肠出血性大肠杆菌EHECO157：H7的分子进化研究
发布时间：2018-08-29 16:07
【摘要】：肠出血性大肠杆菌(EHEC) O157:H7作为一种重要的人畜共患传染病病原菌,其感染具有暴发流行趋势、强烈的致病性与致死性和抗生素治疗可加剧病情的危险性等特点,已成为全球性的公共卫生问题,对人们的健康构成了巨大威胁。此外,EHEC O157菌培养容易、感染力强、传播途径多样,使O157菌极有可能作为未来军事斗争的细菌武器和生物恐怖战剂;O157菌的烈性致病因子还有可能用于基因武器的构建。美国疾病控制中心已将其列为B类生物恐怖病原体严加防范。因此,无论从公共卫生还是生物反恐的需要出发,加强其流行病学研究对预防和控制O157感染具有重大的战略意义。 病原体在进化的过程中通过自然突变、转导和转化等方式发生遗传物质的改变,而引起性状(毒力、耐药性等)的改变。EHEC O157:H7是由055:H7进化而来的,大多数毒力基因都是在进化过程中获得的,对其引起人类疾病发挥关键的作用;同时毒力基因大多数处于移动性遗传物质上,很容易重新组合产生新的致病菌株,而导致O157感染的暴发流行。因此,从分子水平对EHEC O157:H7的流行病学进行研究,观察其分子变异和进化,可为O157感染的暴发提供预警信号以应对可能出现的流行事件。 本研究在对我国分离获得的EHEC O157:H7菌株进行微生物学、血清学、生化以及分子生物学(16S rRNA)检测等全面鉴定的基础上,采用PCR检测其主要毒力基因(stx1/2、hlyA、tccP)、MLST测定和分析其保守的管家基因(aspc, icdA, uidA,clpX,mdh, fadD ,lysp )核苷酸序列,通过系统生物学和分子进化分析,以研究EHEC O157:H7分子进化和流行规律,为寻找其感染暴发流行的预警信号提供线索和依据。 主要完成了以下工作: 1.对1999~2007年江苏省疾病预防控制中心建立的O157菌株资源库以及2005~2007年重庆市主城区和三峡库区收集的O157菌株库中的105株EHEC O157:H7菌株从分离培养、血清学试验、生化试验、16S rRNA检测等进行了全面鉴定。 2.对上述105株EHEC O157:H7菌株基因组的7个管家基因(aspc, icdA, uidA,clpX,mdh, fadD ,lysp )的核苷酸序列进行了测定,获得了7个ST型分别为ST836、ST837、ST838、ST839、ST840、ST841和ST842。其中ST837、ST840、ST841属于谱系1,ST836、ST838、ST839和ST842属于谱系2。 3.对上述105株EHEC O157:H7菌株基因组的毒力基因(stx1/2、hlyA、tccP)进行了PCR检测和分析,结果:所有菌株均检出了stx2基因,stx2的检出率为100%,仅1个菌株(99G144)同时携带stx1和stx2基因, stx1的检出率为0.95%;HlyA在105株的EHEC 0157:H7中检出率是100%;在105株EHEC 0157:H7中共检测出包括3个、4个和5个重复片段三种不同重复片段的tccp基因,其中三个重复片段的有13株(12.4%),4个重复片度的有6株(5.7%),5个重复片段的有86株(82.0%),同时,人源的菌株均为5个重复片段,动物源菌株70%菌株属于5个重复片段。 4.将获得的7个新的ST基因型与EcMLST数据库比对,采用最小进化树和分解树进行溯源分析,发现ST836是这105株的来源菌株,与来源于Sakai的ST66联系紧密,而ST66又与源于EDL933的ST69属于同一群,可以认为ST836与ST69、ST66是来源于同一祖先的。初步推测在中国爆发流行的EHEC O157:H7起源可能是由最早爆发的美国EDL933株经日本传播到我国的。 5.对不同分离地点和宿主来源菌株的STs分配频率分析发现:ST842仅来源于人源的,ST838、ST840、ST839这3个STs型仅见于动物源菌株。从菌株的数量来看,ST837的菌株最多,其次是ST836,两者占到85%。 结论: 上述研究工作为建立我国EHEC O157:H7菌株基因信息库奠定了一定的基础,同时为研究我国EHEC O157:H7的分子进化和流行规律、寻找其感染暴发流行的预警信号提供了有利的线索和依据。
[Abstract]:Enterohemorrhagic Escherichia coli (EHEC) O157:H7, as an important pathogen of zoonosis, has become a global public health problem and poses a great threat to people's health. In addition, EHEC infection has the characteristics of outbreak and epidemic, strong pathogenicity and lethality, and antibiotic treatment can aggravate the risk of the disease. O157 is easy to cultivate, highly infectious, and has a wide range of transmission routes. O157 bacteria are highly likely to be used as bacterial weapons and bioterrorism warfare agents in future military struggles. O157 virulent pathogens may also be used in the construction of genetic weapons. The U.S. Centers for Disease Control (CDC) has classified it as a B bioterrorism pathogen to be strictly guarded against. In order to prevent and control O157 infection, it is of great strategic significance to strengthen the epidemiological study of public health or biological anti-terrorism.
EHEC O157:H7 is evolved from 055:H7. Most virulence genes are acquired in the process of evolution and play a key role in causing human diseases. Most of the force genes are on mobile genetic material and are easy to recombine to produce new pathogenic strains, leading to an outbreak of O157 infection. Therefore, the study of the epidemiology of EHEC O157:H7 at the molecular level and the observation of its molecular variation and evolution can provide early warning signals for the outbreak of O157 infection to respond to possible epidemics. Event.
In this study, the main virulence genes (stx1/2, hlyA, tccP) and conserved housekeeping genes (aspc, icdA, uidA, clpX, mdh, fadD, lysp) of EHEC O157:H7 strain isolated in China were detected by PCR and analyzed by MLST on the basis of microbiology, serology, biochemistry and molecular biology (16S rRNA) detection. The acid sequence was analyzed by phylogenetic and molecular evolutionary methods to study the molecular evolution and epidemic regularity of EHEC O157:H7, and to provide clues and evidence for finding the early warning signals of the outbreak of EHEC O157:H7 infection.
The following works have been completed:
1. The Resource Bank of O157 strains established by Jiangsu Center for Disease Control and Prevention from 1999 to 2007 and 105 strains of EHEC O157:H7 strains collected from the O157 strain bank collected from the main urban areas of Chongqing and the Three Gorges Reservoir Area from 2005 to 2007 were identified from isolation and culture, serological test, biochemical test and 16S rRNA detection.
2. Seven housekeeping genes (aspc, icdA, uidA, clpX, mdh, fadD, lysp) of the 105 strains of EHEC O157:H7 genome were sequenced and seven ST-types were obtained, which were ST836, ST837, ST838, ST839, ST840, ST841 and ST842. ST837, ST840, ST841 belonged to lineage 1, ST836, ST838, ST839 and ST842.
3. The genomic virulence genes (stx1/2, hlyA, tccP) of 105 strains of EHEC O157:H7 were detected and analyzed by PCR. The results showed that Stx2 gene was detected in all strains. The detection rate of Stx2 gene was 100%. Only one strain (99G144) carried stx1 and Stx2 genes simultaneously. The detection rate of stx1 was 0.95%. HlyA was 100% in 105 strains of EHEC 0157:H7. A total of 105 strains of EHEC 0157:H 7 were found to contain three, four and five different repeat fragments of tccp gene, of which 13 (12.4%) were three repeat fragments, 6 (5.7%) were four repeat fragments, 86 (82.0%) were five repeat fragments, and 70% were five heavy strains from animal origin. Repeat the fragment.
4. Seven new ST genotypes were compared with EcMLST database. Minimum evolutionary tree and decomposition tree were used for traceability analysis. ST836 was found to be the source strain of 105 strains, closely related to ST66 from Sakai, and ST66 belonged to the same group as ST69 from EDL933. ST836 and ST69, ST66 were considered to be from the same ancestor. It is speculated that the origin of EHEC O157:H7 epidemic outbreak in China may be transmitted to China by the earliest outbreak of EDL 933 strain from the United States through Japan.
5. The distribution frequency of ST842 from different isolation sites and hosts showed that ST842 was only from human sources, and ST838, ST840 and ST839 were only found in animal-derived strains.
Conclusion:
The above work laid a foundation for the establishment of the gene information database of EHEC O157:H7 strain in China, and provided a useful clue and basis for the study of molecular evolution and epidemiology of EHEC O157:H7 strain in China, and for the search of early warning signals for the outbreak of EHEC O157:H7 infection.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R378
本文编号：2211712
[Abstract]:Enterohemorrhagic Escherichia coli (EHEC) O157:H7, as an important pathogen of zoonosis, has become a global public health problem and poses a great threat to people's health. In addition, EHEC infection has the characteristics of outbreak and epidemic, strong pathogenicity and lethality, and antibiotic treatment can aggravate the risk of the disease. O157 is easy to cultivate, highly infectious, and has a wide range of transmission routes. O157 bacteria are highly likely to be used as bacterial weapons and bioterrorism warfare agents in future military struggles. O157 virulent pathogens may also be used in the construction of genetic weapons. The U.S. Centers for Disease Control (CDC) has classified it as a B bioterrorism pathogen to be strictly guarded against. In order to prevent and control O157 infection, it is of great strategic significance to strengthen the epidemiological study of public health or biological anti-terrorism.
EHEC O157:H7 is evolved from 055:H7. Most virulence genes are acquired in the process of evolution and play a key role in causing human diseases. Most of the force genes are on mobile genetic material and are easy to recombine to produce new pathogenic strains, leading to an outbreak of O157 infection. Therefore, the study of the epidemiology of EHEC O157:H7 at the molecular level and the observation of its molecular variation and evolution can provide early warning signals for the outbreak of O157 infection to respond to possible epidemics. Event.
In this study, the main virulence genes (stx1/2, hlyA, tccP) and conserved housekeeping genes (aspc, icdA, uidA, clpX, mdh, fadD, lysp) of EHEC O157:H7 strain isolated in China were detected by PCR and analyzed by MLST on the basis of microbiology, serology, biochemistry and molecular biology (16S rRNA) detection. The acid sequence was analyzed by phylogenetic and molecular evolutionary methods to study the molecular evolution and epidemic regularity of EHEC O157:H7, and to provide clues and evidence for finding the early warning signals of the outbreak of EHEC O157:H7 infection.
The following works have been completed:
1. The Resource Bank of O157 strains established by Jiangsu Center for Disease Control and Prevention from 1999 to 2007 and 105 strains of EHEC O157:H7 strains collected from the O157 strain bank collected from the main urban areas of Chongqing and the Three Gorges Reservoir Area from 2005 to 2007 were identified from isolation and culture, serological test, biochemical test and 16S rRNA detection.
2. Seven housekeeping genes (aspc, icdA, uidA, clpX, mdh, fadD, lysp) of the 105 strains of EHEC O157:H7 genome were sequenced and seven ST-types were obtained, which were ST836, ST837, ST838, ST839, ST840, ST841 and ST842. ST837, ST840, ST841 belonged to lineage 1, ST836, ST838, ST839 and ST842.
3. The genomic virulence genes (stx1/2, hlyA, tccP) of 105 strains of EHEC O157:H7 were detected and analyzed by PCR. The results showed that Stx2 gene was detected in all strains. The detection rate of Stx2 gene was 100%. Only one strain (99G144) carried stx1 and Stx2 genes simultaneously. The detection rate of stx1 was 0.95%. HlyA was 100% in 105 strains of EHEC 0157:H7. A total of 105 strains of EHEC 0157:H 7 were found to contain three, four and five different repeat fragments of tccp gene, of which 13 (12.4%) were three repeat fragments, 6 (5.7%) were four repeat fragments, 86 (82.0%) were five repeat fragments, and 70% were five heavy strains from animal origin. Repeat the fragment.
4. Seven new ST genotypes were compared with EcMLST database. Minimum evolutionary tree and decomposition tree were used for traceability analysis. ST836 was found to be the source strain of 105 strains, closely related to ST66 from Sakai, and ST66 belonged to the same group as ST69 from EDL933. ST836 and ST69, ST66 were considered to be from the same ancestor. It is speculated that the origin of EHEC O157:H7 epidemic outbreak in China may be transmitted to China by the earliest outbreak of EDL 933 strain from the United States through Japan.
5. The distribution frequency of ST842 from different isolation sites and hosts showed that ST842 was only from human sources, and ST838, ST840 and ST839 were only found in animal-derived strains.
Conclusion:
The above work laid a foundation for the establishment of the gene information database of EHEC O157:H7 strain in China, and provided a useful clue and basis for the study of molecular evolution and epidemiology of EHEC O157:H7 strain in China, and for the search of early warning signals for the outbreak of EHEC O157:H7 infection.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R378
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