Longitudinal Urinary Microbiome Studies: A Need to Transition to Voided Urine

It is now established that the bladder is not sterile; it contains communities of microbes (microbiota). While the healthy bladder microbiota have been defined using expanded quantitative urine culture (EQUC) and urines obtained by transurethral catheterization (TUC), longitudinal and population studies have not previously been possible. These studies cannot be done using TUC urines, as it would be impractical to catheterize participants daily. Instead, we must transition to using voided urines.In order to make this switch, we have addressed three main issues raised by using voided urine. First, we showed that EQUC out performs standard urine culture on voided urines in a clinically relevant population. Next, we defined the constituents of the urethral and peri-urethral microbiota in an attempt to deconstruct voided urine. Finally, we identified a cleaner catch method. Combined, these studies provide crucial insight for interpreting voided urine for urinary microbiome research

Elucidating Enterococcal Mechanisms Underlying Recurrent Urinary Tract Infections

Urinary tract infection (UTI) is one of the most common bacterial infections globally and can lead to life-threatening complications. UTI disproportionately affects females, with the majority of females being treated for at least one UTI in their lifetime. Of the females who develop UTI in their lifetime, almost one third will develop recurrence within six months after initial treatment and many will struggle with UTI recurrence for long spans of their lives. Although recurrent UTI has previously been attributed to persistence of Escherichia coli, recent studies utilizing more sensitive detection methods have shown Enterococcus faecalis is strongly associated with recurrent and chronic lower urinary tract symptoms. Because E. faecalis is largely regarded as a commensal organism pushed to an opportunistic pathogenic lifestyle in specific cases and the fact that roughly half of all E. faecalis-UTI cases are missed by standard detection methods, mechanisms underlying colonization and pathogenesis is understudied in the bladder. Currently, the understanding of enterococcal UTI is based on a small fraction of urinary E. faecalis isolates. This knowledge gap prevents the urobiome field from accurately identifying and studying enterococcal factors important for bladder colonization and establishment of persistent infection. Thus, this dissertation highlights the importance of E. faecalis in various lower urinary tract conditions and compares genetic profiles of urogenital E. faecalis isolates to determine factors that are enriched in cases of asymptomatic colonization, sporadic UTI, and recurrent UTI. Upon identification of bacterial factors unique to recurrent UTI isolates, additional experiments utilizing bladder organoids investigate their impact on the host response and overall environment

Comparative genomic analysis of clinical Enterococcus faecalis distinguishes strains isolated from the bladder

Abstract Background Enterococcus faecalis is the most commonly isolated enterococcal species in clinical infection. This bacterium is notorious for its ability to share genetic content within and outside of its species. With this increased proficiency for horizontal gene transfer, tremendous genomic diversity within this species has been identified. Many researchers have hypothesized E. faecalis exhibits niche adaptation to establish infections or colonize various parts of the human body. Here, we hypothesize that E. faecalis strains isolated from the human bladder will carry unique genomic content compared to clinical strains isolated from other sources. Results This analysis includes comparison of 111 E. faecalis genomes isolated from bladder, urogenital, blood, and fecal samples. Phylogenomic comparison shows no association between isolation source and lineage; however, accessory genome comparison differentiates blood and bladder genomes. Further gene enrichment analysis identifies gene functions, virulence factors, antibiotic resistance genes, and plasmid-associated genes that are enriched or rare in bladder genomes compared to urogenital, blood, and fecal genomes. Using these findings as training data and 682 publicly available genomes as test data, machine learning classifiers successfully distinguished between bladder and non-bladder strains with high accuracy. Genes identified as important for this differentiation were often related to transposable elements and phage, including 3 prophage species found almost exclusively in bladder and urogenital genomes. Conclusions E. faecalis strains isolated from the bladder contain unique genomic content when compared to strains isolated from other body sites. This genomic diversity is most likely due to horizontal gene transfer, as evidenced by lack of phylogenomic clustering and enrichment of transposable elements and prophages. Investigation into how these enriched genes influence host-microbe interactions may elucidate gene functions required for successful bladder colonization and disease establishment

Cataloging the phylogenetic diversity of human bladder bacterial isolates

Abstract Background Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, such as the gut and oral cavity. Results To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, we present a bladder-specific bacterial isolate reference collection comprising 1134 genomes, primarily from adult females. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial isolate reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2% of the genera found when re-examining previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis finds that the taxonomies and functions of the bladder microbiota share more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder Escherichia coli isolates and 387 gut Escherichia coli isolates support the hypothesis that phylogroup distribution and functions of Escherichia coli strains differ dramatically between these two very different niches. Conclusions This bladder-specific bacterial isolate reference collection is a unique resource that will enable bladder microbiota research and comparison to isolates from other anatomical sites