腸出血性大腸桿菌EHECO157：H7的分子進(jìn)化研究
發(fā)布時(shí)間：2018-08-29 16:07
【摘要】：腸出血性大腸桿菌(EHEC) O157:H7作為一種重要的人畜共患傳染病病原菌,其感染具有暴發(fā)流行趨勢、強(qiáng)烈的致病性與致死性和抗生素治療可加劇病情的危險(xiǎn)性等特點(diǎn),已成為全球性的公共衛(wèi)生問題,對人們的健康構(gòu)成了巨大威脅。此外,EHEC O157菌培養(yǎng)容易、感染力強(qiáng)、傳播途徑多樣,使O157菌極有可能作為未來軍事斗爭的細(xì)菌武器和生物恐怖戰(zhàn)劑;O157菌的烈性致病因子還有可能用于基因武器的構(gòu)建。美國疾病控制中心已將其列為B類生物恐怖病原體嚴(yán)加防范。因此,無論從公共衛(wèi)生還是生物反恐的需要出發(fā),加強(qiáng)其流行病學(xué)研究對預(yù)防和控制O157感染具有重大的戰(zhàn)略意義。 病原體在進(jìn)化的過程中通過自然突變、轉(zhuǎn)導(dǎo)和轉(zhuǎn)化等方式發(fā)生遺傳物質(zhì)的改變,而引起性狀(毒力、耐藥性等)的改變。EHEC O157:H7是由055:H7進(jìn)化而來的,大多數(shù)毒力基因都是在進(jìn)化過程中獲得的,對其引起人類疾病發(fā)揮關(guān)鍵的作用;同時(shí)毒力基因大多數(shù)處于移動(dòng)性遺傳物質(zhì)上,很容易重新組合產(chǎn)生新的致病菌株,而導(dǎo)致O157感染的暴發(fā)流行。因此,從分子水平對EHEC O157:H7的流行病學(xué)進(jìn)行研究,觀察其分子變異和進(jìn)化,可為O157感染的暴發(fā)提供預(yù)警信號以應(yīng)對可能出現(xiàn)的流行事件。 本研究在對我國分離獲得的EHEC O157:H7菌株進(jìn)行微生物學(xué)、血清學(xué)、生化以及分子生物學(xué)(16S rRNA)檢測等全面鑒定的基礎(chǔ)上,采用PCR檢測其主要毒力基因(stx1/2、hlyA、tccP)、MLST測定和分析其保守的管家基因(aspc, icdA, uidA,clpX,mdh, fadD ,lysp )核苷酸序列,通過系統(tǒng)生物學(xué)和分子進(jìn)化分析,以研究EHEC O157:H7分子進(jìn)化和流行規(guī)律,為尋找其感染暴發(fā)流行的預(yù)警信號提供線索和依據(jù)。 主要完成了以下工作: 1.對1999~2007年江蘇省疾病預(yù)防控制中心建立的O157菌株資源庫以及2005~2007年重慶市主城區(qū)和三峽庫區(qū)收集的O157菌株庫中的105株EHEC O157:H7菌株從分離培養(yǎng)、血清學(xué)試驗(yàn)、生化試驗(yàn)、16S rRNA檢測等進(jìn)行了全面鑒定。 2.對上述105株EHEC O157:H7菌株基因組的7個(gè)管家基因(aspc, icdA, uidA,clpX,mdh, fadD ,lysp )的核苷酸序列進(jìn)行了測定,獲得了7個(gè)ST型分別為ST836、ST837、ST838、ST839、ST840、ST841和ST842。其中ST837、ST840、ST841屬于譜系1,ST836、ST838、ST839和ST842屬于譜系2。 3.對上述105株EHEC O157:H7菌株基因組的毒力基因(stx1/2、hlyA、tccP)進(jìn)行了PCR檢測和分析,結(jié)果:所有菌株均檢出了stx2基因,stx2的檢出率為100%,僅1個(gè)菌株(99G144)同時(shí)攜帶stx1和stx2基因, stx1的檢出率為0.95%;HlyA在105株的EHEC 0157:H7中檢出率是100%;在105株EHEC 0157:H7中共檢測出包括3個(gè)、4個(gè)和5個(gè)重復(fù)片段三種不同重復(fù)片段的tccp基因,其中三個(gè)重復(fù)片段的有13株(12.4%),4個(gè)重復(fù)片度的有6株(5.7%),5個(gè)重復(fù)片段的有86株(82.0%),同時(shí),人源的菌株均為5個(gè)重復(fù)片段,動(dòng)物源菌株70%菌株屬于5個(gè)重復(fù)片段。 4.將獲得的7個(gè)新的ST基因型與EcMLST數(shù)據(jù)庫比對,采用最小進(jìn)化樹和分解樹進(jìn)行溯源分析,發(fā)現(xiàn)ST836是這105株的來源菌株,與來源于Sakai的ST66聯(lián)系緊密,而ST66又與源于EDL933的ST69屬于同一群,可以認(rèn)為ST836與ST69、ST66是來源于同一祖先的。初步推測在中國爆發(fā)流行的EHEC O157:H7起源可能是由最早爆發(fā)的美國EDL933株經(jīng)日本傳播到我國的。 5.對不同分離地點(diǎn)和宿主來源菌株的STs分配頻率分析發(fā)現(xiàn):ST842僅來源于人源的,ST838、ST840、ST839這3個(gè)STs型僅見于動(dòng)物源菌株。從菌株的數(shù)量來看,ST837的菌株最多,其次是ST836,兩者占到85%。 結(jié)論: 上述研究工作為建立我國EHEC O157:H7菌株基因信息庫奠定了一定的基礎(chǔ),同時(shí)為研究我國EHEC O157:H7的分子進(jìn)化和流行規(guī)律、尋找其感染暴發(fā)流行的預(yù)警信號提供了有利的線索和依據(jù)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R378
本文編號：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R378
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