| OBJECTIVEInfections caused by Candida species manifest in a number of diseases. For the past few years, Candida albicans and Candida parapsilosis are still two of the leading Candida species causing infections worldwide. Microbial biofilm is a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a matrix of extracellular polymeric substances. Biofilms are ubiquitous in nature and are characterized by their recalcitrance towards antimicrobial treatment. The increase of drug resistance and invasion caused by biofilm formation brings enormous challenges to the management of Candida infection. Therefore, there is a continuous need for the discovery of new antimicrobial agents that are effective against biofilms.The previous resμlts show that aspirin, one of the oldest and most widely used anti-inflammatory drugs, dramatically decreases biofilm formation by C. albicans. Alem et al found aspirin was active against growing and fully mature (48h) biofilms and its effect was dose related. However, the significant effects of aspirin on growth and biofilm formation of Candida spp. were achieved only with suprapharmacological concentrations of the drug, which limits its clinical application. Amphotericin B (AMB), a polyene macrolide agent, used as "the gold standard" antifungal drug since1960s, is a crucial agent in the management of serious systemic fungal infections. In spite of its proven track record, its well-known side effects and toxicity will sometimes require discontinuation of therapy despite a life-threatening systemic fungal infection. Aspirin, which possesses a weak and broad-spectrum antimicrobial activity towards some planktonic and biofilm cultures, may be useful in combined therapy with conventional antifungal agents to reduce their dose and improve their efficacy, while amphotericin B, needs to be combined with agents that are cheaper, more effective, more tolerable, and less toxic, particularly less nephrotoxic than AMB deoxycholate. Considering these, combination between aspirin and amphotericin B is an excellent choice for clinical use.Vaginal candidiasis is a frequent and common distressing disease affecting up to75%of the women of fertile age; most of these women have recurrent episodes. It has long been appreciated that mucosal infections are in essence biofilms. Common themes have emerged between the biofilm in vitro and in vivo. Unique features have emerged as well, such as the influence of host interactions on mucosal biofilms, complex interactions with bacterial flora, and biofilm-associated echinocandin resistance.The first part of our work was needed to illustrate whether the combianation of aspirin and AMB have a strong synergistic action to candidiasis in vitro or not. The second part of our work was aimed at assessing the anti-fungal activity and the combined effects of aspirin and AMB in an experimental infection of vaginal candidiasis. The final part of our work was to investigate the underlying mechanism of the synergistic action.METHODS1. In vitro As there have been controversies over assessing the nature and intensity of drug interactions, The combined use of the most commonly used methods, the fractional inhibitory concentration index (FICI) and a newly developed method, the ΔE model, which uses the concentration-effect relationship over the whole concentration range instead of using the MIC index alone enables the interpretation of results more reliable. As an attractive tool for studying the pharmacodynamics of antimicrobial agents, time-kill curves can provide detailed information about antimicrobial efficacy as a function of both time and concentration. In the meantime, the XTT reduction assay used for data generation instead of the traditional methods of visual reading and colony counting make it possible to evaluate fungal biofilm growth accurately.We investigated the minimal inhibitory concentration (MIC) and combined effects of aspirin and amphotericin B against the planktonic cells and biofilm cells of C. albicans and C. parapsilosis by microdilution method and the checkerboard microdilution method, and compared the differences of drug efficacy in different states. Furthermore, we obtained the detailed information about antimicrobial efficacy as a function of both time and concentration by time-killing test to assess the pharmacodynamics of each antimicrobial agent and the combined effects. New methods and interpretation models such as ΔE model were employed in comparison with FICI.2. Mechanism:We study the role of aspirin against candida biofilms related gene expression, and the mechanism of biofilm inhibition was discussed preliminarily3. In vivo:Mice were maintained under pseudoestrus condition prior to infection, and then candida albicans was inoculated and colonization on the vaginal mucosa. Treatment with different combination drug, The fungus burden was quantitated (CFU) by cultural a vaginal lavage fluid. Differences between asprin treated, AMB treated and ASA-AMB treated mice were evaluated by viable count data of the fungus burden in the vagina and the inhibitory effect of vaginal candida in mice were compared using the Student’s t-test (two-tailed).RESULTS1. In vitro1.1By testing the drug alone, in planktonic cells, aspirin has weak effect on the tested strains and AMB has strong fungicidal effect, while in biofilm cells, the highest level of resistance to AMB is observed, with the MIC (IC50) to the corresponding strain increased up to64and128-fold after biofilm formation, respectively, based on MICs by XTT. However, aspirin’s fungistatic activity in biofilm cells seems to change little in comparison to planktonic cells, which indicates dramatic antibiofilm activity. In terms of planktonic cells, the MICs of either individual agent were reduced by one to two dilutions against the tested strains, while remarked reductions were observed for AMB against biofilm cells when combined with aspirin. The geometric mean (GM) of the MIC to AMB and aspirin decreased up to32and16-fold, respectively, based on FIC indices.1.2In the checkerboard microtiter plate format, strong synergism was observed in biofilm cells of all three strains analyzed by FICI and ΔE. The two models correlated very well. The FICI indexes were far less than0.5, while all the ΣSYNs of the three tested strains were beyond900%, which indicate a super strong synergistic action between aspirin and amphotericin B. As to planktonic cells, synergisms were observed in ATCC22019and YEM30, Indifference was observed in CCA10.1.3The concentration-dependent synergistic action of aspirin and amphotericin B against biofilm cells that proved by checkerboard microdilution assay was confirmed by time-kill curves. Furthermore, the curves also revealed the time-dependent synergistic action between them. In aspirin alone, discernible improvement in the extent of fungistatic activity and the slope function of the time-kill curve to each strain was noted as the amount of drug in solution increased. Marked concentration-dependent fungistatic activity was also observed, what’s more, the rate and extent of fungistatic activity varied over time and the time to achieve a fungistatic endpoint was shortened as the dose increased. In combination, the addition of amphotericin B in low dose to various concentrations of aspirin resulted in strikingly improvement in extent of activity and trend toward a shorter time to the fungistatic endpoint versus single agents.2. We studied how the aspirin influences the expression of gene which related to biofilm formation. The experimental results show that the expression of GCA1is raised, and CDC35, CSR1, EFG1, HWP1decreased.3. In vivoWe determinated the fungus load of vaginal lavage fluid after different combination drugs treated in mice, the fungal load in the vagina were measured at the9,11,13,16and24days post-infection (2,4,6,9,15days after treatment), there is a significant reduction of Candida load in mice treated with ASA-AMB with respect to asprin and AMB treated alone or diluent saline treated mice starting from11days post infection(4days after treatment), and this beneficial effect as maintained until22days post infection (15days after treatment), even6days after therapy discontinued (p <0.05). CONCLUSION1. Established a method to evaluate synergistic effect of aspirin and amphotericin B combined use against candida biofilm based on FICI and delta E method2. We demonstrated that aspirin can intervene in candida biofilm by raising GCA1gene expression and cut CDC35, CSR1, EFG1, HWP1gene expression when aspirin combined application with amphotericin B against candida biofilm play a synergistic antibacterial effect.3. Found that a strong synergistic antimicrobial effect of aspirin and amphotericin B combined application against vaginal candida in mice.Based on the above study,a new way of thinking have provided for the treatment of refractory fungal infection. |
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