are needed to investigate if the symptom state of the urinary tract is associated with these two different groups.

Since the discovery of the bladder microbiome (urobiome), interest has grown in learning whether urobiome characteristics have a role in clinical phenotyping and provide opportunities for novel therapeutic approaches for women with common forms of urinary incontinence.
This study aimed to test the hypothesis that the bladder urobiome differs among women in the control cohort and women affected by urinary incontinence by assessing associations between urinary incontinence status and the cultured urobiome.
With institutional review board oversight, urine specimens from 309 adult women were collected through transurethral catheterization. These women were categorized into 3 cohorts (continent control, stress urinary incontinence [SUI], and urgency urinary incontinence [UUI]) based on their responses to the validated Pelvic Floor Distress Inventory (PFDI) questionnaire. Among 309 women, 150 were in the continent control cohort, 50 were in the SUI cohort, and 109 were in the UUI cohort. Symptom severity was assessed by subscale scoring with the Urinary Distress Inventory (UDI), subscale of the Pelvic Floor Distress Inventory. Microbes were assessed by expanded quantitative urine culture protocol, which detects the most common bladder microbes (bacteria and yeast). Microbes were identified to the species level by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Alpha diversity indices were calculated for culture-positive samples and compared across the 3 cohorts. The correlations of UDI scores, alpha diversity indices, and species abundance were estimated.
Participants had a mean age of 53 years (range 22-90); most were whites (65%). Women with urinary incontinence were slightly older (control, 47; SUI, 54; UUI, 61). By design, UDI symptom scores differed (control, 8.43 [10.1]; SUI, 97.95 [55.36]; UUI, 93.71 [49.12]; P<.001). Among 309 participants, 216 (70%) had expanded quantitative urine culture-detected bacteria; furthermore, the urinary incontinence cohorts had a higher detection frequency than the control cohort (control, 57%; SUI, 86%; UUI, 81%; P<.001). In addition, the most frequently detected species among the cohorts were as follows: continent control, Lactobacillus iners (12.7%), Streptococcus anginosus (12.7%), L crispatus (10.7%), and L gasseri (10%); SUI, S anginosus (26%), L iners (18%), Staphylococcus epidermidis (18%), and L jensenii (16%); and UUI, S anginosus (30.3%), L gasseri (22%), Aerococcus urinae (18.3%), and Gardnerella vaginalis (17.4%). However, only Actinotignum schaalii (formerly Actinobaculum schaalii), A urinae, A sanguinicola, and Corynebacterium lipophile group were found at significantly higher mean abundances in 1 of the urinary incontinence cohorts when compared with the control cohort (Wilcoxon rank sum test; P<.02), and no individual genus differed significantly between the 2 urinary incontinence cohorts. Both urinary incontinence cohorts had increased alpha diversity similar to continent control cohort with indices of species richness, but not evenness, strongly associated with urinary incontinence.
In adult women, the composition of the culturable bladder urobiome is associated with urinary incontinence, regardless of common incontinence subtype. Detection of more unique living microbes was associated with worsening incontinence symptom severity. Culturable species richness was significantly greater in the urinary incontinence cohorts than in the continent control cohort.

To characterise the bladder microbiota of continent adult women.
Cross-sectional study of adult women who contributed catheterised urine samples, completed validated symptom questionnaires, and provided demographic data.
US academic medical centre.
Well-characterised continent adult women.
Participants contributed symptoms questionnaires, demographic data, and catheterised urine samples that were analysed by enhanced urine culture methodology and 16S rRNA gene sequencing.
Associations between demographics and microbial community state structures (urotypes, defined by the dominant taxon of each specimen).
The bladder microbiota (urobiome) of a control group of 224 continent women were characterised, demonstrating variability in terms of urotype. The most common urotype was Lactobacillus (19%), which did not differ with any demographic. In contrast, the Gardnerella (P < 0.001) and Escherichia (P = 0.005) urotypes were more common in younger and older women, respectively.
For urobiome research, enhanced culture methods and/or DNA sequencing are the preferred techniques for bacterial detection. The interpretation of clinical tests, such as the standard urine culture, should incorporate the knowledge that some women have Gardnerella or Escherichia urotypes without evidence of any clinical disorder. Clinical care strategies should preserve or restore the beneficial effects of the native urobiome, as disruption of that microbial community could result in unintended vulnerability to uropathogen invasion or opportunistic pathogen overgrowth. Longitudinal studies of urobiome responses to therapies should be encouraged.
In continent adult women bladder microbiome composition differs by age, with relevance for clinical practice.