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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: A. mellifera; Antibiotic resistance genes; Apis cerana; Gut resistome; Horizontal transfer; IncQ.

Fig. 1

The map showing the location of 18 sampling sites from 14 provinces across China. Detailed information of the geographic location and sampling time is shown in Dataset S1

Fig. 2

Abundance and composition of ARGs in A. cerana and A. mellifera guts. a Stacked bar charts showing relative abundance of different classes of ARGs in each bee individual. b, c Box plots showing the total class (b) and group c number of ARGs identified in each sample. d The total abundance of ARGs normalized by the amount of gut bacterial cells. e The Shannon diversity of each sample at the level of ARG group. f PCoA based on Bray-Curtis distances of group-level resistome compositions. Boxplots (left panel) indicate the distribution of each group of honeybee along the first principal coordinate (PCoA 1). g, h Procrustes analysis between the microbiota taxonomic compositions and the profiles of resistome. g The change in the ordination position of resistome (dotted ends) and the microbiota (non-dotted ends) is displayed. The correlation coefficients and significance were calculated with the protest function in vegan. M² indicates the sum of squared distances between matched sample pairs. h The residual line plot enables easier residual size comparison, showing the differences in the microbiome-resistome association between A. cerana gut samples. Different letters (a, b, c, d) above each bar stand for statistical differences between sampling sites (least-significant difference (LSD) test, P < 0.05)

Fig. 3

Core and location-specific ARG components of A. cerana. a Relative abundance of the core ARGs detected in > 50% of A. cerana samples (core of country-wide) and those detected in > 50% samples only from certain locations (core of specific locations). Other ARGs occurred only in < 50% samples of any sampling sites. b Distribution of the six group of ARGs (Core of country-wide) in bees from different locations. Different letters (a, b, c, d) above each bar stand for statistical differences between A. cerana sampling sites (LSD test, P < 0.05). c The presence of the core ARG groups specific to different locations

Fig. 4

Core and transferrable ARGs are carried by different gut symbionts specific to A. cerana and A. mellifera. a Pearson correlations between the prevalence of each group of core ARGs (both core of country-wide and specific locations) and the number of bacterial species carrying this ARG group in all A. cerana samples. The shaded area represents the 95% confidence region. b Different classes of ARGs are carried mainly by the core bacterial genera specific to A. cerana. Each arc represents the link between a group of ARGs from each class and the bacterial species in each genus. The pie charts show the distribution of bacterial genera in each class of ARGs. The sizes of the pie charts are proportional to the prevalence of each class of ARGs, and the thickness of the lines is proportional to the number of connections (ARG group-bacterial species). c Box plots showing the number of transferrable ARGs per honeybee gut sample. Different letters (a, b, c, d) above each bar stand for statistical differences between sampling sites (LSD test, P < 0.05). d The presence (pink squares) and absence (black squares) transferrable ARGs in different bacterial genus in A. cerana and A. mellifera gut samples. Gram-negative are marked with grey shades

Fig. 5

The sul2-strA-strB gene cluster in bee gut bacteria originates from different IncQ-family plasmids. Genetic map of gut bacteria contigs carrying the sul2-strA-strB gene cluster compared with the scaled linear map of the RSF1010 (a) and the pMS260 (b) plasmids of IncQ-family. Homologous ori genes, ARGs, and genes required for mobilization and replication are indicated by different colors. Point mutations and insertions when comparing with the plasmid sequences are indicated on each contig of assembled contigs in samples from different sampling locations. (The position of the mutations and insertions are indicated on each assembled contig from different samples. Dashes represent deletion in the nucleotide sequence)

Fig. 6

IncQ plasmid transmission between honeybee-specific gut symbionts. a Experimental procedure for in vitro conjugation. IncQ plasmid pBTK519 (Kan^R) was used for transferability test between different donor and recipient strains. Donor strains of E. coli MFDpir and G. apis W8126 harboring the pBTK519 were co-incubated with strains from three honeybee gut bacterial genus, Gilliamella, Snodgrassella and Bartonella, as recipients. BHI agar supplemented with kanamycin and another designated antibiotics were used for transconjugants selection. b Experimental results of in vitro conjugation. The phylogenetic tree was constructed by maximum-likelihood method (RAxML) based on the whole genomes of isolated strains. The donor G. apis strain W8126 is shown in red