gidA基因缺失对貉源大肠杆菌生物学特性的影响

Effect of gidA gene detection on the biological characteristics of Escherichia coli strain from raccoon dog

为研究葡萄糖抑制的分裂蛋白A(GidA)在貉源大肠杆菌致病机制中的作用,本研究利用λ-Red同源重组技术构建貉源致病性大肠杆菌LCE-1(WT)gidA基因缺失株LCE-1ΔgidA(KO)和回补株LCE-1ΔgidA/gidA(RS),均采用PCR和测序鉴定正确后,进一步分析各菌株的生长特性、生化特性,结果显示,在LB培养基中3株菌的生长速度均无明显差异;KO菌株的部分生化特性如肌醇、α-葡萄糖苷酶、β-葡萄糖苷酶指标均发生变化,RS菌株指标基本恢复。通过试管法及微量结晶紫法分析评估3株菌生物被膜(BF)的形成能力,结果显示,与WT菌株和RS菌株相比,KO菌株BF形成能力极显著下降(P<0.0001)。体外模拟并检测在酸应激、碱应激、热应激以及氧化应激条件下这3株菌的生长情况,结果显示,gidA基因缺失能够显著提高大肠杆菌对酸应激、碱应激、热应激和氧化应激的敏感性。通过K-B纸片法对这3株菌的耐药性进行测定,结果显示,与WT菌株相比,KO菌株对头孢类、喹诺酮类、氨基糖苷类和青霉素类药物的敏感性增强。利用WT和KO菌株分别感染小鼠进行致病性试验,结果显示,WT和KO菌株对小鼠的LD50分别为1.58×10^(5)cfu/mL和3.16×10^(6)cfu/mL,与WT菌株相比,KO菌株的毒力下降大于1个数量级。本研究为进一步研究gidA基因对貉源大肠杆菌致病机制的影响奠定了基础。

To investigate the role of glucose-inhibited fission protein A(GidA)in the pathogenesis of Escherichia coli(E.coli)from raccoon dog,theλ-Red homologous recombination technology was used to constructed the gidA gene deletion mutant LCE-1ΔgidA(KO)based on the pathogenic isolate LCE-1(WT)from raccoon dog,and the complementary strain LCE-1ΔgidA/gidA(RS)was constructed as well,and all were identified by PCR and sequencing.On this basis,the effect of gidA gene deletion on the growth and biochemical characteristics of E.coli was further analyzed,and it showed that there was no significant difference in the growth rate of the three strains of bacteria in LB medium,some biochemical characteristics of KO strain such as inositol,α-glucosidase andβ-glucosidase indicators altered,RS strain indicator base resporse.The biofilm(BF)formation ability of the three strains was assessed by tube staining test and microcrystalline violet analysis,and the results showed that the BF formation ability of the KO strain highly significant decreased compared to the WT and RS strains(P<0.0001).The growth of these three strains under acid stress,alkaline stress,heat stress or oxidative stress was simulated and examined in vitro,and the results showed that the gidA gene deletion significantly increased the susceptibility of E.coli to acid stress,alkaline stress,heat stress,and oxidative stress.The rsults of the K-B drug resistance test of these three strains showed that the KO strain had increased susceptibility to cephalosporins,quinolones,aminoglycosides and penicillins compared to the WT strain.Mice were infected with the WT and KO strains for pathogenicity test,and it showed that the LD50 of the WT strain was 1.58×105cfu/mL,and the LD50 of the KO strain was 3.16×106cfu/mL,and the virulence of the KO strain decreased by greater than one order of magnitude compared to the WT strain.This study laid a foundation for further research on the effect of gidA gene on the pathogenesis of E.coli from raccoon dog.