Verification of oxytetracycline-resistant American foulbrood pathogen Paenibacillus larvae in the United States
Verification of oxytetracycline-resistant American foulbrood pathogen Paenibacillus larvae in the United States
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DOI：
10.1006/jipa.1999.4888
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发表时间：
2000-01-01
影响因子：
3.4
通讯作者：
Doi, RH
中科院分区：
文献类型：
--
作者：
Miyagi, T;Peng, CYS;Doi, RH
美洲蜜蜂幼虫腐臭病 (AFB) 是最常见、最致命的幼虫疾病，它会大量消灭受感染的蜂群，对全世界养蜂业造成严重损害。 AFB 是由微需氧革兰氏阳性芽孢杆菌幼虫类芽孢杆菌（以前称为幼虫芽孢杆菌）引起的。四十年来，这种疾病一直通过使用单一抗生素——盐酸土霉素（土霉素）得到控制（Shimanuki 等，1992）。最近的报告（Alippi，1996）表明，阿根廷 AFB 的重新流行可能是由于 P. 幼虫具有抗药性。近年来，尽管美国许多养蜂人都经历过使用盐酸土霉素（OTC）控制AFB感染蜂群的无效情况，但全国调查结果并未发现对OTC具有抗性的蜂幼虫（Shimanuki和Knox，1994）。我们在这里首次验证了来自 AFB 感染幼虫的 OTC 抗性菌株的存在。育雏巢于 1998 年从明尼苏达州的一个蜂群收集，该蜂群曾对土霉素治疗 AFB 没有反应。本实验中使用的从死亡幼虫残骸中分离 P. 幼虫孢子的方法和程序，以及随后从单细胞菌落中生长的菌株的分离和表征以及致病性测试均按照 Williams 等人之前的描述进行。 （1998）。 Bauer-Kirby 纸盘扩散法（Bauer 等，1966）按照 NCCLS（1997）的一般准则，通过 OTC 确定当前分离的 P. 幼虫菌株（UCD-P-MN-98 菌株）的抑菌圈，将 UCD-P-MN-98 菌株与从同一明尼苏达梳子收获的混合孢子分离物生长的细菌培养物进行比较，并与菌株进行比较。 NRRL B-3650（获自爱荷华州爱荷华市爱荷华大学 D. P. Stahly 博士）。还对当前分离的菌株进行了酒石酸泰乐菌素（Sigma Chemical Co.，St. Louis，MO）的抑制区。通过肉汤稀释药敏试验测定OTC对UCD-PMN-98的最小抑制浓度(MIC)。目前的结果总结在表 1 中，并与之前发布的数据进行了比较。数据清楚地表明，与敏感菌株 NRRL B-3650、来自阿根廷的 OTC 抗性菌株（Alippi，1996）、从加利福尼亚分离的早期两个菌株（Leighton，1981）以及 1994 年 USDA 研究相比，UCD-P-MN-98 菌株对 OTC 具有抗性。目前测试的菌株对泰乐菌素的敏感性没有观察到差异。目前的结果证实了养蜂人的怀疑，即蜂箱中存在 OTC 抗性 AFB 病原体。鉴于目前的发现，迫切需要研究具有与四环素类药物不同的药理机制的抗生素，例如青霉素和大环内酯类（Okayama等，1994）、黄霉素和维吉尼亚霉素（Stahly和Ryan，1995）、泰乐菌素（Peng等，1996）和其他新抗生素，以用于未来控制AFB以保护蜜蜂群落。关于 UCD-P-MN-98 特性的详细研究将在别处报道。
American foulbrood disease (AFB) of honeybees is the most common and virulent brood disease, causing severe damage to the beekeeping industry worldwide by decimating the infected bee colonies. AFB is caused by the microaerophilic, Gram-positive, spore-forming bacterium Paenibacillus larvae (formerly Bacillus Larvae). The disease has been under control for four decades by use of a single antibiotic-oxytetracycline hydrochloride (Terramycin) (Shimanuki et al., 1992). Recent reports (Alippi, 1996) indicated that the resurgence of AFB in Argentina was probably due to drug-resistant P. larvae. In recent 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. larvae (Shimanuki and Knox, 1994). We verify here for the first time the existence 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 history of failing to respond to Terramycin treatment for AFB. Methods and procedures 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 diffusion 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 isolate harvested from the same Minnesota comb and to strain NRRL B-3650 (obtained from Dr. D. P. Stahly, University of Iowa, Iowa City, IA). Zone of inhibition to tylosin tartrate (Sigma Chemical Co., St. Louis, MO) was also conducted for the currently isolated strain. The minimal inhibition concentration (MIC) of OTC to UCD-PMN-98 was determined by the broth dilution susceptibility 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 strain is resistant to OTC as compared to susceptible strain NRRL B-3650, OTC-resistant strains from Argentina (Alippi, 1996), the earlier two strains isolated from California (Leighton, 1981), and the 1994 USDA study. No difference was observed in the susceptibility to tylosin in the currently tested strains. The present results confirm what the beekeepers have suspected, i.e., the presence of OTC-resistant AFB pathogen in the beehives. In wake of the current find, it is urgent that antibiotics having a pharmacological mechanism different from that of the tetracyclines, e.g., penicillins and macrolides (Okayama et al., 1994), flavomycin and virginiamycin (Stahly and Ryan, 1995), tylosin (Peng et al., 1996), and other new antibiotics, be investigated for future control of AFB to protect honeybee colonies. A detailed study on the characterization of UCD-P-MN-98 will be reported elsewhere.