碳青霉烯类耐药菌NDM-1质粒获取的危险因素和周围基因环境研究

发布时间：2018-02-28 23:05

本文关键词： NDM-1基因耐药菌株危险因素基因周围环境　出处：《南昌大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景和目的:区域性播散是造成质粒编码NDM-1酶(New Delhi metallo-β-lactamase1,即新德里金属-β-内酰胺酶-1)广泛流行的主要原因,然而其相关的危险因素和基因周围环境尚未见系统性报道。本研究通过收集筛选我院产NDM-1酶的碳青霉烯耐药菌,在调查bla_(NDM-1)基因的流行状况的基础上,对含NDM-1基因耐药菌株感染的临床危险因素与预后进行分析,提取含bla_(NDM-1)基因的质粒进行全基因组测序和生物信息学分析研究,系统探讨NDM-1基因的流行状况,bla_(NDM-1)基因耐药菌株的影响因素和预后,进一步分析质粒中NDM-1的基因周围环境,并与国内外相关研究进行比较。对揭示NDM-1耐药基因的流行、易感因素和进化趋势具有重要意义。为制订科学、有效的预防和控制措施提供实验依据。方法:1、收集和筛选我院临床标本中对碳青霉烯类抗生素不敏感的革兰氏阴性杆菌(剔除同一患者同一部位的重复菌株),应用PCR技术进行bla_(NDM-1)基因阳性标本筛查,将保存的鉴定为bla_(NDM-1)基因阳性的混合菌液传代培养,并将传代培养的菌液行bla_(NDM-1)基因筛查,由于分离的菌种数目多,菌种复杂,含有革兰氏阳性和阴性细菌,应用BIOLOG自动微生物鉴定系统进行鉴定同时扩增细菌16sr RNA进行测序,明确含bla_(NDM-1)基因的菌种,应用E-test法检测菌株抗菌药物敏感性、MIC值及金属酶表型筛选实验。通过耐药性和金属酶表型筛选实验,获得上述含bla_(NDM-1)基因的耐药菌株数据,结合临床资料进行分析。2、运用病例对照研究方法,将含NDM-1酶细菌感染的患者为A组,另按l:1:1的比例配对,分离出碳青霉素类不敏感细菌(NDM-1基因阴性)的患者为B组和碳青霉烯类抗生素敏感的细菌的患者为C组进行研究,采用病历信息收集回顾性研究的方法,制作调查表后查阅出院病历,调查患者的住院科室、姓名、性别、年龄、住院时间、基础疾病、危险因素、临床结局等资料,通过单因素和多因素Logstic回归分析碳青霉烯类耐药菌NDM-1质粒获取的危险因素。3、质粒接合试验验证bla_(NDM-1)基因遗传稳定性,对于分离的能够稳定遗传bla_(NDM-1)基因的菌株,Southern blot对bla_(NDM-1)进行基因定位。提取质粒DNA,将满足要求(含有NDM-1基因)的5个质粒分别构建基因组测序文库,将bla_(NDM-1)质粒进行高通量测序,对测序结果进行拼接及比对,进行质粒gap的填补及差异序列验证,并注释质粒系列的基因。将得到的质粒序列输入到NCBI在线网站中进行Blast比对,利用Mauve软件进行对比详细查看不同点。结果:1、1735株碳青霉烯类抗生素不敏感细菌中,一共筛选到含bla_(NDM-1)基因菌株54株,其中肺炎克雷伯菌中筛到阳性菌株44株,鲍曼不动杆菌株中筛到阳性菌株8株,大肠杆菌中筛到阳性菌株2株,bla_(NDM-1)基因阳性菌株体外药敏实验显示,bla_(NDM-1)基因阳性菌株对大多数的抗生素的耐药率都超过了50%,尤其是对耐碳青霉烯类抗生素耐药率非常高。改良Hodge实验提示有1735株碳青霉烯抗生素不敏感菌株中出现512株阳性。54株bla_(NDM-1)基因阳性菌株的临床资料显示其来自43位患者,住院科室主要分布在烧伤科、呼吸科、ICU;主要标本来源于痰液、尿液、血液。2、A组和B组对绝大多数抗生素表现出非常高的耐药率,C组对大部分抗生素的耐药率均较低,其中A组与B组统计分析比较具有统计学差异的抗生素为亚胺培南、阿米卡星、左亚氟沙星;耐碳青霉素类抗生素菌株(A+B组)和对碳青霉素类抗生素敏感菌株(C组)单因素分析,得出以下方面具有差异性:住院时间、分离出该菌株前两个月内应用广谱抗菌药物大于7天、从外院转入、分离出该菌株前14天内使用过抗生素类药物、发热峰值(平均℃)、抗生素使用种类、治疗中使用替加环素、好转出院(%)、治疗失败(%)和自动出院(%),并进行多因素Logstic回归分析得出分离出该菌株前两个月内应用广谱抗菌药物大于7天和分离出该菌株前14天内使用过抗生素类药物是耐碳青霉烯类抗生素菌株的独立危险因素;对A组和B组进行单因素分析,既往使用碳青霉素类抗生素和发生感染到死亡的时间具有差异性,并进行多因素Logstic回归分析得出既往使用碳青霉烯类抗生素史为碳青霉烯类耐药菌NDM-1质粒获取的独立危险因素。3、含bla_(NDM-1)基因菌株直接提取的质粒和大肠J53菌株进行接合的质粒,进行Southern blot杂交显色,均证实bla_(NDM-1)基因大部分存在于质粒中,且含bla_(NDM-1)基因菌株与大肠J53菌株进行质粒结合成功率为50%,提示携带NDM-1基因的质粒可以高效地在肠杆菌科类细菌之间发生转移,为细菌的耐药流行播散提供了传播载体。将5株经接合实验验证的质粒基因组建库成功后,进行质粒测序质量评价和基因组拼装,共得到2个完整的质粒基因组序列,即p12和p11106。选取7个物种的相关质粒序列,分别通过Mauve软件与的质粒序列做相似性图。通过上述基因环境比较,发现:1、p11106和p12质粒与p NDM-BTR非常相似;2、p11106、p12质粒和另外六个质粒存在包含(orf00032-orf00043)20-30kb区域的差异;3、NDM-1基因位于(orf00032-orf00043)的中间位置,区域的上端的有两个tnp A基因,以及在其下游发现36kb区域的orf00052(tnp A)。结论:1、bla_(NDM-1)基因在区域性医院存在一定的耐药流行,在我院以肺炎克雷伯杆菌为主;提示肺炎克雷伯杆菌在bla_(NDM-1)基因的传播过程中起到了重要作用,可能是bla_(NDM-1)基因的保存宿主;2、碳青霉烯类耐药菌NDM-1质粒获取的危险因素为既往有碳青霉素类抗生素使用史;3、p11106和p12质粒可能具有p NDM-BTR质粒的特征,含NDM-1质粒存在orf00032-orf00043差异性,且具有多态性,NDM-1基因位于orf00032-orf00043的中间位置,联系上端的两个tnp A基因和下游36kb区域的orf00052(tnp A),提示NDM-1可能是质粒通过转座获得,并长期保留在质粒上。
[Abstract]:Background and objective: regional spread is caused by plasmid encoding NDM-1 enzyme (New Delhi metallo- beta -lactamase1, namely New Delhi metallo beta lactamase -1) mainly due to widespread, but around the related risk factors and gene environment have not been reported. This study collected bacteria resistant ene carbapenems screening the NDM-1 enzyme production in our hospital, in the investigation of bla_ (NDM-1) based on the epidemic status of genes, the clinical risk factors for infection of resistant strains containing NDM-1 gene and prognosis were analyzed and extracted with bla_ (NDM-1) gene plasmid for whole genome sequencing and bioinformatics analysis, system to study the epidemic situation of NDM-1 gene bla_, (NDM-1) and prognostic factors influencing gene of resistant strains, further analysis of the NDM-1 gene plasmid in surrounding environment, and related research at home and abroad were compared. To reveal the NDM-1 resistant gene flow OK, the susceptible factors is of great significance and evolutionary trends. For the formulation of science, to provide experimental basis for effective prevention and control measures. Methods: 1, gram negative bacteria are not sensitive to carbapenem antibiotics in our hospital were collected and the clinical specimens (repeated strains removed the same with the same parts of the application). PCR bla_ (NDM-1) gene screening positive samples, will be preserved and identified as bla_ (NDM-1) gene positive bacteria were cultured and subcultured bacteria bla_ (NDM-1) gene screening, because the number of bacteria isolated, species complex, containing both Gram positive and gram negative bacteria, the application of BIOLOG automatic microorganism identification system identification and amplification of bacterial 16sr RNA sequencing, bla_ (NDM-1) gene containing specific strains, strains detected by E-test antimicrobial susceptibility, and metal enzyme phenotype screening test MIC value. A resistance and metal enzyme phenotype screening test, the bla_ (NDM-1) gene data of resistant strains, the clinical data were analyzed using.2, case-control study, NDM-1 containing bacterial infection in patients with A group, the other according to the ratio of l:1:1 pairing, separating carbon penicillin insensitive bacteria (NDM-1 gene negative) of patients with B group and carbapenem antibiotic sensitive bacteria were C group were studied, using medical information collection methods retrospective study, making the questionnaire after access to hospital medical records of patients in hospital investigation department, name, gender, age, duration of hospitalization, underlying diseases, risk factors clinical outcome data by univariate and multivariate Logstic regression analysis of.3 risk factors for carbapenem resistant strains of NDM-1 plasmid, bla_ plasmid conjugation test (NDM-1) genetic stability, for separation Stable genetic bla_ (NDM-1) gene of blot strain, Southern bla_ (NDM-1) gene mapping. The extraction of plasmid DNA, will meet the requirements (containing NDM-1 gene) of 5 plasmids were constructed for genome sequencing library, bla_ (NDM-1) plasmid for high-throughput sequencing, splicing and comparison of the sequencing results, fill the difference and sequence verification of plasmid gap, and plasmid genes. Note the input plasmid sequence is obtained to compare Blast NCBI online website, compared with different view using Mauve software. Results: 11735 strains were not sensitive to carbapenem antibiotics in bacteria, were screened from bla_ (NDM-1) gene 54 strains of Klebsiella pneumoniae screened positive strains and 44 strains of Bauman Acinetobacter strains screened positive strains and 8 strains of Escherichia coli were screened 2 positive isolates, bla_ (NDM-1) gene positive strains in vitro Experimental results show that bla_ (NDM-1) gene positive strains resistant to most antibiotics rate more than 50%, especially very high on carbapenem resistance rate of modified Hodge. The experiments indicated that 512 strains of positive.54 strains bla_ 1735 carbapenem non susceptible antibiotic strains (NDM-1) clinical data of gene show the positive strains from 43 patients in hospital departments, mainly in the Department of burns, Department of respiration, ICU; mainly collected from sputum, urine, blood.2, A group and B group to most antibiotics showed very high resistance, C group of resistance to most antibiotics were low, including A statistical analysis group and B group had significant difference compared to antibiotics such as imipenem, Amikacin, left sub loxacin; resistance to carbon penicillin strain (A+B group) and the carbon penicillin sensitive strain (group C) single factor analysis to Has the difference out of the following aspects: the hospitalization time, isolated two strains of broad-spectrum antibacterial drugs before the month more than 7 days, transferred from outside the hospital, 14 days before the separation of the strain in the use of antibiotics, fever peak (average C), use of antibiotics, the use of tigecycline therapy and discharged (%), treatment failure (%) and automatic discharge (%), and analyze the application of broad-spectrum isolated two months before the strain of antibacterial drugs more than 7 days and 14 days before the separation of the strain in the use of antibiotics is the independent risk factors of carbapenem resistant strains multi factor Logstic regression; single factor analysis of A group and B group, previous use of penicillin and carbon infection to death time is different, and multivariate Logstic regression analysis of previous use of carbapenem antibiotics as carbon black history .3 independent risk factors get penem type of drug-resistant plasmid NDM-1, containing bla_ (NDM-1) gene plasmid strains directly extracted and Escherichia J53 strains were joined by Southern plasmid, blot hybridization, confirmed that bla_ (NDM-1) genes exist in most plasmid, and containing bla_ (NDM-1) gene and Escherichia coli strains J53 strains were combined with a success rate of 50%, suggesting that plasmid carrying NDM-1 gene can be efficiently transferred between Enterobacteriaceae bacteria, provides a carrier for bacterial drug-resistance spread. 5 strains after successful joint experimental verification of the plasmid genome database, plasmid sequencing quality evaluation and genome assembly, CO get the complete genome sequences of 2 plasmids, namely p12 and p11106. selected plasmid sequences of 7 species, respectively, and the plasmid sequence by Mauve software to do similarity graph through the gene environment than. A, found: 1, p11106 and p12 plasmid and P NDM-BTR are very similar; 2, p11106, p12 and other six plasmids contained the plasmid (orf00032-orf00043) differences in 20-30kb area; 3, NDM-1 (orf00032-orf00043) gene is located in the middle position, the upper end of the region two TNP A gene, and found a region of 36KB orf00052 (TNP A) in the lower reaches. Conclusion: 1, bla_ (NDM-1) gene drug-resistance in certain regional hospital, dominated by Klebsiella pneumoniae in our hospital; that of Klebsiella pneumoniae in bla_ (NDM-1) gene transmission played an important role in the process, may bla_ (NDM-1) gene preservation host; 2, the risk factors for carbapenem resistant strains of NDM-1 plasmid had a history of using carbon penicillin; 3, characteristics of p11106 and p12 may have p plasmid NDM-BTR plasmid, NDM-1 plasmid containing orf00032-orf00043 and differences. The polymorphism of NDM-1 gene is located in the middle position of orf00032-orf00043, which links the two TNP A genes at the upper end and orf00052 (TNP A) in the downstream 36KB region, suggesting that NDM-1 may be acquired through transposition of plasmid and remain in plasmids for a long time.

【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R446.5

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