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.
Highlights
American foulbrood (AFB) is a highly contagious bacterial disease of honey bee brood and causes the collapse of honey bee colonies (Genersch, 2010)
After 5, 10, 15 and 25 cycles of the non-selective broth culture steps, serial dilutions of the Bacteria Isolated From Japanese Honey and Their Antimicrobial Susceptibility
To investigate whether the putative macrolide resistance genes found in mobile genetic elements (MGEs) (Figure 3 and Table 1) have the potential to confer macrolide resistance to P. larvae, we introduced them into P. larvae using pMX2-TA and assessed antimicrobial susceptibility of the transformants
Summary
American foulbrood (AFB) is a highly contagious bacterial disease of honey bee brood and causes the collapse of honey bee colonies (Genersch, 2010). The etiological agent of AFB is the Gram-positive, rod-shaped bacterium Paenibacillus larvae. Strains of this bacterium have been classified into five ERIC types (ERIC I–V) by repetitiveelement PCR (Genersch et al, 2006; Beims et al, 2020) and 30 sequence types (STs) by multilocus sequence typing (MLST) (Morrissey et al, 2015). Among the five ERIC genotypes, ERIC I and II are the major types among the strains isolated from AFB cases. In Japan, ERIC I and II are the only genotypes found in isolates from AFB cases, and isolation of ERIC II strains has recently increased (Ueno et al, 2018). Similar to other Paenibacillus bacteria, P. larvae forms spores, which are the only infectious form of this organism (Genersch, 2010)
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