| Background:With the increasing number of AIDS patients and malignant neoplasms, more use of catheterization and invasive endoscopic treatment technology, the application of efficiency and broad-spectrum antibiotics and immunosuppressants or hormone after organ transplantation, pathogenic fungi has become an important cause of nosocomial infections pathogen. Widely clinical application of fluconazole for fungal infection prevention and treatment of first-line therapy, the clinical isolation rate of non-albicans Candida species whice have high intrinsic resistance or acquired resistance to fluconazole increased gradualy, and the clinical detection rate of Candida glabrata which has a higher resistance to fluconazole accounts second place in all Candida species; Although the detection rate of Candida krusei that possesses natural resistance to fluconazole is not high, but they bring about challenges for the treatment of non- albicans Candida infections, and this has aroused people’s concern and attention. Comprehensively considering the high resistance and increasing detection rate of non-albicans Candida, Candida glabrata and Candida krusei resistant to fluconazole were selected to be the tested Candida species. Currently clinically available antifungal agents is limited, the expensive price or drug toxicity limits their application in clinical practice, nevertheless the development of a new antifungal drug time-consuming and costly. Therefore, more and more researchers have devoted to the research on the combined application of the antifungal drugs and the components of Chinese medicine, in order to find the components of Chinese medicine whice can increase the sensitivity of fluconazole against non-albicans Candida, and reverse the resistance of non-albicans Candida to fluconazole. Objective:The purpose of this study was to evaluate the static and dynamic antifungal effects of berberine, Pseudolaric acid B, shikonin, eugenol, baicalin, gallic acid combined with fluconazole against the fluconazole-resistant Candida glabrata and Candida krusei in vitro, and to explore the possible mechanism of the synergistic effect.Methods:According to the scheme determination of antifungal drug sensitivity of M27-A3, screen out the isolates of Candida glabrata and Candida krusei resistant to fluconazole, and the static and dynamic antifungal effects of components of traditional Chinese medicine combined with fluconazole against the fluconazole-resistant Candida glabrata and Candida krusei was researched by the checkerboard method and time kill curve method respectively in vitro. MICs for planktonic fungal in 24h and SMICs for biofilms in 24h of components of traditional Chinese medicine and fluconazole when used alone and in combination were determined by the naked eye observation and by determinating the absorbance of the two drugs in the 492nm wavelength through microplate reader respectively, two-drug’s combination effects against planktonic fungal and biofilm were evaluated by fractional inhibitory concentration index (FICI) and percentages of growth difference (△E); After the fungal suspension was exposed to the tested drugs 0,6 12,24, and 48h, colony counting method was implemented, then design the abscissa as the incubation time of fungal suspension after exposed to the tested drugs, and plot the vertical ordinate as the colony number of 1mL fungal suspension commonly used in numerical, to evaluate the dynamic effect of combined drugs by the time -kill curve obtained. As to study the mechanism of antifungal drugs combined, we investigated the influence of effective components of traditional Chinese medicine of gallic acid on Candida glabrata and Candida krusei resistant strains with respect to the early adhesion during biofilm formation, and assessed the effect of calcium channel blockers on gallic acid increased sensitivity to fluconazole against Candida glabrata and Candida krusei resistant to fluconazole.Results:The antifungal effect results of the components of traditional Chinese medicine when used alone show that Pseudolaric acid B or eugenol has almost no antifungal effect against Candida glabrata and Candida krusei resistant to fluconazole (MIC80 value is 128～256pμg/mL), shikonin, baicalin, gallic acid showed only weak antifungal effect against non-albicans Candida (MIC80 value is 16～64μg/mL), berberine when used alone showed strong antifungal effect against non-albicans Candida (MIC80 value is 2～8μg/mL).The antifungal effect results of the components of traditional Chinese medicine combined with fluconazole show that there is independent effects when Berberine, Pseudolaric acid B, shikonin, eugenol was used in combination with fluconazole; Baicalin combined with fluconazole showed strong synergistic effect against drug-resistant Candida krusei isolates; When using gallic acid and fluconazole in combination they would have significant synergistic antifungal effect against Candida glabrata and Candida krusei resistant strains, the MIC value of fluconazole reduced to 1/4～1/8 of the original after adding with gallic acid, FICI value was 0.375; Gallic acid can increase the susceptibility of fluconazole against fluconazole-resistant Candida glabrata and Candida krusei; The dose-effect relationship results also confirmed that with the concentration of gallic acid increasing, the synergism is also growing.The time-kill assay experimental results further confirmed the synergy when fluconazole was used in combination with gallic acid and the log10 CFU/mL was decreased by 2.1 and 2.25 at 24h compared with fluconazole used alone, respectively.The experiments also prove that gallic acid has the obvious inhibitory action on early adhesion during biofilm formation against Candida glabrata and Candida krusei resistant to fluconazole, and this would be the preliminary study on the mechanism of synergistic effect of gallic acid on fluconazole.Conclusion:Berberine in single use showed strong antifungal effect against non-albicans Candida, but when used in combination with fluconazole showed independent effects, further research is worthy to investigate the antifungal effect of berberine against non-albicans Candida. Fluconazole combined with gallic acid showed a synergistic effect to planktonic and biofilm of fluconazole-resistant Candida glabrata and Candida krusei, gallic acid has the obvious inhibitory effect on early adhesion during biofilm formation against fluconazole-resistant non-albicans Candida. The experimental results showed that the combined application of gallic acid and fluconazole may reverse the resistance of Candida glabrata and Candida krusei to fluconazole, this will have an important significance for the prevention and treatment of catheter-related clinical non-albicans Candida infections. This study provides ideas for the development of antifungal drugs, and may become a new method in the treatment of fungal infections of non-albicans Candida. |
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