Characterizing the Microbiome of ’Sterile’ Organs in Experimental Mice

Abstract Recent studies have shown that microorganisms can be present in organs or blood traditionally considered sterile, both in healthy and diseased individuals. In this study, we investigated the presence of microorganisms in the “sterile” organs (brain, heart, kidney, liver, lung, and spleen) of commercially available experimental mice using culturomics and metagenomics. To avoid potential environmental contamination, strict disinfection assays were performed. Among 104 mice of the C57BL/6J, BALB/c, and ICR strains purchased from three major experimental animal suppliers in China, 23 mice (22.1%) exhibited a high microbial burden (>1 x 10 4 CFU/g tissue) in at least one organ, and 42 mice (40.4%) exhibited >1 x 10 3 CFU/g tissue in at least one organ. In total, 216 microbial species were identified through culturomics. Of them, 56 microbial species were found in the organs of at least three mice of the 42 mice (>1 x 10 3 CFU/g tissue in at least one organ). Metagenomics analysis of 10 high-burden mice verified the existence of microbes in the mouse organs and identified 262 species. Several species, including Acinetobacter sp., Alcaligenes faecalis , and Ligilactobacillus murinus , were identified as the most abundant microbes across both culturomics and metagenomics assays. 16S FISH staining assays verify the presence of microbes in the mouse organs. Feces Metagenomics analysis and detection of the wide distribution of L. murinus in the mouse mesenteric lymph nodes (MLNs) imply that microbial cells could be translocated from the gut to the MLNs and then to the remote tissues. Together, these findings reveal the presence of non-pathogenic microorganisms in the “sterile” tissues of experimental mice, raising concerns about potential variability in experimental outcomes when using such animals in research.