Species level resolution of female bladder microbiota from 16S rRNA amplicon sequencing

Abstract

The human bladder contains bacteria 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 is typically restricted to genus-level identification. Our primary aim 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. ImportanceSpecies-level information may deepen our understanding of associations between bladder microbiota and bladder conditions, such as lower urinary tract symptoms and urinary tract infections. The capability to identify bacterial species depends on large databases of sequences, algorithms that leverage statistics and available computer hardware, and knowledge of bacterial genetics and classification. Taken together, this is a daunting body of knowledge to become familiar with before the simple question of bacterial identity can be answered. Our results show the choice of taxonomic database and variable region of the 16S rRNA gene sequence makes species level identification possible. We also show this improvement can be achieved through the more careful application of existing methods and use of existing resources.