宁波沿海陆源排污口细菌结构多样性和铁代谢相关的毒力基因的研究

RESEARCH OF THE STRUCTURE DIVERSITY OF BACTERIA FORM NINGBO COASTAL OUTFALL AND VIRULENCE GENES ASSOCIATED WITH IRON METABOLISM

应用多重PCR技术对肠杆菌铁代谢相关毒力基因fhuA、irp2、sltA和sodB进行同步检测,研究宁波沿海陆源排污口人畜共患致病菌群落结构在人类活动影响下的分布差异及对环境因子变化的响应趋势。通过16S rRNA基因的PCR扩增和限制性片段长度多态性(restriction fragment length polymorphism,RFLP)分析技术,研究了东海重要陆源排污口的细菌群落多样性;建立肠杆菌铁载体外膜受体蛋白fhuA、铁调节蛋白irp2、类志贺样毒素sltA、含铁超氧物歧化酶sodB基因多重PCR体系,并用该体系检测铁代谢相关毒力基因在陆源排污口的分布情况。2011年3、5、8、10月份,在宁波沿海陆源排污口中共分离纯化1000余株、98种细菌,16S rRNA基因序列分析表明,它们分属于4个门5个纲13个科21个属,其中?-变形杆菌纲(67株,68.4%)和芽孢杆菌纲(16株,16.3%)分别为变形菌门、厚壁菌门中主要纲。98株细菌中,fhuA+irp2+sodB+基因型检出率为35.71%;fhuA+irp2+sltA+的基因型检出率为25.27%;irp2的基因型检出率为13.19%;fhuA基因型检出率为12.09%;fhuA+irp2+基因型检出率为9.89%;sltA+sodB+基因型检出率为8.79%;fhuA+irp2+sodB+sltA+基因型检出率为8.79%。

To analysis the trend of the zoonotic pathogens structure diversity from Ningbo coastal outfall related to the human activates and environmental factors, multiple PCR was used to detect the virulence genes associated with the iron metabolism such as fhuA, irp2, sltAand sodBwhich are in Enterobacter. The structure diversity of bacteria form Ningbo coastal outfall was analyzed through PCR amplification of 16S rRNA and restriction fragment length polymorphism （RFLP） analysis technology; the multiple PCR system on the Enterobacterrail carrier outer membrane receptor protein fhuA, iron regulatory protein irp2, Shiga-like toxin sltA, super oxide dismutase content with iron sodBwas established; then the metabolism related virulence genes in the distribution of Ningbo coastal outfall was detected by using this system. More than 1000 strains, 98 kinds of bacteria were isolated and purified from Ningbo coastal outfall in March, May, October and December in 2011, the sequence analysis of 16S rRNA showed that, these points belong to 4 doors 5 classes 13 families and 21 species respectively, among them, gamma proteus classes （67 strains, 68.4%） and bacillus classes （16 strains, 16.3%） were the major classes in Proteobacteria and Firmicutes. Among the 98 strains of bacteria, the detection rate of fhuA＋irp2＋sodBgenotype was 35.71%, fhuA＋irp2＋sltA＋ was 25.27%, irp2was 13.19%, fhuAwas 12.09%, fhuA＋irp2＋ was 9.89%, sltA＋sodB＋ was 8.79%, fhuA＋irp2＋sodB＋sltA＋ was 8.79%.