Investigating the gut bacteria structure and metabolism of hibernating bats through 16S rRNA high-throughput sequencing and culturomics

The gut microbiota of bats is vital for their health and ecosystem roles, yet studies on hibernating bats in southwest China, particularly in the unique karst landscape of Guizhou, are limited. We captured three hibernatingbat species—Pipistrellus (PB), Hipposideros (HB), and Myotis (MB)—in Liping County, collecting rectal samples for 16S rRNA amplicon sequencing. Data processing involved Trimmomatic, Flash, and Qiime2 for OTU standardization and species annotation via the Greengenes database. Differential abundance was analyzed using LEfSe, and diversity metrics were assessed through alpha and beta diversity analyses. The HB group was predominantly composed of Proteobacteria(80.99%), while MB and PB exhibited diverse compositions with significant OTU richness (729 in MB). Notable genera included Hafniaand Yersinia in HB, and Cosenzaea myxofaciensin MB. High proportions of unclassified taxa were observed, particularly in HB (83.81%). Functional predictions indicated metabolic pathways, with a significant representation of Human Diseases in PB. Culturomics revealed the successful cultivation of Huaxiibacter chinensisand Enterobacter chengduensisfrom bats for the first time. These findings highlight the ecological complexity and diversity of hibernatingbat gut microbiota, emphasizing the need for cultivation-based approaches to explore unknown bacterial species.