本文關鍵詞： NDM-1基因 耐藥菌株 危險因素 基因周圍環(huán)境　出處：《南昌大學》2017年博士論文　論文類型：學位論文

【摘要】：研究背景和目的:區(qū)域性播散是造成質(zhì)粒編碼NDM-1酶(New Delhi metallo-β-lactamase1,即新德里金屬-β-內(nèi)酰胺酶-1)廣泛流行的主要原因,然而其相關的危險因素和基因周圍環(huán)境尚未見系統(tǒng)性報道。本研究通過收集篩選我院產(chǎn)NDM-1酶的碳青霉烯耐藥菌,在調(diào)查bla_(NDM-1)基因的流行狀況的基礎上,對含NDM-1基因耐藥菌株感染的臨床危險因素與預后進行分析,提取含bla_(NDM-1)基因的質(zhì)粒進行全基因組測序和生物信息學分析研究,系統(tǒng)探討NDM-1基因的流行狀況,bla_(NDM-1)基因耐藥菌株的影響因素和預后,進一步分析質(zhì)粒中NDM-1的基因周圍環(huán)境,并與國內(nèi)外相關研究進行比較。對揭示NDM-1耐藥基因的流行、易感因素和進化趨勢具有重要意義。為制訂科學、有效的預防和控制措施提供實驗依據(jù)。方法:1、收集和篩選我院臨床標本中對碳青霉烯類抗生素不敏感的革蘭氏陰性桿菌(剔除同一患者同一部位的重復菌株),應用PCR技術進行bla_(NDM-1)基因陽性標本篩查,將保存的鑒定為bla_(NDM-1)基因陽性的混合菌液傳代培養(yǎng),并將傳代培養(yǎng)的菌液行bla_(NDM-1)基因篩查,由于分離的菌種數(shù)目多,菌種復雜,含有革蘭氏陽性和陰性細菌,應用BIOLOG自動微生物鑒定系統(tǒng)進行鑒定同時擴增細菌16sr RNA進行測序,明確含bla_(NDM-1)基因的菌種,應用E-test法檢測菌株抗菌藥物敏感性、MIC值及金屬酶表型篩選實驗。通過耐藥性和金屬酶表型篩選實驗,獲得上述含bla_(NDM-1)基因的耐藥菌株數(shù)據(jù),結合臨床資料進行分析。2、運用病例對照研究方法,將含NDM-1酶細菌感染的患者為A組,另按l:1:1的比例配對,分離出碳青霉素類不敏感細菌(NDM-1基因陰性)的患者為B組和碳青霉烯類抗生素敏感的細菌的患者為C組進行研究,采用病歷信息收集回顧性研究的方法,制作調(diào)查表后查閱出院病歷,調(diào)查患者的住院科室、姓名、性別、年齡、住院時間、基礎疾病、危險因素、臨床結局等資料,通過單因素和多因素Logstic回歸分析碳青霉烯類耐藥菌NDM-1質(zhì)粒獲取的危險因素。3、質(zhì)粒接合試驗驗證bla_(NDM-1)基因遺傳穩(wěn)定性,對于分離的能夠穩(wěn)定遺傳bla_(NDM-1)基因的菌株,Southern blot對bla_(NDM-1)進行基因定位。提取質(zhì)粒DNA,將滿足要求(含有NDM-1基因)的5個質(zhì)粒分別構建基因組測序文庫,將bla_(NDM-1)質(zhì)粒進行高通量測序,對測序結果進行拼接及比對,進行質(zhì)粒gap的填補及差異序列驗證,并注釋質(zhì)粒系列的基因。將得到的質(zhì)粒序列輸入到NCBI在線網(wǎng)站中進行Blast比對,利用Mauve軟件進行對比詳細查看不同點。結果:1、1735株碳青霉烯類抗生素不敏感細菌中,一共篩選到含bla_(NDM-1)基因菌株54株,其中肺炎克雷伯菌中篩到陽性菌株44株,鮑曼不動桿菌株中篩到陽性菌株8株,大腸桿菌中篩到陽性菌株2株,bla_(NDM-1)基因陽性菌株體外藥敏實驗顯示,bla_(NDM-1)基因陽性菌株對大多數(shù)的抗生素的耐藥率都超過了50%,尤其是對耐碳青霉烯類抗生素耐藥率非常高。改良Hodge實驗提示有1735株碳青霉烯抗生素不敏感菌株中出現(xiàn)512株陽性。54株bla_(NDM-1)基因陽性菌株的臨床資料顯示其來自43位患者,住院科室主要分布在燒傷科、呼吸科、ICU;主要標本來源于痰液、尿液、血液。2、A組和B組對絕大多數(shù)抗生素表現(xiàn)出非常高的耐藥率,C組對大部分抗生素的耐藥率均較低,其中A組與B組統(tǒng)計分析比較具有統(tǒng)計學差異的抗生素為亞胺培南、阿米卡星、左亞氟沙星;耐碳青霉素類抗生素菌株(A+B組)和對碳青霉素類抗生素敏感菌株(C組)單因素分析,得出以下方面具有差異性:住院時間、分離出該菌株前兩個月內(nèi)應用廣譜抗菌藥物大于7天、從外院轉(zhuǎn)入、分離出該菌株前14天內(nèi)使用過抗生素類藥物、發(fā)熱峰值(平均℃)、抗生素使用種類、治療中使用替加環(huán)素、好轉(zhuǎn)出院(%)、治療失敗(%)和自動出院(%),并進行多因素Logstic回歸分析得出分離出該菌株前兩個月內(nèi)應用廣譜抗菌藥物大于7天和分離出該菌株前14天內(nèi)使用過抗生素類藥物是耐碳青霉烯類抗生素菌株的獨立危險因素;對A組和B組進行單因素分析,既往使用碳青霉素類抗生素和發(fā)生感染到死亡的時間具有差異性,并進行多因素Logstic回歸分析得出既往使用碳青霉烯類抗生素史為碳青霉烯類耐藥菌NDM-1質(zhì)粒獲取的獨立危險因素。3、含bla_(NDM-1)基因菌株直接提取的質(zhì)粒和大腸J53菌株進行接合的質(zhì)粒,進行Southern blot雜交顯色,均證實bla_(NDM-1)基因大部分存在于質(zhì)粒中,且含bla_(NDM-1)基因菌株與大腸J53菌株進行質(zhì)粒結合成功率為50%,提示攜帶NDM-1基因的質(zhì)�？梢愿咝У卦谀c桿菌科類細菌之間發(fā)生轉(zhuǎn)移,為細菌的耐藥流行播散提供了傳播載體。將5株經(jīng)接合實驗驗證的質(zhì)�；蚪M建庫成功后,進行質(zhì)粒測序質(zhì)量評價和基因組拼裝,共得到2個完整的質(zhì)粒基因組序列,即p12和p11106。選取7個物種的相關質(zhì)粒序列,分別通過Mauve軟件與的質(zhì)粒序列做相似性圖。通過上述基因環(huán)境比較,發(fā)現(xiàn):1、p11106和p12質(zhì)粒與p NDM-BTR非常相似;2、p11106、p12質(zhì)粒和另外六個質(zhì)粒存在包含(orf00032-orf00043)20-30kb區(qū)域的差異;3、NDM-1基因位于(orf00032-orf00043)的中間位置,區(qū)域的上端的有兩個tnp A基因,以及在其下游發(fā)現(xiàn)36kb區(qū)域的orf00052(tnp A)。結論:1、bla_(NDM-1)基因在區(qū)域性醫(yī)院存在一定的耐藥流行,在我院以肺炎克雷伯桿菌為主;提示肺炎克雷伯桿菌在bla_(NDM-1)基因的傳播過程中起到了重要作用,可能是bla_(NDM-1)基因的保存宿主;2、碳青霉烯類耐藥菌NDM-1質(zhì)粒獲取的危險因素為既往有碳青霉素類抗生素使用史;3、p11106和p12質(zhì)�？赡芫哂衟 NDM-BTR質(zhì)粒的特征,含NDM-1質(zhì)粒存在orf00032-orf00043差異性,且具有多態(tài)性,NDM-1基因位于orf00032-orf00043的中間位置,聯(lián)系上端的兩個tnp A基因和下游36kb區(qū)域的orf00052(tnp A),提示NDM-1可能是質(zhì)粒通過轉(zhuǎn)座獲得,并長期保留在質(zhì)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.

【學位授予單位】：南昌大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R446.5

【相似文獻】

相關期刊論文 前2條

1 葉皓;周永君;黃國嬌;殷建華;;NDM-1型金屬β-內(nèi)酰胺酶的生物信息學分析[J];西南國防醫(yī)藥;2012年11期

2 張薇;梁慧;劉麗萍;彭國鈞;胡紅焱;;洛菲不動桿菌株多藥耐藥NDM-1基因的鑒定[J];武警醫(yī)學;2013年04期

相關會議論文 前1條

1 邱峰;;新型金屬β-內(nèi)酰胺酶NDM-1及其臨床防治[A];2010年中國藥學大會暨第十屆中國藥師周論文集[C];2010年

相關博士學位論文 前1條

1 向天新;碳青霉烯類耐藥菌NDM-1質(zhì)粒獲取的危險因素和周圍基因環(huán)境研究[D];南昌大學;2017年

相關碩士學位論文 前4條

1 劉果;細菌間NDM-1質(zhì)粒的水平傳播研究[D];中國人民解放軍軍事醫(yī)學科學院;2016年

2 孫秋;中國首例產(chǎn)NDM-1陰溝腸桿菌感染病例研究[D];重慶醫(yī)科大學;2013年

3 路榮;NDM-1肺炎克雷伯菌裂解性噬菌體的分離鑒定及其對小鼠菌血癥的治療研究[D];吉林大學;2013年

4 周亞俊;NDM-1的純化、表征以及在細菌活細胞體內(nèi)活性的實時檢測及其抑制研究[D];西北大學;2015年

，

本文編號：1549335