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

Abstract

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.

Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice

“…Modern techniques such as metagenomics and next-generation sequencing (NGS) assays reveal a wide array of host-associated organisms that can influence the outcome of disease as well as potential microbial effects on the studied phenotype that are currently less understood. 7,27,38 As the host microbiome can lead to biotransformation, modified absorption, and altered toxicity of compounds, incorporation of these tools in preclinical studies may be critical to the characterization of the microbial populations within study animals. 40 We experienced large-scale morbidity and mortality within a group of NSG mice that was unassociated with any known murine pathogens.…”

Perspectives of FTIR as Promising Tool for Pathogen Diagnosis, Sanitary and Welfare Monitoring in Animal Experimentation Models: A Review Based on Pertinent Literature

“…This technique can analyze proteins, nucleic acids, lipids, and carbohydrates in a biological sample, proving to be very sensitive for detecting small changes in the molecular structure, such as the secondary structure of proteins, the mutation of nucleic acids, and the peroxidation of phospholipids [4][5][6]. This tool can be combined with animal experimentation as it plays a crucial role in the development of science, to act by monitoring the well-being and health of animals, which are parameters that directly impact the quality, reproducibility, and reliability of the data generated [7,8].…”

“…To reduce the number of animals, sanitary quality, genetics, and well-being must be premises for user researchers and for all professionals involved in research, from animal husbandry to euthanasia [12,13]. The breeding of laboratory animals requires operating conditions that guarantee the welfare of the animals and their health, following the recommendations of the competent agencies [7,8]. This care is also in line with the principle of the 3Rs [7,14].…”