Honeybees play a vital role in the ecological environment and agricultural economy. Increasing evidence shows that the gut microbiome greatly influences the host’s health. Therefore, a thorough understanding of gut bacteria composition can lead to the development of probiotics specific for each development stage of honeybees. In this study, the gut microbiota at different developmental stages (larvae, pupae, and adults) of the honeybees Apis cerana in Hanoi, Vietnam, was assessed by sequencing the V3–V4 region in the 16S rRNA gene using the Illumina Miseq platform. The results indicated that the richness and diversity of the gut microbiota varied over the investigated stages of A. cenara. All three bee groups showed relative abundance at both phylum and family levels. In larvae, Firmicutes were the most predominant (81.55%); however, they decreased significantly along with the bee development (33.7% in pupae and 10.3% in adults) in favor of Proteobacteria. In the gut of adult bees, four of five core bacteria were found, including Gilliamella apicola group (34.01%) Bifidobacterium asteroides group (10.3%), Lactobacillus Firm-4 (2%), and Lactobacillus Firm-5 (1%). In contrast, pupae and larvae lacked almost all core bacteria except G. apicola (4.13%) in pupae and Lactobacillus Firm-5 (4.04%) in larvae. This is the first report on the gut microbiota community at different developmental stages of A. cerana in Vietnam and provides potential probiotic species for beekeeping.

Beneficial bacteria are vital for maintaining honeybee health by outcompeting pathogenic microorganisms, boosting immunity, and enhancing resilience to diseases. Identifying the specific bacterial strains associated with honeybees enables the development of targeted probiotics that can improve the health of bees and humans. The present study describes the isolation and identification of bacterial strains from Apis cerana honeybees in Hanoi, Vietnam, utilizing a culture-based method, Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, and 16S rRNA sequencing. MALDI-TOF analysis revealed several beneficial bacterial species, including Lactobacillus kunkeei, Lactobacillus plantarum, Pediococcus pentosaceus, Leuconostoc mesenteroides, Leuconostoc citreum, Bacillus subtilis, and Bacillus megaterium. Antimicrobial spectrum analysis showed that 16 out of the 23 identified isolates exhibited inhibitory effects against tested bacteria. Selected isolates with broad antimicrobial spectra, including L. kunkeei, L. plantarum, P. pentosaceus, L. mesenteroides, L. citreum, and B. subtilis, were further validated through 16S rRNA gene sequencing. The results confirmed the identity of these strains, emphasizing the probiotic potential of L. kunkeei, L. plantarum, L. mesenteroides, L. citreum, P. pentosaceus, and B. subtilis for honeybee health. Our findings provide valuable insights into the bacterial diversity and antimicrobial properties associated with honeybees, suggesting their use as probiotics in beekeeping and beyond.