Identification of genetic determinants of antimicrobial resistance and virulence in Canadian isolates of Melissococcus plutonius

European foulbrood (EFB), a bacterial disease affecting honey bee larvae, is a re-emerging threat for the Canadian beekeeping industry, with EFB outbreaks anecdotally reported to be increasing in severity and incidence (1). The bacterium responsible for this disease, Melissococcus plutonius, was detected in 37% of honey bee colonies in Alberta by molecular methods. In combination with integrated pest management (IPM) techniques, the antibiotic oxytetracycline is used by Canadian beekeepers to treat EFB in their colonies. Considering that antimicrobial resistance has been reported in Paenibacillus larvae, the bacterium responsible for American foulbrood, there is concern that antimicrobial resistance in M. plutonius may explain the reported increase in EFB disease in Canadian apiaries. To investigate this hypothesis, we performed whole genome sequencing of 37 Canadian isolates of M. plutonius, collected from 2007-2021, as well as 8 American isolates of M. plutonius, to investigate for the presence of antimicrobial resistance (AMR) genes, in combination with antimicrobial susceptibility testing. Additionally, using pan-genome analysis, we characterized the genetic diversity of M. plutonius isolates from Canada and the USA, and investigated these isolates for the presence of putative virulence genes. Specifically, we screened M. plutonius isolates for the plasmid-encoded melissotoxin A (mtxA) gene, which is hypothesized to encode a toxin that may increase the pathogenicity of M. plutonius to honey bee larvae.
We examined the genomes of 37 Canadian isolates of M. plutonius (15 from British Columbia, 2 from Alberta, 4 from Saskatchewan, 16 from Quebec) and 8 American isolates (2 from each of Michigan, Oregon, Texas, and Utah). Preliminary phylogenetic analysis of these North American isolates revealed the isolates clustered in two groups which were distinct from M. plutonius isolates sequenced previously from Japan, Switzerland, Norway, England, and the USA. One of the isolates from Saskatchewan (2020SK1) was found to represent a novel genetic sequence type (8), supporting the hypothesis that Canadian apiaries may harbor new genetic variants of this pathogen. 49% of M. plutonius isolates in this study were found to carry the mtxA gene, while 51% did not, suggesting that spread of M. plutonius strains carrying mtxA may not explain anecdotal reports of increased clinical severity of EFB in Canada, although additional field data is necessary to investigate further.
To date, no canonical/well-established AMR genes have been identified in the 45 M. plutonius genomes analyzed. In contrast, oxytetracycline (OTC)-resistance was observed in 21/22 isolates examined for antimicrobial susceptibility. OTC-resistant isolates had MICs ranging from 8-64 µg/ml OTC, which were in excess of the susceptibility breakpoint of 2.5 µg/ml OTC established by the Clinical and Laboratory Standards Institute (CLSI). Only one Canadian M. plutonius isolate (2017QU1) was considered susceptible to OTC with an MIC of 4 µg/ml, which is less than two-fold greater than the CLSI breakpoint value. Although CLSI breakpoint values are not established for tylosin (TYL) and lincomycin (LMC) for M. plutonius, all isolates examined had MIC values of 4 µg/ml or less for these antimicrobials, suggesting that the M. plutonius isolates were susceptible to TYL and LMC. Importantly, TYL and LMC are not licensed for treatment of EFB in North America.
The population of M. plutonius isolates in Canadian honey bee colonies is genetically distinct from other regions of the world, and contains at least one previously undescribed genetic variant. Based on laboratory testing, Oxytetracycline(OTC)-resistance is common among Canadian isolates of M. plutonius; however, to date, whole genome sequence analysis of these isolates has failed to identify AMR genes to explain this resistance phenotype. Despite the observed OTC-resistance of M. plutonius in vitro, OTC may still be effective for EFB treatment in combination with IPM strategies and the inherent social immune defenses of honey bee colonies.
Future studies are needed to study the most effective antimicrobial dosing strategies for treatment of EFB in both the laboratory and the field ss well as to investigate the genetic diversity of Canadian M. plutonius isolates, their pathogenicity to honey bee larvae. Taken together, our current and future research efforts will enhance antimicrobial stewardship and management of EFB within the Canadian beekeeping industry and, in turn, improve the productivity and profitability of Canadian honey bee colonies.