Urine sterility and the bacteria that routine cultures missed
Urine sterility met a concrete obstacle in 2018: a collection of 149 bacterial strains grown from bladder samples. Krystal Thomas-White and colleagues isolated and sequenced those organisms from catheter-collected urine supplied by 77 women. Of those women, 38 had no urinary symptoms and 39 had symptoms. The Nature Communications study archived 67 isolates from the asymptomatic participants and 82 from those with symptoms.
The collection covered 78 species. Comparing genomes also revealed highly similar strains in the bladder and vagina of the same women, including Lactobacillus crispatus and Lactobacillus iners. Those organisms extended the overlap beyond bacteria associated with disease. For urine sterility, cultivation supplied a crucial distinction: the organisms had grown, rather than merely leaving detectable genetic material. The team preserved bacterial isolates alongside their genome sequences, creating a reference collection for research into what these organisms do. Their interpretation was an interconnected microbial community across neighboring anatomical sites.
Earlier investigations had exposed the limits of ordinary bacterial inventories. In 2013, Debbie Lewis and colleagues examined urine from 16 adults: 10 women and six men, aged 26 to 90. They used sequencing of a bacterial marker gene, 16S rRNA, to identify organisms represented in the samples. They detected 94 bacterial genera, of which only 31 were likely to be cultivated routinely by standard methods. The other 63 were either outside routine cultivation or would not normally receive an individual identification in routine laboratory reporting.
Collection imposed a limit on that result. These were midstream specimens, which could include organisms from the urethra or surrounding tissues. The participants lacked lower urinary tract symptoms but were hospital outpatients attending for other reasons, making them an imperfect stand-in for the wider population. The investigators acknowledged both limitations. Their challenge to urine sterility concerned a detectable microbial population in collected specimens; the sampling route left the precise origin of every organism less certain.
Lisa Karstens and colleagues approached anatomical origin more directly at Oregon Health and Science University. Their 2016 pilot study collected catheter specimens from 10 women with normal bladder function and 10 with urgency urinary incontinence. Bacterial DNA was successfully sequenced from 95% of the samples. The team found multiple bacterial types in both groups, with 14 showing differences in relative abundance between them. Here, a microbial signal was present even among participants recruited specifically for normal bladder function.
The results also resisted a tidy bacterial scorecard. Overall measures of diversity did not differ significantly between the two groups, although lower diversity correlated with greater symptom severity within the incontinence group. Individual specimens varied considerably. The investigators described the work as a pilot requiring validation in larger populations. The groups were also balanced for menopausal status and estrogen use, factors that had differed between groups in some earlier investigations.
Scale increased sharply in a study published by Junjie Qin and colleagues in 2021. Their team recruited 1,172 Chinese adults aged 45 to 86 from three regions, with specimens collected during 2016. After quality controls excluded seven samples, 1,165 remained. The investigation included negative controls to help identify signals arising from contamination, a particular difficulty when the biological material contains relatively little bacterial material. Candidate sequences were filtered before the investigators described the microbial communities.
The researchers deliberately used the term genitourinary microbiome. Midstream urine could contain organisms from across the urinary system and nearby genital regions, so the label reflected the material actually sampled. Several different community types appeared, commonly dominated by genera including Prevotella, Streptococcus, Lactobacillus, or Gardnerella. Composition differed between men and women and could vary with age. After filtering, 2,475 distinct sequence variants remained in the dataset.
Even the computer analysis became part of the historical record. In 2022, Nazema Siddiqui and colleagues revisited sequencing data from 123 women with mixed urinary incontinence and 84 controls. Using updated processing and reference resources, they recovered bacterial classifications that differed from the earlier analysis. Only 40% of the newly identified classifications had also been identified originally. Yet the classifications shared by both methods represented more than 99% of the sequence abundance. Much of the disagreement concerned organisms represented by relatively few sequences.
For urine sterility studies, the updated approach accounted for sequencing error when grouping genetic variants, replacing an earlier method based on similarity. The reanalysis also changed some clinical associations: relationships that had appeared to differ by age group were absent after the updated analysis incorporated different adjustment variables. Associations between certain bacterial communities and mixed incontinence remained. The investigators also found that sequencing different regions of the same marker gene recovered different sets of bacterial classifications.
- Thomas-White et al. (2018), Culturing of female bladder bacteria reveals an interconnected urogenital microbiota — Nature Communications — www.nature.com
- Lewis et al. (2013), The human urinary microbiome; bacterial DNA in voided urine of asymptomatic adults — Frontiers in Cellular and Infection Microbiology — www.frontiersin.org
- Karstens et al. (2016), Does the Urinary Microbiome Play a Role in Urgency Urinary Incontinence and Its Severity? — Frontiers in Cellular and Infection Microbiology — www.frontiersin.org
- Qin et al. (2021), Characterization of the Genitourinary Microbiome of 1,165 Middle-Aged and Elderly Healthy Individuals — Frontiers in Microbiology — www.frontiersin.org
- Siddiqui et al. (2022), Updating Urinary Microbiome Analyses to Enhance Biologic Interpretation — Frontiers in Cellular and Infection Microbiology — www.frontiersin.org