Effect Of BlaNDM-1 Gene On Biological Characteristics And Pathogenicity Of Enterobacter Cloacae

Objective(s):Enterobacter cloacae is an opportunistic pathogen,one of the normal intestinal flora,which can cause a variety of extraintestinal infections.The drug resistance gene blaNDM-1 encodes New Delhi metal-β-lactamase-1(NDM-1),which is one of the main mechanisms of carbapenem-resistant Enteroba.cter cloacae.The purpose of this study was to clarify the effect of drug-resistant gene blaDM-1 on the biological characteristics and pathogenicity of Enterobacter cloacae,and to provide the basis for the prevention,control and clinical diagnosis and treatment of carbapenem-resistant Enterobacter cloacae producing NDM-1.Methods:1.Plasmid stability test:The antimicrobial susceptibility test was carried out After 200 passages of Enterobacter cloacae T2(NDM-1 group),Enterobacter cloacae T2 blaNDM-1 gene knockout strain(NDM-1 group)and Enterobacter cloacae ATCC13047(ST group).The blaNDM-1 gene was detected to determine whether the plasmid carrying blaNDM-1 gene would be lost in the passage or not.2.Study on the biological characteristics and pathogenicity of drug-resistant gene blaNDM-1 on Enterobacter cloacae:Firstly,we studied the influence of blaNDM-1 gene on the biological characteristics of Enterobacter cloacae T2,Enterobacter cloacae T2 blaNDM-1 gene knockout strain and Enterobacter cloacae ATCC 13047.Then,the effect of drug resistance gene blaNDM-1 on the pathogenicity of Enterobacter cloacae was studied at cellular and animal levels.2.1 Biological characterization of enterobacter cloacae:including drug resistance and virulence gene amplification,motility tests,biofilm-forming ability,and serum resistance tests.The experimental groups included Enterobacter cloacae T2 carrying drug resistance gene blaNDM-1,Enterobacter cloacae T2 blaNDM-1 gene knockout strain and Enterobacter cloacae ATCC13047.The common carbapenems resistance genes and virulence genes carried by the three Enterobacter cloacae strains were amplified by PCR,and verified by sequencing.2.2Cell assay:Adhesion invasion assay,LDH concentration determination.2.3 Animal experiment:the death number of ICR mice with peritonitis within a week,the pathological characteristics of liver,lung and small intestine of peritonitis mice,the amount of bacteria carried in spleen,and the contents of cytokines IL-1β,IL-6 and TNF-α produced by liver,lung and spleen.3.Statistical analysis:IBM SPSS 20.0 statistical software package was used for data analysis.Results:1.Plasmid stability test:after 200 passages,Enterobacter cloacae T2 still carried blaNDM-1 gene,Enterobacter cloacae T2 blaNDM-1 gene knockout strain did not carry blaNDM-1 gene,and Enterobacter cloacae ATCC13047 also did not carry blaNDM-1 gene.The results showed that the plasmid carrying blaNDM-1 gene was not lost and remained stable.2.Effect of drug resistance gene blaNDM-1 on biological characteristics and pathogenicity of Enterobacter cloacae.2.1 Study on biological characteristics of Enterobacter cloacae:①Detection of drug resistance genes and virulence genes:Enterobacter cloacae T2 only carried carbapenem-resistant gene blaNDM-1,and other carbapenem-resistant genes blaKPC-2,blaIMP-4,blaVIM-1,blaOXA-48 were all negative.Enterobacter cloacae T2 blaNDM-1 gene knockout strain and Enterobacter cloacae ATCC13047 were all negative for carbapenem-resistant genes.The same virulence genes clpB,icmf,acrA were found in Enterobacter cloacae T2 and Enterobacter cloacae T2 blaNDM-1 knockout strains.Other virulence genes VasD/Lip,wcam,fimH,mrkD and mrkB and csgB were negative.The virulence genes clpB,icmf,acrA,VasD/Lip and wcam were positive,but fimH,mrkD,mrkB and csgB were not detected in Enterobacter cloacae ATCC13047.②The movement diameter of Enterobacter cloacae T2 with blaNDM-1 resistance gene was 0.785±0.127cm,that of Enterobacter cloacae T2 blaNDM-1 resistance gene knockout strain was 1.445±0.255cm,and that of Enterobacter cloacae ATCC13047 was 1.370 ±0.270cm.The movement diameter of Enterobacter cloacae T2 blaNDM-1 resistance gene knockout strain and Enterobacter cloacae ATCC13047 was higher than that of Enterobacter cloacae T2 with blaNDM-1 resistance gene.The difference was statistically significant.③At 24h,48h and 72h,the absorbance of biofilm of Enterobacter cloacae T2 carrying blaNDM-1 resistance gene and Enterobacter cloacae T2 blaNDM-1 resistance gene knockout strain had no significant difference,they were higher than that of negative control group,but lower than that of Enterobacter cloacae ATCC13047.④Serum resistance test results showed that the survival rate of Enterobacter cloacae T2 carrying blaNDM-1 resistance gene was 0.624±0.112,the survival rate of Enterobacter cloacae T2 blaNDM-1 resistance gene knockout strain was 0.476±0.143,and the survival rate of Enterobcter cloacae ATCC13047 was 0.533±0.166.There was no statistical difference between the three groups.2.2Cell assay:①After co-incubation of Enterobacter cloacae and RAW264.7 for 3 hours,the results of adhesion test showed that there was significant difference between Enterobacter cloacae T2 blaNDM-1 resistance gene knockout strain and Ent.erobacter cloacae ATCC 13 047,but there was no significant difference among other groups.There was no significant difference in invasion test among the three groups.After 24 hours of co-incubation,adhesion test and invasion test showed that there was significant difference between Enterobacter cloacae T2 blaNDM-1 resistance gene knockout strain and Enterobacter cloacae ATCC13047.There was significant difference between Enterobacter cloacae T2 carrying blaNDM-1 resistance gene and Enterobacter cloacae ATCC 13047.There was no significant difference between Enterobacter cloacae T2 carrying blaNDM-1 resistance gene and Enterobacter cloacae T2 blaNDM-1 resistance gene knockout strains.②The determination of LDH concentration showed that there was no statistical difference in the concentration of LDH in the cell supernatant of the three groups2.3Animal experiments:①When the experimental group was intraperitoneally injected with 7.5×108 CFU and 0.4ml bacterial liquid,the negative control group was injected with 0.4ml LB liquid medium,there was no significant change in the negative control mice within a week.There was no statistically significant difference in the survival curves among the three experimental groups The death number of mice infected with Enterobacter cloacae ATCC13047 was the lowest,and the death number of mice infected with Enterobacter cloacae T2 carrying blaNDM-1 resistance gene was the highest.②ompared with the negative control group,liver,lung and small intestine tissues of mice in the three experimental groups showed different degree of inflammatory pathological changes 24 hours after infection with Enterobacter cloacae,but there was no statistical difference in pathological grade.③At 24h,48h and 72h after infection,the spleen bacterial load of Enterobacter cloacae ATCC13047 infected mice was lower than that of Enterobacter cloacae T2 and Enterobacter cloacae T2 blaNDM-1 resistant gene knockout strains infected mice.However,there was no significant difference between Enterobacter cloacae T2 carrying blaNDM-1 resistance gene and Enterobacter cloacae T2 blaNDM-1 resistance gene knockout strain infected mice.④There was no significant difference in the contents of IL-1 β,IL-6 and TNF-α in liver,lung and spleen between Enterobacter cloacae T2 group carrying blaNDM-1 resistance gene and Enterobacter cloacae T2 blaNDM-1 resistance gene knockout group.Conclusion(s):1.The drug-resistant gene blaNDM-1 can weaken the movement ability of Enterobacter cloacae,but does not affect the biofilm formation and serum resistance ability of Enterobacter cloae.2.Mice infected with Enterobacter cloacae carrying the resistant gene blaNDM-1 had a slightly higher mortality rate than the other groups,but the resistant gene blaNDM-1 did not increase the virulence of enterobacter cloacae.Moreover,it did not affect the contents of cytokines IL-1β,IL-6 and TNF-α secreted in the visceral organs of peritonitis mice.