Bacterial community associated with worker honeybees () affected by European foulbrood.

Background: is an entomopathogenic bacterium that causes European foulbrood (EFB), a honeybee ( L.) disease that necessitates quarantine in some countries. In Czechia, positive evidence of EFB was absent for almost 40 years, until an outbreak in the Krkonose Mountains National Park in 2015. This occurrence of EFB gave us the opportunity to study the epizootiology of EFB by focusing on the microbiome of honeybee workers, which act as vectors of honeybee diseases within and between colonies.

Methods: The study included worker bees collected from brood combs of colonies (i) with no signs of EFB (EFB0), (ii) without clinical symptoms but located at an apiary showing clinical signs of EFB (EFB1), and (iii) with clinical symptoms of EFB (EFB2). In total, 49 samples from 27 honeybee colonies were included in the dataset evaluated in this study. Each biological sample consisted of 10 surface-sterilized worker bees processed for DNA extraction. All subjects were analyzed using conventional PCR and by metabarcoding analysis based on the 16S rRNA gene V1-V3 region, as performed through Illumina MiSeq amplicon sequencing.

Results: The bees from EFB2 colonies with clinical symptoms exhibited a 75-fold-higher incidence of than those from EFB1 asymptomatic colonies. was identified in all EFB1 colonies as well as in some of the control colonies. The proportions of , , , , and were higher in EFB2 than in EFB1, whereas was significantly higher in EFB2 than in EFB0. and , and, exhibited higher proportion in EFB1 than in EFB2 and EFB0. The occurrence of and were higher in EFB0 than in EFB2 and EFB1. incidence was highest in EFB2.

Conclusions: High-throughput Illumina sequencing permitted a semi-quantitative analysis of the presence of within the honeybee worker microbiome. The results of this study indicate that worker bees from EFB-diseased colonies are capable of transmitting due to the greatly increased incidence of the pathogen. The presence of sequences in control colonies supports the hypothesis that this pathogen exists in an enzootic state. The bacterial groups synergic to both the colonies with clinical signs of EFB and the EFB-asymptomatic colonies could be candidates for probiotics. This study confirms that is a secondary invader to ; however, other putative secondary invaders were not identified in this study.