In Vitro Interaction And Mechanism Research Of Fluconazole In Combination With Antibacterial Agents Against Candida Albicans

In the past decades, the widespread use of potent broad spectrum antibiotics, glucocorticoid, immunosuppressant, cytotoxic drugs, as well as the increase of immunoimpression patients (such as AIDs, organ transplantation, and the ones who are undergoing cancer), have induced the incidence of invasive fungal infections (IFIs). Among the pathogens, C.albicans is the most important one, with a mortality of 40%. Challenges of treat the IFIs have turned out as the prolong use of FLC has also induced a resistance in Candida spp. The mechanisms of C. albicans to traditional antifungal agents are complicated, as they are regulated by multiple genes and the mechanisms may have interactions in themselves. Anyway, the resistance mechanisms of C. albicans to traditional antifungal agents are not a simple reason. Now, drugs combinations have become one of the hottest and most effective strategies to solve resistant fungi infections.The antifungal effect of minocycline has been already referred to when used alone or in association with other antimicrobial drugs. It is also well accepted that Fosfomycin (FOM), Macrolides, and Fluoroquinolones (FQNs) have destroyed the clinical important bacterial pathogens of Pseudomonas aeruginosa biofilm. This fact interests us that if they were combined with FLC, a similar effect would be also seen in the biofilms of C. albicans? In the present study, we eventually designed a series of concentrations of the antibiotics combined with FLC to evaluate their effect on biofilms of C. albicans. Some probably related mechanisms of the synergistic effect were also studied.1. Antifungal activity of fluconazole combined with antibacterial agentsAccording to the method described in CLSI M27-A2, a series concentrations of Minocycline (MINO), Rifampin (RIF), Ofloxacin (OFLX), Azithromycin(AMZ) and Fosfomycin (FOM) were designed from 32-0.5μg/ml,32-0.5μg/ml,8-0.125μg/ml,32-0.5μg/ml, and 64-1μg/ml, respectively. The concentrations of FLC were from 0.5-512μg/ml. both FLC-resistant strain and susceptive strain were tested. The obtained results suggested that MINO show a little antifungal effect, and no antifungal effect was observed in the other antibiotics. The interactions of FLC combined with the antibiotics were performed by chequeboard method and were interpreted by FICI and△E. We found out that the combinations only showed synergistic effect on resistant strain. And by comparing the interpretation of FICI and AE, we found that△E may be the better one to evaluate the interaction of antifungal with non-antifungal.A time-killing method was also employed to evaluate the dynamic activity of the studied antibiotics on FLC resistant C. albicans (CA10). At determined time point (0,6,12,24,48h), viable count and XTT assay were used to obtain the number of cells. The results reflected that the combinations of FLC with RIF, MINO, and OFLX had a better antifungal effect than FLC used alone, yet AMZ and PHO couldn't enhance the antifungal effect of FLC.2. The effect of drug combinations on FLC penetrating the biofilms of C. albicans-"sandwich" method.We found that the combinations of FLC with MINO, RIF, and AMZ could enhance FLC penetrating CA10 (resistant) biofilms, as the inhibition zones produced by the combinations were bigger than the one of FLC used alone. The same effect was only observed on the high concentrations of OFLX, yet antagonistic action was obtained in the lower concentrations, as the inhibition zones produced by combinations were smaller than the one of FLC used alone. No effect was observed in the combination of FLC with FOM. For the susceptive strain, no obvious synergistic effect was observed, which suggested the biofilms formed by CA10 may be one of the mechanisms of CA10.3. The effect of combinations on the metabolic activity of the cells in biofilms evaluated by FDA method.To justify the results got from the "sandwich" method, metabolic activity of the cells in biofilms was further tested by FDA method. The results suggested that the combinations of FLC with MINO, RIF, and OFLX do have effect on enhancing FLC penetrating the biofilms of CA10. At the same time, the cell activities of AMZ and FOM combined FLC groups almost have no different with FLC used alone.4. Relationship between sensitization of minocycline and intracellular calciumFluo-3/AM is a trans-membrane fluorescent indicator, which can immediately give out fluorescence after bounding to Ca2+, and reflect the square pulse changes of intracellular calcium. Flow cytometry was introduced to test the effect of drugs combinations on intracellular calcium of CA10. CA10 was incubated in YPD liquid medium until log phase growth, then washed by D-Hanks buffer and resuspended to 107CFU/ml. Cells were loaded with isovolumic Fluo-3/AM at 37℃and 120rpm for 30min to a final concentration of 3μM. Results showed that the sensitization of MINO may due to the release of calcium from calcium store, and extremely raised intracellular calcium.5. The impact of calcium regulation on drug interactionCa2+ is a very important second messager for cell functions. It is still uncertain whether the increased calcium attributed to the intracellular calcium release or extracellular calcium influx. So we further studied the calcium regulators on the antifungal effect of FLC combined with minocycline on plate against CA10,CA137, CA8,CA14. Moderate combined antifungal effect of FLC and minocycline was also observed, especially to the resistant strains, while addition of low concentrations of CaCl2 reduced the inhibitory effect of combination. That means the increase of calcium in the external environment produced a facilitating role. When calcium channel blocker BEN was added to FLC, almost no antifungal effect was found compared to the control, but the presence of BEN significantly enhanced the effect of FLC combined with minocycline for both FLC resistant and susceptible strains. EDTA also shown an obvious enhancement to the combination antifungal effect of FLC and minocycline, the only difference was that EDTA shown a little weaker impact. There were no significant differences in FLC, BEN and EDTA treating alone group compared with the control group.6. The effect of minocycline on FLC uptake and effluxThe effect of minocycline on FLC uptake and efflux were evaluated using rhodamine 6G (Rh6G) as substrate. The decrease of drug accumulation in cells attributed to low uptake and high efflux was one mechanism of fungal resistance. Our results showed that the uptake (within 50 min) of Rh6G was similar in both FLC sensitive and resistant strains when they were incubated in glucose free conditions. When glucose was added (after 50 min), intracellular concentrations of Rh6G were sharply decreased. Addition of minocycline did not increase the Rh6G uptake and decrease the efflux in both FLC sensitive and resistant strains.