| Objective The purpose of this study was to detect the sensitivity of Enterobacteriaceae bacteria to carbapenem antibiotics and drug resistance genes and to know the molecular epidemiology of carbapenem-resistant Enterobacteriaceae.To grasp the prevalence of the drug resistance gene bla NDM subtype in the region.Exploring the effect of strains with NDM-1 on apoptosis and identify the effect of NDM-1 on resistance of Escherichia coli and the change of bacterial expression profile after overexpression.Methods WHONET5.6 software was used to screen the bacteria resistant to IPM in Enterobacteriaceae isolated from a top three comprehensive teaching hospital from 2014 to2016.The minimum inhibitory concentration(MIC)of bacteria against carbapenems was determined by broth dilution method.CRE resistance genes(bla NDM,bla KPC and bla OXA-48,etc.)and CRE resistance-related gene(bla CTX-M-15)were detected by PCR,and bla NDM subtypes were identified by sequencing.BL21(DE3)Escherichia coli competent cells were transformed by constructing pet28a(+)-NDM-1 overexpression vector,and the expression of NDM-1 in competent cells was detected by Coomassie blue staining and q RT-PCR;optimizing the expression of NDM-1 by orthogonal experimental design based on three factors and three levels.K-B method(paper diffusion method)was used to detect the resistance of pet28a(+)-NDM-1-BL21(DE3)strain to imipenem and other antibiotics.Changes of the pet28a(+)-NDM-1-BL21(DE3)after imipenem combined with berberine in strain sensitivity were observed.The pet28a(+)-NDM-1-BL21(DE3),pet28a(+)-NDM-1 and BL21(DE3)strains were infected with RAW264.7 cells,respectively,and the apoptosis model was constructed.The expressions of Caspase-9、Apaf-1、Bax and Bcl-2 of apoptosis-related genes were detected by q RT-PCR.Pet28a(+)-NDM-1-BL21(DE3),pet28a(+)-BL21(DE3)and BL21(DE3)strains were subjected to transcriptome sequencing to observe the up-regulation or down-regulation of the related gene after NDM-1 overexpressed in BL21(DE3).Results 92 strains of Enterobacteriaceae(CRE)resistant to carbapenems were counted and screened by WHONET5.6 software.It was found that 92 strains of CRE mainly came from hepatobiliary surgery,ICU and vascular surgery,and the samples were mainly from bile,sputum and aseptic midstream urine.Among them,the drug resistance rate to amikacin was the lowest(12%),the drug resistance rates to other antibiotics were higher,and the drug resistance rates to cefuroxime and cefazoline were as high as 100%.PCR was used to detect drug resistance genes.The detection rates of bla NDM,bla CTX-M-15,bla TEM and bla SHV were high.No bla OXA-48 resistance gene was detected.Sequencing showed that 35 of 59 NDM positive bacteria were bla NDM-1,and 24 were bla NDM-5.After constructing NDM-1overexpression vector,Escherichia coli BL21(DE3)was transformed and its protein expression and m RNA expression were detected.It was found that protein and m RNA expression were present under several induction conditions,and the best induction condition was culture for 4 hours after the addition of IPTG.It was detected that the drug resistance of pet28a(+)-NDM-1-BL21(DE3)changed from sensitivity to resistance to four antibiotics after the over-expression of NDM-1 by K-B method.Combined drug sensitivity test showed that imipenem combined with berberine on pet28a(+)-NDM-1-BL21(DE3)is additive,but has no effect on the clinical isolate E.coli-73.After transfection of RAW264.7 cells with pet28a(+)-NDM-1-BL21(DE3),apoptosis-related genes were detected.It was found that NDM-1 overexpressing strains and negative control strains can both cause apoptosis,and the expression levels of apoptosis-related genes in the two groups are different at different time(P<0.05).The RNA-seq results of pet28a(+)-NDM-1-BL21(DE3),pet28a(+)-BL21(DE3)and BL21(DE3)indicated that there were 290 differentially expressed genes in pet28a(+)-NDM-1-BL21(DE3)compared with pet28a(+)-BL21(DE3),122 of which were up-regulated and 168 were down-regulated.Among the 290 differentially expressed genes,18 genes have been functionally annotated and related to bla NDM-1 expression.These genes are mainly involved in "nucleotide binding" and most are "ABC transporters",such as opp D,gln Q and fec B genes encoding ABC transporter..Conclusions(1)The prevalence of drug resistance genes of CRE in 2014-2016 indicate that CREs have high resistance to multiple antibiotics such as cefuroxime and cefazolin.The drug resistance gene bla NDM has a higher positive rate in CRE,while the bla NDM-1 subtype is predominant in this region.(2)NDM-1 can enhance the resistance of Escherichia coli to IPM,MEM,CZO and CAZ.Berberine combined with imipenem has a certain antibacterial effect on NDM-1 overexpressing bacteria.(3)The expression of NDM-1 in Escherichia coli can lead to the up-regulation of apoptosis-related genes Apaf-1,Caspase-9 and Bcl-2/Bax in cells infected with the bacteria,thus affecting the effect of the bacteria on apoptosis.(4)The expression of bla NDM-1 in BL21(DE3)strain can regulate the expression of genes related to bacterial resistance and metabolism such as opp D,gln Q and fec B and has a strong effect on host cells. |
