广东省鼠伤寒沙门菌的分子流行病学研究

发布时间：2018-01-18 06:07

本文关键词：广东省鼠伤寒沙门菌的分子流行病学研究　出处：《南方医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景食源性疾病是各种致病因子通过摄食进入人体内引起的一类感染性或中毒性疾病,每年全球仅食源性腹泻就造成几百万儿童死亡,即使在发达国家也至少有三分之一的人患食源性疾病,沙门菌(Salmonella)是主要的病原体之一。随着生产生活模式的改变,地域之间人员、食品往来、物资流通更加广泛,食品加工、供给及饮食方式的多样化,每年世界范围包括我国在内都会有沙门菌引起的食源性疾病报道,所造成的公共卫生问题不容忽视,卫生经济负担逐渐引起相关部门的重视。沙门菌有2500多个血清型,其中鼠伤寒(Salmonella enterica serovar Typhimurium, S. Typhimurium)是引起人类胃肠炎或食物中毒最常见,最重要的血清型之一,寄生于人体和爬行类动物的肠道,是一种重要的人畜共患病原菌,临床主要表现为胃肠炎型、败血症等沙门菌病,由该菌引起的食源性疾病暴发在国内外经常发生,并造成重大的经济损失。广东省每年都会有鼠伤寒沙门菌引起的食物中毒事件报道,腹泻暴发事件由以前的点源性集中暴发,越来越多地转变为跨地区的“散在暴发”,使污染源及传播途经的发现越来越依赖于分子分型手段的辅助及确认。目前已有比较成熟的国际通用标准化脉冲场凝胶电泳(Pulse-Field Gel Electrophoresis, PFGE)分型方案、第二代分子分型技术多位点可变数目串联重复序列分析(Multiple-locus Variable-numbers Tandem Repeat Analysis, MLVA)以及英国华威大学公布的7个管家基因位点的多位点序列分析(Multiple Locus Sequence Typing, MLST)分型方法,并成功应用于多起由鼠伤寒沙门菌引起的食源性疾病暴发的溯源调查。为了建立以实验室为基础的主动监测系统,完善分子分型网络库,广东省自2009年正式启动沙门菌加强监测(Enhance Salmonella Surveillance, ESS),对散发的腹泻患者进行监测,通过监测发现鼠伤寒沙门菌为本省非伤寒沙门菌感染性腹泻病例的优势菌型。本研究旨在广东地区开展鼠伤寒沙门菌分子分型监测研究,对2009-2011年ESS的鼠伤寒沙门菌临床分离株进行PFGE和MLVA分子分型,以获取菌株的核酸指纹基础数据,并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,为发现鼠伤寒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,科学防控鼠伤寒沙门菌所致的食源性疾病提供决策依据,提高食源性疾病的检测和防控能力。研究目的 1、了解广东省2009-2011年沙门菌加强监测系统的现状,主要分析优势血清型鼠伤寒的流行病学特征、抗生素敏感性和多重耐药的特点。 2、对三年加强监测收集的鼠伤寒沙门菌进行PFGE分型,对菌株带型的聚集和分布规律进行分析,以发现带型的流行病学规律,为监测和暴发研究提供基础的分子分型数据库。 3、参考PulseNet公布的鼠伤寒沙门菌MLVA分型标准方案,进行相关地优化,并将其应用于加强监测系统的分型分析,对MLVA型别的亲缘进化关系和分布规律进行分析,并与PFGE分型进行比较,综合评价MLVA的分型能力,以阐明该方法实际应用的价值和意义,实现简便、快速的分型技术提高实验室主动监测能力的目标。研究方法 1、采用WHO推荐的改良K-B纸片扩散法对广东省2009-2011年ESS的鼠伤寒沙门菌进行抗生素敏感性实验。根据美国2012版CLSI选择12种抗生素纸片和判读敏感性结果,最后使用WHONET5.4进行抗生素敏感性试验数据分析。菌株的流行病学资料和检测情况录入Excel2003,并将数值导入SPSS13.0软件进行统计分析,对2009-2011年加强监测系统沙门菌的阳性检出率状况,优势血清型鼠伤寒的地区、时间及人群分布特点,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、参照国际PulseNet的沙门菌PFGE分子分型标准操作方案,对上述沙门菌的优势血清型鼠伤寒进行分子分型。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,建立沙门菌加强监测的PFGE分子分型数据库。结合菌株的流行病学资料,使用描述性分析归纳带型的聚集和分布规律,主要是三间分布规律；并对优势带型的三间分布差异进行卡方检验,同时结合耐药谱型进行关联性分析。 3、参考PulseNet推荐的7个位点MLVA标准操作方案,对反应体系和检测步骤进行相关的优化,并将其应用于加强监测系统鼠伤寒沙门菌的分型分析。使用ABI3730XL测序仪进行PCR产物片段大小的检测,GeneMapper软件对片段大小对应的毛细管荧光信号进行识别,参照普通琼脂糖电泳的结果,对片段大小进行确认,计算相应位点的重复数,BioNumerics软件对重复数进行最小生成树分析,根据分析结果使用既定命名规则对MLVA型别进行编号,建立沙门菌加强监测的MLVA分子分型数据库。结合菌株的流行病学资料,对MLVA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MLVA的分型能力和实际应用价值。研究结果 1、三年监测收集总的样本数为19895份,沙门菌的年均检出率为4.03%(802/19895),鼠伤寒沙门菌的年均检出率、构成比分别为1.38%(275/19895)、34.29%(275/802),鼠伤寒为广东省沙门菌的优势血清型。275株鼠伤寒沙门菌,以2010年的构成比最大(136/275,44.88%),每年的5月、9月为发病高峰,男女比例为1.57：1,不同年份性别间差异无统计学意义(P0.05),0-1岁的婴幼儿为主要发病人群(157/275,57.09%),各年份不同年龄段差异有统计学意义(P0.05),主要来源于广州(95/275,34.55%)、中山(59/275,21.45%)等珠三角城市群。 2、275株鼠伤寒沙门菌抗生素敏感性试验结果显示,对头孢类抗生素敏感性高,均为80%以上,而对环丙沙星耐药率最低(6.2%),对其余8种抗生素高度耐药,耐药率在68.4%-90.5%之间；除了环丙沙星、链霉素,各年份的抗生素耐药率变化趋势一致,不同年份的菌株对氨苄西林、萘啶酸、磺胺甲嗯唑和四环素耐药率差异均有统计学意义(P0.05)；3株鼠伤寒沙门菌(编号ESS221.463.797)对所检测的12种抗生素全部耐受,255株鼠伤寒为多重耐药株(92.7％),AMP+CHL+NAL+SMX+STR+TCY+TMP+GEN为广东省最主要的多重耐药谱型(104/275,37.8%)。 3、275株鼠伤寒沙门菌经Xba I酶切电泳后,相似值为56.09%-100%,分为124种带型、7大聚类簇,分型D值为0.9286,每种带型包含1-71株菌株,平均每种带型有2.22株菌。30种带型在不同地区交叉出现,17种带型在不同年份交叉出现。优势带型为JPXX01.GD0004共71株,主要包括广州21株(29.6%)、中山17株(23.9%)、东莞14株(19.7%),主要集中于2010年(51/71,71.8%),5月(14/71,19.72%)、9月(11/71,15.49%)为高峰期,以0-1岁男婴为主,优势条带的分布符合菌株的流行特征,不同地区、年代、性别以及年龄组间的分布差异有统计学意义(P0.05)；优势带型主要对5-9种抗生素耐药,带型JPXX01.GD0035和JPXX01.GD0047的耐药谱为AMP+CHL+NAL+SMX+STR+TCY+TMP+GEN. 4、275株鼠伤寒沙门菌的MLVA分型结果显示,所有菌株分为143种型别、5个亲缘进化群,D值为0.9665,每种型别有1-42株菌株,平均每种带型包含1.92株菌；29种型别在不同地区交叉出现,14种型别在不同年份交叉出现,优势型别为001(5-10-4-8-24-11-33)42株,流行病学特点与PFGE的优势带型相似；与始祖GDMT001亲缘关系越近,对各种抗生素的耐药性表现越相似,其中001-007(除了004)型对环丙沙星之外的其他抗生素耐药率较高,MLVA型别与耐药谱型AMP+CHL+NAL+SMX+STR+TCY+TMP+GEN完全一致的菌株比较多,尤其是在含单株的型别中比较常见。 5、MLVA与PFGE型能力比较显示,MLVA分型的D值(0.9665)大于PFGE-Xba Ⅰ单酶切分型的D值(0.9286),而与PFGE-Xba Ⅰ-Bln Ⅰ双酶切分辨能力(D值为0.9891)相当；6株001型、4株002型、2株003型、3株005型及2株007型均对应于PFGE-Bln Ⅰ的部分JPXA26.GD0001型及分别对应于部分JPXA26. GD0006、0005、0036、0050、0017型,即MLVA能将Bln I酶切后的条带进一步区分为不同的型别。研究结论 1、鼠伤寒为广东省沙门菌的的优势血清型,2010年的构成比最大,集中分布于广州、中山等珠三角城市群,每年的5月、9月为发病高峰,以0-1岁的男婴患者为主；所有鼠伤寒沙门菌对头孢类抗生素敏感性高,对环丙沙星耐药率最低,对其余8种抗生素高度耐药,多重耐药形式严峻,AMP+CHL+NAL+SMX+STR+TCY+TMP+GEN为最主要的多重耐药谱型。 2、初步建立了广东省ESS的分子分型数据库,PFGE和MLVA分子分型方法均显示广东省鼠伤寒沙门菌具有遗传多样性。型别的聚集和分散符合菌株的总体流行病学特征。 3、两种分型方法的DNA指纹图谱和耐药谱无明显一致性。 4、MLVA的分辨能力高,不仅从宏观上对菌株的相似性进行分析,而且从微观上揭示细菌的亲缘进化关系,在确认鼠伤寒沙门菌引起的暴发事件时,采用MLVA分型方法可满足菌株聚集性分析。
[Abstract]:Research background
Foodborne disease is a kind of infectious or toxic diseases caused by various pathogenic factors feeding into the human body, every year around the world only diarrhea caused the death of millions of children, even in developed countries and at least 1/3 people suffering from foodborne disease, Salmonella (Salmonella) is one of the main pathogens of production. With the change of lifestyle, regional personnel, food contact, material circulation is more extensive, food processing, food supply and diversification of the year all over the world including China have reported foodborne disease caused by Salmonella, public health problems caused by the economic burden of health can not be ignored, gradually attracted the attention of relevant departments. There are more than 2500 serotypes of Salmonella among them, typhimurium (Salmonella enterica serovar Typhimurium, S. Typhimurium) is caused by human gastrointestinal inflammation or food poisoning The most common, one of the most important serotypes, parasitic in humans and reptiles of the animal intestinal tract is an important zoonotic pathogens. The main clinical manifestations of gastroenteritis, septicemia and other diseases caused by the bacteria Salmonella, foodborne disease outbreaks often occur at home and abroad, and cause significant economic losses.
Guangdong Province, every year there are reports of poisoning caused by Salmonella typhimurium food diarrhea outbreak concentrated outbreak by point source of the previous, more and more into the cross region "scattered in the outbreak, the spread of pollution sources and found more and more dependent on aid and confirmation on the molecular typing methods at present. The international standard has been relatively mature of pulsed field gel electrophoresis (Pulse-Field Gel Electrophoresis, PFGE) classification scheme, the second generation of molecular typing techniques of multilocus variable number tandem repeat analysis (Multiple-locus Variable-numbers Tandem Repeat Analysis, MLVA) and multilocus 7 housekeeping genes of British University of Warwick published sequence analysis (Multiple Locus Sequence Typing, MLST) typing method, investigation and successful application of food borne diseases in the outbreak caused by Salmonella typhimurium . in order to establish active monitoring system based on laboratory, improve the molecular typing of network library, Guangdong province since 2009 officially launched to strengthen the monitoring of Salmonella (Enhance Salmonella Surveillance, ESS), the monitoring of patients with sporadic diarrhea, by monitoring found Salmonella typhimurium as the dominant serotype of Salmonella typhi in non infectious diarrhea cases in the province.
The purpose of this study is to Guangdong to carry out Salmonella typhimurium molecular field monitoring, Salmonella typhimurium isolates of 2009-2011 ESS were PFGE and MLVA genotyping, DNA fingerprint based data to obtain the strains, and the analysis of monitoring points of the province hospital diarrhea Genetics Association Lin bed separation between strains, understand the molecular genetic characteristics, to find the outbreak of Salmonella typhimurium may cause the bottom incidence level, for laboratory monitoring, provide technical support and data reference identification investigation and prevention and early warning, provide decision basis for scientific prevention and control of Salmonella typhimurium caused by foodborne diseases, improve food borne disease detection and prevention and control ability.
research objective
1, we need to know the current status of surveillance system for Salmonella in Guangdong Province in the past 2009-2011 years. We mainly analyzed the epidemiological characteristics of the dominant serotype typhus, the sensitivity of antibiotics and the characteristics of multidrug resistance.
2, three years of surveillance and collection of Salmonella typhimurium were carried out for PFGE typing. The accumulation and distribution patterns of strains were analyzed to find out the epidemiological rule of banding pattern, and provide a basic molecular typing database for monitoring and outbreaks.
3, Salmonella typhimurium MLVA reference published by the PulseNet classification scheme, were optimized, and applied to strengthen monitoring system analysis, phylogenetic relationships and distribution of type MLVA were analyzed and compared with PFGE typing, typing MLVA comprehensive evaluation, value and the significance, clarify the practical application of the method is easy to realize fast typing techniques to improve laboratory active monitoring ability.
research method
1, the antibiotic susceptibility tests in Guangdong Province during 2009-2011 ESS in Salmonella typhimurium were recommended by WHO modified K-B Bauer method. According to the 2012 edition of the CLSI paper choose 12 antibiotics and interpretation sensitivity results, and finally the use of WHONET5.4 for antibiotic susceptibility test data analysis. Strains of epidemiological data and detection and input Excel2003. Numerical import SPSS13.0 software for statistical analysis, to strengthen the 2009-2011 monitoring system of the positive rate of Salmonella serovar typhimurium, advantage area, time and population distribution characteristics, descriptive statistical analysis and characteristics of antibiotic sensitivity and multidrug resistance.
2, Salmonella PFGE molecular typing according to the international PulseNet standard for the operation of the program, the advantages of Salmonella serotype Typhimurium was used for molecular typing of strains. After restriction endonuclease digestion, pulsed field gel electrophoresis, analysis system generates image strips using gel imaging, BioNumerics software for image processing, strip recognition. H9812 strain as standard molecular weight calibration. Select the unweighted pair group method with arithmetic mean of band clustering, according to the results of the analysis using the unified naming rules are numbered on the strip, the establishment of Salmonella PFGE molecular typing to strengthen monitoring database. Combined with the epidemiological data of strains, using descriptive analysis with the accumulation and distribution is the main distribution, three; chi square test for the three distribution differences and belt type of advantage, combined with the type of drug resistance spectrum correlation points Analysis.
3, 7 loci MLVA PulseNet recommended reference standard operation plan, optimize the reaction system and inspection procedures, and applied to strengthen the type monitoring system of Salmonella typhimurium analysis. Detection of PCR products amplified using ABI3730XL sequencing, the fragment size of capillary fluorescence signal corresponding to the GeneMapper software according to the common recognition, the results of agarose gel electrophoresis, the fragment size for confirmation, repeat number calculation sites, BioNumerics software for the repeat number of minimum spanning tree are analyzed, according to the analysis results using the established naming rules are numbered for MLVA type, establish Salmonella typing enhanced MLVA molecular monitoring database. Combined with the epidemiological data of strains, genetic the evolutionary relationship of the type of MLVA, analyze the distribution regularity and the relationship with the resistance spectrum, and PFGE classification were compared in To evaluate the classification ability and practical application value of MLVA.
Research results
1, three years of monitoring collection total sample number is 19895, the average annual Salmonella detection rate was 4.03% (802/19895), the average detection rate of Salmonella typhimurium, accounted for 1.38% (275/19895), 34.29% (275/802), the dominant serotype Typhimurium.275 Guangdong Salmonella strains of Salmonella typhimurium, to in 2010 the largest proportion (136/275,44.88%), every May, September is the peak incidence, male to female ratio was 1.57:1, there was no significant difference between the sexes in different years (P0.05), 0-1 years old children as the main disease groups (157/275,57.09%), there was statistical significance each year is different in different ages (P0.05), mainly from Guangzhou (95/275,34.55%), Zhongshan (59/275,21.45%) and the Pearl River Delta city group.
2275 strains of Salmonella typhimurium antibiotic sensitivity test results show that the sensitivity of the cephalosporin antibiotic, was more than 80%, while the lowest rate of ciprofloxacin (6.2%), the other 8 kinds of antibiotics were highly resistant, resistant rate was 68.4%-90.5%; in addition to ciprofloxacin, streptomycin, antibiotic resistance rate of each year is consistent in different years the strains to ampicillin, nalidixic acid, sulfamethoxazole and tetracycline resistance rate differences were statistically significant (P0.05); 3 strains of Salmonella typhimurium (ESS221.463.797) to 12 antibiotics detected all 255 strains of Salmonella typhimurium tolerance, multi drug resistance strains (92.7%), AMP+CHL+NAL+SMX+STR+TCY+TMP+GEN is Guangdong's leading multi drug resistance province profile (104/275,37.8%).
3275 strains of Salmonella typhimurium by Xba I enzyme digestion electrophoresis after the similarity value is 56.09%-100%, divided into 124 types, 7 types of clusters, the D value is 0.9286, each type contains the 1-71 strain, the average of each zone type 2.22 isolates with type.30 in different areas of cross 17, belt type appeared in different years. The advantages of cross type JPXX01.GD0004 with a total of 71 strains, including 21 strains from Guangzhou (29.6%), Zhongshan (23.9%), 17 strains of Dongguan 14 strains (19.7%), mainly in 2010 (51/71,71.8%), May (14/71,19.72%), September (11/71,15.49%) to the peak in 0-1 years old baby boy, the distribution of dominant bands consistent with the epidemiological characteristics of strains in different regions, age, gender and age distribution was statistically significant between group differences (P0.05); the predominant type of antibiotic resistant 5-9 resistant, with type JPXX01.GD0035 and JPXX01.GD0047 spectrum for AMP+CHL+NAL+SMX+STR+TCY+TMP+GEN.
MLVA of the 4275 strains of Salmonella typhimurium typing results showed that all strains were divided into 143 types, 5 phylogenetic groups, the D value is 0.9665, each type of 1-42 strain, the average of each zone type contains 1.92 strains; 29 types of cross in different regions, 14 types in different years cross, the dominant type 001 (5-10-4-8-24-11-33) of 42 strains, the epidemiological characteristics and the advantages of PFGE and similar bands; GDMT001 closer genetic relationship between ancestor, resistant to various antibiotics is similar to the 001-007 (except 004) type of ciprofloxacin outside of other antibiotic resistant rate of type MLVA and type of drug resistance spectrum AMP+CHL+NAL+SMX+STR+TCY+TMP+GEN were identical more, especially in water plant types.
5, compared to MLVA and PFGE type, MLVA type D (0.9665) than PFGE-Xba I single RFLP D value (0.9286), and resolution of PFGE-Xba I and -Bln I double enzyme (D = 0.9891); 6 strains of type 001, type 002 and 4 strains, 2 strains type 003, 3 strains of type 005 and 2 strains of type 007 are corresponding to the PFGE-Bln I JPXA26.GD0001 type and corresponding to the part of the JPXA26. GD00060005003600500017, MLVA Bln can be digested with restriction enzyme I band is further divided into different types.
research conclusion
1, Guangdong Province, Salmonella typhimurium serotypes, 2010 which is the largest, located in Guangzhou, Zhongshan and other Pearl River Delta city group, every May, September is the peak incidence, 0-1 years old male patients; all Salmonella typhimurium to cephalosporin antibiotic sensitivity to ciprofloxacin the lowest rate of other 8 kinds of antibiotics were highly resistant, multi drug resistant forms of severe, AMP+CHL+NAL+SMX+STR+TCY+TMP+GEN is the main type of multi drug resistant spectrum.
2, the molecular typing database of ESS in Guangdong province was preliminarily established. The typing methods of PFGE and MLVA showed that the Salmonella typhimurium in Guangdong province had genetic diversity. The aggregation and dispersion of type were in line with the general epidemiological characteristics of the strain.
3, there was no obvious consistency between the DNA fingerprints and the resistance spectrum of the two types of typing.
4, MLVA has high resolving power. It not only analyzes the similarity of bacteria from macroscopic view, but also reveals the phylogenetic relationship of bacteria from microcosmic aspect. When confirming the outbreak caused by Salmonella typhimurium, MLVA typing method can be applied to analyze the aggregation of bacteria.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R516.3

【参考文献】