| Objective:To explore the effects of berberine(BBR)on the antibiotics sensibility of helicobacter pylori(H.pylori)of multi-drug resistance(MDR)and its correlations with adjustments of Acr AB-Tol C efflux pump genes,in order to provide experimental evidence for the impacts of berberine,effective component of Coptis chinensis on the resistance of H.pylori MDR strains and its mechanisms.Methods:Part 1: H.pylori isolates were obtained from cultures of clinical gastric biopsies of patients who underwent endoscopy.All H.pylori strains were tested resistance for amoxicillin,levofloxacin,clarithromycin and metronidazole by agar dilution method.Then the double-drug resistant(DR)strains were induced to MDR strains in vitro.The expression quantities diffenrence of Acr AB-Tol C efflux pump genes: Hp0605-0607,Hp0969-0971,Hp1327-1329,Hp1487-Hp1489 between MDR group and sensitive group were detected by Real-time fluorescent quantitative polymerase chain reaction(RT-PCR).The correlations between the expression quantities of Acr AB-Tol C efflux pump genes and the sensitivity of H.pylori to antibiotics will be analyzed.Part 2: The MIC values of berberine against MDR strains were determined by agar two-fold dilution method.After the MDR strains were cultured at 1/2 MIC concentration of berberine in liquid for 5 generations,the changes of the MIC values of antibiotics were detected by E-test.A 4-fold decrease or convert to sensitivity in MIC of antibiotics for the MDR strains is considered significant.RT-PCR was used to detect the m RNA expression difference of the efflux pump genes before and after the addition of berberine,whose correlations with the changes of MIC of the MDR strains were also analysed.Genomic DNA in the MDR group before and after the treatment of berberine was extracted to amplify three kinds antibiotics of resistance genes by polymerase chain reaction(PCR),and the gene sequences were analyzed to check the mutations and recovery in MDR strains before and after treatment.Results:Part 1:(1)A total of 128 H.pylori strains were isolated and screened including 25sensitive strains,61 single-drug resistant strains,30 DR strains and 12 clinical isolated MDR strains according to the drug sensitivity assay,23 MDR strains were induced in vitro successfully.The H.pylori resistance rates to amoxicillin,levofloxacin,clarithromycin and metronidazole are 3.9%,28.1%,28.1% and 63.3%,and multi-drug resistance rate is 9.38%;(2)The expression quantities of Hp0605,Hp0606,and Hp1328 in the MDR group are significantly higher than that in the sensitive group(P<0.05).However,there is no obvious correlations between the expression quantities of those genes and the MIC of the resistant antibiotics of MDR strains.There are no high expressions of other genes between two groups.Part 2:(1)Berberine can certainly inhibit the growth of 35 MDR strains and have different MIC values in different MDR strains,which vary from 8?g/m L to 128?g/m L;(2)27 MDR strains with high-level resistance were chosen to detect the effects of berberine on the sensibility of antibiotics of MDR strains.After adding berberine to the MDR trains cultured in liquid,the MIC values of Clarithromycin,Levofloxacin has and Metronidazole have positive changes respectively in 9,11 and 5 MDR strains;(3)The expression quantities of Hp0605,Hp0606,Hp1328 and Hp1488 of MDR group after the effects of berberine remarkably decrease,compared with that before treated with berberine,and the difference has statistical significance,which don't have obvious correlations with changes of MIC values;(4)The sequence results of three kinds of resistance genes:(1)All 10 MDR strains resistant to clarithromycin have mutations in the 23 S r RNA gene,the main mutation forms are T2186 C,G2869A and A2147G;(2)All8 MDR strains resistant to levofloxacin have mutations in the gyr A gene,the dominating mutation forms are C87 A,C261G,G271 A and A272G;(3)All 10 MDR strains resistant to metronidazole have sporadic mutations in the rdx A gene,the dominating mutation forms are A162 G,G179A,C220 T and G229A;(4)Resistance in part of MDR strains can be reversed successfully after adding berberine,with no mutation recovery of the resistance genes.Conclusions:(1)Multi-drug resistance of H.pylori is the result of the combinations of the high expression of efflux pump genes of Hp0605,Hp0606 and Hp1328 as well as specific mutations of the resistance genes.(2)Berberine can partly reverse the resistance of 3 kinds of antibiotics in H.pylori MDRstrains by decreasing the expression quantities of efflux pump genes Hp0605,Hp0606,Hp1328 and Hp1488. |
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