Antimicrobial Resistance Genes in Bacteria Isolated From Japanese Honey, and Their Potential for Conferring Macrolide and Lincosamide Resistance in the American Foulbrood Pathogen Paenibacillus larvae

Abstract

American foulbrood (AFB) is the most serious bacterial disease of honey bee brood. Spores of the causative agent Paenibacillus larvae are ingested by bee larvae via brood foods and germinated cells proliferate in the larval midgut. In Japan, a macrolide antibiotic, tylosin, is used as the approved prophylactic for AFB. Although tylosin-resistant P. larvae has yet to be found in Japan, it may emerge in the future through the acquisition of macrolide resistance genes from other bacteria, and bacteria latent in brood foods, such as honey, may serve as a source of resistance genes. In this study, to investigate macrolide resistance genes in honey, we attempted to isolate tylosin-resistant bacteria from 53 Japanese honey samples and obtained 209 isolates from 48 samples in the presence of 1 μg/ml of tylosin. All isolates were Gram-positive spore-forming bacteria mainly belonging to genera Bacillus and Paenibacillus, and 94.3% exhibited lower susceptibility to tylosin than Japanese P. larvae isolates. Genome analysis of 50 representative isolates revealed the presence of putative macrolide resistance genes in the isolates, and some of them were located on mobile genetic elements (MGEs). Among the genes on MGEs, ermC on the putative mobilizable plasmid pJ18TS1mac of Oceanobacillus strain J18TS1 conferred tylosin and lincomycin resistance to P. larvae after introducing the cloned gene using the expression vector. Moreover, pJ18TS1mac was retained in the P. larvae population for a long period even under non-selective conditions. This suggests that bacteria in honey is a source of genes for conferring tylosin resistance to P. larvae; therefore, monitoring of bacteria in honey may be helpful to predict the emergence of tylosin-resistant P. larvae and prevent the selection of resistant strains.

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

“…The representative strains of P. larvae and M. plutonius listed in Table 1 were used in sensitivity tests of the newly developed multiplex PCR assay. Sixty-five other bacterial strains, including those isolated from Japanese honey [ 43 ] and honey bees [ 3 ], were used in addition to the foulbrood pathogens for specificity tests. Strains ATCC 6344 and ATCC 64, the type strains of Paenibacillus alvei and Brevibacillus laterosporus , respectively, were purchased from the American Type Culture Collection ( Supplementary Table 1 ).…”

“…To assess the sensitivity of the multiplex PCR assay, genomic DNA was manually extracted from representative P. larvae strains cultured on MYPGP agar [ 13 ] at 35°C for two days under air plus 5% CO 2 conditions and M. plutonius strains cultured on KSBHI agar [ 4 ] at 35°C for three days under anaerobic conditions according to the methods described by Okamoto et al . [ 43 ] and Arai et al . [ 4 ], respectively.…”

Evaluating approved and alternative treatments against an oxytetracycline-resistant bacterium responsible for European foulbrood disease in honey bees

“…These genes, which have a colloquial naming system based on discovery, can encode for a series of protective functions such as efflux, ribosomal modification proteins, and antibiotic destruction. In the context of honey bee health, OTC-resistance has been observed in Paenibacillus larvae , the causative agent of AFB disease, resulting from plasmid-encoded tetracycline resistance through the tetL gene 13 . Additionally, a series of Bacillus cereus strains isolated from honey were found to carry tetracycline resistance genes, including tetL and tetK 14 .…”

“…For example, erm genes, a major source of MLS B resistance, encode for rRNA methyltransferases that add one or two methyl groups onto the 23S rRNA, preventing the antibiotics from binding their target 18 . While no TYL or LMC-resistant P. larvae or M. plutonius have been identified, bacteria isolated from honey encode for MLS B resistance genes, including plasmid-borne 23S rRNA methyltransferases such as ermC 13 . Additionally, a recent study found M. plutonius isolates resistant to the macrolide antibiotic, mirosamicin 20 .…”

Paenibacillus amylolyticus osteomyelitis in a Poodle dog: case report and literature review

“…Resistance or low susceptibility of Paenibacillus spp. to clindamycin 22,31,33 and lincomycin 21 has been reported from various hosts and sources, suggesting that intrinsic resistance to lincosamides may exist, although the underlying mechanisms remain to be explored.…”