cpxR基因对大肠杆菌E41株生物学特性及生物被膜形成的影响研究

Effects of cpx R gene on biological characteristics and biofilm formation of Escherichia coli E41

为研究双组份系统cpxR基因缺失对大肠杆菌临床分离野生株E41生物学特性的影响、培养条件与其生物被膜(BF)形成能力的关系及黄芩苷对BF的清除作用,本研究以野生株E41、缺失株E41ΔcpxR、回补株E41ΔcpxR/pcpxR为对象进行试验。比较3株菌的生长特性,结果显示三者生长曲线无显著性差异。比较3株菌的运动性,结果显示E41ΔcpxR的游泳运动和群集运动能力弱于野生株及回补株。采用平板计数检测3株菌的抗鸡血清杀菌能力,结果显示E41ΔcpxR株抗鸡血清杀菌能力弱于野生株及回补株。采用微量肉汤稀释法检测菌株的药物敏感性,结果显示与野生株和回补株相比,卡那霉素、阿米卡星和环丙沙星对缺失株的MIC降低1/2,但氟苯尼考对其MIC升高了2倍。采用结晶紫染色法检测不同成膜载体和培养温度对BF形成的影响,结果显示3株菌均在硅胶载体上的BF形成能力最强,但25℃条件下适于E41和E41ΔcpxR/pcpxR株BF的形成,37℃条件下则更适于E41ΔcpxR株BF的形成。采用平板计数法检测黄芩苷对BF的清除作用,结果显示,不加黄芩苷的空白对照组,E41和E41ΔcpxR/pcpxR株在试验起始时就有菌株开始从BF中游离,而E41ΔcpxR在24 h才有菌株从BF中游离;加入1 MIC和1/2 MIC黄芩苷分别与3株菌共培养条件下,3株菌均在试验起始时即从BF中开始游离。但加入1/4 MIC黄芩苷时,E41和E41ΔcpxR/pcpxR株在试验起始时有菌株从BF中游离,E41ΔcpxR株在12 h开始从BF中游离。本研究结果表明,当cpxR基因缺失后,其运动性和抗血清杀菌能力降低,对氨基糖苷类、喹诺酮类和酰胺醇类药物的敏感性改变,硅胶载体更有利于菌株附着形成BF,黄芩苷对其BF有清除作用。本研究为探究大肠杆菌cpxR基因功能奠定了基础,同时也为BF抑制剂的研发提供了参考依据。

To investigate the effect of the deletion of cpxR gene in the two-component system on the biological characteristics of wild Escherichia coli clinical strain E41,the correlation between the culture conditions and its biofilm forming ability,and the eradication effect of baicalin on biofilm bacteria,wild strain E41,deletion strain E41ΔcpxR,and complementary strain E41ΔcpxR/pcpxR were tested.The growth characteristics of the three strains were compared,and the result showed that there was no significant difference among the three growth curves.Comparing the motility of the three strains,the result showed that the swimming and swarming motility of E41ΔcpxR were weaker than the wild strain and the complementary strain.The bactericidal ability of the chicken antiserum to the three strains was detected by plate counting,and it showed that E41ΔcpxR was weaker than the wild strain and the complementary strain.The drug resistance of the strains was detected by broth microdilution method,and it showed that the MIC of kanamycin,amikacin and ciprofloxacin to the deletion strain decreased to 1/2 of the original,but florfenicol increased by 2 times.Crystal violet staining was used to detect the effects of different biofilm-forming carriers and culture temperatures on biofilm formation.The result showed that the three strains had the strongest biofilm formation ability on silica gel carrier.It was suitable for biofilm formation of E41 and E41ΔcpxR/pcpxR at 25℃,and it was more suitable for E41ΔcpxR at 37℃.The plate counting was used to detect the clearance effect of baicalin on biofilm,and it showed that in the control group without baicalin,E41 and E41ΔcpxR/pcpxR began to dissociate from the biofilm at the beginning of the test,while E41ΔcpxR started to dissociate at 24 hours.Under the co-culture condition of 1MIC and 1/2MIC baicalin,the three strains began to dissociate at the beginning of the experiment.However,when 1/4MIC baicalin was used,E41 and E41ΔcpxR/pcpxR were isolated at the beginning of the test,and E41ΔcpxR was isolated at 12 hours.This study demonstrated that when cpxR gene was deleted,the motility of the bacteria and the bactericidal ability of the antiserum decreased,and its resistance to aminoglycosides,quinolones and amide alcohols was changed.Silica gel carrier was more conducive to the adhesion of strains to form biofilm,and baicalin had a scavenging effect on its biofilm.This study laid a foundation for exploring the function of Escherichia coli cpxR gene,and also provided a reference for the development of BF inhibitors.