广东省2007-2013年肠炎沙门菌耐药特征和分子分型研究

发布时间：2018-06-07 12:22

本文选题：肠炎沙门菌 + 最小抑菌浓度(Minimal　；参考：《南方医科大学》2015年硕士论文

【摘要】：研究背景：沙门菌是革兰氏阴性杆菌,属肠杆菌科,在自然界有广泛宿主分布,存在于大多数动物及人的胃肠道中；也常常存在于水、蛋及蛋制品、肉类及其他食品中。根据O抗原(菌体抗原)及H抗原(鞭毛抗原)的抗原特性进行分类,沙门菌有2500多种血清型；其中鼠伤寒沙门菌、肠炎沙门菌是人兽共患最常见的血清型。患者感染常出现腹泻、腹痛、呕吐等症状；沙门菌感染多为自限性疾病,患者可自愈,但败血症等并发症需要抗生素治疗。目前,临床上治疗沙门菌病时,主要采用喹诺酮类和三代头孢菌素类抗生素,随着使用时间和使用频次的积累,已有相当一部分耐药菌株产生。据美国国家抗生素耐药监测系统(national antimicrobial resistance monitoring system, NARMS)最近的报道显示,22.5%的非伤寒沙门菌至少耐一种抗生素。肠炎沙门菌出现了多重耐药(multidrug resistance,MDR)现象。质粒介导(Plasmid-Mediated Quinolone Resistance,PMQR)是沙门菌耐药的重要机制。目前有3类报道较多的PMQR质粒基因,分别是qnr(编码Qnr蛋白)、aac(6')-Ib-cr(编码AAC(6')Ib-cr蛋白)、qepA(编码QepA蛋白)。PMQR主要介导低水平的耐药,但也可累积突变,引起高水平的耐药。PMQR阳性菌主要以多耐药基因同时出现为主,呈多重耐药表型,且不同地区也有不同的分布。沙门菌污染食品致腹泻暴发由以前的点源性集中暴发,越来越多地转变为跨地区跨州(省)的散在暴发形式出现,使污染源及传播途径的发现更需要分子分型手段的辅助及确认。分子水平上的分型方法一般包括三种：基于DNA序列多态性分析的分型方法、基于扩增特定基因产物的PCR分型方法、以及基于细菌DNA的限制性内切酶分析的分型方法。脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)是20世纪80年代中期发展起来的用于分离大分子量线性DNA分子的一种电泳技术,被誉为细菌分子生物学分型技术的“金标准”。其原理是在琼脂糖凝胶上外加方向、时间与电流大小交替改变的脉冲电场,从而使得DNA分子得到有效地分离。通过比较DNA分子电泳图谱来确认菌株之间的关联。菌株间由于酶切位点突变、插入序列不同等原因会产生不同图谱,一般认为电泳条带差异越少则基因差异越小,菌株亲缘关系则越近；反之则菌株间无相关性,但对亲缘关系较远的菌株分型效果不佳。PFGE适于研究一个暴发事件中,菌株之间的来源关系。多位点序列分型技术(multi-locus sequence typing,MLST)基于核酸序列测定技术,通过测定基因序列的变化反映菌株之间的进化关系,在推论菌株间遗传进化关系和种群相关性等方面有其他分型方法无可比拟的优势。其具有每种细菌的等位基因信息可通过互联网使其全球标准化,具有快速、实验结果可比性高和分辨水平高等优点。由于他们遗传稳定性,他们不能为短期的流行病学研究提供有力的帮助。故MLST适于研究在一个大群体内,一段时期内,菌株之间的亲缘关系。本研究旨在对广东地区开展肠炎沙门菌耐药监测和耐药菌株的分子分型研究,对2007-2013年GSS的肠炎沙门菌临床分离株进行药物敏感性检测(微量肉汤稀释法),并针对目前临床上治疗沙门菌感染主要使用喹诺酮类药物和头孢类药物,特别是Ⅱ代头孢菌素类以及Ⅲ代喹诺酮类的耐药菌株进行耐药基因PMQR的检测和PFGE、MLST分子分型,有利于我们对于临床高频用药的监测追踪,将重点监测结果及时反馈,可以更好地指导临床合理用药。获取的耐药菌株的核酸指纹基础数据,可以分析本省监测点医院腹泻病例临床分离株之间的遗传学关联性,了解其分子遗传特征,为发现肠炎沙门菌可能引起的暴发提供本底发病水平,为实验室监测、鉴定溯源及防控预警提供技术支持和数据参考,提高食源性疾病的检测和防控能力。研究目的：1、了解2007年-2013年广东省人群中感染肠炎沙门菌的耐药情况,绘制耐药谱,分析多重耐药特点。为指导临床医生合理选择使用抗生素,同时为预防和控制肠炎沙门菌引起的感染性腹泻的发生和流行提供科学依据。2、根据耐药结果选择耐氟喹诺酮类和头孢类药物的菌株进行PMQR基因检测,了解耐药基因在广东省肠炎沙门菌中的分布,发现其流行病学规律。3、对耐药肠炎沙门菌菌株进行PFGE分型和MLST分型,对菌株带型的聚集和分布规律进行分析,掌握广东省流行株型别与全球流行现况,以发现带型的流行病学规律,为监测和暴发研究提供基础。研究方法：1、采用CLSI推荐的微量肉汤稀释法对广东省2007-2013年肠炎沙门菌进行抗生素敏感性实验,测定菌株的最小抑菌浓度(MIC值)。使用WHONET 5.3进行抗生素敏感性试验数据分析,并将数值导入SPSS 13.0软件进行统计分析,对2007-2013年GSS系统肠炎沙门菌的阳性检出率状况、三间分布特点,以及抗生素敏感性和多重耐药的特点进行描述性统计分析。2、对耐头孢类和氟喹诺酮的菌株进行PMQR质粒基因qnrA、qnrC、 qnrD、qurS、qepA、 oqxAB、aac(6')-Ib-cr)检测,分析介导肠炎沙门菌对氟喹诺酮耐药的质粒基因在广东省的分布情况及其对MIC值的影响。MIC几何均数值的组间比较采用变量变换后的非配对t检验,计数资料的组间比较采用卡方检验或Fisher确切概率法。检验水准α=0.05,以P0.05为差异有统计学意义。3、参照国际PulseNet的沙门菌PFGE分子分型标准操作方案,对上述耐头孢类和氟喹诺酮类的肠炎沙门菌进行分子分型。菌株先经限制性内切酶消化,脉冲场凝胶电泳后,使用凝胶成像分析系统生成图像条带,BioNumerics软件对图像条带进行识别、处理,H9812菌株作为标准分子量进行校准。选择非加权配对算术平均法进行条带的聚类分析,根据分析结果使用统一命名规则对条带进行编号,建立耐药肠炎沙门菌的PFGE分子分型数据库。4、参考PulseNet推荐的沙门菌7个位点(aroC、dnaN、hemD、hisD、purE、sucA、 thrA)MLST标准操作方案,对反应体系和检测步骤进行相关的优化,测序后根据分析结果使用既定命名规则对ST型别进行编号,建立数据库。结合菌株的流行病学资料,对MLST型别的亲缘进化关系、分布规律以及与耐药谱的关系进行分析,同时与PFGE分型进行比较,综合评价MLST的分型能力和实际应用价值。研究结果：1、2007-2013年共收集散发腹泻病例63687例,分离得到肠炎沙门菌菌株386例(检出率6.06%0)。菌株分离主要来源于广州、东莞,并以珠三角城市群为中心,呈散发趋势,各地区检出率无显著性差异。在分离的386株散发病例肠炎沙门菌中,男女比例为1：0.698；各个年龄段均有感染的可能性,总体以1-4岁的儿童病例为主,占总数的35.23%(136/386),中青年组(18-60岁)病例排行第二,占总数的23.83%(92/386)。全年均可检出,高峰期为6-10月。2、MIC结果显示,肠炎沙门菌对八种抗生素均可能产生耐药。喹诺酮类药物中以第一代喹诺酮类药物萘啶酸(NAL)耐药率最高85.23%(329/386),MIC50值为128μg/ml;以第二代头孢菌素类的头孢西丁耐药率最低(0.52%,2/386),MIC50值为2μg/ml,此外,四环素(TET)的耐药率也较高(30.83%,119/386),MIC50值为2μg/ml。386株菌共有27个耐药谱,以单一耐萘啶酸(NAL)(43.78%,169/386)为主,其次耐四环素加萘啶酸(TET+NAL)(15.03%,58/386)。有63株菌株对3种及3种以上的抗生素耐药,占菌株总数的16.32%(63/386)。3、根据MIC药敏结果筛选出71株耐氟喹诺酮类药物和耐头孢类药物的肠炎沙门菌的PMQR基因检测结果为：总体质粒携带率为63.38%(45/71),其中35株仅携带一种PMQR基因(49.30%,35/71)；9株携带两种PMQR基因(12.68%,9/71)；1株携带四种PMQR基因(1.41%,1/71)。oqxAB检出率最高,达40.85%(29/71),其次为qnrC(9.86%,7/71),qepA未检出。携带PMQR质粒与未携带PMQR质粒的菌株在氯霉素(CHL)和磺胺类药物(TMP/SMZ)的MIC几何均数值有统计学差异,携带PMQR质粒组多重耐药的比例(75.56%,34/45)高于未携带PMQR质粒的菌株比例。4、71株耐药肠炎沙门菌Xba I酶切的聚类分析可分为两个大聚类,PFGE图谱的相似值为56%-100%,共分为PFGE-Xba I 1-35型。最大的型别有8株菌,最小的为1株。5、71株耐药肠炎沙门茵的MLST分型均为同一型别,等位基因序为aroC5、dnaN2、 hemD3、hisD7、purE6、sucA6、thrA11;等位基因谱为ST11型,属于eBG4型。这说明广东省耐药肠炎沙门菌的优势序列型均为ST11型。结论：1、肠炎沙门菌为广东省第二大优势血清型,主要易感人群为14岁的幼儿,夏秋季高发,病例集中分布于珠三角地区。药敏结果显示萘啶酸(NAL)耐药率最高,多重耐药率为16.32%。2、对于耐药菌株的PMQR质粒基因检测结果说明携带PMQR质粒的菌株对比未携带质粒的菌株耐药性更高,且更易发生多重耐药现象。3、MLST的分子分型结果表明,在沙门菌同一血清型内各菌株看家基因碱基序列的高度保守,而PFGE则提示肠炎沙门菌的分化程度不高；对于肠炎沙门菌来说,MLST和PFGE的分辨力均较低,还需用其他分辨力更高的分子分型方法。
[Abstract]:Research background: Salmonella is gram-negative bacilli, a family of Enterobacteriaceae, widely distributed in nature and exists in most animals and human gastrointestinal tract; it also exists in water, egg and egg products, meat and other foods. Classification of O antigen (mycelium antigen) and H antigen (flagellum antigen) antigen characteristics, Salmonella There are more than 2500 serotypes. Among them, Salmonella typhimurium and Salmonella enteritis are the most common serotypes of human zoonosis. Patients often have symptoms such as diarrhea, abdominal pain, vomiting and other symptoms; Salmonella infection is a self limiting disease, and the patients can heal themselves, but the complications such as sepsis need antigreen treatment. At present, the main clinical treatment of Salmonella disease is mainly in clinical treatment. Using the quinolones and three generation cephalosporins, a number of resistant strains have been produced with the accumulation of time and frequency of use. According to the recent reports of national antimicrobial resistance monitoring system (NARMS) in the United States, 22.5% of non typhoid Salmonella is at least one resistance. Multidrug resistance (MDR) has appeared in Salmonella enteritis. Plasmid mediated (Plasmid-Mediated Quinolone Resistance, PMQR) is an important mechanism for the drug resistance of Salmonella. There are 3 kinds of PMQR plasmids which are reported to be qnr (encoded Qnr protein) and AAC (6'). EpA protein).PMQR mainly mediates low level of resistance, but it can also accumulate mutation, causing the high level of drug resistant.PMQR positive bacteria mainly to appear mainly with multidrug-resistant genes and multidrug-resistant phenotype, and there are different distribution in different regions. In the form of outbreaks across regions and provinces, the discovery of pollution sources and channels of transmission requires the assistance and confirmation of molecular typing methods. The classification methods on the molecular level generally include three types: the typing based on DNA sequence polymorphism analysis, the PCR typing method based on the amplification of the specific gene products, and the DN based on the bacteria. A's restriction endonuclease analysis method. Pulsed field gel electrophoresis (PFGE) is an electrophoretic technique developed for the separation of large molecular weight linear DNA molecules in the mid 1980s. It is known as the "gold standard" for bacterial molecular biological typing. The principle is on agarose gel. A pulsed electric field, alternately changing the size of time and current, makes the DNA molecules effectively separated. The correlation between strains is confirmed by comparing the DNA molecular electrophoretic map. The smaller the difference, the closer the strain relationship was, and conversely, there was no correlation between the strains, but the poor genotyping effect of the far relative strains.PFGE was suitable for the study of the source relationship between the strains. The multiple point sequence typing (multi-locus sequence typing, MLST) was based on nucleic acid sequencing technology and through the determination of the basis. The evolution relationship between strains is reflected by the variation of the sequence, and there are unparalleled advantages in other classification methods, such as genetic evolution relationship and population correlation among the strains, and the allelic information of each bacterium can be standardized through the Internet, fast, high experimental results and high resolution. Advantages. Because of their genetic stability, they can not provide strong help for short-term epidemiological studies. Therefore, MLST is suitable for studying the relationship between strains in a large group, for a period of time. This study aims to study the molecular typing of Salmonella enteritis in Guangdong and the molecular typing of resistant strains for 2007-2013 years. GSS clinical isolates of Salmonella enteritis were tested for drug sensitivity (micro broth dilution), and the detection of drug-resistant gene PMQR and PFGE, MLST molecules for the main use of quinolones and cephalosporins, especially the second generation cephalosporins and third generation quinolones, were detected in the clinical treatment of Salmonella infection. The classification is beneficial to the monitoring and tracking of clinical high frequency drugs and the timely feedback of the key monitoring results. It can guide the clinical rational use of drugs better. The basic data of nucleic acid fingerprint of the resistant strains obtained can be used to analyze the genetic association between the clinical isolates of diarrhea cases in the provincial monitoring points and understand its molecular genetic specificity. In order to find the possible background of the outbreak of Salmonella enteritis, it provides technical support and data reference for laboratory monitoring, identification and prevention and control early warning. The purpose of this study is to improve the detection and control ability of food borne diseases. 1. 1. To understand the drug resistance of Salmonella enteritis in Guangdong Province in 2007. In order to guide the clinicians to choose antibiotics reasonably and provide scientific basis for the prevention and control of the occurrence and epidemic of infectious diarrhea caused by Salmonella enteritis,.2, PMQR gene detection was carried out to select the strains resistant to fluoroquinolones and cephalosporins to understand the resistance genes. The distribution of Salmonella enteritidis in Guangdong Province, we found its epidemiological law.3, PFGE typing and MLST typing of Salmonella resistant strains of Salmonella enteritis, analysis of the distribution and distribution of strain band type, grasp the epidemic situation of the epidemic plant type and the global epidemic situation in Guangdong Province, in order to find the pattern of epidemiology, and provide the monitoring and outbreak research. Basic and research methods: 1, the antibiotic susceptibility test of Salmonella enteritis in 2007-2013 years in Guangdong province was carried out by the method of micro broth dilution recommended by CLSI. The minimum inhibitory concentration (MIC value) of the strain was measured. WHONET 5.3 was used to analyze the antibiotic sensitivity test data, and the number of values was introduced into the SPSS 13 software for statistical analysis, to 2007-2013 The positive rate of Salmonella enteritis in GSS system, three distribution characteristics, and the characteristics of antibiotic sensitivity and multidrug resistance were analyzed by.2. The PMQR plasmid gene qnrA, qnrC, qnrD, qurS, qepA, oqxAB, AAC (6') -Ib-cr) for the strains resistant to cephalosporins and fluoroquinolones were detected, and the fluorine was analyzed and mediated by Salmonella enteritis. The distribution of quinolone resistant plasmid gene in Guangdong province and the effect of the MIC value on the value of.MIC were compared with the non paired t test after variable transformation. The comparison of the count data was compared to the chi square test or the Fisher exact probability method. The level of alpha =0.05 was tested, and the P0.05 was statistically significant.3, referring to international Pul. SeNet of Salmonella PFGE molecular classification standard operation scheme, the molecular classification of the above Salmonella enteritis resistant cephalosporins and fluoroquinolones. The strain was digested by restriction endonuclease first, and after pulsed field gel electrophoresis, the image strip was generated by the gel imaging analysis system. The BioNumerics software identified, processed, and H9812 bacteria. The plant was calibrated as a standard molecular weight. The unweighted pairing arithmetic mean method was selected to cluster analysis of strip. According to the results, the PFGE molecular typing database of Salmonella enteritis was numbered and.4 was set up. 7 sites of Salmonella (aroC, dnaN, hemD, hisD, purE, sucA, th) recommended by PulseNet were referred to. RA) MLST standard operation scheme, optimizing the reaction system and detection steps. After sequencing, the ST type is numbered and the database is set up according to the results of the analysis. The relationship of the phylogenetic evolution of the MLST type, the distribution rule and the relationship with the drug resistance spectrum are analyzed with the epidemiological data of the strain. PFGE classification was compared to evaluate the classification ability and practical application value of MLST. Results: 63687 cases of sporadic diarrhea cases were collected in 12007-2013 years, 386 cases of Salmonella enteritis were isolated (detection rate 6.06%0). The isolation of strains was mainly from Guangzhou, Dongguan, and was distributed in the Pearl River Delta city group as the center. There was no significant difference in the detection rate. In the 386 sporadic cases of Salmonella enteritis, the proportion of men and women was 1:0.698, the possibility of infection in each age group was 1-4 years old, accounting for 35.23% (136/386) of the total number of children, and second in the young and middle-aged group (18-60 years old), accounting for 23.83% (92/386). All year all could be detected, high The peak period was 6-10 months.2, MIC results showed that Salmonella enteritis could be resistant to eight antibiotics. The highest quinolones drug resistance rate was 85.23% (329/386), MIC50 value was 128 micron, and second generation cephalosporin (0.52%, 2/386) and 2 g/ml, with the second generation cephalosporin. In addition, the resistance rate of tetracycline (TET) was also higher (30.83%, 119/386), and the MIC50 value of 2 micron g/ml.386 strains had 27 resistance profiles, with single naphthidine (NAL) (43.78%, 169/386), followed by tetracycline and naphthidine acid (TET+NAL) (15.03%, 58/386). 63 strains were resistant to 3 and more than 3 antibiotics, accounting for 16.32% (63/386).3 of the total strain. The results of PMQR gene detection of 71 strains of fluoroquinolone resistant and cephalosporin resistant Salmonella were screened according to the MIC drug sensitivity results: the overall plasmid carrying rate was 63.38% (45/71), 35 of which only carried one PMQR gene (49.30%, 35/71); 9 strains carried two PMQR genes (12.68%, 9/71), and 1 carrying four PMQR genes (1.41%, 1/7). 1) the highest detection rate of.OqxAB was 40.85% (29/71), followed by qnrC (9.86%, 7/71), and qepA was not detected. The PMQR plasmid and the MIC without PMQR plasmid had a statistical difference between the MIC geometry of chloramphenicol (CHL) and the sulfonamides (TMP/SMZ), and the proportion of the multidrug resistance (75.56%, 34/45) carrying the PMQR mass group (75.56%, 34/45) was higher than that of the bacteria without the plasmid carrying plasmid. The cluster analysis of Xba I enzyme cut of Salmonella enteritis resistant to.4,71 strain can be divided into two large clusters. The similarity value of PFGE atlas is 56%-100%, which is divided into PFGE-Xba I 1-35. The largest type has 8 strains, and the minimum of 1 strains of.5,71 strain of Salmonella is the same type, and the allele sequence is aroC5, dnaN2, hemD3. RE6, sucA6, thrA11; the allele gene spectrum is ST11 type, which belongs to eBG4 type. This shows that the dominant sequence of drug resistant Salmonella enteritis in Guangdong province is ST11 type. Conclusion: 1, Salmonella enteritis is the second dominant serotype in the province, the main susceptible population is 14 year old children, in summer and autumn, the case is concentrated in the Pearl River Delta region. The drug sensitivity results show that the case is concentrated in the Pearl River Delta region. The drug resistance of nnidium acid (NAL) was the highest and the multidrug resistance rate was 16.32%.2. For the PMQR plasmid DNA detection results of the resistant strains, the strains carrying PMQR plasmids were more resistant to the strains without plasmid and more susceptible to multidrug resistance.3. The molecular typing of MLST showed that each strain of Salmonella was in the same serotype. Because of the highly conserved base sequence, PFGE suggests that the differentiation degree of Salmonella enteritis is not high; for Salmonella enteritis, the resolution of MLST and PFGE is low, and other molecular typing with higher resolution is needed.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

【参考文献】