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.
Color-coded legend of taxa (families) that were most commonly observed in microbiome analyses. The number in parentheses represents the National Center for Biotechnology Information taxonomic identifier (https://www.ncbi.nlm.nih.gov/) in case the strain nomenclature changes at a future date.
Relative abundance levels for animals receiving rack autowater (RAW) during the study. Each cluster represents a cage of 3 animals that were profiled on days (d)7, d14, d28, and d56. All cages had significant shifts in α-diversity across time points throughout the study; the red dot is placed below the only cage (6A) in the study that did not have significant differences in β-diversity. NOG females are in cages 1 and 2, NOG males are in cages 3 and 4, B6NTac females are in cages 5 and 6, and B6NTac males are in cages 7 and 8.
Relative abundance levels for animals receiving water from disposable plastic pouches (DPP) during the study. Each cluster represents a cage of 3 animals that were profiled at d7, d14, d28 and d56. Black dots are placed below the 2 cages of B6 Females (5H and 6H) that did not have significant differences in α diversity across time points. All other cages had significant shifts in microbiome throughout the study. NOG females are in cages 1 and 2, NOG males are in cages 3 and 4, B6NTac females are in cages 5 and 6, and B6NTac males are in cages 7 and 8.
Relative abundance levels for animals receiving water from reusable water bottles (RWB) during the study. Each cluster represents a cage of 3 animals that were profiled at d7, d14, d28, and d56. Black dots are placed below the 2 cages of NOG animals (1B: females; 3B: males) that did not have significant differences in α diversity across time points. All other cages had significant shifts in microbiome throughout the study. NOG females are in cages 1 and 2, NOG males are in cages 3 and 4, B6NTac females are in cages 5 and 6, and B6NTac males are in cages 7 and 8 and 8B-2.
Contributor Notes