Oxytetracycline resistance of Melissococcus plutonius strains in Japan

Abstract European foulbrood caused by the Gram-positive lanceolate coccus, Melissococcus plutonius, is a major bacterial infection of honey bee brood. Oxytetracycline (OTC) has been used to combat this disease in some countries. Although OTC resistance was not previously reported despite its long-term use, the first case was finally detected in an M. plutonius strain isolated in Canada in 2019. In Japan, OTC has not been approved for use in the control of any honey bee diseases. However, after testing 77 M. plutonius strains isolated in Japan between the 1980s and 2010s, we identified four OTC-resistant strains with a minimum inhibitory concentration of OTC on agar medium of 16 µg/ml. The four strains were isolated in 2008 (1 strain), 2013 (1 strain), and 2015 (2 strains); i.e., before the Canadian OTC-resistant isolate. These results provide insights into the mechanisms underlying the development of OTC-resistant M. plutonius.

European foulbrood (EFB) caused by Melissococcus plutonius is a globally distributed bacterial infectious disease of honey bees (Ellis & Munn, 2005).Oxytetracycline (OTC) has been used to combat EFB in some countries (Hornitzky & Smith, 1999;Waite et al., 2003;Masood et al., 2022).OTC is a broadspectrum antibiotic that inhibits protein synthesis in bacteria.Although it has a long history of use in the apicultural industry, all M. plutonius isolates tested in the UK and Australia were susceptible to the drug, with minimum inhibitory concentrations (MICs) of 3.6-4.2and 1-2 mg/ml, respectively (Hornitzky & Smith, 1999;Waite et al., 2003).However, in 2022, the first OTC-resistant M. plutonius isolate was reported in Canada, where OTC has been used to treat EFB.It was isolated in 2019 (Wood, S. C., personal communication;Wood et al., 2020), and its MIC of OTC was 16 mg/ml on KSBHI agar (Masood et al., 2022).
In Japan, antimicrobials cannot be used to treat EFB or American foulbrood (AFB).Mirosamicin and tylosin have been employed as prophylactics to prevent AFB, but have not been approved for EFB.Nevertheless, mirosamicin-resistant M. plutonius strains have been reported in Japan (Takamatsu et al., 2018).This finding, as well as the detection of OTC-resistant M. plutonius in Canada has raised concerns about OTC resistance in Japanese M. plutonius; therefore, we investigated the OTC susceptibility of Japanese M. plutonius strains using our laboratory collection.
Seventy-seven Japanese M. plutonius strains isolated from Apis mellifera and Apis cerana japonica between 1988 and 2016 (Takamatsu et al., 2018) were used in the present study (Supplementary Table S1).By using the multilocus sequence typing scheme (Haynes et al., 2013), 29, 44, and 4 strains were assigned to clonal complex (CC) 3, CC12, and CC13, respectively (Takamatsu et al., 2014(Takamatsu et al., , 2017(Takamatsu et al., , 2018)).Because the growth of some M. plutonius strains in liquid media is slow and weak even under antibiotic-free conditions (Grossar et al., 2020;Takamatsu D., unpublished observations), the broth microdilution method might not be appropriate as a method to examine MICs in diverse strains.Therefore, in this study, the MICs of OTC (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) were assessed on KSBHI agar (Arai et al., 2012) by agar dilution methods as previously described (Takamatsu et al., 2018).Briefly, M. plutonius strains pre-cultured on KSBHI agar at 35 C for three days were suspended in KSBHI broth (Nakamura et al., 2016) and adjusted to approximately 1-2 Â 10 7 CFU/ml.The bacterial suspensions were spotted (2 ll/spot) onto the KSBHI agar plates containing different concentrations of OTC or OTC-free agar plates (control).After a 72-h incubation of the plates at 35 C under anaerobic conditions, the MICs were determined by reading the lowest concentration of OTC that completely inhibited the growth of inoculated strains on the medium.Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were employed for quality control, and the quality control limits of the MICs of OTC were set as previously described (Takamatsu et al., 2018) (Supplementary Table S2).Fisher's exact test was performed in EZR (Kanda, 2013), and p < 0.05 was set as the threshold for significance.
As shown in Figure 1 and Supplementary Table S1, the MICs of OTC for 19, 54, and 4 M. plutonius strains were 4, 8, and 16 mg/ml, respectively.No significant difference was observed in the rate of strains exhibiting each MIC between CCs (p ¼ 0.322).Waite et al. (2003) regarded strains with MICs of 3.6-4.2mg/ml as susceptible to OTC.In the study by Masood et al. (2022), a strain with MIC of 16 mg/ml was considered to be resistant to OTC.Since the clinical signs of EFB following infection with the Canadian OTC-resistant isolate were attenuated or prevented in vitro when larvae were treated with 1-100 mg/ml OTC (Masood et al., 2022), strains with MIC of 16 mg/ml may not be clinically resistant to OTC.However, in this study, by referring to MIC values from the two previous reports (Waite et al., 2003 [for susceptibility]; Masood et al., 2022 [for resistance]), we tentatively defined the breakpoints of OTC for M. plutonius as follows: susceptible, MIC 4 mg/ml; intermediate, 8 mg/ml; resistant, !16 mg/ ml.Accordingly, three CC3 and one CC12 strain(s) were considered to be resistant to OTC (Figure 1).The four Japanese OTC-resistant strains were isolated in 2008, 2013, and 2015; i.e., before the Canadian OTC-resistant isolate (Supplementary Table S1).The rate of strains exhibiting each MIC significantly differed between the years of isolation (i.e., before 2000, 2001-2010, and 2011-2016) (p < 0.05), and the prevalence of OTC-intermediate strains increased over the years (Figure 2), suggesting the presence of constant selective pressure by OTC.The use of OTC is not approved for EFB in Japan.However, it is not impossible for veterinarians to use unapproved drugs when they deem it unavoidable to control a disease; therefore, the possibility that OTC has been used in Japan cannot be completely denied.It is also important to note that OTC has also been used to prevent infections in various crops in Japan (e.g., bacterial canker of plums and citrus fruits, black rot of cabbage, bacterial shot hole of peach, and bacterial shoot blight of pears), and, thus, honey bees may come into contact with it in the field.Therefore, the use of OTC for agricultural crops may have an impact on reduced OTC susceptibility in Japanese M. plutonius strains as well as the emergence of OTCresistant M. plutonius in this country.Alternatively, OTC-resistant strains may have been brought to Japan along with imported honey bees.However, no OTCresistant M. plutonius strains have been reported from Australia (Hornitzky & Smith, 1999), a major country exporting honey bees to Japan.
The molecular mechanisms underlying tetracycline resistance commonly known in bacteria include efflux, ribosome protection, reduced permeability, ribosome mutations, and enzymatic inactivation (Markley & Wencewicz, 2018).In this study, OTC-resistant and -intermediate strains were found regardless of their genotypes (CCs), suggesting that OTC resistance can occur in all M. plutonius strains; however, our present results alone were not sufficient to predict the resistance mechanism.A survey of 18 publicly available M. plutonius genomes conducted by Masood et al. (2022)  using the Comprehensive Antibiotic Resistance Database also yielded no significant insights into any known tetracycline resistance genes, and OTC-resistant mechanisms in M. plutonius have yet to be elucidated.To elucidate the resistance mechanism, it would be necessary to analyze the genomes not only of resistant strains in Canada and Japan but also of susceptible strains of the same genotype in those regions and to extract common differences between the two phenotypes through a detailed comparison of their genomes.When their resistant mechanisms are suggested by these analyses, recently developed gene manipulation techniques for M. plutonius (Takamatsu et al., 2013(Takamatsu et al., , 2015(Takamatsu et al., , 2020) ) will be useful for testing the hypothesis.
We herein demonstrated that OTC-resistant M. plutonius had already existed before the Canadian OTCresistant isolate was obtained.Although further studies are needed, the presence of OTC-resistant strains in Japan, where the use of this drug is not approved, may provide insights into the mechanisms underlying the development of OTC-resistant M. plutonius.

Figure 1 .
Figure 1.Oxytetracycline (OTC) susceptibility of Japanese Melissococcus plutonius strains on KSBHI agar.The minimum inhibitory concentrations (MICs) of OTC were assessed by agar dilution methods.The percentages of M. plutonius strains belonging to each clonal complex (CC) is indicated by different colors.The number of isolates that showed each MIC is shown on the bars.

Figure 2 .
Figure 2. Chronological distribution of minimum inhibitory concentrations (MICs) of oxytetracycline in Japanese Melissococcus plutonius populations.The number (A) and percentage (B) of M. plutonius strains exhibiting each MIC are indicated by different colors.