急性腹泻患者非伤寒沙门菌的耐药监测和基因分型研究

发布时间：2018-05-01 21:27

本文选题：非伤寒沙门氏菌 + 血清学分型　；参考：《天津医科大学》2014年硕士论文

【摘要】：目的检测本地区非伤寒沙门氏菌(NTS)的血清型和抗生素耐药情况,应用脉冲场凝胶电泳(PFGE)和多位点序列分析(MLST),确定菌株的同源性和优势流行基因型以及菌株间遗传进化关系。为本地区NTS感染的追踪溯源提供数据参考,为NTS感染的预防控制和治疗提供帮助。方法 1、菌株血清学分型和药敏试验：试验菌株分离自天津医科大学第二医院肠道门诊急性腹泻患者粪便标本,收集时间为2012和2013的5-10月份。血清型鉴定按照沙门氏菌血清诊断操作步骤(泰国SA公司提供)进行,根据测定得到的抗原式确定沙门氏菌的血清型。采用K-B纸片扩散法检测非伤寒沙门菌临床株对17种抗菌药物的耐药性。 2、PFGE:提取非伤寒沙门菌菌株全基因组DNA,经限制性内切酶Xbal酶切后进行脉冲场凝胶电泳(PFGE)分析,对于肠炎沙门氏菌再经限制性内切酶Blnl酶切后进行脉冲场凝胶电泳(PFGE)分析。 3.MLST:分别扩增菌株的7个管家基因(thrA, pure, sucA, hisD, aroC, hemD, dnaN)的核心片段(510bp-894bp),将测序结果提交至国际MLST数据库网站(http://www.mlst.net),获得受试菌株的等位基因图谱及其对应的ST型(Sequence Type,ST)。结果 1、共分离45株NTS,血清学分型包括5个血清群、12个血清型,均在A-F群范围内,优势血清群依次为D群和B群,以0：4和0：9为主。血清型包括国内常见的肠炎、鼠伤寒、阿贡纳、山夫登堡、德尔卑、布伦登卢普血清型,国内少见的姆班达卡、蒙得维的亚血清型,以及国内罕见的库卡、格兰扁、菲尔摩雷和奥里塔曼林血清型。优势血清型为肠炎沙门氏菌和鼠伤寒沙门氏菌。 2、45株NTS对萘啶酸敏感率最低,其次是链霉素、庆大霉素、氨苄西林、四环素、哌拉西林、氯霉素。对左氧氟沙星、阿米卡星、阿莫西林/克拉维酸、头孢他啶敏感率大于90%,对环丙沙星、头孢曲松、头孢噻肟、复方新诺明、氨曲南和亚胺培南敏感率为100%。与萘啶酸敏感株相比,萘啶酸耐药株对环丙沙星及左氧氟沙星敏感性下降。菌株全敏感率为24.44%,单药耐药率为44.44%,多重耐药率达17.78%。肠炎血清型主要为单药耐药,而鼠伤寒血清型则以多重耐药较常见,对氨苄西林、氯霉素、四环素、链霉素呈耐药或中介。 3、PFGE产生23种PT型(图谱型),条带数为12到20之间,相似系数为0.56-1,分为4大聚类(A、B、C、D),其中各聚类又包括不同的亚聚类,优势聚类为A1和D1。有11种PT型别包括2株以上分离株,其中肠炎沙门菌PT2和鼠伤寒沙门氏菌PT18为优势PT型,PT2、PT6、PT20、PT22在2012和2013年均被分离出。6株PT2和4株PT18型分别分离自同一日期,6株PT2型肠炎沙门菌被证实为感染暴发。Xbal分为同一型别的11株肠炎沙门菌经BlnI作用后可进一步分为3个型别。用PFGE分型预测血清型,预测与实际血清型一致率占70.9%。 4、MLST将45株试验菌株分为13个STs,其中14株肠炎血清型均为ST11,9株鼠伤寒血清型包括6株ST19和3株ST34,1株库卡血清型和4株山夫登堡血清型均为ST14,4株阿贡纳血清型均为ST13,3株布伦登卢普血清型为ST22,3株德尔卑血清型为1株ST40和2株ST71,2株蒙得维的亚为ST26,2株菲尔摩雷血清型为ST46,1株格兰扁为ST358,1株姆班达卡为ST413,1株奥里塔曼林为ST1516。ST11所占比例最多,其次为ST34。序列型之间遗传关系较远,其中德尔卑血清型包括的序列型ST40和ST71遗传学上不相关,而遗传相关的ST14同时包括了山夫登堡血清型和库卡血清型。结论 1、本地NTS血清型呈现多样性,肠炎血清型和鼠伤寒血清型为主要血清型,检测出一些国内少见(姆班达卡、蒙得维的亚)和罕见(格兰扁、菲尔摩雷和奥里塔曼林)血清型。 2、本组资料显示NTS对三代头孢耐药菌株罕见,但对萘啶酸耐药率较高且多伴有氟喹诺酮(FQs)敏感性降低,对FQs临床经验用药构成威胁。鼠伤寒沙门菌多为MDR,呈现ACST耐药表型。 3、PFGE显示PT2和PT18为优势型别。PT2、PT6、PT20、PT22克隆株跨年度存在,其中PT2型别有跨年度流行现象。有11组相同克隆株的出现,证实了1起肠炎沙门菌感染暴发,发现1起疑似鼠伤寒沙门菌感染暴发。对肠炎沙门菌进行Xbal-Blnl双酶切PFGE可提高分辨能力。PFGE分型对血清学分型有预测价值。 4、MLST分型显示ST11和ST34为本次研究的主要ST型,ST11-PT2型肠炎沙门氏菌和ST34-PT18型鼠伤寒沙门氏菌可能为本地优势克隆株。血清学分型混淆了遗传学上的无关的菌株,ST分型可更好地反应NTS的遗传进化关系。 5、应进一步扩大和加强对本地区NTS耐药监测和分型研究。
[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.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R574.62

【参考文献】