天津地区志贺菌肠毒素基因分布及其PFGE分型的研究
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摘要
目的：
研究天津地区2005～2007年分离的62株志贺菌,对所分离菌株进行生化鉴定、血清学分型和药敏试验,检测肠毒素等毒力基因在志贺菌中的分布,并采用脉冲场凝胶电泳技术对分离的志贺菌进行分子分型,从而为本地区细菌性痢疾的防控工作提供重要依据。
方法：
1.经全自动微生物生化鉴定仪对分离的62株菌株进行生化鉴定和药敏试验,采用玻片凝集试验进行血清学分型。
2.采用PCR方法检测受试菌株中侵袭性质粒基因ipaH以及肠毒素基因set1A、set1B和sen。
3.选用XbaI限制性核酸内切酶对62株志贺菌及标准株沙门菌Braenderup型H9812株染色体酶切后进行脉冲场凝胶电泳分子分型,应用Bionumerics软件处理PFGE结果且绘制出聚类分析图。
结果：
1.经生化鉴定所分离菌株全部为志贺菌,其中福氏志贺菌36株,占58.06%,包括8种亚型,分别为福氏1a,福氏1b,福氏2a,福氏2b,福氏3a,福氏4a,福氏6以及福氏X变种,占全部菌株比例依次为6.45%,6.45%,8.06%,17.74%,6.45%,8.06%,1.61%和3.23%。以福氏2b亚型为优势血清型,占福氏志贺菌株的30,56%：宋内氏志贺菌26株,占41.94%。分离志贺菌均为多重耐药株,绝大多数菌株对氨苄西林,复方新诺明和庆大霉素耐药,但对喹喏酮类药物较为敏感。
2.根据文献设计合成引物,采用PCR方法扩增侵袭性质粒基因ipaH、以及肠毒素基因set1A、set1B和sen,其预期片段大小分别为309bp、147bp、799bp和423bp。62株志贺菌四种毒力基因PCR检出率为ipaH 95.16%(59/62株),set1A 40.32%(25/62株),set1B 37.10%(23/62株),sen 50%(31/62株)。肠毒素基因set1A、set1B、sen分别在福氏2b、福氏2a以及福氏4a等主要血清型别志贺菌中的检出率相对较高,均可达到50%以上,其中在福氏2b中检出率分别为90.91%、63.64%和90.91%,在福氏2a中检出率分别为100%、80%和60%,在福氏4a中检出率分别为100%、50%和100%；而在宋内氏志贺菌中未检出肠毒素基因set1A、set1B, sen的检出率仅为11.54%。经χ2检验处理表明肠毒素基因set1A、set1B和sen在福氏志贺菌与宋内氏志贺菌中的分布差别具有统计学意义(P<0.005)。31株sen基因阳性菌株的ipaH基因也均为阳性。36株福氏志贺菌肠毒素毒力基因显示了6种不同的基因组合,尤以携带3种和2种不同基因的组合为主,占所有被检福氏志贺菌的80.56%,其中set1A、set1B、sen三者并存的菌株为优势组合,占全部菌株的41.67%。
3.62株志贺菌株的脉冲场凝胶电泳结果经Bionumerics软件分析获得14种不同的型别。其中宋内氏志贺菌带谱为4种(SⅠ～SⅣ),福氏志贺菌带谱为10种(FⅠ～FⅩ)。宋内氏志贺菌中SⅠ,型有23株,又可分为两种亚型,其余3种型别各1株。福氏志贺菌中FⅠ、FⅡ、FⅢ、FⅣ和FⅩ型各1株；FⅤ、FⅥ和FⅨ型各2株；FⅧ型共4株；FⅦ型21株,又可以分为6种主要亚型。
结论：
1.2005～2007年天津地区流行的志贺菌主要为福氏志贺菌和宋内氏志贺菌,其中福氏2b为福氏志贺菌中的优势血清型。值得注意的是宋内氏志贺菌呈上升趋势。分离志贺菌均为多重耐药菌株,但对喹喏酮类药物敏感,可作为治疗用药参考。
2.志贺菌肠毒素基因set1A、set1B和sen在不同血清型的志贺菌中分布有明显差异。set1A和set1B存在于福氏志贺菌多种血清型中,以福氏2b、福氏2a和福氏4a检出率较高。sen广泛分布于各型福氏志贺菌中,在宋内氏志贺菌亦有少量分布。志贺菌肠毒素基因的多种组合提示肠毒素毒力因子之间可能存在一定的协同作用。
3.天津地区流行的宋内氏志贺菌可能源于亲缘关系很近的克隆系(SⅠ基因型),福氏志贺菌PFGE分子分型呈现出明显的多态性,源自多个克隆系,同一种血清型菌株的进化来源则比较简单。
Objective
To study Shigella strains isolated in Tianjin from the year 2005 to 2007. The strains are identified by biochemical method,serotyping and susceptibility test. Shigella enterotoxin genes are detected by PCR to know its distribution and these Shigella strains are typed molecularly by pulsed field gel electrophoresis.
Methods
1. All the strains are identified by biochemical method, serotyping and susceptibility test;
2. Virulence genes such as Shigella enterotoxin genes set1A、set1B、sen and ipaH in Shigella are detected by PCR to know the distribution;
3. The Shigella strains are typed molecularly by pulsed field gel electrophoresis. Restriction enzyme XbaI is used for the molecular typing. PFGE patterns were analyzed by Bionumerics (Version 6.0) software to perform cluster analysis.
Results
1. Shigella flexneri were 36 strains and divided into 8 subtype:F1a, F1b, F2a, F2b, F3a, F4a, F6, Fx; they were accounted for 6.45%,6.45%,8.06%,17.74%, 6.45%,8.06%,1.61%,3.23% and 41.94% in order. F2b was the dominance serum group and accounted for 30.56% of Shigella flexneri. Shigella Sonnei were 26 strains without subtype and accounted for 41.94% of all Shigella strains. Shigella inTianjin was multidrug resistant strains.It was resistant to penicillins, TMP-SMZ, gentamicin and still sensitive to quinolones.
2. The positive rate of ipaH、set1A、set1B and sen were 95.16%(59/62),40.32% (25/62),37.10%(23/62) & 50%(31/62) in order. The average positive rate of enterotoxin genes were 50% above and most of the genes were presented in F2b、F2a and F4a strains. set1A and set1B were not detected in Shigella Sonnei. The positive rate of sen were only 11.54%.There were large difference between distribution of enterotoxin genes of Shigella flexneri and Shigella Sonnei byχ2-test.There were 6 gene groups of virulence factors, accounted for 80.56%. The groups including all enterotoxins were the the dominance combination, accounted for 41.67%.
3. All the strains were divided into 14 genotypes. There were 4 genotypes for Shigella Sonnei; they were SⅠ-SⅣ.The other 10 genotypes belonged to Shigella flexneri; they were FⅠ-FⅩ.
Conclusion
1. Serum agglutination test showed that the strains mainly were Shigella Sonnei and Shigella flexneri in Tianjin area from the year 2005 to 2007. F2b was the dominance serum group of Shigella flexneri. Shigella Sonnei grew up every year. Shigella sonnei should be given more monitoring and control. Shigella was multidrug resistant strains.It was sensitive to quinolones and we can use it.
2. There were large differences on distribution of enterotoxins in different strains. setlA and set1B presented in diverse serum groups of Shigella flexneri, especially in F2b、F2a and F4a strains. sen were presented in all Shigella strains. Combination of the virulence genes referred to the association among the virulence factors.
3. Shigella Sonnei strains isolated in Tianjin had very close relationship; S1 was the dominance strain in Tianjin area from 2005 to 2007. Shigella flexneri came from different clones.
研究天津地区2005～2007年分离的62株志贺菌,对所分离菌株进行生化鉴定、血清学分型和药敏试验,检测肠毒素等毒力基因在志贺菌中的分布,并采用脉冲场凝胶电泳技术对分离的志贺菌进行分子分型,从而为本地区细菌性痢疾的防控工作提供重要依据。
方法：
1.经全自动微生物生化鉴定仪对分离的62株菌株进行生化鉴定和药敏试验,采用玻片凝集试验进行血清学分型。
2.采用PCR方法检测受试菌株中侵袭性质粒基因ipaH以及肠毒素基因set1A、set1B和sen。
3.选用XbaI限制性核酸内切酶对62株志贺菌及标准株沙门菌Braenderup型H9812株染色体酶切后进行脉冲场凝胶电泳分子分型,应用Bionumerics软件处理PFGE结果且绘制出聚类分析图。
结果：
1.经生化鉴定所分离菌株全部为志贺菌,其中福氏志贺菌36株,占58.06%,包括8种亚型,分别为福氏1a,福氏1b,福氏2a,福氏2b,福氏3a,福氏4a,福氏6以及福氏X变种,占全部菌株比例依次为6.45%,6.45%,8.06%,17.74%,6.45%,8.06%,1.61%和3.23%。以福氏2b亚型为优势血清型,占福氏志贺菌株的30,56%：宋内氏志贺菌26株,占41.94%。分离志贺菌均为多重耐药株,绝大多数菌株对氨苄西林,复方新诺明和庆大霉素耐药,但对喹喏酮类药物较为敏感。
2.根据文献设计合成引物,采用PCR方法扩增侵袭性质粒基因ipaH、以及肠毒素基因set1A、set1B和sen,其预期片段大小分别为309bp、147bp、799bp和423bp。62株志贺菌四种毒力基因PCR检出率为ipaH 95.16%(59/62株),set1A 40.32%(25/62株),set1B 37.10%(23/62株),sen 50%(31/62株)。肠毒素基因set1A、set1B、sen分别在福氏2b、福氏2a以及福氏4a等主要血清型别志贺菌中的检出率相对较高,均可达到50%以上,其中在福氏2b中检出率分别为90.91%、63.64%和90.91%,在福氏2a中检出率分别为100%、80%和60%,在福氏4a中检出率分别为100%、50%和100%；而在宋内氏志贺菌中未检出肠毒素基因set1A、set1B, sen的检出率仅为11.54%。经χ2检验处理表明肠毒素基因set1A、set1B和sen在福氏志贺菌与宋内氏志贺菌中的分布差别具有统计学意义(P<0.005)。31株sen基因阳性菌株的ipaH基因也均为阳性。36株福氏志贺菌肠毒素毒力基因显示了6种不同的基因组合,尤以携带3种和2种不同基因的组合为主,占所有被检福氏志贺菌的80.56%,其中set1A、set1B、sen三者并存的菌株为优势组合,占全部菌株的41.67%。
3.62株志贺菌株的脉冲场凝胶电泳结果经Bionumerics软件分析获得14种不同的型别。其中宋内氏志贺菌带谱为4种(SⅠ～SⅣ),福氏志贺菌带谱为10种(FⅠ～FⅩ)。宋内氏志贺菌中SⅠ,型有23株,又可分为两种亚型,其余3种型别各1株。福氏志贺菌中FⅠ、FⅡ、FⅢ、FⅣ和FⅩ型各1株；FⅤ、FⅥ和FⅨ型各2株；FⅧ型共4株；FⅦ型21株,又可以分为6种主要亚型。
结论：
1.2005～2007年天津地区流行的志贺菌主要为福氏志贺菌和宋内氏志贺菌,其中福氏2b为福氏志贺菌中的优势血清型。值得注意的是宋内氏志贺菌呈上升趋势。分离志贺菌均为多重耐药菌株,但对喹喏酮类药物敏感,可作为治疗用药参考。
2.志贺菌肠毒素基因set1A、set1B和sen在不同血清型的志贺菌中分布有明显差异。set1A和set1B存在于福氏志贺菌多种血清型中,以福氏2b、福氏2a和福氏4a检出率较高。sen广泛分布于各型福氏志贺菌中,在宋内氏志贺菌亦有少量分布。志贺菌肠毒素基因的多种组合提示肠毒素毒力因子之间可能存在一定的协同作用。
3.天津地区流行的宋内氏志贺菌可能源于亲缘关系很近的克隆系(SⅠ基因型),福氏志贺菌PFGE分子分型呈现出明显的多态性,源自多个克隆系,同一种血清型菌株的进化来源则比较简单。
Objective
To study Shigella strains isolated in Tianjin from the year 2005 to 2007. The strains are identified by biochemical method,serotyping and susceptibility test. Shigella enterotoxin genes are detected by PCR to know its distribution and these Shigella strains are typed molecularly by pulsed field gel electrophoresis.
Methods
1. All the strains are identified by biochemical method, serotyping and susceptibility test;
2. Virulence genes such as Shigella enterotoxin genes set1A、set1B、sen and ipaH in Shigella are detected by PCR to know the distribution;
3. The Shigella strains are typed molecularly by pulsed field gel electrophoresis. Restriction enzyme XbaI is used for the molecular typing. PFGE patterns were analyzed by Bionumerics (Version 6.0) software to perform cluster analysis.
Results
1. Shigella flexneri were 36 strains and divided into 8 subtype:F1a, F1b, F2a, F2b, F3a, F4a, F6, Fx; they were accounted for 6.45%,6.45%,8.06%,17.74%, 6.45%,8.06%,1.61%,3.23% and 41.94% in order. F2b was the dominance serum group and accounted for 30.56% of Shigella flexneri. Shigella Sonnei were 26 strains without subtype and accounted for 41.94% of all Shigella strains. Shigella inTianjin was multidrug resistant strains.It was resistant to penicillins, TMP-SMZ, gentamicin and still sensitive to quinolones.
2. The positive rate of ipaH、set1A、set1B and sen were 95.16%(59/62),40.32% (25/62),37.10%(23/62) & 50%(31/62) in order. The average positive rate of enterotoxin genes were 50% above and most of the genes were presented in F2b、F2a and F4a strains. set1A and set1B were not detected in Shigella Sonnei. The positive rate of sen were only 11.54%.There were large difference between distribution of enterotoxin genes of Shigella flexneri and Shigella Sonnei byχ2-test.There were 6 gene groups of virulence factors, accounted for 80.56%. The groups including all enterotoxins were the the dominance combination, accounted for 41.67%.
3. All the strains were divided into 14 genotypes. There were 4 genotypes for Shigella Sonnei; they were SⅠ-SⅣ.The other 10 genotypes belonged to Shigella flexneri; they were FⅠ-FⅩ.
Conclusion
1. Serum agglutination test showed that the strains mainly were Shigella Sonnei and Shigella flexneri in Tianjin area from the year 2005 to 2007. F2b was the dominance serum group of Shigella flexneri. Shigella Sonnei grew up every year. Shigella sonnei should be given more monitoring and control. Shigella was multidrug resistant strains.It was sensitive to quinolones and we can use it.
2. There were large differences on distribution of enterotoxins in different strains. setlA and set1B presented in diverse serum groups of Shigella flexneri, especially in F2b、F2a and F4a strains. sen were presented in all Shigella strains. Combination of the virulence genes referred to the association among the virulence factors.
3. Shigella Sonnei strains isolated in Tianjin had very close relationship; S1 was the dominance strain in Tianjin area from 2005 to 2007. Shigella flexneri came from different clones.
引文
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