几种肠道菌中细胞致死性肿胀毒素的检测及其细胞毒性初步研究

Investigation on Constitution of Cytolethal Distending Toxin Gene in Some Intestinal Bacteria and Cell Toxicity thereof

目的了解肠出血性大肠杆菌、非典型致泻性大肠杆菌、志贺菌及部分肠杆菌中CDT毒素基因存在情况及其细胞毒性。方法用PCR方法检测cdt基因,并将阳性菌株培养上清与HeLa细胞共同孵育检测细胞形态的改变,初步评价CDT毒素的表达及其细胞毒性。结果在肠出血性大肠杆菌O157∶NM中,cdt基因检出率为16.28%(7/43),其中3株为cdt-III型,其他4株为cdt-I型;携带HPI毒力岛的非典型致泻性大肠杆菌中cdt基因检出率为6.25%(5/80);在志贺菌中,cdt基因检出率为2.67%(4/150),非典型致泻性大肠杆菌和志贺菌检出的cdt基因全部为cdt-I型。而在O157∶H7大肠杆菌、枸橼酸杆菌和沙门菌未检出cdt基因。所有cdt基因阳性菌株均使HeLa细胞出现典型的细胞肿胀变化,效应滴度介于1∶2到1∶16之间。结论CDT毒素只在肠出血性大肠杆菌O157∶NM、携带HPI毒力岛的非典型致泻性大肠杆菌和志贺菌中检出,且检出率均比国外同类菌株低,其存在是否与疾病严重程度相关及其细胞毒性机制尚须进一步研究予以证明。

Objective To investigate the constitution of cytolethal distending toxin （CDT） in Escherichia coli strains, Shigella strains and part of Enterobacteriaceae strains, and the cell toxicity thereof. Methods The presence of cdt gene was tested by polymerase chain reaction （PCR）, and preliminary evaluation of the expression of CDT toxin and the toxicity thereof were performed by the observation of changes in cell morphology through co - incubation of supernatant of positive strain culture and Hela cells. Results Cdt gene was found in 7 including 3 cdt - Ⅲ and 4 cdt - Ⅰ strains from 43 （ 16. 28% ） E. coli O157： NM strains, 4 from 150 （2.67%） Shigella reference strains and 5 from 80 （6.25%） E. coli strains harboring high pathogenicity island of Yersinia enterocolitica, but not found in other members of Enterobacteriaceae, such as Salmonella and Citrobacter. All 16 cdt - positive strains produced active CDT which was evidenced by a progressive distension in Hela cells for up to 4 days after exposure to culture filtrates of the cdt - positive strains. The CDT titres ranged from 1 ： 2 to 1 ： 16. Conclusion The data indicate that cdt is present with lower frequency by test only in E. coli O157： NM, Shigella and high pathogenicity island of Yersinia enterocolitica -harboring E. coli. However, further studies are required to evaluate the relevance of the presence of cdt and severity of the disease, as well as the mechanism of CDT cell toxicity.