Recent advances in the analysis of microbial communities colonizing the human body have identified a resident microbial community in the human urinary tract (UT). Compared to many other microbial niches, the human UT harbors a relatively low biomass. Studies have identified many genera and species that may constitute a core urinary microbiome. However, the contribution of the UT microbiome to urinary tract infection (UTI) and recurrent UTI (rUTI) pathobiology is not yet clearly understood. Evidence suggests that commensal species within the UT and urogenital tract (UGT) microbiomes, such as Lactobacillus crispatus, may act to protect against colonization with uropathogens. However, the mechanisms and fundamental biology of the urinary microbiome-host relationship are not understood. The ability to measure and characterize the urinary microbiome has been enabled through the development of next-generation sequencing and bioinformatic platforms that allow for the unbiased detection of resident microbial DNA. Translating technological advances into clinical insight will require further study of the microbial and genomic ecology of the urinary microbiome in both health and disease. Future diagnostic, prognostic, and therapeutic options for the management of UTI may soon incorporate efforts to measure, restore, and/or preserve the native, healthy ecology of the urinary microbiomes.

No abstract

Glycosaminoglycans (GAGs) are linear polysaccharides and are among the primary components of mucosal surfaces in mammalian systems. The GAG layer lining the mucosal surface of the urinary tract is thought to play a critical role in urinary tract homeostasis and provide a barrier against urinary tract infection (UTI). This key component of the host-microbe interface may serve as a scaffolding site or a nutrient source for the urinary microbiota or invading pathogens, but its exact role in UTI pathogenesis is unclear. Although members of the gut microbiota have been shown to degrade GAGs, the utilization and degradation of GAGs by the urinary microbiota or uropathogens had not been investigated. In this study, we developed an in vitro plate-based assay to measure GAG degradation and utilization and used this assay to screen a library of 37 urinary bacterial isolates representing both urinary microbiota and uropathogenic species. This novel assay is more rapid, inexpensive, and quantitative compared to previously developed assays, and can measure three of the major classes of human GAGs. Our findings demonstrate that this assay captures the well-characterized ability of Streptococcus agalactiae to degrade hyaluronic acid and partially degrade chondroitin sulfate. Additionally, we present the first known report of chondroitin sulfate degradation by Proteus mirabilis, an important uropathogen and a causative agent of acute, recurrent, and catheter-associated urinary tract infections (CAUTI). In contrast, we observed that uropathogenic Escherichia coli (UPEC) and members of the urinary microbiota, including lactobacilli, were unable to degrade GAGs.

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data may include mobile genetic elements, such as plasmids and extrachromosomal phage, that contribute to the dissemination of antimicrobial resistance and further complicate treatment of urinary tract infection (UTI). In addition to providing fine resolution of genome structure, complete, closed genomes allow for the detailed comparative genomics and evolutionary analyses. The generation of complete genomes de novo has long been a challenging task due to limitations of available sequencing technology.Paired-end Next Generation Sequencing (NGS) produces high quality short reads often resulting in accurate but fragmented genome assemblies. On the contrary, Nanopore sequencing provides long reads of lower quality normally leading to errorprone complete assemblies. Such errors may hamper genome-wide association studies or provide misleading variant analysis results. Therefore, hybrid approaches combining both short and long reads have emerged as reliable methods to achieve highly accurate closed bacterial genomes. Reported herein is a comprehensive method for the culture of diverse urinary bacteria, species identification by 16S rRNA gene sequencing, extraction of genomic DNA (gDNA), and generation of short and long reads by NGS and Nanopore platforms, respectively. Additionally, this method describes a bioinformatic pipeline of quality control, assembly, and gene prediction algorithms for the generation of annotated complete genome sequences. Combination of bioinformatic tools enables the selection of high quality read data for hybrid genome assembly and downstream analysis. The streamlined approach for the hybrid de novo genome assembly described in this protocol may be adapted for the use in any culturable bacteria.

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data may include mobile genetic elements, such as plasmids and extrachromosomal phage, that contribute to the dissemination of antimicrobial resistance and further complicate treatment of urinary tract infection (UTI). In addition to providing fine resolution of genome structure, complete, closed genomes allow for the detailed comparative genomics and evolutionary analyses. The generation of complete genomes de novo has long been a challenging task due to limitations of available sequencing technology.Paired-end Next Generation Sequencing (NGS) produces high quality short reads often resulting in accurate but fragmented genome assemblies. On the contrary, Nanopore sequencing provides long reads of lower quality normally leading to errorprone complete assemblies. Such errors may hamper genome-wide association studies or provide misleading variant analysis results. Therefore, hybrid approaches combining both short and long reads have emerged as reliable methods to achieve highly accurate closed bacterial genomes. Reported herein is a comprehensive method for the culture of diverse urinary bacteria, species identification by 16S rRNA gene sequencing, extraction of genomic DNA (gDNA), and generation of short and long reads by NGS and Nanopore platforms, respectively. Additionally, this method describes a bioinformatic pipeline of quality control, assembly, and gene prediction algorithms for the generation of annotated complete genome sequences. Combination of bioinformatic tools enables the selection of high quality read data for hybrid genome assembly and downstream analysis. The streamlined approach for the hybrid de novo genome assembly described in this protocol may be adapted for the use in any culturable bacteria.

Community-acquired urinary tract infection (UTI) is among the most common bacterial infections observed in humans. Postmenopausal women are a rapidly growing and underserved demographic group who are severely affected by rUTI with a >50% recurrence rate. In this population, rUTI can persist for years, reducing quality of life and imposing a significant healthcare burden. rUTI is most often treated by long-term antibiotic therapy, but development of antibiotic resistance and allergy leave physicians with fewer treatment options. The female urobiome has been identified as a key component of the urogenital environment. However, structural and functional changes in the urobiome underlying rUTI susceptibility in postmenopausal women are not well understood. Here, we used strictly curated, controlled cross-sectional human cohorts of postmenopausal women, urobiome whole genome (shotgun) metagenomic sequencing (WGMS), advanced urine culturing techniques, extensive biobanking of >900 patient-derived urinary bacterial and fungal isolates, and mass spectrometry-based estrogen profiling to survey the urobiome in rUTI patients during infection relapse and remission as well as healthy comparators with no lifetime history of UTI. Our results suggest that a history of rUTI strongly shapes the taxonomic and functional ecology of the urobiome. We also find a putative protective commensal population, consisting of species known to convey protection against bacterial vaginosis such as Lactobacillus crispatus, within the urobiome of women who do not experience UTI. Integration of clinical metadata detected an almost exclusive enrichment of putative protective species belonging to the genus, Lactobacillus, in women taking estrogen hormone therapy (EHT). We further show that the urobiome taxonomic ecology is shaped by EHT, with strong enrichments of putatively protective lactobacilli, such as L. crispatus and L. vaginalis. Integrating quantitative metabolite profiling of urinary estrogens with WGMS, we observed robust associations between urobiome taxa, such as Bifidobacterium breve and L. crispatus, and urinary estrogen conjugate concentrations, suggesting that EHT strongly alters the taxonomic composition of the female urobiome. We have further used functional metagenomic profiling and patient-derived isolate phenotyping to identify microbial metabolic pathways, antimicrobial resistance genes (ARGs), and clinically relevant antimicrobial resistance phenotypes enriched between disease-states. Our data suggest distinct metabolic and ARG signatures of the urobiome associated with current rUTI status and history. Taken together, our data suggests that rUTI history and estrogen use strongly shape the functional and taxonomic composition of the urobiome in postmenopausal women.

Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI). This disease disproportionately affects women and frequently develops into recurrent UTI (rUTI) in postmenopausal women. Here, we report the complete genome sequences of seven UPEC isolates obtained from the urine of postmenopausal women with rUTI.

Enterococcus raffinosus is an understudied member of its genus possessing a characteristic megaplasmid contributing to a large genome size. Although less commonly associated with human infection compared to other enterococci, this species can cause disease and persist in diverse niches such as gut, urinary tract, blood, and environment. Few complete genome assemblies have been published to date for E. raffinosus. In this study, we report the complete assembly of the first clinical urinary E. raffinosus strain, Er676, isolated from a postmenopausal woman with history of recurrent urinary tract infection (rUTI). We additionally completed the assembly of clinical type strain ATCC49464. Comparative genomic analyses reveal inter-species diversity driven by large accessory genomes. The presence of a conserved megaplasmid indicates it is a ubiquitous and vital genetic feature of E. raffinosus. We find that the E. raffinosus chromosome is enriched for DNA replication and protein biosynthesis genes while the megaplasmid is enriched for transcription and carbohydrate metabolism genes. Prophage analysis suggests that diversity in the chromosome and megaplasmid sequences arises, in part, from horizontal gene transfer. Er676 demonstrated the largest genome size reported to date of E. raffinosus and the highest probability of human pathogenicity. Er676 also possesses multiple antimicrobial resistance genes (ARGs), of which all but one are encoded on the chromosome, and has the most complete prophage sequences. Complete assembly and comparative analyses of the Er676 and ATCC49464 genomes provide important insight into the inter-species diversity of E. raffinosus that enables its ability to colonize and persist in the human body. Investigating genetic factors that contribute to this species pathogenicity will lend valuable tools to combat diseases caused by this opportunistic pathogen.