A novel multiplex PCR assay to detect and distinguish between different types of <i>Paenibacillus larvae</i> and <i>Melissococcus plutonius</i>, and a survey of foulbrood pathogen contamination in Japanese honey

Abstract

Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.

Detection of macrolide resistance genes, <i>ermC</i> and <i>ermB</i>, in Japanese honey using real-time PCR assays

“…The spore suspensions were then added to honey samples to obtain final solutions of 1,000, 100, 10, and 1 spore(s)/mL honey. Genomic DNA was extracted from 5 mL of each spore-spiked honey sample using the Johne Pure Spin kit (FASMAC Co., Ltd., Atsugi, Japan), according to Okamoto et al [10]. Realtime PCR assays were performed as described earlier, and the detection limit for both genes was found to be 10 spores/mL honey.…”

“…Although the effect of ermB on P. larvae was less than that of ermC , the ermB gene also decreased the susceptibility of P. larvae to macrolides [ 9 ]. As P. larvae was suggested to be widely distributed in apiaries in Japan [ 10 ], tylosin-resistant P. larvae could easily develop if such macrolide-resistant bacteria are already present in beehives and apiaries, and the developed resistant P. larvae would be selected by using the prophylactic. However, bacteria possessing ermC and ermB genes have only been isolated from one of each of the fifty-three honey samples analyzed using cultivation methods in a previous study [ 9 ].…”

“…To study the distribution of ermC and ermB in Japanese honey, 116 honey samples collected from 2017 to 2020, were analyzed in this study ( Supplementary Table 2 ). DNA was extracted from 5 mL of each honey sample as previously described [ 10 ]. Genomic DNA of J18TS1 and J45TS6 (positive controls) was extracted using InstaGene™ Matrix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer’s instructions.…”

“…DNA was extracted from the sediments of 5 mL of honey using the Johne Pure Spin kit (FASMAC Co., Ltd., Atsugi, Japan), and 1 µL of the extracted DNA from each sample was used as a template in real-time PCR assay to detect ermC / ermB. The detection rate of P. larvae from the honey samples was retrieved from a previous study conducted by our research group [ 10 ]. N. D., not detected.…”

“…P. larvae becomes tylosin-resistant if it acquires the ermC gene [ 9 ]; therefore, the co-contamination of honey with ermC and P. larvae suggests the potential risk of the emergence of macrolide-resistant P. larvae in beehives and apiaries. To further investigate this risk, we retrieved the P. larvae -positive/negative data of the same 116 honey samples analyzed in an earlier study conducted by our research group [ 10 ] and compared it with the ermC- positive/negative data obtained from the present study. Surprisingly, both ermC and P. larvae were detected in 71.55% of honey samples ( Fig.…”