First report on isolation and characterization of Bacillus Sp. associated with honey bee brood disease.

Bacterial infections in honey bee brood pose a significant threat to bee populations, leading to decreased honey production and disrupting critical crop pollination networks. While Paenibacillus larvae and Melissococcus plutonius are well-established as major pathogens of honey bee eggs and larvae, the presence of other harmful bacteria in the Khyber Pakhtunkhwa (KPK) province of Pakistan remains largely unexplored. Molecular characterization of Bacillus species and B. cereus isolates revealed the presence of key virulence genes, including the cry gene, known for its insecticidal properties. The ability of B. cereus to produce cytotoxins, haemolysins, and enterotoxins raises concerns about its impact on larval immunity and survival. Similarly, B. mycoides, a close relative of B. cereus, was identified in diseased broods, highlighting its potential involvement in microbial dysbiosis within the hive. Reports from North America and Europe have also linked these Bacillus species to declining honey bee health, emphasizing the need for further investigation into their pathogenic mechanisms. This study investigated bacterial contamination in broods collected from infected beehives across various cities of KPK. Biochemical and molecular analyses revealed a widespread presence of bacteria the Bacillus species, as the emerging most dominant, followed by Bacillus cereus. Phylogenetic analysis indicated a close evolutionary relationship between Bacillus species, and Bacillus cereus, highlighting their shared spore-forming characteristics. This research is the first to report the involvement of Bacillus species in infecting honey bee eggs and larvae, shedding light on a previously unrecognized threat to apiculture and pollination in the region.