Geographical resistome profiling in the honeybee microbiome reveals resistance gene transfer conferred by mobilizable plasmids

The spread of antibiotic resistance genes (ARGs) has been of global concern as one of the greatest environmental threats. The gut microbiome of animals has been found to be a large reservoir of ARGs, which is also an indicator of the environmental antibiotic spectrum. The conserved microbiota makes the honeybee a tractable and confined ecosystem for studying the maintenance and transfer of ARGs across gut bacteria. Although it has been found that honeybee gut bacteria harbor diverse sets of ARGs, the influences of environmental variables and the mechanism driving their distribution remain unclear. We characterized the gut resistome of two closely related honeybee species, Apis cerana and Apis mellifera, domesticated in 14 geographic locations across China. The composition of the ARGs was more associated with host species rather than with geographical distribution, and A. mellifera had a higher content of ARGs in the gut. There was a moderate geographic pattern of resistome distribution, and several core ARG groups were found to be prevalent among A. cerana samples. These shared genes were mainly carried by the honeybee-specific gut members Gilliamella and Snodgrassella. Transferrable ARGs were frequently detected in honeybee guts, and the load was much higher in A. mellifera samples. Genomic loci of the bee gut symbionts containing a streptomycin resistance gene cluster were nearly identical to those of the broad-host-range IncQ plasmid, a proficient DNA delivery system in the environment. By in vitro conjugation experiments, we confirmed that the mobilizable plasmids could be transferred between honeybee gut symbionts by conjugation. Moreover, "satellite plasmids" with fragmented genes were identified in the integrated regions of different symbionts from multiple areas. Our study illustrates that the gut microbiota of different honeybee hosts varied in their antibiotic resistance structure, highlighting the role of the bee microbiome as a potential bioindicator and disseminator of antibiotic resistance. The difference in domestication history is highly influential in the structuring of the bee gut resistome. Notably, the evolution of plasmid-mediated antibiotic resistance is likely to promote the probability of its persistence and dissemination. Video Abstract.