Verification of oxytetracycline-resistant American foulbrood pathogen Paenibacillus larvae in the United States

American foulbrood disease (AFB) of honeybees is the most common and virule nt brood disease, causing severe damage to the beekeeping industry worldwid e by decimating the infected bee colonies. AFB is caused by the microaeroph ilic, Gram-positive, spore-forming bacterium Paenibacillus larvae (formerly Bacillus Larvae). The disease has been under control for four decades by u se of a single antibiotic-oxytetracycline hydrochloride (Terramycin) (Shima nuki et al., 1992). Recent reports (Alippi, 1996) indicated that the resurg ence of AFB in Argentina was probably due to drug-resistant P. larvae. In r ecent years, although many beekeepers in United States have experienced the ineffectiveness of using oxytetracycline HCl (OTC) to control AFB-infected colonies, the results of a national survey did not find OTC-resistant P. l arvae (Shimanuki and Knox, 1994). We verify here for the first time the exi stence of an OTC-resistant strain of P. larvae from AFB-infected brood. The brood comb was collected in 1998 from a Minnesota bee colony having a hist ory of failing to respond to Terramycin treatment for AFB. Methods and proc edures used in the present experiment to isolate P. larvae spores from dead brood remains and subsequent isolation and characterization of the strain grown from a single-cell colony and pathogenicity tests were carried out as described previously by Williams et al. (1998). The Bauer-Kirby disk diffu sion method (Bauer et al,, 1966), following the general guidelines of NCCLS (1997), was performed to determine the zone of inhibition of the presently isolated strain of P. larvae (strain UCD-P-MN-98) by OTC, The UCD-P-MN-98 strain was compared to the bacterial culture grown from a mixed spore isola te harvested from the same Minnesota comb and to strain NRRL B-3650 (obtain ed from Dr. D. P. Stahly, University of Iowa, Iowa City, IA). Zone of inhib ition to tylosin tartrate (Sigma Chemical Co., St. Louis, MO) was also cond ucted for the currently isolated strain. The minimal inhibition concentrati on (MIC) of OTC to UCD-PMN-98 was determined by the broth dilution suscepti bility test. The present results are summarized in Table 1 and compared to previously published data. The data clearly show that the UCD-P-MN-98 strai n is resistant to OTC as compared to susceptible strain NRRL B-3650, OTC-re sistant strains from Argentina (Alippi, 1996), the earlier two strains isol ated from California (Leighton, 1981), and the 1994 USDA study. No differen ce was observed in the susceptibility to tylosin in the currently tested st rains. The present results confirm what the beekeepers have suspected, i.e. , the presence of OTC-resistant AFB pathogen in the beehives. In wake of th e current find, it is urgent that antibiotics having a pharmacological mech anism different from that of the tetracyclines, e.g., penicillins and macro lides (Okayama et al., 1994), flavomycin and virginiamycin (Stahly and Ryan , 1995), tylosin (Peng et al., 1996), and other new antibiotics, be investi gated for future control of AFB to protect honeybee colonies. A detailed st udy on the characterization of UCD-P-MN-98 will be reported elsewhere.