Comparative metagenomic characterization of gut microbiota and antibiotic resistome in multi-facility SPF mice

Specific pathogen-free (SPF) mice are pivotal preclinical models linking basic microbiology to clinical translation, yet comprehensive high-resolution profiling of their gut microbiome, especially antibiotic resistance genes (ARGs), remains limited. To address this gap, metagenomic sequencing was conducted on cecal contents from C57BL/6 and BALB/c SPF mice from five Shanghai laboratory animal facilities, generating 141 Gbp high-quality sequencing data. From 1,761,909 predicted genes, 1,048,575 non-redundant genes were identified for analysis. Taxonomic annotation identified Bacillota (73.0%), Bacteroidota (16.6%), and Actinomycetota (2.9%) as dominant phyla. At the genus level, microbial communities varied markedly across facilities, with Muribaculaceae prevailing in SHA/SHD and Blautia or Enterococcus enriched in SHB/SHE. Beta diversity analysis showed communities clustered by facility, indicating breeding environment had a stronger impact on gut microbiota diversity than host strain. KEGG, COG, and GO functional annotation revealed broad metabolic and molecular diversity. Antibiotic resistome profiling identified 11 ARG categories, predominantly associated with glycopeptides (18.1%) and tetracycline (11.3%) resistance. The most enriched ARG carriers were Pseudomonadota (acrD, emrB, mdtB etc.), Bacillota (tet(44), tet(M), tet(O) etc.), Bacteroidota (tet(Q), mel, tet(X) etc.), and Actinomycetota (rpoB, ileS). Furthermore, ARGs resistance mechanisms varied between facilities with distinct beta-diversity clustering: SHB and SHE mice mainly employed antibiotic target alteration against glycopeptides, whereas SHA, SHD, and SHC-C57BL/6 primarily utilized antibiotic target protection against tetracyclines. This study presents a high-resolution comparison of gut microbiota and ARGs in SPF mice from multiple facilities, highlighting facility-dependent microbial and resistome variation and providing valuable references for preclinical microbiological standardization and risk assessment.