目录
摘 要 I
关键词 I
Abstract II
引言
第一章 前言 1
1.1 猪链球菌与细菌耐药性研究现状 2
1.1.1 猪链球菌概况 2
1.1.2 细菌耐药性的产生与水平传播 2
1.1.3 细菌对替米考星耐药的研究进展 4
1.1.4 细菌对万古霉素耐药的研究进展 4
1.1.5 细菌对利奈唑胺耐药的研究进展 5
1.2 研究目的及意义 5
1.3 研究内容与可行性分析 6
第二章 猪链球菌对替米考星、万古霉素和利奈唑胺的耐药性和相关耐药基因的检测及其基因环境分析 7
2.1 实验材料及来源 7
2.1.1 样品来源 7
2.1.2 抗菌药物 7
2.1.3 培养基及其配制 7
2.1.4 主要生化试剂 7
2.1.5 主要仪器设备 7
2.2 实验方法与结果处理 8
2.2.1 猪链球菌的分离纯化 8
2.2.2 猪链球菌的鉴定 8
2.2.3 药敏试验 9
2.2.4 替米考星、万古霉素和利奈唑胺相关耐药基因的检测 9
2.2.5 WGS测序和序列分析 10
2.3 结果与分析 10
2.3.1 猪链球菌的分离鉴定 10
2.3.2 猪链球菌对替米考星、万古霉素和利奈唑胺的耐药率分析 11
2.3.3 不同来源菌株的耐药率比较 11
2.3.4 猪链球菌的耐药谱分析 11
2.3.5 耐药基因ermB、vanG和optrA的检测率 12
2.3.6 ermB、vanG和optrA的基因环境及相关可移动基因元件分析 13
2.4 讨论 15
第三章 结论与展望 17
参考文献 18
致谢 21
发表文章 22
猪链球菌对替米考星、万古霉素和利奈唑胺的耐药性以及相关耐药基因检测分析
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要
本研究从江苏北部地区猪场分离鉴定了103株猪链球菌，通过微量肉汤稀释法检测了替米考星、万古霉素和利奈唑胺对这些菌株的最小抑菌浓度（MIC），用PCR方法检测了替米考星耐药基因ermB、万古霉素耐药基因vanG和利奈唑胺耐药基因optrA，并分析了相关耐药基因的基因环境。结果显示103株菌对替米考星、利奈唑胺和万古霉素的耐药率分别为90.29%（93/103）、16.50%（17/103）和3.88%（4/103），耐药基因的检出率分别为ermB 94.17%（97/103）、optrA 24.27%（25/103）、vanG 3.88%（4/103）。分别对1株万古霉素和替米考星耐药并含有ermB、vanG的菌株，以及1株对3种抗菌药均耐药并同时含有ermB、optrA、vanG的多重耐药菌株进行了全基因组测序，测序结果显示上述3种耐药基因均由可移动基因元件携带，为ICESa2603家族的整合与接合性元件和类似Φm46.1的前噬菌体。该研究结果提示猪链球菌不仅对兽医临床常用的抗菌药替米考星产生耐药性，还对人医临床使用但兽医临床未批准使用的万古霉素和利奈唑胺产生耐药性，且相关耐药基因均由可移动基因元件携带，存在水平传播风险，需引起重视。
ABSTRACT
The aim of the study is to monitor the resistance rates of Streptococcus suis (S. suis) to tilmicosin, vancomycin, and linezolid as well as to clarify the related mobile genetic elements which contribute to the transfer of ermB, vanG, and optrA. 103 strains of Streptococcus suis were identified from northern Jiangsu Province. The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) of tilmicosin, vancomycin, and linezolid against those strains. The distribution of resistance genes ermB, vanG, and optrA was detected by PCR. The genetic environment of resistance genes was analyzed by Whole Genome Sequencing (WGS). Results showed that resistant rates of these strains to tilmicosin, linezolid, and vancomycin were 90.29% (93/103), 16.50% (17/103) and 3.88% (4/103), respectively. Detection rates of ermB, optrA and vanG were 94.17% (97/103), 24.27% (25/103), and 3.88% (4/103), respectively. WGS was applied to two stains, one is resistant to vancomycin and telmicosin which harbors ermB and vanG, and the other harbours ermB, optrA, and vanG and is resistant to all three antimicrobials. Sequencing results showed that three kinds of resistance genes in both strains were all carried by mobile genetic elements ICEs belonging to ICESa2603 family, and a prophage similar to Φm46.1. In conclusion, the results indicate that Streptococcus suis is resistant not only to tilmicosin, which is commonly used in veterinary medicine, but also to vancomycin and linezolid, which are critical antimicrobials usually used in human medicine which are not approved to be used in veterinary medicine. Moreover, those resistance genes are all carried by mobile genetic elements, which has risks of horizontal transmission and should be paid much attention to.

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