【作者】 张煜；

【导师】 王建英； 王晶妍；

【作者基本信息】 内蒙古科技大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 布鲁氏菌病是人畜共患的传染病，是由布鲁氏菌侵染机体而引发的自然防疫源性疾病。该病抗菌药物治疗的疗程较长，患者难以坚持用药，易导致耐药情况出现，且有部分患者在患有布鲁氏菌病的同时还伴随着其它细菌感染症状发生。如何选用抗菌药物有效地治疗布鲁氏菌病已是亟待解决的问题。本研究通过分离、鉴定布鲁氏菌病病人血液中的细菌，以判断病人在感染布鲁氏菌的同时是否存在其它细菌感染情况的发生；同时测定临床常用抗菌药物及新型抗菌药物对布鲁氏菌的最低抑菌浓度（MIC）、防突变浓度（MPC），为临床治疗提供抗菌药物用药参考。本文以从乌兰察布市四子王旗中心医院及大口子村提取的布鲁氏菌病确诊病例及疑似病例的8个血样作为样本，对血液中的细菌进行分离；提取菌体基因组DNA，用布鲁氏菌属的特异性引物eri进行PCR鉴定以判断所分离出的细菌是否为布鲁氏菌属；对基因组DNA进行16S rDNA PCR扩增，将PCR产物16S rDNA测序并将测序结果输入至NCBI数据库中进行序列比对，结果共分离出19株、9种不同类型细菌。因此在治疗布鲁氏菌病时应先进行血液细菌培养，以确定所感染的菌体类型，从而选出最优的抗菌药物用药方案。采用CLSI（美国临床实验室标准化协会）规定的肉汤稀释法对布氏杆菌活疫苗S2株进行抗菌药物的MIC检测。各种药物的MIC值分别为：硫酸庆大霉素0.470μg/mL、硫酸卡那霉素1.362μg/mL、利福平1.821μg/mL、硫酸链霉素1.700μg/mL、四环素2.710μg/mL、莫西沙星0.298μg/mL。与其余5种药物相比，第四代喹诺酮类药物莫西沙星的MIC值最低，抑菌能力最强。氨基糖苷类抗菌药物中，硫酸庆大霉素的MIC值最低，硫酸链霉素的MIC值最高。通过琼脂稀释法，分别进行了硫酸庆大霉素、硫酸卡那霉素、利福平、硫酸链霉素、莫西沙星对布氏杆菌活疫苗S2株的MPC及耐药选择性指数SI的检测。以上五种药物中莫西沙星的MPC值最低，利福平的MPC值最高；硫酸庆大霉素的SI值最低，利福平的SI值最高。莫西沙星、硫酸庆大霉素抑制细菌耐药的能力要优于硫酸卡那霉素、利福平、硫酸链霉素。研究表明：MPC值低的药物其SI值不一定也低，同类型抗菌药物的MPC、SI值存在差异。因此对于作用机理相类似的同类抗菌药物而言，仍然需要检测药物的MPC、SI值；在选择治疗药物时需同时参考抗菌药物的MPC及SI值，选用MPC、SI值均较低的药物进行治疗。更多还原

【Abstract】 Brucellosis is a zoonosis which is caused by Brucella bacteria infection. The antibiotictherapy requires long time treatment that makes it difficult for patients to adhere to themedication; this situation will easily lead to the emergence of drug resistance. In themeanwhile, some patients suffering from brucellosis may have symptoms of other bacterialinfection. Therefore, how to choose antimicrobial agents for the effective treatment ofbrucellosis is a problem which is required to solve immediately. In this study, the separationand identification of bacteria in the blood of brucellosis patients were conducted to diagnosewhether a patient infected by Brucella would have symptoms of other bacterial infections atthe same time. For the purpose of prevention of drug resistance, the minimum inhibitoryconcentration (MIC) and mutant prevention concentration (MPC) for a Brucella bacterialstrain were determined. This work will provide reference for the clinical treatment ofantibacterial drugs in the future.In this article, eight blood samples of brucellosis confirmed cases and suspected casesfrom Siziwangqi Central Hospital in Wulanchabu City and Dakouzi Village were sampled asresearch materials and the bacteria in the blood were separated; the bacterial genomic DNAwas extracted to determine whether the isolated bacteria were the genus of Brucella throughBrucella auele specific primer eri and PCR analysis; The16S rDNA were PCR amplifiedand sequenced. Sequences were compared to the NCBI database, showing that the19strainsof bacteria belonged to9different species, including Bacillus pumilus, Bacillus thuringiensis,Arthrobacter phenanthrenivorans, Bacillus megaterium, Pseudomonas stutzeri, Delftiaacidovorans, Bordetella avium, Brevundimonas subvibrioides and Acinetobacter baumannii.Therefore, blood culture should be carried out first for the drug dosage schedules and for thechoosing types of the drug to be used in the treatment of brucellosis.The MICs of six drugs on Brucella living vaccine S2strain were determined usingCLSI Broth Filution method as follows: gentamicin sulfate0.470μg/mL, kanamycin sulfate1.362μg/mL, the rifampicin1.821μg/mL, streptomycin sulfate1.700μg/mL, tetracycline2.710μg/mL, moxifloxacin0.298μg/mL respectively. Compared with the other five drugs,the fourth generation fluoroquinolone moxifloxacin had the lowest MIC value, indicatingthat it possesses the strongest antimicrobial activity. Among the Aminoglycoside antibioticsdrugs, gentamicin sulfate has the lowest MIC and streptomycin sulfate possesses the highestMIC value. The MPCs and drug resistance selectivity index (SI) on Brucella living vaccine S2strain were determined using the agar dilution method for gentamicin sulfate, kanamycinsulfate, rifampicin, streptomycin sulfate and moxifloxacin. Among the five drugs,moxifloxacin had the lowest MPC value and rifampicin had the highest MPC value;gentamicin sulfate had the minimum SI and rifampin had the highest SI, indicating thatmoxifloxacin and gentamicin sulfate have better ability to prevent emergence of drugresistance mutants than kanamycin sulfate, rifampicin and streptomycin sulfate do. Our datashowed that drugs with the lower MPC values did not necessarily have the lower SI values.Furthermore, antibacterial drugs in the same categories with a similar mechanism of actionmay have different MPC and SI values; thus their MPC and SI values need to be determinedindividually, and the variation of MPC and SI for the drugs should be taken intoconsideration at the same time for choosing antimicrobial agents in clinics.更多还原