| Objective: Widespread use of antibacterial drugs,increasing environmental pollution and interacting microorganisms and other pressure factors have accelerated the emergence of multidrug resistant(MDR)bacteria,especially Carbapenem-resistant enterobacteriaceae(CRE)is the most dangerous.Raoultella ornithinolytica B1645-1 is the first CRE strain isolated from northwest Hubei province carrying New delhi metallo-β-lactamase-1(NDM-1)gene,which poses a serious threat to the clinic in this region.Therefore,it is necessary to study its drug resistance mechanism and infection control.In this study,we used the CRE strains carrying bla NDM-1 from clinical specimens as the main research object to screen the active ingredients from traditional Chinese medicines,explore the possible mechanism of reversing the drug sensitivity of the strains,so as to provide theoretical basis and practical application options for clinical control of its infection.Methods: 1.To collect blood,urine,sputum and other specimens from inpatients in Dongfeng hospital affiliated to Hubei University of medicine from 2013 to 2019.Concentional agar culture method was used to isolate fermentative Enterobacter bacteria such as Escherichia coli,Klebsiella pneumoniae,Klebsiella oxytoca,R.ornithinolytica,Serratia and Enterbacter cloacae.DL-96Ⅱ automatic identification system preliminarily identified the strains and detected its drug resistance.16 S r DNA further identified the genus and species to screen out the CRE bacteria.Polymerase chain reaction(PCR)method was used to detect β-lactamase genes carried by CRE bacteria.2.The selected CRE strains carrying bla NDM-1 gene were selected as the target bacteria to study the ability of luteoloside to reverse its drug resistance.The gradient dilution method was used to detect the MIC of luteoloside inhibiting the CRE strain.Checkerboard dilution method to detect the combined antibacterial experiment of luteoloside and imipenem.Optical and scanning electron microscope(SEM)to detect the morphological changes of bacteria after the intervention of luteoloside.Quantitative Real-time PCR(q RT-PCR)to detect the expression of resistance genes bla NDM-1,bla TEM-1 and bla CTX-M-9.The DNA template and DNA Taq polymerase were added into the luteoloside reaction system to detect the reversion of 16 S r DNA,bla NDM-1,bla TEM-1 and bla CTX-M-9 genes.The molecular docking further simulated the coordination of luteoloside with DNA polymerase.3.The effect of luteoloside on bacterial infection of CRE in BALB/c mice.1.0×1010cfu/m L R.ornithinolytica B1645-1 gastrointestinal infection BALB/c mice,set up the normal control group,infected group,3 mg/kg/d luteoloside group and 6 mg/kg/d luteoloside group.The main physiological and biochemical indicators of the mice were detected on the second,third and sixth days of infection,including body weight,pathological changes of intestinal tissue,tumor necrosis factor α(TNF-α),interleukin-12(IL-12),serum alkaline phosphatase(AKP)and 5-hydroxytryptamine(5-HT).Results: 1.A total of 26 CRE strains were isolated and identified in this study,and the proportion of carbapenemase gene was 76.92%.2.The antibacterial experiment of the active ingredient of traditional Chinese medicine showed that luteoloside could significantly inhibit the growth of CRE strains such as R.ornithinolytica B1645-1,and the bacteria showed obvious changes such as broken cells and long chains after the intervention of luteoloside.q RT-PCR showed luteoloside could inhibit the expression of bla NDM-1,bla TEM-1 and bla CTX-M-9 in some CRE strains.Polymerase replenishment experiments showed that replenished DNA Taq polymerase could restore the inhibition of luteoloside.Molecular docking showed that luteoloside could form stable combination with DNA polymerase III.3.The results of luteoloside against CRE bacterial infection in BALB/c mice: 1)The serum TNF-α concentration of mice increased significantly from the second day compared with other groups.On the sixth day of infection,the serum IL-12 concentration of mice in the 6 mg/kg/d luteoloside group increased the most,which was similar to the change of 5-HT concentration.On the sixth day of infection,the serum AKP concentration of mice in the 3 mg/kg/d luteoloside group(59.60±2.57 ng/L)and 6 mg/kg/d luteoloside group(78.78±0.68 ng/L)both increased significantly compared with the normal control group(50.91±0.22 ng/L)and the infection group(49.36±4.64 ng/L).2)The results of intestinal HE in mice showed that all the experimental groups had inflammatory changes on the second day of infection.On the third day of infection,the 6 mg/kg/d luteoloside group had infiltration of inflammatory cells and bacterial groups in the mucosa and myometrium was significantly improved compared with the day before.On the sixth day of infection,there was only a small amount of inflammatory cell infiltration in the mucosa and muscle layer in the 3 mg/kg/d luteoloside group and the intestinal inflammation in the 6 mg/kg/d luteoloside group was almost completely restored.Conclusion: 1.The resistance of bacteria to carbapenems is related to the carbapenemase genes(bla KPC,bla GES,bla NDM-1,bla IMP and bla OXA,etc.)they carry;2.Luteoloside may significantly inhibit the growth of bla NDM-1 positive CRE bacteria by destroying the bacterial cell wall and bacterial DNA polymerase,inhibiting the resistance of bla NDM-1,bla TEM-1 and bla CTX-M-9 genes.3.Luteoloside can significantly improve the intestinal pathological inflammation of BALB/c mice infected with R.ornithinolytica B1645-1,which provides a basis for the clinical development of new β-lactamase inhibitors. |
PDF Full Text Request