Microbiota and environmental health monitoring of mouse colonies by metagenomic shotgun sequencing

Metagenomic next-generation sequencing (mNGS) allows the monitoring of microbiota composition of murine colonies employed for scientific purposes in a single test by assessing the composition of gut microbiome and the detection of pathogens from fecal pellets. In this study, we tested the potential use of mNGS for monitoring both microbiota composition and the presence of pathogens through Environmental Health Monitoring, by using exhaust dust collection filters derived from individually ventilated cages (IVC) systems. mNGS analysis was performed on nucleic acids isolated from filters collecting air from the exhaust of: (1) cages with mice housed in a non-pathogen free facility; (2) animal-free cages with clean chow and bedding from the same facility; (3) cages housing mice from a specific-pathogen free (SPF) facility. mNGS results revealed correspondence between microbiome composition from fecal pellets and filter, including pathogenic bacteria (Helicobacter hepaticus, Helicobacter typhlonius, Chlamydia muridarum, Rodentibacter pneumotropicus, Citrobacter rodentium), intestinal protozoa (Tritrichomonas muris, Spironucleus muris) nematoda (Aspiculuris tetraptera) and eukaryotic parasites (Myocoptes musculinus), present in the colony. Entamoeba muris and Syphacia obvelata were detected in fecal pellets but not in filter. The animal free exhaust dust filter, exposed to clean cages (no mice) placed in the IVC after removal of all mice, exhibited the presence of the same pathogens due to contaminated connecting pipes, confirming the sensitivity of the approach. Conversely, the filter from SPF colony revealed the absence of pathogens. The current use of exhaust dust collection filters in health surveillance requires multiple molecular tests to identify specific pathogens and does not provide information on the colony microbiome. This work provides the proof-of-principle that assaying exhaust dust collection filters by mNGS for microbiota monitoring of laboratory mice is feasible. In its daily application, results suggest the usefulness of the test in SPF facilities, where pathogenic micro-organisms are expected to be absent. mNGS analysis of exhaust dust collection filters allows the analysis of multiple cages, reducing the number of tests required for pathogen detection and corresponding costs, and avoiding the use of sentinel mice.