Gut microbiota of insectivorous bats in Japan: host-, location- and season-associated community structure and exploratory screening for bacterial pathogen targets

Background The gut microbiota of bats is increasingly studied for its ecological and public health significance, yet factors shaping gut microbial communities, such as host species, environmental location, and ecological variation, remain poorly understood in insectivorous bats in Japan. We characterised the gut microbiota of three insectivorous bat species ( Miniopterus fuliginosus , Myotis macrodactylus , and Rhinolophus ferrumequinum ) collected from three sites in Yamaguchi Prefecture, using 16S rRNA gene (V3–V4) High-throughput amplicon sequencing. We further screened for pathogenic bacteria using species-specific nested PCR. Results The gut microbiota was dominated by Pseudomonadota (63.6%) and Bacillota (22.3%). A global core of ten genera accounted for 74.5% of total relative abundance across all three bat species. Nested PCR detected DNA of Chlamydophila psittaci , Bacillus cereus , and Shiga toxin 1-producing Escherichia coli . PERMANOVA revealed that both host species (R² = 0.166, p = 0.001) and sampling location (R² = 0.132, p = 0.001) significantly influenced community composition based on Bray-Curtis dissimilarity, while alpha diversity did not differ significantly among species or locations. Functional profiles predicted by PICRUSt2 showed no significant differentiation among species ( p = 0.114) or locations ( p = 0.412), suggesting potential functional redundancy despite taxonomic variation. Co-occurrence network analysis revealed species- and location-specific patterns in the connectivity of core and potentially pathogenic taxa. Conclusions This study provides the first detection of Chlamydophila psittaci DNA in bats, alongside other pathogenic species, Bacillus cereus and Shiga toxin-producing Escherichia coli . In addition, we provide a comprehensive characterisation of the gut microbiome of insectivorous bats in Japan and show that both host species and sampling location significantly influence gut community composition. Meanwhile, predicted metabolic functions remained conserved, consistent with potential functional redundancy. The detection of pathogenic bacteria underscores the value of integrating wildlife microbiome surveillance into One Health frameworks. Future studies incorporating culture-based methods and whole-genome sequencing would strengthen the assessment of zoonotic potential.