中国大肠杆菌O157:H7暴发相关菌株的遗传学和毒力分析
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摘要
肠出血性大肠杆菌(Enterohermorrhagic Escherichia coli, EHEC)0157：H7是出血性肠炎(Hemorrhagic colitis, HC)、血小板减少性紫癜(Thrombotic thrombocytopenia purpura, TTP)和溶血性尿毒综合症(Hemolytic uremic syndrome, HUS)的病原体,1982年被发现以来,世界许多国家和地区都有不同规模的暴发。1999年4-8月间,我国苏皖交界地区发生了大肠杆菌0157：H7感染的暴发,报告急性尿毒综合症患者195人,死亡177人,估计感染20000余人。这是迄今为止世界上发病人数最多、病死人数最多的一次大肠杆菌0157：H7暴发。但中国暴发相关菌株的分析一直未能解释流行病学和临床特点。
我们使用包括15个看家基因的多位点序列分型(Multi-Locus Sequencing Typing, MLST) (http://www.shigatox.net/mlst)方法,可以把124株中国分离菌株分成5个序列型(ST型)：ST23包含101株菌,ST24包含3株菌,ST96包含16株菌,ST97包含3株菌,ST98只有1株菌。其中,ST96-97是新发现的ST型。1999年暴发期间所有从患者分离的5株菌都属于ST96。ST96还包括6株1999年暴发地区从家畜家禽中分离的菌株,1株1992年从山东省患者分离的菌株,4株2001年从云南食品中分离的菌株。有趣的是,2001年以后,我国未能继续分离到ST96型别的菌株。
为了进一步了解我国1999年暴发相关菌株的特点,我们对中国分离的124株菌和13株国外菌株进行了聚类分析。E-burst分析表明,大肠杆菌055序列群以ST26为founder,大肠杆菌0157序列群以ST23 (Sakai菌株)为founder,中国1999年暴发相关菌株Xuzhou21(ST96)与菌株Sakai有两个等位基因的差异,ST97型别的菌株与Sakai有一个等位基因的差异,ST96型别的菌株与ST97型别的菌株有一个等位基因的差异,ST98型别的菌株与Sakai也存在一个等位基因的差异。以数据库中11克隆群中的5个大肠杆菌055：H7菌株作为外群,用最大简约法(MP)构建系统发生树,新发现的3个ST型(ST96-98)与其余5个ST型别ST23, ST24, ST25, ST84,ST101形成了一个克隆群。研究发现,和1982年美国暴发相关菌株EDL933、1996年日本暴发相关菌株Sakai、以及2006年美国暴发相关菌株TW14359不同,1999年中国暴发相关菌株Xuzhou21和1996年日本暴发相关菌株遗传学关系较近,和1982年、2006年美国暴发相关菌株EDL933和TW14359遗传学关系较远。使用基因组序列构建的系统发生树和使用15个管家基因序列构建的系统发生树的结果基本一致。结果提示,在世界上引起2万人规模暴发的大肠杆菌0157：H7菌株Xuzhou21和Sakai在遗传学上密切相关。
那么,在遗传学上密切相关的暴发相关菌株Sakai和Xuzhou21是否在表现型上具有和其他菌株不同的特点呢?为此,我们对实验室保存的暴发相关菌株ELD933、Sakai和Xuzhou21进行了生物学特性的比较研究,包括酸抗性、产生志贺毒素2和刺激外周血单核细胞产生细胞因子的能力。我们发现,将菌株在pH3.0的酸性溶液中作用2小时后,Xuzhou21和Sakai的存活率分别为45.7%,46.7%,EDL933为23.6%。表明Xuzhou21和Sakai对酸的抗性相似。对酸抗性相关基因rpoS的测序分析发现,Sakai的rpoS序列与已发表的两株耐受性较强菌株AF182102和AF182107的rpoS序列完全相同。与Sakai比较,菌株EDL933有一个点突变(G917T),导致编码区内终止子的提前出现。菌株Xuzhou21有一个点突变(A904G),天冬氨酸变成了甘氨酸。对志贺毒素Stx2的表达水平研究发现,在未加入抗生素诱导的情况下,Sakai和Xuzhou21菌株的Stx2的mRNA表达水平分别是EDL933菌株的3.7倍和7.5倍；而加入丝裂霉素C(mitomycinC)诱导3小时后,Sakai、Xuzhou21菌株的Stx2的mRNA表达水平,分别是EDL933的32.7和68.6倍。将细菌灭活,与PBMC细胞共同孵育4小时后,发现EDL933、Sakai和刺激产生IL-1β的含量分别为207.45±11.08、234.84±±7.87、315.34±23.71pg/ml,刺激产生IL-6的含量分别为317.38±10.25、545.06±±33.69、565.31±22.09 pg/ml,产生IL-8的含量分别为2601.12±111.08、3568.51±123.97、4837.67±333.49 pg/ml。与EDL933相比,Xuzhou21和Sakai能够刺激PBMC产生较高水平的IL-1β、IL-6、IL-8,差别有统计学意义。
简言之,我们的研究发现,在1996年日本和1999年苏皖引起万人规模的大肠杆菌0157：H7暴发的菌株Sakai和Xuzhou21在遗传学上密切相关,可分为一个克隆群,它们也具有较高的酸抗性,产生高水平的志贺毒素2,能够刺激PBMC产生高水平的细胞因子IL-1B、IL-6、IL-8等。这些特性可能和它们能够引起大暴发有关。
Enterohermorrhagic Escherichia coli O157:H7 can cause hemorrhagic colitis (HC), thrombotic thrombocytopenia purpura (TTP) and life-threatening complications, hemolytic uremic syndrome (HUS). EHEC O157:H7 strains were first identified in 1982 and have since been rapidly becoming popular all over the world. During the period from April to August 1999, a massive outbreak of E. coli O157:H7 occurred in Jiangsu and Anhui Province of China, with 195 hospitalized patients who were clinically diagnosed as hemolytic-uremic syndrome (HUS) and 177 deaths. The sero-epidemiological study suggested that there were about 20,000 persons infected during the outbreak. This is the largest outbreak of human infections of E.coli on the world, resulted in 195 HUS patients and 177 deaths. However, the genetic charactics of the outbreak associated isolates have not been well understood.
By using the 15-locus multilocus sequence typing (MLST) scheme (http://www.shigatox.net/mlst), we analyzed 124 strains of E.coli O157:H7 isolated in China in various years and from various geographic locations. The 15 locus MLST scheme differentiated the 124 isolates into five sequence types (STs).101 of 124 strains were typed as ST23, which included 1996 Japan outbreak associated strain Sakai. All of the 5 strains isolated fron patients during 1999 China outbreak were typed as ST96, a novel sequence type has not be reported. The ST96 has 16 strains, including 5 isolated from animals during 1999 outbreak, one isolated from patients in Shandong province in 1992 and 4 from food samples in Yunnan province in 2001. Interestingly, no strain of ST96 has been isolated after 2001 in China.
Based on the sequences of 15 locus of MLST scheme, we found that 1999 China outbreak associated strain Xuzhou 21 was distantly related with1996 Japan outbreak associated strain Sakai,1992 and 2006 American outbreak associated strain EDL933 and TW14359. When whole genemo sequences were used to study the genetic ralatideness of those outbreak strains, we found that 1999 China outbreak associated strain Xuzhou21 was closed related with 1996 Japan outbreak associated strain Sakai. Since both of Sakai and Xuzhou 21 caused two largest outbreaks of human infections, their potential charactritics for causing big outbreak were further studied.
When the organisms were exposed to acidic conditions at pH 3.0 for 2 hours, we found that the survival rate for EDL933 was 23.6%, significantly lower than 46.7% and 45.7% for Sakai and Xuzhou21 respectively. The difference has statistical significance. We found that the rpoS sequence of Sakai were identical to those of two co-called high acid resistant strains AF182102 and AF182107, which was associated with the high acid resistance. And, the rpoS sequence of EDL933 had a change at nucleotide 917, resulting in an early stop codon.
We then measured the level of transcriptional of Shiga toxin 2 (stx2) gene in strains of Xuzhou21, EDL933 and Sakai using real-time quantitative PCR, which was demonstrated to be associated with development of HUS. The transcript levels of Sakai and Xuzhou21 were 3.7 and 7.5 times higher than that of EDL933. After induction of mitomycin C, the expressed level of stx2 in EDL933 was increased 4.5 times, but it was increased as high as 32.7,68.6 times in Sakai and Xuzhou21 respectively. When co-incubated with human peripheral blood mononuclear cells (PBMCs), it was found that the level of cytokines,IL-6 and IL-8 stimulated by strain EDL933、Sakai and Xuzhou21 were 207.45±11.08,234.84±7.87 and 315.34±23.71pg/ml for IL-1β,317.38±10.25,545.06±33.69 and 565.31±22.09 pg/ml for IL-6; and 2601.12±11.08、3568.51±123.97、4837.67±333.49 pg/ml for IL-8. In general, the level of expression of those 3 cytokines induced by Xuzhou21 is similar to that induced by Sakai, but is singnificantly higher than that induced by EDL933.
In short, we found that the strains of E.coli O157:H7 associated with 1999 China outbreak belonged to a novel sequence type 96. Genome sequence analyziz indicated that the 1999 China outbreak associated strain Xuzhou21 was phylogenetically related with 1996 Japan outbreak associated strain Sakai, but distant from 1982 and 2006 American outbreak associated strain EDL933 and TW14359. And furthomore, we found that the two large outbreak associated strains Xuzhou21 and Sakai are more acid resistant, producing higher amount of Shiga toxin 2 and stimulated host cells to producing higher level of cytokines, namely IL-1 β, IL-6 and IL-8. Therefore, we proposed Sakai and Xuzhou 21 as the strains with potential to cause large outbreak.
我们使用包括15个看家基因的多位点序列分型(Multi-Locus Sequencing Typing, MLST) (http://www.shigatox.net/mlst)方法,可以把124株中国分离菌株分成5个序列型(ST型)：ST23包含101株菌,ST24包含3株菌,ST96包含16株菌,ST97包含3株菌,ST98只有1株菌。其中,ST96-97是新发现的ST型。1999年暴发期间所有从患者分离的5株菌都属于ST96。ST96还包括6株1999年暴发地区从家畜家禽中分离的菌株,1株1992年从山东省患者分离的菌株,4株2001年从云南食品中分离的菌株。有趣的是,2001年以后,我国未能继续分离到ST96型别的菌株。
为了进一步了解我国1999年暴发相关菌株的特点,我们对中国分离的124株菌和13株国外菌株进行了聚类分析。E-burst分析表明,大肠杆菌055序列群以ST26为founder,大肠杆菌0157序列群以ST23 (Sakai菌株)为founder,中国1999年暴发相关菌株Xuzhou21(ST96)与菌株Sakai有两个等位基因的差异,ST97型别的菌株与Sakai有一个等位基因的差异,ST96型别的菌株与ST97型别的菌株有一个等位基因的差异,ST98型别的菌株与Sakai也存在一个等位基因的差异。以数据库中11克隆群中的5个大肠杆菌055：H7菌株作为外群,用最大简约法(MP)构建系统发生树,新发现的3个ST型(ST96-98)与其余5个ST型别ST23, ST24, ST25, ST84,ST101形成了一个克隆群。研究发现,和1982年美国暴发相关菌株EDL933、1996年日本暴发相关菌株Sakai、以及2006年美国暴发相关菌株TW14359不同,1999年中国暴发相关菌株Xuzhou21和1996年日本暴发相关菌株遗传学关系较近,和1982年、2006年美国暴发相关菌株EDL933和TW14359遗传学关系较远。使用基因组序列构建的系统发生树和使用15个管家基因序列构建的系统发生树的结果基本一致。结果提示,在世界上引起2万人规模暴发的大肠杆菌0157：H7菌株Xuzhou21和Sakai在遗传学上密切相关。
那么,在遗传学上密切相关的暴发相关菌株Sakai和Xuzhou21是否在表现型上具有和其他菌株不同的特点呢?为此,我们对实验室保存的暴发相关菌株ELD933、Sakai和Xuzhou21进行了生物学特性的比较研究,包括酸抗性、产生志贺毒素2和刺激外周血单核细胞产生细胞因子的能力。我们发现,将菌株在pH3.0的酸性溶液中作用2小时后,Xuzhou21和Sakai的存活率分别为45.7%,46.7%,EDL933为23.6%。表明Xuzhou21和Sakai对酸的抗性相似。对酸抗性相关基因rpoS的测序分析发现,Sakai的rpoS序列与已发表的两株耐受性较强菌株AF182102和AF182107的rpoS序列完全相同。与Sakai比较,菌株EDL933有一个点突变(G917T),导致编码区内终止子的提前出现。菌株Xuzhou21有一个点突变(A904G),天冬氨酸变成了甘氨酸。对志贺毒素Stx2的表达水平研究发现,在未加入抗生素诱导的情况下,Sakai和Xuzhou21菌株的Stx2的mRNA表达水平分别是EDL933菌株的3.7倍和7.5倍；而加入丝裂霉素C(mitomycinC)诱导3小时后,Sakai、Xuzhou21菌株的Stx2的mRNA表达水平,分别是EDL933的32.7和68.6倍。将细菌灭活,与PBMC细胞共同孵育4小时后,发现EDL933、Sakai和刺激产生IL-1β的含量分别为207.45±11.08、234.84±±7.87、315.34±23.71pg/ml,刺激产生IL-6的含量分别为317.38±10.25、545.06±±33.69、565.31±22.09 pg/ml,产生IL-8的含量分别为2601.12±111.08、3568.51±123.97、4837.67±333.49 pg/ml。与EDL933相比,Xuzhou21和Sakai能够刺激PBMC产生较高水平的IL-1β、IL-6、IL-8,差别有统计学意义。
简言之,我们的研究发现,在1996年日本和1999年苏皖引起万人规模的大肠杆菌0157：H7暴发的菌株Sakai和Xuzhou21在遗传学上密切相关,可分为一个克隆群,它们也具有较高的酸抗性,产生高水平的志贺毒素2,能够刺激PBMC产生高水平的细胞因子IL-1B、IL-6、IL-8等。这些特性可能和它们能够引起大暴发有关。
Enterohermorrhagic Escherichia coli O157:H7 can cause hemorrhagic colitis (HC), thrombotic thrombocytopenia purpura (TTP) and life-threatening complications, hemolytic uremic syndrome (HUS). EHEC O157:H7 strains were first identified in 1982 and have since been rapidly becoming popular all over the world. During the period from April to August 1999, a massive outbreak of E. coli O157:H7 occurred in Jiangsu and Anhui Province of China, with 195 hospitalized patients who were clinically diagnosed as hemolytic-uremic syndrome (HUS) and 177 deaths. The sero-epidemiological study suggested that there were about 20,000 persons infected during the outbreak. This is the largest outbreak of human infections of E.coli on the world, resulted in 195 HUS patients and 177 deaths. However, the genetic charactics of the outbreak associated isolates have not been well understood.
By using the 15-locus multilocus sequence typing (MLST) scheme (http://www.shigatox.net/mlst), we analyzed 124 strains of E.coli O157:H7 isolated in China in various years and from various geographic locations. The 15 locus MLST scheme differentiated the 124 isolates into five sequence types (STs).101 of 124 strains were typed as ST23, which included 1996 Japan outbreak associated strain Sakai. All of the 5 strains isolated fron patients during 1999 China outbreak were typed as ST96, a novel sequence type has not be reported. The ST96 has 16 strains, including 5 isolated from animals during 1999 outbreak, one isolated from patients in Shandong province in 1992 and 4 from food samples in Yunnan province in 2001. Interestingly, no strain of ST96 has been isolated after 2001 in China.
Based on the sequences of 15 locus of MLST scheme, we found that 1999 China outbreak associated strain Xuzhou 21 was distantly related with1996 Japan outbreak associated strain Sakai,1992 and 2006 American outbreak associated strain EDL933 and TW14359. When whole genemo sequences were used to study the genetic ralatideness of those outbreak strains, we found that 1999 China outbreak associated strain Xuzhou21 was closed related with 1996 Japan outbreak associated strain Sakai. Since both of Sakai and Xuzhou 21 caused two largest outbreaks of human infections, their potential charactritics for causing big outbreak were further studied.
When the organisms were exposed to acidic conditions at pH 3.0 for 2 hours, we found that the survival rate for EDL933 was 23.6%, significantly lower than 46.7% and 45.7% for Sakai and Xuzhou21 respectively. The difference has statistical significance. We found that the rpoS sequence of Sakai were identical to those of two co-called high acid resistant strains AF182102 and AF182107, which was associated with the high acid resistance. And, the rpoS sequence of EDL933 had a change at nucleotide 917, resulting in an early stop codon.
We then measured the level of transcriptional of Shiga toxin 2 (stx2) gene in strains of Xuzhou21, EDL933 and Sakai using real-time quantitative PCR, which was demonstrated to be associated with development of HUS. The transcript levels of Sakai and Xuzhou21 were 3.7 and 7.5 times higher than that of EDL933. After induction of mitomycin C, the expressed level of stx2 in EDL933 was increased 4.5 times, but it was increased as high as 32.7,68.6 times in Sakai and Xuzhou21 respectively. When co-incubated with human peripheral blood mononuclear cells (PBMCs), it was found that the level of cytokines,IL-6 and IL-8 stimulated by strain EDL933、Sakai and Xuzhou21 were 207.45±11.08,234.84±7.87 and 315.34±23.71pg/ml for IL-1β,317.38±10.25,545.06±33.69 and 565.31±22.09 pg/ml for IL-6; and 2601.12±11.08、3568.51±123.97、4837.67±333.49 pg/ml for IL-8. In general, the level of expression of those 3 cytokines induced by Xuzhou21 is similar to that induced by Sakai, but is singnificantly higher than that induced by EDL933.
In short, we found that the strains of E.coli O157:H7 associated with 1999 China outbreak belonged to a novel sequence type 96. Genome sequence analyziz indicated that the 1999 China outbreak associated strain Xuzhou21 was phylogenetically related with 1996 Japan outbreak associated strain Sakai, but distant from 1982 and 2006 American outbreak associated strain EDL933 and TW14359. And furthomore, we found that the two large outbreak associated strains Xuzhou21 and Sakai are more acid resistant, producing higher amount of Shiga toxin 2 and stimulated host cells to producing higher level of cytokines, namely IL-1 β, IL-6 and IL-8. Therefore, we proposed Sakai and Xuzhou 21 as the strains with potential to cause large outbreak.
引文
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