牛支原体对氟喹诺酮及氨基糖苷类抗生素耐药机制的初步研究

发布时间：2018-04-26 21:39

本文选题：牛支原体 + 氟喹诺酮类抗生素　；参考：《吉林农业大学》2017年硕士论文

【摘要】：牛支原体(Mycoplasma bovis,M.bovis)是引起牛呼吸系统疾病的(Bovine respiratory disease,BRD)主要病原之一,该病原引发的疾病主要采用大环内酯类、氟喹诺酮类、氨基糖苷类抗生素进行治疗,但随着抗生素在兽医临床上的广泛使用,M.bovis对上述药物逐渐产生了耐药性。近年来,M.bovis对其主要治疗药物的耐药性所引起的抗感染失败给我国畜牧养殖业造成了较严重的经济损失。本研究采用改良微量稀释法检测临床分离的32株M.bovis对氟喹诺酮及氨基糖苷类抗生素的最低抑菌浓度(Minimal inhibitory concentration,MIC),结果表明,部分M.bovis已对氟喹诺酮类抗生素产生了耐药性(其MIC为4或8μg/mL),分离的耐药菌株占此次临床分离M.bovis的19%;临床分离的32株M.bovis均对氨基糖苷类抗生素敏感或中度耐药,可能存在耐药风险。分别成功诱导了对氟喹诺酮类抗生素高度耐药的M.bovis和对氨基糖苷类抗生素高度耐药的M.bovis,其中,经体外诱导获得的对一种氟喹诺酮类抗生素高度耐药的M.bovis对其它两种受试氟喹诺酮类抗生素均存在交叉耐药现象,经体外诱导获得的对一种氨基糖苷类抗生素高度耐药的M.bovis对其它两种受试氨基糖苷类抗生素存在部分交叉耐药现象。在此基础上,本研究对临床分离的M.bovis氟喹诺酮类抗生素敏感株、耐药株及体外诱导获得的高度耐药株进行氟喹诺酮类抗生素靶位突变分析,结果表明,临床分离的耐药菌株其氟喹诺酮类抗生素耐药决定区(Quinolone resistance determining region,QRDR)均存在gyrA(Ser83Phe)或parC(Ser80Ile)的氨基酸突变;体外诱导的高度耐药株中QRDR突变类型则以gyrA(Ser83Phe/Tyr或Glu87Lys)和parC(Ser80Ile或Asp84Asn/Tyr)的氨基酸发生突变为主;耐药性逆转试验结果表明,1/4 MIC外排泵抑制剂羰基氰氯苯腙(carbonyl cyanide m-chlorophenyl hydrazone,CCCP)和维拉帕米(verapamil,VP)作用前后受试菌对环丙沙星、恩诺沙星、诺氟沙星的MIC值未发生变化,推断M.bovis中不存在过量表达的以氟喹诺酮类抗生素为底物的主动外排系统。本研究对临床分离的M.bovis氨基糖苷类抗生素敏感株、中度耐药株及体外诱导获得的高度耐药株进行氨基糖苷类抗生素靶位突变分析,结果表明,临床分离的氨基糖苷类抗生素敏感株及中度耐药株在氨基糖苷类抗生素耐药决定区(Aminoglycosides resistance determining region,ARDR)均不存在碱基突变;经体外诱导获得的对庆大霉素、卡那霉素、大观霉素高度耐药的M.bovis在其16S rRNA(rrs3和rrs4)的ARDR中分别存在A1409T、A1408G、C1192A的碱基突变,但上述经体外诱导获得的氨基糖苷类抗生素高度耐药株在编码核糖体蛋白S5的rpsE基因上未检测到有意义的碱基突变;对临床分离的32株M.bovis进行16S rRNA甲基化酶基因及氨基糖苷类抗生素耐药基因检测,结果表明,临床分离的M.bovis均携带aphA1、strB磷酸转移酶基因及aacA4乙酰转移酶基因。此外,耐药性逆转试验结果表明,1/4 MIC外排泵抑制剂CCCP和VP作用前后受试菌对庆大霉素、卡那霉素、大观霉素的MIC值亦未发生变化,推断M.bovis中不存在过量表达的以氨基糖苷类抗生素为底物的主动外排系统。
[Abstract]:Mycoplasma bovis (M.bovis) is one of the main pathogens causing Bovine respiratory disease (BRD). The disease caused by this pathogen is mainly treated with macrolide, fluoroquinolone, aminoglycoside antibiotics, but with the extensive use of antibiotics in veterinary clinic, M.bovis against the above drugs In recent years, the anti infection failure caused by M.bovis's resistance to the main therapeutic drugs has caused serious economic loss to the livestock breeding industry in China. In this study, the minimal inhibitory concentration of 32 clinical isolates of M.bovis against fluoroquinolone and aminoglycoside antibiotics was detected by modified microdilution method. Minimal inhibitory concentration, MIC), the results showed that partial M.bovis had produced resistance to fluoroquinolone antibiotics (its MIC was 4 or 8 mu g/mL), and the isolated drug resistant strains accounted for 19% of the clinically isolated M.bovis; 32 strains of clinical isolates were both sensitive to aminoglycoside antibiotics or moderately resistant, and may have the risk of resistance. M.bovis and M.bovis, highly resistant to fluoroquinolones, were not successfully induced, in which the highly resistant M.bovis of a fluoroquinolone antibiotic induced in vitro was cross resistant to two other fluoroquinolones, and was induced in vitro. M.bovis, which is highly resistant to an aminoglycoside antibiotic, is partially cross resistant to the other two kinds of aminoglycoside antibiotics. On the basis of this, the clinical isolates of M.bovis fluoroquinolone sensitive strains, drug-resistant strains and in vitro induced highly resistant strains were carried out with fluoroquinolone antibiotics. The results of target mutation analysis showed that the Quinolone resistance determining region (QRDR) in the drug-resistant isolates of the clinical isolates had the amino acid mutations of gyrA (Ser83Phe) or parC (Ser80Ile), and the QRDR mutation types of the highly resistant strains in vitro were gyrA. The amino acid mutation of er80Ile or Asp84Asn/Tyr was dominant, and the resistance reversal test showed that the MIC values of ciprofloxacin, enrofloxacin and norfloxacin were not changed before and after the effect of the carbonyl cyano-chlorobenzene hydrazone (carbonyl cyanide m-chlorophenyl hydrazone, CCCP) and Vera Pammy (verapamil, VP) on the pump inhibitor of 1/4 MIC. There was no excessive expression of the active efflux system with fluoroquinolone as the substrate. In this study, a clinically isolated M.bovis aminoglycoside antibiotic sensitive strain, moderately resistant strain and highly resistant strains in vitro were analyzed for the aminoglycoside antibiotic target mutation analysis. The results showed that the clinical isolates were separated from ammonia. There was no base mutation in the aminoglycoside antibiotic resistant determinant region (Aminoglycosides resistance determining region, ARDR) in the amino glycoside antibiotic sensitive and moderately resistant strains, and the highly resistant M.bovis of gentamicin, kanamycin and macromycin in the ARDR of 16S rRNA (rrs3 and rrs4) in vitro, respectively, respectively. There was a base mutation in A1409T, A1408G, and C1192A, but the above - mentioned aminoglycoside resistant highly resistant strains of the above - induced aminoglycoside resistant strains did not detect significant base mutations in the rpsE gene encoding ribosomal protein S5, and 32 strains of clinical isolates of M.bovis were detected by 16S rRNA methylase gene and aminoglycoside antibiotic resistance gene. The results showed that the clinical isolates of M.bovis all carried aphA1, strB phosphotransferase gene and aacA4 acetyltransferase gene. In addition, the drug resistance reversal test showed that the MIC values of the tested bacteria to gentamicin, kanamycin and mycophenycin before and after the action of CCCP and VP of the 1/4 MIC pump inhibitor were also not changed. It was inferred that there was no existence in M.bovis. Over expression of the active efflux system based on aminoglycoside antibiotics.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.62

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