体外诱导禽巴氏杆菌链霉素耐药株及耐药机制的初步分析

汪最 孔令严 邵华斌 卢琴 罗青平 杨玉莹

摘要：為了研究多杀性巴氏杆菌（Pasteurella multocida，Pm）对链霉素（Streptomycin，Str）药物敏感性变化及其耐药机制，采用体外诱导法对禽源Pm亲本敏感株（C48-1）进行诱导后成功获得Str耐药株，并对Str耐药株的最小抑菌浓度（MIC）、生化特性、生长曲线、耐药稳定性、耐药基因点突变以及耐药逆转性进行了研究。结果表明，与亲本株相比，Str耐药株生化特性与生长曲线无显著差异，MIC值由8 μg/mL上升至1 024 μg/mL；发现耐药基因StrA、StrB，且核糖体基因rpsL编码的氨基酸发生了Lys43→Thr突变；耐药逆转性试验中羰基氰氯苯腙（CCCP）将Str耐药株MIC值降为16 μg/mL。逐步递增药物浓度可以诱导禽源Pm对氨基糖苷类药物的耐药性，其耐药机理为磷酸转移酶类对氨基糖苷类抗生素结构的修饰作用，核糖体靶位点突变和外排泵的过量表达。

关键词：禽源多杀性巴氏杆菌；体外诱导；链霉素；耐药基因；位点突变；耐药机制

中图分类号：S852.61+5 文献标识码：A 文章编号：0439-8114（2018）08-0096-04

DOI：10.14088/j.cnki.issn0439-8114.2018.08.024

Characterization of Sreptomycin-resistant Avian Pasteurella multocida in Vitro Induction and Preliminary Analysis of Resistance Mechanism

WANG Zui1，2，KONG Ling-yan2，SHAO Hua-bin2，LU Qin2，LUO Qing-ping2，YANG Yu-ying1

（1.College of Animal Sciences，Yangtze University，Jingzhou 434025，Hubei，China；2.Institute of Animal Husbandry and Veterinary，Hubei Academy of Agricultural Sciences/Key Laboratory of Prevention and Control Agents for Animal Bacteriosis（Ministry of Agriculture），

Wuhan 430064，China）

Abstract： In order to study the drug susceptibility to sreptomycin（Str） and its drug resisitance mechanism of avian Pasteurella multocida（Pm），a Str-resistant avian Pm was obtained via in vitro induction with gradually increasing drug concentrations to the standard strain named C48-1. The minimal inhibitory concentration（MIC），biochemical characteristics， growth curve，stability of drug resistance，drug resistance gene site mutation and drug resistance reversal of the Str-resistant strain was analyzed. The results showed that compared with the parent strain， the biochemical characteristics and growth curve had no significant difference，the MIC of the Str-resistant strain was increased from 8 μg/mL to 1 024 μg/mL. What more，the drug resistance gene StrA and StrB were found and the amino acid sequence encoded by the rpsL gene had an amino acid exchange of Lys43→Thr. In the drug resistance reversal test， carbonyl cyanide-m-chlorophenylhydrazone （CCCP） reduced the MIC of the Str-resistant strain to 16 μg/mL. In total，it shows that gradually increasing drug concentration can induce Pm resistance to aminoglycosides in vitro. Its mechanism is the modification of aminoglycoside antibiotics by phospho-transferase，the mutation of ribosomal protein and the over expression of efflux pumps.

Key words： avian Pasteurella multocida； in vitro induction； sreptomycin； drug resistance gene； site mutation； resistance mechanism

禽源多杀性巴氏杆菌（Pasteurella multocida，Pm）是引起禽霍乱的致病菌，该病属于急性、热性、高致病性传染病[1]。在中国引起禽霍乱的病原菌主要是荚膜血清A型Pm[2]。目前对于该病的控制措施，主要采用药物防治，尤其是抗生素如链霉素、磺胺嘧啶等效果较好，但耐药菌株的出现对该病的防控带来了严峻挑战[3]。

氨基糖苷类药物属于最早投入临床使用的抗菌药物之一，由于长期、频繁、大剂量使用，近年来临床分离出的Pm对氨基糖苷类药物出现普遍耐药现象，部分分离株对Str等氨基糖苷类药物已达到了高度耐药[4]。……