| Objective1.To investigate the distribution and drug resistance of the pathogenic strains of recent vulvovaginal candidiasis.2. To explore the effective components of Xianglian compound in the aspect of anti fungal and anti drug resistance, and the mechanism of them to reverse drug resistance to provide new methods and ideas for the study of Chinese medicine in anti fungi in the future, especially for the research of drug resistant fungi.Methods1. Retrospectively, survey the clinical manifestations, leucorrhea test, fungal culture and drug sensitivity test of the recent patients with the diagnosis of vulvovaginal candidiasis in our hospital, in order to realise recent pathogenic strains distribution and drug resistance situation of vulvovaginal candidiasis (VVC) and Recurrent Vulvovaginal Candidiasis (RVVC) patients. Survey of recent diagnosis of vulvovaginal candidiasis patients’ clinical manifestations, leucorrhea, fungal culture and drug sensitivity results, in order to investigate recent simple vulvovaginal candidiasis and recurrence of the vulva vaginal candidiasis patients of pathogenic bacteria distribution and drug resistance.2. Xianglian lotion, eugenol, berberine and the mixture of eugenol and berberine were used respectively to induct and culture Candida albicans drug resistant standard strain CA17 and clinical isolates drug-resistant strains in order to observe the reversal effect to drug resistance of the induced drug to drug resistant Candida albicans, and to compare the differences between the different groups.3. The changes of expression levels of the efflux pump genes, CDR1, CDR2 and MDR1, of the drug-resistant strains and their off springs reversed drug resistancewere were detected by using fluorescence quantitative RT-PCR technology, in order to investigate the effect of Xianglian lotion, eugenol and berberine to the efflux pump gene.4. Detect the changes of the function of efflux fluorescent dye Rhodamine 6G of the drug resistant primary strains and thair reversal sensitive progeny by using flow cytometry, to investigate the effect of Xianglian lotion, eugenol and berberine to efflux pump protein function.Results1. In recent period, The main pathogenic species is remain Candida albicans (69.8%), meanwhile Candida glabrata (27.6%) was the most common Candida species in non-candida albicans. the highest rate of drug resistance is itraconazole (14.7%) of the total 5 kinds of antifungal drugs, followed by f luconazole (6.9%). The distribution of pathogenic species and drug resistance rate of VVC and RVVC patients were not significantly different. The resistant rate of Candida albicans to fluconazole was higher than that of non Candida albicans, and there was no significant difference in the rate of drug resistance to other drugs. The detection of cross resistance of 3 kinds of zole drugs is obvious.2. Three clinically isolated resistant strains can be induced in the continuous induction of 6-8 generations to reverse drug resistance by Xianglian lotion, eugenol, berberine and the mixture of eugenol and berberine. And the MIC is stable within the sensitive range by continuous culture for three generations. And the three strains of clinical drug-resistant strains which were carried out two kinds of culture media, hadn’t achieve reversal in the 10 generations, the above drugs. Resistant strains CA17 hadn’t achieved reversal of drug resistancet within 30 generations.3.The expression levels of CDR1, CDR2,and MDR1 of clinical resistant strain were significantly higher than that of the clinical sensitive strains. The above efflux pump genes’expression of drug sensitive sub generation in these 4 groups which were induced by induction drug were decreased significantly compared to the primary drug-resistant strains, in which Xianglian lotion group and the mixture of eugenol and berberine group decreased more significantly than eugenol and berberine solo. The expression of the 3 genes in Eugenol group ratio decreased more significantly than berberine group.4. The efflux function of Candida albicans standard resistant strains CA17 is stronger than clinical resistant strains, but mainly manifested in glucose (which is the energy source of the case). The efflux function of R6G were significantly higher in clinical drug resistant group than that of clinical sensitive strains with or without energy source. The efflux rate of sensitive sub clinical resistant strains in the generation were decreased in free energy source conditions by inducing with Xianglian lotion, berberine and the mixture of eugenol and berberine, and there was no significant difference in Eugenol group. The efflux rate decreased significantly compared with the resistant group after adding glucose. Xianglian lotion had the strongest inhibitory effect on efflux pumping function followed by the mixture of eugenol and berberine, and there is no obvious difference to eugenol, berberine respectively.Conclusion1. The dominant pathogenic strain of WC and RVVC patients is still Candida albicans, followed by Candida glabrata. The main strain which is resistant to fluconazole is still Candida albicans, which changes little compared with the previous epidemiological investigation. But itraconazole resistance rate is higher than fluconazole for the first time.2. Xianglian lotion, eugenol, berberine and the mixture of eugenol and berberine have a role in reversal of drug resistance of clinical drug-resistant strains, and the earlist reversal appeared in Xianglian lotion group.3.Induced by Xianglian lotion, eugenol, berberine and the mixture of eugenol and berberine, the expression levels of the three efflux pump resistance related gene, CDR, CDR2, MDR1, of the clinical drug resistant strains of Candida albicans can be reduced significantly. And the efflux function of efflux pump protein encoded by the three genes was inhibited. It is conferred that part of the effective ingredients including eugenol and berberine in Xianglian lotion have effects in anti drug resistant by reducing the efflux pump gene expression and inhibiting of fungal cell efflux of antifungal drugs.4. Effect of Xianglian solution was better than the two Chinese medicine monomer solo in the inhibition of efflux pump genes expression and efflux pump proteins function. But in some cases it is close to the two monomer mixture. We confer that eugenol, berberine in Xianglian lotion have synergistic anti drug resistance, and the role of other components of Xianglian solution needs to be further verified. |
PDF Full Text Request