肺炎链球菌SWU02全基因组测定及其整合性接合元件分析

发布时间：2018-05-14 14:03

本文选题：肺炎链球菌 + 多重耐药　；参考：《西南医科大学》2017年硕士论文

【摘要】：肺炎链球菌(Streptococcus pneumoniae,Spn)是呼吸道感染的主要病原菌之一,已可以引起脑膜炎、胸膜炎、败血症等。以青霉素为代表的β内酰胺类药物和以红霉素为代表的大环内酯类药物,曾是抗肺炎链球菌感染的一线药物,而两类药物的耐药率不断攀升,给临床治疗带来了极大困难。本研究通过比较肺炎链球菌耐药特征,分析高毒力菌株全基因组和整合性接合元件,研究其耐药机制。目的:调查2015年10月至2016年6月某医院临床所分离62株肺炎链球菌(Streptococcus pneumoniae,Spn)的耐药特征,为临床正确使用抗生素提供依据;了解肺炎链球菌SWU02全基因组基本特征,分析其耐药基因;分析肺炎链球菌SWU02全基因组的整合性接合元件(Integrative and Conjugative Element,ICE)的遗传特征,研究其耐药播散机制。方法:(1)收集2015年10月至2016年6月某医院临床分离的62株肺炎链球菌,用法国梅里埃VITEK-2 Compact全自动微生物鉴定仪鉴定菌种,用E-test方法检测其对14种临床常用抗菌药物的耐药情况。(2)通过第三代高通量测序技术对肺炎链球菌SWU02进行全基因测序,原始测序结果进行拼接、组装、基因预测、基因功能注释、耐药基因和毒力基因分析。用多点位序列分型技术(MLST)分析肺炎链球菌SWU02的ST型别,在NCBI Gen Bank数据库查找并下载肺炎链球菌常见血清型序列19A、19F、23F、6A/B、14B/C、15B/C,通过BLAST比对确定肺炎链球菌SWU02血清型。(3)用隐马尔科夫模型识别肺炎链球菌SWU02的整合性接合元件,NCBI数据库和ICEmerg数据库检索肺炎链球菌整合性接合元件(ICE)的参考序列,将肺炎链球菌SWU02整合性接合元件序列与其进行比较。结果:(1)62株肺炎链球菌对青霉素(PEN)和红霉素(ERY)耐药率为100%,对四环素(TE)、阿奇霉素(AZM)、阿莫西林(AMC)、复方新诺明(SXT)耐药率较高,分别为95.2%、75.8%、72.6%、71.0%。对左氧氟沙星(LEV)、莫西沙星(MXF)、利奈唑胺(LNZ)、万古霉素(VAN)耐药率较低,分别为12.9%、11.3%、6.4%、0.0%、0.0%。(2)测序结果表明,SWU02基因组含有2092148 bp个碱基,G+C碱基占39.77%。共挖掘出2120个各种编码基因,2012个CDS(蛋白质编码区),93个假基因,13个r RNAs(包括5S,16S,23S),58个t RNAs,6个nc RNA。主要耐药基因包括对喹诺酮类抗生素耐药基因gyr A、gyr B,对大环内酯类抗生素耐药基因erm B、mef A、mre C、mre D,对四环素类抗生素耐药基因tet A、tet C、tet M。另外含有12个经典的肺炎链球菌毒力基因psp C、lyt A、lyt B、rrg A、nana、iga、ply、hys A stk P、clp P、cps2A、pcs B。序列提交到NCBI数据库得到登录号为CP018347,遗传进化分析,该菌株与登录号为CP006844菌株遗传距离最近。SWU02经多点位序列分型和血清分型,确定型别为ST271,19F,该型别以多重耐药和同时携带erm B和mef基因为主要特点。(3)通过比较发现该菌株在ORF91到ORF115区域存在整合性接合元件(ICE)结构,该ICE属于Tn916家族,序列全长25881bp,GC含量38.7%,含有25个ORF,所编码蛋白依次为:DUF961、DUF961、Fts K、rst A1、erm B、Transposase、conjugal transfer protein、Ard A、membrane protein、ATP/GTP、conjugative transposon、Nlp C/P60、conjugative transposon、tet M、umu C、Ims C、Yol D、mel、hypothetical protein、Mef(A)、DNA recombinase hypothetical protein、helix-turn-helix protein、DNA-binding protein、Xis、int。结论:(1)所分离的肺炎链球菌对β-内酰胺类抗菌药物耐药率为32.6-72.3%,大环内酯类抗菌药物耐药率为75.8-100%,四环素类抗生素耐药率为95.2%,耐药情况严峻。(2)全基因测序表明,肺炎链球菌SWU02基因组全长2092148 bp,G+C碱基占39.77%,含有Erm B、Tet(M)、Mef(E)等耐药基因和12个毒力基因,可能导致该菌株对多类抗菌药物多重耐药并表现出高毒力。(3)SWU02(ST271,19F)ICE属于TN916家族,同时携带有mef(A)erm B,tet M,青霉素耐药相关蛋白ATP/GTP耐药基因,此外还含有转座子可能与耐药基因的播散有关。
[Abstract]:Streptococcus pneumoniae (Spn) is one of the main pathogens of respiratory tract infection, which can cause meningitis, pleuritis, septicemia, etc.. Beta lactam, represented by penicillin and macrolide represented by erythromycin, have been a first-line drug for Streptococcus pneumoniae infection, and the drug resistance of the two types of drugs By comparing the drug resistance characteristics of Streptococcus pneumoniae, this study analyzed the whole genome and integrated conjugation components of highly virulent strains and studied the mechanism of drug resistance. Objective: To investigate the 62 strains of Streptococcus pneumoniae (Streptococcus pneumoniae, Spn) isolated from a hospital from October 2015 to June 2016. The characteristics of drug resistance provide the basis for the correct clinical use of antibiotics; understand the basic characteristics of the whole genome of Streptococcus pneumoniae SWU02, analyze its resistance genes, analyze the genetic characteristics of the integrated SWU02 Conjugative Element (ICE) of the whole genome of Streptococcus pneumoniae (Integrative and Conjugative Element, ICE), and study the mechanism of its resistance to spread. Methods: (1) collect the 2015 62 strains of Streptococcus pneumoniae isolated in a hospital from October to June 2016 were identified by the French MLIR VITEK-2 Compact automatic microbial identification instrument, and the resistance to 14 commonly used antibiotics was detected by E-test. (2) the whole gene sequencing of Streptococcus pneumoniae SWU02 was sequenced by third generation high-throughput sequencing technology. The sequencing results were stitching, assembling, gene prediction, gene function annotation, drug resistance gene and virulence gene analysis. The ST type of Streptococcus pneumoniae SWU02 was analyzed by multipoint sequence typing (MLST), and the common serotype 19A, 19F, 23F, 6A/B, 14B/C, 15B/C, and 15B/C were found and downloaded in the NCBI Gen Bank database. The serotype of Streptococcus pneumoniae SWU02 was determined. (3) the integrated conjugation element of Streptococcus pneumoniae SWU02 was identified by the hidden Markov model, the NCBI database and the ICEmerg database were used to retrieve the reference sequence of the integrated Streptococcus pneumoniae conjugation element (ICE), and the sequence of SWU02 integrated connector of Streptococcus pneumoniae was compared with that of the Streptococcus pneumoniae. Results: (1) 62 strains of pneumonia chain The resistance rate of staphylococci to penicillin (PEN) and erythromycin (ERY) was 100%. The resistance rate to tetracycline (TE), azithromycin (AZM), amoxicillin (AMC) and compound neorammine (SXT) was higher, 95.2%, 75.8%, 72.6% respectively. The resistance rate of 71.0%. to levofloxacin (LEV), moxifloxacin (MXF), linezolid (LNZ), vancomycin (VAN) was lower, 12.9%, 11.3%, 6.4%, 0, respectively. .0%, 0.0%. (2) sequencing results show that the SWU02 genome contains 2092148 BP bases, and G+C bases occupy 39.77%. with 2120 various encoding genes, 2012 CDS (protein coding regions), 93 pseudogenes, 13 R RNAs (5S, 16S, 23S), and 58 drug-resistant genes including the quinolone resistance genes. ERM B, MEF A, MRE C, MRE D, Tet A of tetracycline antibiotics, Tet C. Evolutionary analysis, the genetic distance between the strain and the CP006844 strain is the nearest.SWU02 by multipoint sequence typing and serotyping, and the type is ST271,19F. This type is characterized by multiple resistance and simultaneous carrying of ERM B and MEF base. (3) it is found that the strain has an integrated junction element (ICE) in the ORF91 to ORF115 region. The ICE belongs to the Tn916 family, the sequence full length 25881bp, the GC content 38.7%, contains 25 ORF, the encoded proteins are DUF961, DUF961, Fts K, RST A1. Ical protein, Mef (A), DNA recombinase hypothetical protein, helix-turn-helix protein, DNA-binding protein, conclusion: (1) the resistance rate of isolated Streptococcus pneumoniae to beta lactam antibiotics, the resistance rate of macrolide antibiotics and the resistance rate of tetracycline antibiotics is 95.2%. (2) total gene sequencing showed that the genome of Streptococcus pneumoniae SWU02 genome was 2092148 BP, G+C base accounted for 39.77%, Erm B, Tet (M), Mef (E) and 12 virulence genes, which could lead to multi resistance and high virulence of the strain. (3) SWU02 (ST271,19F) ICE belonged to the family. RM B, Tet M, the ATP/GTP gene of penicillin resistance associated protein, and the transposon may also be associated with the spread of drug-resistant genes.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378.1

【参考文献】