Hive-associated bacteria improve honeybee survival during <i>Paenibacillus larvae</i> infection

Honeybees (<i>Apis mellifera</i> L.) are the most common pollinators of crops. Honeybee larvae are susceptible to <i>Paenibacillus larvae</i>, a spore-forming Gram-positive bacterium that causes American Foulbrood (AFB), one of the most destructive brood diseases worldwide. Existing antibiotic treatments are undesirable due to increasing pathogen resistance and their residual accumulation in bee products. Consequently, there is increasing interest in biological agents and natural strategies for AFB control. However, most studies remain limited to <i>in vitro</i> or <i>in vivo</i> experiments and rarely evaluate impacts on adult bees. In this study, we systematically investigated hive-associated bacteria for their potential to enhance larval survival during <i>P. larvae</i> infection. Bacteria isolated from AFB-infected combs were sequenced and screened for antagonistic activity. Several <i>Bacillus</i> species-including <i>B. zhangzhouensis</i>, <i>B. subtilis</i>, <i>B. amyloliquefaciens</i>, <i>B. licheniformis</i>, and <i>B. mojavensis</i>-inhibited one or more <i>P. larvae</i> strains. Notably, the lysate of <i>B. licheniformis</i> suppressed all tested strains; Further characterization revealed that its main antibacterial component consists of heat-stable proteins between 30-50 kDa. Two <i>Bacillus</i> strains were selected for larval infection assays using laboratory-reared honeybee larvae: both significantly improved survival by 42% and 71%, respectively. To evaluate potential effects on adult bees, newly emerged workers were caged and fed <i>B. licheniformis</i> ASx lysate. Their survival differed from that of untreated controls but remained comparable to the Terra-Pro-fed bees, a Terramycin-based AFB treatment, and their gut microbiome remained similar to that of untreated controls. Overall, our findings suggest that hive-associated <i>Bacillus</i> species offer promising, low-impact candidates for AFB disease management.