本文選題：非傷寒沙門氏菌 + 血清學(xué)分型��； 參考：《天津醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的 檢測(cè)本地區(qū)非傷寒沙門氏菌(NTS)的血清型和抗生素耐藥情況,應(yīng)用脈沖場(chǎng)凝膠電泳(PFGE)和多位點(diǎn)序列分析(MLST),確定菌株的同源性和優(yōu)勢(shì)流行基因型以及菌株間遺傳進(jìn)化關(guān)系。為本地區(qū)NTS感染的追蹤溯源提供數(shù)據(jù)參考,為NTS感染的預(yù)防控制和治療提供幫助。 方法 1、菌株血清學(xué)分型和藥敏試驗(yàn)：試驗(yàn)菌株分離自天津醫(yī)科大學(xué)第二醫(yī)院腸道門診急性腹瀉患者糞便標(biāo)本,收集時(shí)間為2012和2013的5-10月份。血清型鑒定按照沙門氏菌血清診斷操作步驟(泰國(guó)SA公司提供)進(jìn)行,根據(jù)測(cè)定得到的抗原式確定沙門氏菌的血清型。采用K-B紙片擴(kuò)散法檢測(cè)非傷寒沙門菌臨床株對(duì)17種抗菌藥物的耐藥性。 2、PFGE:提取非傷寒沙門菌菌株全基因組DNA,經(jīng)限制性內(nèi)切酶Xbal酶切后進(jìn)行脈沖場(chǎng)凝膠電泳(PFGE)分析,對(duì)于腸炎沙門氏菌再經(jīng)限制性內(nèi)切酶Blnl酶切后進(jìn)行脈沖場(chǎng)凝膠電泳(PFGE)分析。 3.MLST:分別擴(kuò)增菌株的7個(gè)管家基因(thrA, pure, sucA, hisD, aroC, hemD, dnaN)的核心片段(510bp-894bp),將測(cè)序結(jié)果提交至國(guó)際MLST數(shù)據(jù)庫(kù)網(wǎng)站(http://www.mlst.net),獲得受試菌株的等位基因圖譜及其對(duì)應(yīng)的ST型(Sequence Type,ST)。 結(jié)果 1、共分離45株NTS,血清學(xué)分型包括5個(gè)血清群、12個(gè)血清型,均在A-F群范圍內(nèi),優(yōu)勢(shì)血清群依次為D群和B群,以0：4和0：9為主。血清型包括國(guó)內(nèi)常見的腸炎、鼠傷寒、阿貢納、山夫登堡、德爾卑、布倫登盧普血清型,國(guó)內(nèi)少見的姆班達(dá)卡、蒙得維的亞血清型,以及國(guó)內(nèi)罕見的庫(kù)卡、格蘭扁、菲爾摩雷和奧里塔曼林血清型。優(yōu)勢(shì)血清型為腸炎沙門氏菌和鼠傷寒沙門氏菌。 2、45株NTS對(duì)萘啶酸敏感率最低,其次是鏈霉素、慶大霉素、氨芐西林、四環(huán)素、哌拉西林、氯霉素。對(duì)左氧氟沙星、阿米卡星、阿莫西林/克拉維酸、頭孢他啶敏感率大于90%,對(duì)環(huán)丙沙星、頭孢曲松、頭孢噻肟、復(fù)方新諾明、氨曲南和亞胺培南敏感率為100%。與萘啶酸敏感株相比,萘啶酸耐藥株對(duì)環(huán)丙沙星及左氧氟沙星敏感性下降。菌株全敏感率為24.44%,單藥耐藥率為44.44%,多重耐藥率達(dá)17.78%。腸炎血清型主要為單藥耐藥,而鼠傷寒血清型則以多重耐藥較常見,對(duì)氨芐西林、氯霉素、四環(huán)素、鏈霉素呈耐藥或中介。 3、PFGE產(chǎn)生23種PT型(圖譜型),條帶數(shù)為12到20之間,相似系數(shù)為0.56-1,分為4大聚類(A、B、C、D),其中各聚類又包括不同的亞聚類,優(yōu)勢(shì)聚類為A1和D1。有11種PT型別包括2株以上分離株,其中腸炎沙門菌PT2和鼠傷寒沙門氏菌PT18為優(yōu)勢(shì)PT型,PT2、PT6、PT20、PT22在2012和2013年均被分離出。6株P(guān)T2和4株P(guān)T18型分別分離自同一日期,6株P(guān)T2型腸炎沙門菌被證實(shí)為感染暴發(fā)。Xbal分為同一型別的11株腸炎沙門菌經(jīng)BlnI作用后可進(jìn)一步分為3個(gè)型別。用PFGE分型預(yù)測(cè)血清型,預(yù)測(cè)與實(shí)際血清型一致率占70.9%。 4、MLST將45株試驗(yàn)菌株分為13個(gè)STs,其中14株腸炎血清型均為ST11,9株鼠傷寒血清型包括6株ST19和3株ST34,1株庫(kù)卡血清型和4株山夫登堡血清型均為ST14,4株阿貢納血清型均為ST13,3株布倫登盧普血清型為ST22,3株德爾卑血清型為1株ST40和2株ST71,2株蒙得維的亞為ST26,2株菲爾摩雷血清型為ST46,1株格蘭扁為ST358,1株姆班達(dá)卡為ST413,1株奧里塔曼林為ST1516。ST11所占比例最多,其次為ST34。序列型之間遺傳關(guān)系較遠(yuǎn),其中德爾卑血清型包括的序列型ST40和ST71遺傳學(xué)上不相關(guān),而遺傳相關(guān)的ST14同時(shí)包括了山夫登堡血清型和庫(kù)卡血清型。 結(jié)論 1、本地NTS血清型呈現(xiàn)多樣性,腸炎血清型和鼠傷寒血清型為主要血清型,檢測(cè)出一些國(guó)內(nèi)少見(姆班達(dá)卡、蒙得維的亞)和罕見(格蘭扁、菲爾摩雷和奧里塔曼林)血清型。 2、本組資料顯示NTS對(duì)三代頭孢耐藥菌株罕見,但對(duì)萘啶酸耐藥率較高且多伴有氟喹諾酮(FQs)敏感性降低,對(duì)FQs臨床經(jīng)驗(yàn)用藥構(gòu)成威脅。鼠傷寒沙門菌多為MDR,呈現(xiàn)ACST耐藥表型。 3、PFGE顯示PT2和PT18為優(yōu)勢(shì)型別。PT2、PT6、PT20、PT22克隆株跨年度存在,其中PT2型別有跨年度流行現(xiàn)象。有11組相同克隆株的出現(xiàn),證實(shí)了1起腸炎沙門菌感染暴發(fā),發(fā)現(xiàn)1起疑似鼠傷寒沙門菌感染暴發(fā)。對(duì)腸炎沙門菌進(jìn)行Xbal-Blnl雙酶切PFGE可提高分辨能力。PFGE分型對(duì)血清學(xué)分型有預(yù)測(cè)價(jià)值。 4、MLST分型顯示ST11和ST34為本次研究的主要ST型,ST11-PT2型腸炎沙門氏菌和ST34-PT18型鼠傷寒沙門氏菌可能為本地優(yōu)勢(shì)克隆株。血清學(xué)分型混淆了遺傳學(xué)上的無(wú)關(guān)的菌株,ST分型可更好地反應(yīng)NTS的遺傳進(jìn)化關(guān)系。 5、應(yīng)進(jìn)一步擴(kuò)大和加強(qiáng)對(duì)本地區(qū)NTS耐藥監(jiān)測(cè)和分型研究。
[Abstract]:objective
Detection of the serotype and antibiotic resistance of non typhimurium (NTS) in the region, using pulse field gel electrophoresis (PFGE) and multipoint sequence analysis (MLST) to determine the homology of the strain and the dominant genotypes and the relationship between the genetic evolution of the strains. It provides the data reference for tracing the source of NTS infection in the region, and is the precondition for the infection of NTS. Control and treatment provide help.
Method
1, the strain serotyping and drug sensitivity test: the test strains were isolated from the stool specimens of acute diarrhea patients in the Second Hospital Affiliated to Tianjin Medical University intestinal outpatient clinic, and the collection time was 2012 and 2013 of the month of 5-10. The serotype identification was carried out according to the serological diagnostic procedures of Salmonella (Thailand SA company), and determined according to the determination of the antigen formula. The serotype of Salmonella was detected by K-B disk diffusion method to detect the resistance of clinical isolates of non typhoid Salmonella to 17 kinds of antibiotics.
2, PFGE: was extracted from the whole genome DNA of the non typhi Salmonella strain and was analyzed by pulsed field gel electrophoresis (PFGE) after the restriction endonuclease Xbal enzyme digestion. The pulse field gel electrophoresis (PFGE) was used to analyze the restriction endonuclease Blnl enzyme of Salmonella enteritis after the restriction enzyme digestion.
3.MLST: amplified the core fragment (510bp-894bp) of 7 housekeeping genes (thrA, pure, sucA, hisD, aroC, hemD, dnaN), respectively. The sequencing results were submitted to the international MLST database website (http://www.mlst.net) to obtain the allelic map of the tested strain and its corresponding ST type.
Result
1, a total of 45 strains of NTS were separated. The serotype consisted of 5 serogroups and 12 serotypes, all in the A-F group. The dominant serogroups were D and B in sequence. The serotypes included common enteritis, typhus, Agona, mountain Fu den, Del hump, brunenlup serotype, and rare mBAND Da card, Montevideo. Serotype, and rare type of serum in KUKA, Glenn, Phil Morel and olimasman in China. The dominant serotypes are Salmonella enteritidis and Salmonella typhimurium.
The sensitivity of 2,45 strain NTS to nanidixic acid was the lowest, followed by streptomycin, gentamicin, ampicillin, tetracycline, piperidin, chloramphenicol. The susceptibility to levofloxacin, Amikacin, amoxicillin / clavulanic acid, ceftazidime was greater than 90%, and the sensitivity of ciprofloxacin, ceftriaxone, cefotaxime, compound sulfamethoxime, amamenin and imipenem was sensitive to ciprofloxacin, ceftriaxone, cefotaxime, and compound Sulfamethoxine 100%. was more sensitive to ciprofloxacin and levofloxacin than that of naphthidine acid sensitive strains. The total sensitivity of the strains was 24.44%, the single drug resistance rate was 44.44%. The multidrug resistance rate of the serotype of 17.78%. enteritis was mainly single drug resistance, and the serotype of the rat typhus was more common with multidrug resistance, and ampicillin, chloramphenicol and tetracycline. Streptomycin is resistant or mediator.
3, PFGE produces 23 types of PT type (Atlas) with a band number of 12 to 20, and the similarity coefficient is 0.56-1, which is divided into 4 clusters (A, B, C, D), and each cluster includes a different sub cluster. The dominant clustering is A1 and D1., and there are 11 kinds of PT types including 2 or more isolates, among which, the enteritis Salmonella PT2 and Salmonella typhimurium are the dominant type. 2.6 strain PT2 and 4 PT18 types were isolated from the same date respectively in 2012 and 2013. 6 strains of Salmonella enteritis PT2 were confirmed to be infected with the same type. 11 strains of Salmonella enteritis can be further divided into 3 types after the action of the same type. The serotype was predicted with PFGE typing, and the consistency between the serotype and the actual serotype was 70.9%..
4, MLST divided 45 strains of experimental strains into 13 STs, of which 14 enteritis serotypes were ST11,9 strains of chill serotypes including 6 ST19 and 3 ST34,1 strains of KUKA serotypes and 4 serotype of Mount mongburgh were ST14,4 strains of the ST13,3 strain of brenden lupper serotype as ST22,3 strain Del inferior serotype for 1 strains of ST40 and 2 ST71,2 The type of Phil mormine serotype of ST26,2 strain of ST26,2 strain was ST46,1 strain, Glan flattened ST358,1 strain, and ST413,1 strain of alteman forest was the largest proportion of ST1516.ST11, followed by the distant genetic relationship between ST34. sequence type, and the sequence type ST40 and ST71 were not genetically related to the sequence type ST40 and ST71, and the genetic correlation was related. ST14 also includes scheffenberg serotype and KUKA serotype.
conclusion
1, the serotypes of local NTS showed diversity, serotypes of enteritis serotypes and serotypes of typhus were the main serotypes, and some rare serotypes were detected in China (Montevideo) and rare (Glan, Phil Morel and olarimarin) serotypes.
2, the data in this group showed that NTS was rare for the three generation cephalosporin resistant strains, but the resistance to nadyl acid was higher and the sensitivity of multiple fluoroquinolones (FQs) decreased, which threatened the clinical experience of FQs. The Salmonella typhimurium was mostly MDR, presenting ACST resistant phenotype.
3, PFGE showed that PT2 and PT18 were the dominant type.PT2, PT6, PT20, and PT22 clones across the year, among which PT2 types had a cross year epidemic. There were 11 groups of identical clones, which confirmed the outbreak of 1 Salmonella enteritis and 1 suspected Salmonella typhimurium infection. The Xbal-Blnl double enzyme cut PFGE for Salmonella enteritis could improve the score. The typing of.PFGE has predictive value for serological typing.
4, MLST typing showed that ST11 and ST34 were the main ST types of this study. Salmonella enteritis and Salmonella typhimurium of type ST34-PT18 may be the local dominant clones. The serotype of the serotype confuses the unrelated strains in genetics, and the ST genotyping can better respond to the genetic evolution of NTS.
5, we should further expand and strengthen the surveillance and typing of NTS in the region.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R574.62

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本文編號(hào)：1831102