临床分离胥伐成格隆沙门菌的抗菌药物敏感性和毒力基因研究

发布时间：2018-03-17 01:18

本文选题：胥伐成格隆沙门菌　切入点：微生物敏感性试验　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的研究临床分离胥伐成格隆沙门菌的抗菌药物敏感性、分子分型、毒力基因携带和患者临床特征,为了解胥伐成格隆沙门菌病原学特点、预防与控制其感染以及临床合理使用抗菌药物提供依据。方法1.收集2014年5月至10月我院肠道门诊急性腹泻患者的临床资料,采集患者粪便标本进行沙门菌分离培养、生化鉴定、血清型分型。2.对鉴定得到的胥伐成格隆沙门菌采用K-B法和MIC法进行11种抗菌药物敏感性检测。3.多位点序列分型(MLST):扩增胥伐成格隆沙门菌的7个管家基因(thrA,purE,sucA,hisD,aroC,hemD,dnaN),将测序结果与国际数据库网站比较后获得等位基因图谱和ST型。4.脉冲场凝胶电泳分型(PFGE):对胥伐成格隆沙门菌基因组DNA采用限制性内切酶XbaI酶切,经过脉冲场凝胶电泳后采用BioNumerics软件对电泳条带进行聚类分析。5.分析患者病史和临床表现以及与菌株分子分型的关系。6.毒力基因PCR检测:以沙门菌毒力基因岛(SPI)1~5中的代表性基因(SPI1:invA、sitC、hilA;SPI2:sseL、sifA;SPI3:mgtC;SPI4:siiE;SPI5:sopB)、毒力质粒基因(pefA、prot6E、spvB)、调控基因(phoP)为目的基因,对菌株进行特异引物PCR扩增。结果1.共分离到胥伐成格隆沙门菌16株(14.8%),占同期全部非伤寒沙门菌(108株)的14.8%。2.16株胥伐成格隆沙门菌对11种抗菌药物氨苄西林、氯霉素、链霉素、复方新诺明、四环素、萘啶酸、环丙沙星、左氧氟沙星、头孢曲松、头孢他啶、厄他培南均100%敏感。3.16株胥伐成格隆沙门菌的7个管家基因等位基因谱完全一致,依次是43、47、49、16、41、15、3,均为ST241型。4.以相似度100%为标准,16株胥伐成格隆沙门菌共获得3种PFGE带型,包括A型14株、B型1株和C型1株。A型与B型相似度为94.2%,二者与C型相似度为77.8%。5.16例胥伐成格隆沙门菌感染患者男性10例、女性6例,中位年龄35.5岁,均因急性腹泻就诊,临床诊断包括细菌性痢疾3例和胃肠炎13例,其中1例患者伴有高热。有8例患者共同进餐后发病,就诊时间集中于2014年6月17日至19日的两天之内,患者粪便分离菌株PFGE带型完全一致,均为A型。6.16株胥伐成格隆沙门菌SPI1~5代表性基因(invA、sitC、hilA、sseL、sifA、mgtC、siiE、sopB)和调控基因(phoP)100%阳性,毒力质粒基因(pefA、prot6E、spvB)均为阴性。结论1.2014年本地区出现多例胥伐成格隆沙门菌临床感染,其中包含了一起感染暴发。2.本研究检测到多种PFGE带型ST241型胥伐成格隆沙门菌临床株,对常用抗菌药物尚保持敏感,但携带SPI1~5毒力基因和调节基因,提示其具有高致病力,对公众健康构成威胁。3.应关注胥伐成格隆沙门菌临床感染在我国的变化趋势和危害,开展持续监测和研究。
[Abstract]:Objective to study the antimicrobial susceptibility, molecular typing, virulence gene carrying and clinical characteristics of Scheffarn's Salmonella schevaronii in order to understand its etiological characteristics. Prevention and control of infection and rational use of antimicrobial agents. Methods 1. Clinical data of patients with acute diarrhea in intestinal outpatient department from May 2014 to October were collected and stool samples were collected for isolation and culture of Salmonella. Biochemical identification, Serotype typing .2. using K-B method and MIC method to detect the susceptibility of 11 antimicrobial agents to the identified Salmonella schevaronii. Multilocus sequence typing: amplification of seven housekeeping genes of Schuffat into Salmonella grossurum DaroChemDdna nna, and sequencing. Results compared with the international database website, allelic map and St type. 4. Pulsed field gel electrophoresis (PGE) typing: the genomic DNA of Salmonella schevatensis was digested by restriction endonuclease XbaI. After pulse field gel electrophoresis, BioNumerics software was used to cluster analysis of the electrophoresis bands. 5. Analysis of patient's history, clinical manifestation and relationship with strain molecular typing. 6. Detection of virulence gene PCR: in Salmonella virulence gene island. The representative gene SPI1: invAt sitCor hila, SPI2: sseLLnsifAn SPI3: MGtCn SPI4: siiEn SPI5: sopB, virulent plasmid gene pefAbuprot6EspvB, regulatory gene phoP, is the target gene. Results 1. A total of 16 strains of Salmonella vulgaris, which accounted for 108 strains of non-typhoid salmonella, were isolated from the bacteria by PCR. 2. 16 strains of Salmonella xulfengensis were isolated for 11 antimicrobial agents ampicillin, chloramphenicol, streptomycin. The alleles of seven housekeeping alleles of compound sulfamine, tetracycline, naphthalic acid, ciprofloxacin, levofloxacin, ceftriaxone, ceftazidime and ertapenem were identical in alleles of 7 alleles of Salmonella xuvalensis. The sequence was 43, 47, 49, 41C153, all of which were ST241 type. 4. According to similarity degree 100%, 16 strains of Salmonella xufangchengensis were found to have three PFGE banding patterns. The similarity between type A and type C was 94.2, and the similarity between them was 77.8.5.16 cases of schistosomiasis infection were male 10 cases, female 6 cases, median age 35.5 years old, all of them were treated with acute diarrhea, the average age was 35.5 years old, the average age was 35.5 years old, and the similarity between the two strains was 77.8% and 5.16 cases respectively, including 10 males and 6 females, with a median age of 35.5 years, and a median age of 35.5 years. Clinical diagnosis included 3 cases of bacillary dysentery and 13 cases of gastroenteritis, of which 1 case was accompanied by high fever. The PFGE banding patterns of fecal isolates from patients were identical and were all positive for type A. 6.16 SPI1~5 representative genes of Salmonella schevaronii, AhilsseSse, sifsif, mgtCsii, sopB, and the regulatory gene, phopt1, 100%, respectively, and the positive rate was 100%, and the positive rate was 100% (P < 0.05), and the positive rate was 100% (P < 0.05). The virulence plasmid gene was negative. Conclusion 1. There were many clinical infections of Salmonella schevarinensis in 2014, including an outbreak of infection. In this study, a variety of clinical strains of ST241 type of PFGE were detected, and the results showed that there were many clinical strains of Schulonella schevaronii in 2014, and the results were as follows: (1) in this study, there were many clinical infections of Salmonella schevaronii in this area, including an outbreak of infection. SPI1~5 virulence gene and regulatory gene are still sensitive to common antimicrobial agents, which indicates that it is highly virulent and poses a threat to public health .3.We should pay attention to the trend and harm of clinical infection of Salmonella xuvalchengensis in China. Carry out continuous monitoring and research.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5;R516.3

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