洋葱伯克霍尔德菌医院血流感染暴发的分子流行病学研究

发布时间：2018-04-26 02:20

本文选题：医院感染 + 血流感染　；参考：《天津医科大学》2007年硕士论文

【摘要】： 医院感染发病率日益增加，危害严重。医院感染的暴发，导致病死率高，且延长住院时间，增加住院费用，影响恶劣。控制医院感染暴发的关键是如何早期发现，及时控制。表型分型方法包括生化分型、抗生素药敏谱分型等方法，存在诸多不足，如分辨力低，影响因素多，重复性较差等。分子分型技术应用于医院感染暴发的流行病学调查有重要意义。本研究对近期某院术后28株洋葱伯克霍尔德菌医院血流感染临床株进行表型和基因分型研究。探讨应用分子流行病方法调查洋葱伯克霍尔德菌医院血流感染暴发的价值。第一部分洋葱伯克霍尔德茵血流感染临床株的抗生素药敏谱分析目的：了解28株洋葱伯克霍尔德菌医院血流感染临床株的抗生素药敏谱。方法：应用琼脂纸片扩散法(K-B法)和微量肉汤稀释法(MICs)检测28株临床株对15种抗生素的抗生素药敏谱。结果：洋葱伯克霍尔德菌医院血流感染临床株，对15种药物敏感实验的结果表明，全部菌株对四环素、阿米卡星、庆大霉素耐药，对氟喹诺酮、碳氢酶烯类、复方新诺明、米诺环素、头孢曲松、头孢他啶、哌拉西林敏感，对头孢哌酮舒巴坦钠、头孢吡肟、头孢氨噻肟的敏感性存在微小差异。第二部分洋葱伯克霍尔德菌血流感染临床株的recA基因分型研究目的：28株洋葱伯克霍尔德菌医院血流感染临床株的recA基因分型研究。方法：进行recA基因分型研究： 1．对28株洋葱伯克霍尔德菌医院血流感染临床株进行recA基因的PCR扩增。 2．应用限制性内切酶HaeⅢ对recA基因PCR扩增片段进行PCR-RFLP分析。 3．recA基因PCR扩增片段的序列分析。结果：应用PCR-RFLP技术对28株临床株进行recA基因分型，结果表明全部临床株recA基因的PCR扩增阳性，PCR产物经HaeⅢ限制性内切酶反应，出现一致的限制性内切酶反应谱型，符合RFLP-J型，即B．stabilis(基因型Ⅳ)。recA基因PCR扩增片段的序列分析，进一步证实为B．stabilis(基因型Ⅳ)。第三部分洋葱伯克霍尔德菌血流感染临床株的脉冲场凝胶电泳(PFGE)分析目的：了解应用脉冲场凝胶电泳(PFGE)在洋葱伯克霍尔德菌医院血流感染暴发调查中的作用。方法：从28株分离株中随机选取一株未经recA基因测序的样本B140(抗生素药敏谱型Ⅰ)和一株经recA基因测序的样本B258(抗生素药敏谱型Ⅴ)，应用限制性内切酶SpeⅠ进行全基因组DNA的脉冲场凝胶电泳(PFGE)分析。结果：临床株B140和B258全基因组DNA的限制性内切酶反应图谱具有同样的条带数，且相应条带大小相同，依照Tenover等的标准，判断为同一克隆。结论： 1．本次研究的致病菌区别于区别于囊性纤维化(CF)患者的呼吸系统流行的多重耐药菌株。依据对头孢哌酮舒巴坦钠、头孢吡肟、头孢氨噻肟的敏感性存在的微小差异，，28株临床株的抗生素药敏谱表现为七型。 2．应用PCR-RFLP技术对28株临床株进行recA基因分型，结果表明全部临床株recA基因的PCR扩增阳性，PCR产物经HaeⅢ限制性内切酶反应，出现一致的限制性内切酶反应谱型，符合RFLP-J型，即B．stabilis(基因型Ⅳ)。recA基因PCR扩增片段的序列分析，进一步证实为B．stabilis(基因型Ⅳ)。 3．从分离株中，随机选取一株未经recA基因测序的样本B140(抗生素药敏谱型Ⅰ)和一株经recA基因测序的样本B258(抗生素药敏谱型Ⅴ)，进行脉冲场凝胶电泳(PFGE)分析，结果表明2株临床株的全基因组DNA的限制性内切酶SepⅠ反应图谱，有同样的条带数，且相应条带大小相同，依照Tenover等的标准，判断2株临床株为同一克隆。 4．综上研究，经抗生素药敏谱分型、recA基因的PCR-RFLP分型和序列分析、以及脉冲场凝胶电泳分型研究，可以判定2005年6月至7月某医院手术后发生的28例败血症患者为B．stabilis(基因型Ⅳ)的医院血流感染暴发。 5．recA基因的PCR-RFLP分型和脉冲场凝胶电泳分型对洋葱伯克霍尔德菌的分型比较，均显示相同的分型结果，对医院感染暴发的调查及追踪溯源有重大价值。以经济和易操作性的角度，对洋葱伯克霍尔德菌医院感染暴发的调查，recA基因的PCR-RFLP分型更具推广价值。 6．本研究医院血流感染暴发中，洋葱伯克霍尔德菌作为致病菌应引起高度重视。
[Abstract]:The incidence of nosocomial infection is increasing and the harm is serious. The outbreak of nosocomial infection leads to high mortality, prolongs hospitalization time and increases hospitalization expenses. The key of controlling nosocomial infection outbreaks is how to discover early and control timely. The methods of phenotypic typing include biochemical typing, antibiotic susceptibility typing, and so on, there are many shortcomings. Such as low resolution, many influencing factors and poor repeatability, etc. molecular typing technology is of great significance in the epidemiological investigation of nosocomial infection outbreaks.
In this study, the phenotypic and genotyping of 28 clinical strains of Burke and Holder fungus nosocomial flow infection in a hospital of onions were studied in this study. The value of molecular epidemiological method was investigated to investigate the outbreak of blood flow infection in Burke and Holder fungus hospital.
Part 1 antibiotic susceptibility spectrum analysis of clinical isolates of Burke Holder blood stream infected with onion
Objective: to understand the antibiotic susceptibility spectrum of 28 strains of Burke Holder fungus hospital infection.
Methods: agar disk diffusion method (K-B) and micro broth dilution (MICs) were used to detect antibiotic susceptibility spectrum of 28 clinical isolates to 15 antibiotics.
Results: the clinical strain of the bloodstream infection of the onions Burke Holder hospital, the results of 15 drug sensitive experiments showed that all strains were resistant to tetracycline, Amikacin, gentamicin, fluoroquinolone, carbenolene, minocycline, ceftriaxone, ceftazidime, piperacillin, cefoperazone, and cefoperazine, and cefoperazone, sulbactam, and cefoperazone. The sensitivity of pyrazoxime and cefotaxime is slightly different.
The second part is recA genotyping of clinical isolates of Burke Holder infected blood stream in onion.
Objective: To study the recA genotyping of 28 strains of clinical isolates of Burke Holder disease in onion hospital.
Methods: the study of recA genotyping:
1. PCR amplification of recA gene was carried out on 28 strains of clinical isolates of Burke Holder fungus hospital infection.
2. restriction endonuclease Hae III was used to carry out PCR-RFLP analysis of recA gene PCR amplification fragment.
Sequence analysis of PCR amplified fragment of 3.recA gene.
Results: the recA genotyping of 28 strains of clinical strains was carried out by PCR-RFLP technique. The results showed that the PCR amplification of recA gene was positive in all clinical strains. The PCR product, through the Hae III restriction endonuclease reaction, appeared a consistent restriction endonuclease response spectrum type, which conformed to the RFLP-J type, that is, the sequence of B.stabilis (genotype IV).RecA gene PCR amplification fragment. It is further confirmed that B.stabilis (genotype IV).
The third part is the pulsed field gel electrophoresis (PFGE) analysis of clinical isolates of Burke Holder blood stream infection in onion.
Objective: To investigate the role of pulsed field gel electrophoresis (PFGE) in investigating the outbreak of bloodstream infection in Burke Holder fungus hospital.
Methods: a sample of B140 (antibiotic susceptibility type I) and a sample of B258 (antibiotic susceptibility type V), which were sequenced by recA gene, were randomly selected from 28 isolates, and the restriction endonuclease Spe I was used to analyze the whole genome DNA by pulse field gel electrophoresis (PFGE).
Results: the restriction endonuclease response map of B140 and B258 genome DNA has the same number of bands, and the corresponding band size is the same. According to the standard of Tenover and so on, it is judged to be the same clone.
Conclusion:
1. the pathogenic bacteria in this study were distinguished from the multidrug-resistant strains prevalent in the respiratory system of patients with cystic fibrosis (CF). According to the slight differences in the sensitivity of cefoperazone sulbactam, cefepime, cefotaxime, the antibiotic susceptibility of 28 clinical strains was shown to be type seven.
2. the recA genotyping was performed on 28 strains of clinical strains by PCR-RFLP technology. The results showed that the PCR amplification of the recA gene was positive in all clinical strains. The PCR product, through the Hae III restriction endonuclease reaction, appeared a consistent restriction endonuclease response spectrum type, which conformed to the sequence analysis of RFLP-J type, namely, B.stabilis (genotype IV).RecA gene PCR amplification fragment. One step was confirmed as B.stabilis (genotype IV).
3. from the isolates, a sample of B140 (antibiotic susceptibility type I) and a sample B258 (antibiotic susceptibility profile V), which were sequenced by recA gene, were randomly selected and analyzed by pulse field gel electrophoresis (PFGE). The results showed that the restriction endonuclease Sep I reaction Atlas of the total genomic DNA of the total genome of 2 strains of clinical strains was similar. The number of bands and the size of the corresponding bands were the same. According to the criteria such as Tenover, 2 clinical isolates were identified as the same clone.
4. in a comprehensive study, 28 cases of septicemia in a hospital from June 2005 to July were diagnosed as B.stabilis (genotype IV) in hospital blood flu outbreak, after the antibiotic susceptibility typing, PCR-RFLP typing and sequence analysis of recA gene, and pulse field gel electrophoresis typing.
The comparison of the typing of 5.recA gene PCR-RFLP and pulse field gel electrophoresis on the typing of Burke and Holder bacteria in onion showed the same typing results. It was of great value for the investigation and tracing of the nosocomial outbreak. The investigation of the outbreak of the nosocomial infection of the onion Burke and Holder bacteria, the P of the recA gene in the perspective of economic and easy operation The CR-RFLP classification is more valuable.
6. in the study of outbreak of nosocomial bloodstream infection, onion Burke Holder bacteria should be highly regarded as pathogenic bacteria.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R181.3

【引证文献】