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.