牛呼吸道多杀性巴氏杆菌的分离鉴定及胡黄汤对其耐药性的消除作用

Isolation and identification of Pasteurella multocida from bovine respiratory tract and evaluation of the attenuating effects of Huhuang decoction on its drug resistance

摘要:

【背景】 多杀性巴氏杆菌(Pasteurella multocida)是导致我国牛只患呼吸道疾病的主要病原，越来越多的多杀性巴氏杆菌表现出多重耐药性。【目的】 了解不同地区牛呼吸道多杀性巴氏杆菌的流行情况和耐药性，探究中药对耐药多杀性巴氏杆菌耐药性的消除效果。【方法】 对不同地区采集的病牛肺组织和鼻拭子进行病原菌分离培养、生化鉴定、分子生物学鉴定；K-B法检测分离菌株的药物敏感性，利用PCR法检测分离菌株耐药基因携带情况；之后进行耐药性消除试验，采用微量稀释法测定胡黄汤及其组分对分离株的最小抑菌浓度(minimum inhibitory concentration, MIC)，将菌株与1/2 MIC、1/4 MIC、1/8 MIC浓度药物分别作用24、48、72 h后，观测菌株药物敏感性变化。【结果】 分离获得10株疑似多杀性巴氏杆菌，在血琼脂培养基上生长良好，无溶血现象，表面可见光滑凸起、圆形、灰白色的均一菌落，瑞氏染色呈两极着色的短杆菌，生化鉴定结果与多杀性巴氏杆菌一致，特异性基因kmt1扩增片段大小与预期一致；荚膜血清分型结果显示，8株分离株为A型，2株为D型。药敏试验结果显示，7株分离菌对链霉素耐药，耐药率为70%，对庆大霉素、氧氟沙星、阿莫西林等耐药率分别为60%、60%、50%。对阿米卡星、卡那霉素、环丙沙星、诺氟沙星、恩诺沙星和复方新诺明的耐药率为10%-40%，其中有5株为多重耐药菌株，占分离菌株总数的50%。耐药基因检测结果显示，所有菌株均携带strA、strB、bla_ROB-1耐药基因，6株携带aac(6')-Ib-cr耐药基因，检出率为60%，Sul1、tetB和tetH耐药基因的检出率分别为50%、30%、10%。消除试验显示，胡黄汤和黄芩水煎液对菌株阿米卡星、卡那霉素、环丙沙星、恩诺沙星的耐药性有不同程度的消除作用，并呈现浓度和时间依赖性，1/2 MIC药物浓度干预72 h后的耐药性消除效果最好。【结论】 共分离出10株多杀性巴氏杆菌，其中有50%的菌株呈现多重耐药性，表明耐药菌株已是临床感染株的大多数，胡黄汤对多重耐药多杀性巴氏杆菌有消除作用，其中黄芩是起消除作用的主要成分。

Abstract:

[Background] Pasteurella multocida is the major pathogen causing bovine respiratory diseases in China, and strains with multidrug resistance keep emerging. [Objective] To investigate the prevalence and drug resistance of bovine respiratory tract-derived P. multocida strains from different regions and evaluate the attenuating effects of traditional Chinese medicine (TCM) on the resistance of drug-resistant strains. [Methods] The pathogenic bacteria were isolated from the lung tissue samples and nasal swabs of diseased cattle and identified via biochemical and molecular methods. The K-B method was adopted to test the antimicrobial susceptibility, and resistance genes were identified by PCR. Subsequently, the test for attenuating effects on drug resistance was conducted. The minimum inhibitory concentrations (MICs) of Huhuang decoction and its components against the isolates were determined by the microdilution method. Strains were exposed to antimicrobials at 1/2, 1/4, and 1/8 MIC for 24, 48, and 72 h, and changes in their antimicrobial susceptibility were assessed. [Results] Ten suspected P. multocida strains were isolated, showing smooth, convex, and gray-white colonies on blood agar, with no hemolysis. Wright's staining showed that the cells were rod, with both ends stained. The biochemical test results of the strains were consistent with those of P. multocida, and the amplification product of kmt1 showed the expected length. Capsular serotyping identified 8 strains as type A and 2 as type D. Antimicrobial susceptibility test results revealed high resistance rates to streptomycin (70%), gentamicin (60%), ofloxacin (60%), and amoxicillin (50%). The resistance rates to amikacin, kanamycin, ciprofloxacin, norfloxacin, enrofloxacin, and trimethoprim-sulfamethoxazole ranged from 10% to 40%. Five strains showcased multi-drug resistance, accounting for 50% of the total strains. The resistance genes strA, strB, and bla_ROB-1 were identified in all the isolates, while aac(6')-Ib-cr, Sul1, tetB, and tetH were found in 60%, 50%, 30%, and 10% of strains, respectively. Huhuang decoction and Scutellariae Radix decoction attenuated the resistance to amikacin, kanamycin, ciprofloxacin, and enrofloxacin in a concentration- and time-dependent manner. The most pronounced attenutation of resistance was observed after 72 h-exposure to the decoctions at 1/2 MIC. [Conclusion] A total of 10 P. multocida strains were isolated, among which 50% of the strains showed multi-drug resistance, indicating that multi-drug resistant strains constituted the majority of clinical isolates. Huhuang decoction demonstrated significant attenuating effects against the multidrug resistance of P. multocida, with Scutellariae Radix identified as the primary active component responsible for this effect.