CONICET | Buscador de Institutos y Recursos Humanos

AFB disease caused by the spore-forming bacterium Paenibacillus larvae is the most devastating disease affecting honey bee brood. In many countries antibiotic applications are used as preventive and curative treatments for AFB with the consequent risk of honey contamination and development of bacterial resistance. The use of natural biocides against AFB that do not interfere with the normal balance of the environment will improve the quality of honeys. For these reasons, the objective of the present work was to characterize antagonistic compounds produced by Bacillus cereus strains isolated from honey that were biologically active against P. larvae in vitro. Antagonistic compounds were obtained from cell-free supernatants of stationary phase cultures of B. cereus growing on TS broth and concentrated by NH4SO4 precipitation, ultrafiltration (3 KDa) and butanol extraction (0.7 volumes). After butanol evaporation under reduced pressure, the compounds were suspended in PBS. Antimicrobial activity was assessed by an agar spot test against P. larvae ATCC 9545. Effect of enzymes, pH and temperatures were also evaluated. By using a dialysis membrane, molecular mass of the compounds were estimated between 3 and 8 KDa. The antibacterial activity was sensitive to high temperatures (60 min at 70 to 121 ˚C) and pH ranges from 8 to 10 (1h at 37 ˚C) but remained unaltered after proteolytic enzyme treatments such as trypsin, pepsin and proteinase K (1h at 37 ºC); temperatures of 60 ºC for 60 min and pH ranges from 3 to 7 (1h at 37 C). Sensitivity to papaine, lipase and chymotrypsine was strain-dependent. According to the MIC and MBC values obtained, we concluded that the compounds were bactericide. Aquose phase butanol extraction evaporated under reduce pressure was also inhibitory, demonstrating that antagonistic activity are related to several compounds of diverse nature.