| Objective: The aim of this study was to make clear the mechanism of reversed Multi-resistant Staphylococcus aureus by Ampelopsin.And the antibacterial effect of APS on Multi-resistant Staphylococcus aureus in vitro and in vivo was carried out.Moreover,it was expected to provide a theoretical basis for the search of drugs for effective treatment of Multi-resistant Staphylococcus aureus.Methods: 1.The method of multistep inducement and double dilution was used when taken penicillin as control.In order to evaluation whether APS was easy to results drug resistant in standar staphylococcus aureus,minimal inhibitory concentration(MIC)was measured before and after inducd with clinical SA.2.Antibacterial experiments in vitro:(1)Drug susceptibility test and mec A gene test were performed on 6 clinical isolation of SA to determine the type of drug resistance.(2)Double broth dilution method was used to detect the MIC and minimal bactericidal concentration(MBC)of 6 clinical isolation of SA by APS.(3)In order to screen the tested strains,antibacterial drug susceptibility test was performed on 6 clinical isolates after pretreatment with APS.(4)In order to screen effective antibiotic of reversal resistance by APS,the MIC,MBC,and FICI of penicillin,tetracycline,gentamicin,chloramphenicol to SA that pretreated with APS was measured.(5)?-lactam resistance genes(bla Z,bla I,bla R1),active efflux resistance genes(qac A/B,nor A),and the aminoglycoside-resistance genes(aac(6')-aph(2"),ant(4')-Ia,aph(3')-III)was detected by One-step RT-PCR afer only treatment with APS or combined with penicillin,gentamycin,respectively.3.Antibacterial experiments in vivo:(1)The A600 and plate colony counting was measured to drawn the growth curve of Multi-resistant Staphylococcus aureus.(2)To establish a Multi-resistant Staphylococcus aureus model of abdominal cavity infection,Mul-1 strain was injected by intrapertoneal.Peritoneal fluid bacterial counts,leukocyte counts were recored by plate countor colony count.And IL-1?,IL-6,TNF-?,and IL-10 IL-12,IFN-? levels was dectected to observe the protective effect of APS on intraperitoneal infection in Multi-resistant Staphylococcus aureus.Result: 1.The sensitive standard SA induced by penicillin or APS in vitro after 288 h,that was observed resistant to penicillin at a concentration of 32ŚMIC,however,no resistance to APS was observed.2.In vitro antibacterial test results:(1)Drug susceptibility test and mec A gene detection results showed that Mul-1,Mul-3,Mul-4 were multidrug resistant SA,Mul-2 was single drug resistant SA,Mul-5,Mul-6 were MRSA.(2)Double broth dilution method indicated that the MIC or MBC of APS alone against the 6 clinical isolates of SA were 125?g/m L,62.65?g/m L,125?g/m L,125?g/m L,62.5?g/m L,62.5?g/m L,respectively.(3)Mul-1 was selected as the test strain.(4)The checkerboard dilution method determined the MIC of penicillin,tetracycline,gentamycin,and chloramphenicol against Mul-1 strains were 29.25?g/m L,40?g/m L,40 ?g/m L,and 78.13?g/m L,respectively,and the MBC were 117?g/m L,320?g/m L,160?g/m L,>1250?g/m L;The MIC and MBC of penicillin,tetracycline,gentamycin,and chloramphenicol had no effectwith pretreatment of Mul-1 strain with 1/2 MIC,1/4 MIC,and 1/8 MIC APS afer 24 h,48h,and 72h;The FICI of APS(15.625?g/m L)combined with penicillin,tetracycline,gentamycin,and chloramphenicol were 0.37,0.63,1.13,1.13 for multidrug resistant SA,respectively.And the FICI of APS(31.25?g/m L)combined with these four antibiotics were 0.50,0.38,1.25,0.75,respectively.The FICI of APS(62.5 ?g/m L)combined with these four antibiotics were 0.51,0.63,0.53,0.56,respectively.The MIC,MBC and FICI of penicillin,tetracycline,gentamycin,chloramphenicol,chloramphenicol and chloramphenicol were not affected after the pretreatment of Mul-1 strains for 24 h,48h and 72 h respectively.(5)The results of PCR showed 31.25 ?g/m L and 15.625 ?g/m L of APS alone,3.66 ?g/m L and 0.915 ?g/m L of penicillin alone,15.625 ?g/m L of APS combined with 1.83?g/m L of penicillin,7.813?g/m L of APS combined with 0.915?g/ml of penicillin can significantly reduce the expression of bla Z gene;The combination of 31.25 ?g/m L of APS alone,15.625 ?g/m L of APS combined with 1.83 ?g/m L of penicillin,and 7.813?g/m L of APS combined with 0.915?g/m L of penicillin can significantly reduce the expression of bla I gene,15.625 ?g/m L of APS alone,3.66?g/m L and 0.915 ?g/m L of penicillin alone can significantly increase the expression of bla I gene.1.25 ?g/m L of APS alone,15.625 ?g/m L of APS combined with 1.83?g/m L of penicillin,7.813?g/m L of APS combined with 0.915?g/m L of penicillin,and 0.915?g/m L of penicillin alone could significantly reduce the expression of bla R1 gene,3.66 ?g/m L,1.83 ?g/m L of penicillin alone significantly reduced bla R1 gene expression.15.525?g/m L,7.813?g/m L of APS alone and 10?g/m L,5?g/m L of gentamycin alone significantly increased the expression of aac(6')-aph(2')gene,31.25?g/m L of APS combined with 20?g/m L of gentamycin,15.625?g/m L of APS combined with 10?g/m L of gentamicin significantly reduced the expression of aac(6')-aph(2')gene.31.25?g/m L of APS alone and 31.25?g/m L of APS combined with 20?g/m L gentamicin can significantly reduce the expression of ant(4')-Ia gene.No expression was found for the aph(3')-III gene,which was also induced by drug treatment.31.25?g/m L of APS alone,gentamicin alone,15.625?g/m L of APS combined with 1.83?g/m L of penicillin and 7.813?g/m L of APS combined with 0.915 ?g/m L of penicillin,15.625 ?g/m L of APS combined with 10?g/ml of gentamicin,7.813?g/m L of APS combined with 5?g/m L of gentamicin can significantly reduce the expression of nor A gene.The penicillin alone can significantly increase the expression of nor A gene.31.25 ?g/m L and 15.625 ?g/m L of APS alone,1.83 ?g/m L and 0.915?g/m L of penicillin alone,gentamicin alone,15.625 ?g/m L of APS combined with 1.83 ?g/m L of penicillin,7.813 ?g/m L of APS combined with 0.915?g/m L of penicillin,15.625 ?g/m L of APS combined with 10 ?g/m L of gentamycin,7.813 ?g/m L of APS combined with 5 ?g/m L of gentamycin all reduced qac A/B gene.No expression was found for the msr A gene,nor was it induced after drug treatment.3.The results of experiments in vivo showed that leukocytes was siginificantly reduced in the peritoneal fluid of the model group(P<0.01);the classification of neutrophils,eosinophils was obvious.The leukocytes in the group were significantly higher than those in the model group.The levels of TNF-?,IL-1?,IL-10 and IL-12 secreted by the model mice were significantly higher(P<0.05?P<0.01),and the abdominal cavity in mice.The increase in the number of liquid bacteria(P<0.01)caused inflammation of the body to start.Most of the drug-administered groups significantly reduced the levels of inflammatory cytokines in the peritoneal fluid of mice infected with the abdominal cavity,and the number of bacteria was significantly reduced.Conclusion: To standard SA was not easy to induced resistance by APS.Synergistic or additive antibacterial effect in vitro combined with penicillin,tetracycline,gentamycin and chloramphenicol was observed.APS,penicillin and gentamicin reversed the multi-drug resistant SA resistance by regulating the expression of relevant drug resistance genes and active efflux resistance genes,and then antibiotic susceptibility was recovered.In the abdominal cavity infected with multi-drug resistant SA,the immune response function could be enhanced by APS through increasing the number of leukocytes and the levels of inflammatory factors(TNF-?,IL-1?,IL-10,IL-12).The elimination and killing ability of bacteria can achieve the effect of preventing multi-drug resistant SA infection. |
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