Abstract
ABSTRACTThe human bladder contains bacteria, even in the absence of infection. Interest in studying these bacteria and their association with bladder conditions is increasing. However, the chosen experimental method can limit the resolution of the taxonomy that can be assigned to the bacteria found in the bladder. 16S rRNA amplicon sequencing is commonly used to identify bacteria in urinary specimens, but it is typically restricted to genus-level identification. Our primary aim here was to determine if accurate species-level identification of bladder bacteria is possible using 16S rRNA amplicon sequencing. We evaluated the ability of different classification schemes, each consisting of combinations of a reference database, a 16S rRNA gene variable region, and a taxonomic classification algorithm to correctly classify bladder bacteria. We show that species-level identification is possible and that the reference database chosen is the most important component, followed by the 16S variable region sequenced.IMPORTANCE Accurate species-level identification from culture-independent techniques is of importance for microbial niches that are less well characterized, such as that of the bladder. 16S rRNA amplicon sequencing, a common culture-independent way to identify bacteria, is often critiqued for lacking species-level resolution. Here, we extensively evaluate classification schemes for species-level bacterial annotation of 16S amplicon data from bladder bacteria. Our results show that the proper choice of taxonomic database and variable region of the 16S rRNA gene sequence makes species level identification possible. We also show that this improvement can be achieved through the more careful application of existing methods and resources. Species-level information may deepen our understanding of associations between bacteria in the bladder and bladder conditions such as lower urinary tract symptoms and urinary tract infections.
Highlights
The human bladder contains bacteria, even in the absence of infection
All 78 bladder bacterial species were present in the Silva and NCBI 16S Microbial databases, whereas only 21 species were present in the Greengenes database
Our findings show that use of the V2-V3 and V1-V3 regions of the 16S rRNA gene allowed for the correct identification of the most bladder bacterial species when msystems.asm.org 9 combined with the NCBI 16S database and either classifier
Summary
The human bladder contains bacteria, even in the absence of infection. Interest in studying these bacteria and their association with bladder conditions is increasing. Culturing specific bacterial species is time-consuming and laborious This limitation has been circumvented by adopting culture-independent methods of sequencing DNA directly from an environmental sample, such as shotgun metagenomic sequencing and targeted amplicon sequencing, the latter most commonly involving the 16S rRNA gene [17, 18]. Targeted amplicon sequencing of the 16S rRNA gene is a common method for identifying bladder bacteria in a large-scale manner [20, 21] This is true in urine, where the low microbial biomass [9, 22] often results in smaller bacterial DNA quantities than what is required for shotgun metagenomic sequencing [23]. Bioinformatics are used to process the resulting sequences and identify the taxonomy of the bacteria [24]
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