Using whole- genome sequencing to assess the diversity of Paenibacillus larvae within an outbreak and a beekeeping operation

The spore- forming bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees (Apis mellifera). In the present study, we used whole- genome sequencing (WGS) to investigate an extensive outbreak of AFB in northwestern Slovenia in 2019. A total of 59 P. larvae isolates underwent WGS, of which 40 originated from a single beekeeping operation, to assess the diversity of P. larvae within the beekeeping operation, apiary and colony. By applying a casespecific single- linkage threshold of 34 allele differences (AD), whole- genome multilocus sequence typing (wgMLST) identified two outbreak clusters represented by ERIC II- ST11 clones. All isolates from a single beekeeping operation fell within cluster 1 and the median pairwise AD between them was 10 (range=1-22). The median pairwise AD for apiaries of the same beekeeping operation ranged from 8 to 11 (min.=1, max.=22). For colonies of the same apiary and honey samples from these colonies, the median pairwise AD ranged from 8 to 14 (min.=1, max.=20). The maximum within- cluster distance was 33 pairwise AD for cluster 1 and 44 for cluster 2 isolates. The minimum distance between the outbreak- related and non- related isolates was 37 AD, confirming the importance of associated epidemiological data for delineating outbreak clusters. The observed transmission events could be explained by the activities of honeybees and beekeepers. The present study provides insight into the genetic diversity of P. larvae at different levels and thus provides information for future AFB surveillance.