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.

Escherichia albertii is a Gram-negative, facultative anaerobic bacillus recognized as a zoonotic foodborne pathogen. Aquatic E. albertii populations likely coexist with protist hosts. Dynamic interactions between pathogenic bacteria and their protist predators may extend beyond shared growth spaces and involve host-directed cytotoxicity. E. albertii carries cytotoxicity-associated genes such as cdt, stx, and astA. This study aimed to elucidate the role of cdtB in E. albertii interaction within the Tetrahymena thermophila CU427 strain. Cytolethal distending toxins (CDTs) are tripartite protein exotoxins; CdtB requires CdtA and CdtC to bind to the glycosphingolipids ganglioside GM1 and GM3 and induce full cytotoxicity in mammalian cells. Ganglioside GM1 was highly distributed in CU427 in our study, whereas GM3 showed no comparable distribution, suggesting that ganglioside GM1 may act as a receptor for CdtB in Tetrahymena cells. However, cholera toxin B subunit treatment failed to inhibit E. albertii cytotoxicity against CU427. A LIVE/DEAD assay using Carboxyfluorescein succinimidyl ester/Propidium Iodide staining was conducted for CU427 infected with E. coli expressing recombinant E. albertii CdtB. CU427 viable cell number decreased half-fold after 24 h in a similar tendency with E. albertii infection. These results indicate that CdtB is involved in E. albertii cytotoxicity against protist hosts, particularly Tetrahymena, and suggest that the cytotoxic mechanism, especially its interaction with host cells, differs from that in mammalian cells. Thus, this study provides deeper insights into the involvement of CdtB in the E. albertii cytotoxicity mechanism.