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, seasonal and ecological variations, 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 high-throughput 16S rRNA gene (V3–V4) amplicon sequencing. We further screened selected samples for bacterial pathogen targets using nested PCR. Results The gut microbiota was dominated by Pseudomonadota (63.6%) and Bacillota (22.3%), and ten genera in the proposed global core accounted for 74.5% of total relative abundance. In a multivariable marginal PERMANOVA model on Bray-Curtis distances including species, location, season, sex, and weight, bacterial community composition was significantly associated with host species (R² = 0.10, p = 0.005), geographical location (R² = 0.06, p = 0.005), and season (R² = 0.05, p = 0.015). Alpha diversity measured by Shannon index was statistically significant among different seasons, p = 0.023, but not in host species and sampling locations. PICRUSt2-inferred functional profiles showed no significant difference among species ( p = 0.114) or locations ( p = 0.412). Co-occurrence network analysis suggested species- and location-specific patterns in the connectivity of core taxa. Exploratory screening for pathogenic bacteria identified several bacterial taxa of potential clinical and zoonotic relevance, including Bacillus_A , corresponding to the Bacillus cereus group. Conclusions We provide a characterisation of the gut microbiome of insectivorous bats in Japan and identify host-, location- and season-associated differences in community composition, while acknowledging that these factors were partly confounded. The identification of bacterial taxa of potential clinical and zoonotic relevance highlights the value of integrating gut microbiome profiling with exploratory bacterial surveillance in wildlife. The PICRUSt2 and co-occurrence results are hypothesis-generating rather than direct evidence of microbial function or ecological interaction. Further studies are needed to characterize the bacterial taxa identified and assess their potential One Health relevance.