Influence of Water Delivery Method on the Gut Microbiome in Laboratory Mice (Mus musculus)

Nuances related to the milieu of the gastrointestinal tract have led to investigations of environmental (or extrinsic) factors, like feed sources and fluid intake, and their influences on the gut microbiome in research animals. Water is typically provided to laboratory mice either by reusable water bottle (RWB), housing rack automatic water (RAW) delivery, or single-use disposable plastic pouch (DPP). In this study, the influence of differing water delivery methods on gut microbiome stability was evaluated in immunocompetent (n = 36 B6; 18 male [M]:18 female [F]) and immunocompromised (n = 36 NOG; 18 M:18 F) strains of mice. Mice were housed on a single IVC rack in sex-specific groups and provided with autoclaved caging and bedding, irradiated feed, and chlorinated, reverse-osmosis water provided by one of 3 delivery methods (8 cages per method). Access to the room was restricted to select personnel to conduct animal care and sample collection tasks. Fecal pellets (n = 2) were collected from each animal every other week, and water samples were collected weekly for analysis. Over the course of the study, bacteria were detected in 11% of the RWB samples (7 of 63) and 4% of the RAW samples (1 of 25). DPP samples were consistently free of bacterial contamination. Shotgun metagenomics and statistical analyses revealed overt shifts in gut microbiota in the majority of mice throughout the study (21 of 25 cages). Histologic examinations of organs from representative clinically normal study mice (n = 12) were unremarkable. With minimal exceptions, microbiome shifts were statistically significant across cage cohorts, despite attempts to control experimental variables. This study is the first to demonstrate that the water delivery method does not impart a significant influence on gut microbiota stability in research rodents and highlights the need to document water type, treatment, and delivery method as extrinsic factors in reporting animal studies.