本文選題：肺炎鏈球菌 + 多重耐藥��； 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：肺炎鏈球菌(Streptococcus pneumoniae,Spn)是呼吸道感染的主要病原菌之一,已可以引起腦膜炎、胸膜炎、敗血癥等。以青霉素為代表的β內(nèi)酰胺類藥物和以紅霉素為代表的大環(huán)內(nèi)酯類藥物,曾是抗肺炎鏈球菌感染的一線藥物,而兩類藥物的耐藥率不斷攀升,給臨床治療帶來了極大困難。本研究通過比較肺炎鏈球菌耐藥特征,分析高毒力菌株全基因組和整合性接合元件,研究其耐藥機(jī)制。目的:調(diào)查2015年10月至2016年6月某醫(yī)院臨床所分離62株肺炎鏈球菌(Streptococcus pneumoniae,Spn)的耐藥特征,為臨床正確使用抗生素提供依據(jù);了解肺炎鏈球菌SWU02全基因組基本特征,分析其耐藥基因;分析肺炎鏈球菌SWU02全基因組的整合性接合元件(Integrative and Conjugative Element,ICE)的遺傳特征,研究其耐藥播散機(jī)制。方法:(1)收集2015年10月至2016年6月某醫(yī)院臨床分離的62株肺炎鏈球菌,用法國梅里埃VITEK-2 Compact全自動(dòng)微生物鑒定儀鑒定菌種,用E-test方法檢測其對(duì)14種臨床常用抗菌藥物的耐藥情況。(2)通過第三代高通量測序技術(shù)對(duì)肺炎鏈球菌SWU02進(jìn)行全基因測序,原始測序結(jié)果進(jìn)行拼接、組裝、基因預(yù)測、基因功能注釋、耐藥基因和毒力基因分析。用多點(diǎn)位序列分型技術(shù)(MLST)分析肺炎鏈球菌SWU02的ST型別,在NCBI Gen Bank數(shù)據(jù)庫查找并下載肺炎鏈球菌常見血清型序列19A、19F、23F、6A/B、14B/C、15B/C,通過BLAST比對(duì)確定肺炎鏈球菌SWU02血清型。(3)用隱馬爾科夫模型識(shí)別肺炎鏈球菌SWU02的整合性接合元件,NCBI數(shù)據(jù)庫和ICEmerg數(shù)據(jù)庫檢索肺炎鏈球菌整合性接合元件(ICE)的參考序列,將肺炎鏈球菌SWU02整合性接合元件序列與其進(jìn)行比較。結(jié)果:(1)62株肺炎鏈球菌對(duì)青霉素(PEN)和紅霉素(ERY)耐藥率為100%,對(duì)四環(huán)素(TE)、阿奇霉素(AZM)、阿莫西林(AMC)、復(fù)方新諾明(SXT)耐藥率較高,分別為95.2%、75.8%、72.6%、71.0%。對(duì)左氧氟沙星(LEV)、莫西沙星(MXF)、利奈唑胺(LNZ)、萬古霉素(VAN)耐藥率較低,分別為12.9%、11.3%、6.4%、0.0%、0.0%。(2)測序結(jié)果表明,SWU02基因組含有2092148 bp個(gè)堿基,G+C堿基占39.77%。共挖掘出2120個(gè)各種編碼基因,2012個(gè)CDS(蛋白質(zhì)編碼區(qū)),93個(gè)假基因,13個(gè)r RNAs(包括5S,16S,23S),58個(gè)t RNAs,6個(gè)nc RNA。主要耐藥基因包括對(duì)喹諾酮類抗生素耐藥基因gyr A、gyr B,對(duì)大環(huán)內(nèi)酯類抗生素耐藥基因erm B、mef A、mre C、mre D,對(duì)四環(huán)素類抗生素耐藥基因tet A、tet C、tet M。另外含有12個(gè)經(jīng)典的肺炎鏈球菌毒力基因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數(shù)據(jù)庫得到登錄號(hào)為CP018347,遺傳進(jìn)化分析,該菌株與登錄號(hào)為CP006844菌株遺傳距離最近。SWU02經(jīng)多點(diǎn)位序列分型和血清分型,確定型別為ST271,19F,該型別以多重耐藥和同時(shí)攜帶erm B和mef基因?yàn)橹饕攸c(diǎn)。(3)通過比較發(fā)現(xiàn)該菌株在ORF91到ORF115區(qū)域存在整合性接合元件(ICE)結(jié)構(gòu),該ICE屬于Tn916家族,序列全長25881bp,GC含量38.7%,含有25個(gè)ORF,所編碼蛋白依次為: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。結(jié)論:(1)所分離的肺炎鏈球菌對(duì)β-內(nèi)酰胺類抗菌藥物耐藥率為32.6-72.3%,大環(huán)內(nèi)酯類抗菌藥物耐藥率為75.8-100%,四環(huán)素類抗生素耐藥率為95.2%,耐藥情況嚴(yán)峻。(2)全基因測序表明,肺炎鏈球菌SWU02基因組全長2092148 bp,G+C堿基占39.77%,含有Erm B、Tet(M)、Mef(E)等耐藥基因和12個(gè)毒力基因,可能導(dǎo)致該菌株對(duì)多類抗菌藥物多重耐藥并表現(xiàn)出高毒力。(3)SWU02(ST271,19F)ICE屬于TN916家族,同時(shí)攜帶有mef(A)erm B,tet M,青霉素耐藥相關(guān)蛋白ATP/GTP耐藥基因,此外還含有轉(zhuǎn)座子可能與耐藥基因的播散有關(guān)。
[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.

【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378.1

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