大肠杆菌肠菌素对空肠弯曲菌的促生长作用研究

Effects of Escherichia coli Enterobactin on Growth Promotion of Campylobacter jejuni

[目的]调查鸡肠道内大肠杆菌分泌肠菌素的水平,以及其对空肠弯曲菌的促生长作用。[方法]利用间接竞争ELISA(ic-ELISA)方法检测临床分离的30株鸡肠道大肠杆菌分离株的肠菌素产量;利用λRed同源重组系统构建大肠杆菌entB基因缺失突变株,并用ic-ELISA方法测定大肠杆菌亲本株和突变株的肠菌素产量;检测亲本株和突变株在不同限铁培养基中的生长曲线;将大肠杆菌亲本株和突变株培养上清接种空肠弯曲菌,探究大肠杆菌肠菌素对空肠弯曲菌生长的影响。[结果]30株鸡肠道大肠杆菌分离株分泌肠菌素水平介于11~126μmol/L之间。利用λRed同源重组系统成功构建2株大肠杆菌entB基因缺失突变株。定性结果显示,大肠杆菌亲本株在CAS培养基中产生黄色晕圈,而entB基因缺失株不产生晕圈。ic-ELISA检测结果显示,亲本株分泌肠菌素浓度为100~150μmol/L,而基因缺失株未检测出肠菌素。大肠杆菌亲本株和entB基因缺失株在富铁培养基中生长无显著差异(P>0.05),在限铁培养基中达到平台期的D 600 nm值分别为0.6和0.3。促生长试验结果显示,大肠杆菌亲本株培养上清接种空肠弯曲菌24和36 h,菌落数分别为7.8和8.2 lg CFU/mL,而entB基因缺失株培养上清接种后菌落数分别为7.0和7.3 lg CFU/mL。[结论]大肠杆菌具有分泌肠菌素的能力,且其在限铁环境下能促进空肠弯曲菌的生长,试验结果为深入研究肠菌素介导的肠道细菌之间的相互作用奠定基础。

[Objective]The purpose of this experiment was to investigate the levels of enterobactin secreted by Escherichia coli isolated from the chicken intestines and the effects of enterobactin on Campylobacter jejuni growth promotion.[Method]The indirect competitive ELISA(ic-ELISA)method was used to detect the production of enterobactin in 30 chicken enteric Escherichia coli isolates.TheλRed homologous recombination system was used to construct the entB gene deletion mutant strain of Escherichia coli,and the enterobactin production of the parental and mutant strains was determined by ic-ELISA.The growth curves of the parental and mutant strains were determined in different iron-limited media.The supernatants from the parental and mutant strains were used to inoculate Campylobacter jejuni for investigating the effects of entericin secreted by Escherichia coli on the growth of Campylobacter jejuni.[Result]The levels of enterobactin secreted by 30 chicken intestinal Escherichia coli isolates ranged from 11 to 126μmol/L.2 entB gene deletion mutant strains of Escherichia coli were successfully constructed usingλRed homologous recombination system.The qualitative results showed that Escherichia coli parental strains produced the yellow halo in the CAS medium,while no halo was observed for the entB gene deletion strain.ic-ELISA results showed that Escherichia coli parental strains produced enterobactin in the culture supernatants with a concentration of 100-150μmol/L,while no enterobactin was detected for the entB gene deletion strain.In the iron-rich medium,there was no significant difference in the growth of Escherichia coli parental and entB gene deletion strains(P>0.05).The D 600 nm values that reached the plateau in iron-limited medium were 0.6 and 0.3,respectively.The growth promotion results showed that the colony numbers of Campylobacter jejuni in the culture supernatant of Escherichia coli parental strain reached 7.8 and 8.2 lg CFU/mL at 24 and 36 h of inoculation,respectively.By comparison,the colony numbers in the culture supernatant of the entB gene deletion strain were 7.0 and 7.3 lg CFU/mL,respectively.[Conclusion]Escherichia coli had the ability to secrete enterobactin for promoting the growth of Campylobacter jejuni in iron-limited environments.This study laid a foundation for further investigation of enterobactin-mediated interactions between intestinal bacteria.