| Biofilm is a group of organisms that adhering on the tissues or biological suface of the medical material, and its structure is the cells enclosed by the extracellular matrix. In the past decades, with the increasing cases of malignancy, autoimmune disease, AIDS, and the widespread use of broad-spectrum antibiotics, the incidence rates of fungal infections are arising. With the widely applications of ventilator and medical biological material especially, the generation rates of biofilm related fungal infections are increasing. In a clinical, the infections are always related with polymicrobial microbial biofilm. Candida albicans is the fourth leading cause of bloodstream infection and the third most commonly isolated organism from intravascular catheters, and it is associated with the highest incidence of mortality of62%. Staphylococcus epidermidis is a kind of normal flora living with human, but it is also an important source of causing cross-infection. Since S. epidermidis is easy to form biofilm on biomaterials, it becomes a main pathogen that cause medical equipment related infection. It has been found that the infection that related with these two micrograms is usually accompany with the formation of biofilms, which lead the infection to be persist, recurrent, and difficult to cure. It is important to look for a therapeutic approach for the treatment of polymicrobial biofilm infections.Since fluconazole is a useful antifungal agent, cheap, and with little side effects, it is widely used clinically. However, Long-term therapy with fluconazole facilitates the emergence of fluconazole-resistance. In addition, C. albicans commonly exist in biofilms and easily attach on both mucosal and plastic surfaces of indwelling devices. Lots of approaches have been used to improve the effect of antimicrobial therapy for resistant isolates, and one of them is drug combination. In preliminary experiment in our laboratory, we found synergism against planktonic C.albicans by combination of fluconazole and doxycycline. However, little is known about the effect of combined effect against polymicrobial biofilms of Candida and polymicrobial biofilms of C.andida and S. epidermidis.The in vitro combined effect of fluconazole and doxycycline against polymicrobial biofilms of C. albicans and S. epidermidis was studied in this research, and three aspects of mechanism including expression of resistance gene, influences of intracellular calcium level, and changes of cell cycle were investigated. All of them could be of great significance in finding new antifungal agents and a new antifungal mechanism of biofilms infection.1. Screening of growth medium for C.albicans and S.epidermidisFor solid medium, streak plate method was used to observe the growth state of C.albicans and S.epidermidis after cultivated for24h and48h in different mediums. In addition, we evaluated the impact of sheep blood and calcium on the growth of the isolates, and they were found could not promote the growth of the fungi and bacteria simultaneouly. The following ratio is the best choose:yeast extract0.25%, beef powder0.25%, peptone1.25%, agar1.625%, glucose0.5%, and sodium chloride0.375%. For liquid medium, yeast suspension and different proportions of the liquid medium were added in96well plates, and the plates were incubated at35℃for24h and48h. The following ratio is the best choose:RPMI16400.52%, MOPS1.7265%, sodium bicarbonate0.1%, glucose0.225%, tryptone0.85%, plant peptone0.15%sodium chloride0.25%, dipotassium hydrogen phosphate0.125%. The foundings of this study provides a reliable basis to further explore of effect and mechanism of combined drugs against polymicrobial biofilms.2. Combined effect of fluconazole and doxycycline against C.albicans biofilm.Microdulution method was selected according to the provition of CLSI M27-A3to determine the effect of a certain concentration of doxycycline (0.5-64μg/mL) with fluconazole against planktonic cells and biofilm (formed over4,8, and12h) of C.albicans, and ten isolates with different sensitivities were studied in this research. The drug interaction was evaluated by FICI and△E models. For planktonic cells, the synergistic effects were different among isolates. Strong synergisms were observed against resistant and dose dependent sensitive isolates, however, little synergism was observed against sensitive isolates, and antagonism was observed in a few wells. For biofilms, a consistent synergistic effect of fluconazole and doxycycline was interpreted by FICI and AE models against C. albicans biofilms formed over4,8,12h. However, as the biofilms matured and its complexity increased, the synergism weakened. The effect against biofilm was weaker than planktonic cells. These results have significance of searching for new antifungal agents and looking for new methods to overcome C. albicans biofilm infection.3. Combined effect of fluconazole and doxycycline against polymicrobial biofilm of C.albicans and S.epidermidis.In this study, C.albicans and S.epidermidis with different seneitivities were selected to form polymicrobial biofilm. The experiment was carried out by microdilution, and the effect against planktonic cells was also performed as a contrast to the result of biofilms. XTT assay was selected to determine the activity of biofilm, and the interactions of two drugs were interpreted by two models of FICI andAE. Synergism were observed against all the polymicrobial planktonic cells and biofilms, and the effects varied due to the C. albicans with different susceptibilities to fluconazole, which suggested that the synergistic effect of fluconazole and doxycycline mainly acted on C.albicans instead of S. epidermidis.4. Effect evaluation of the combined drugs against C.albicans and S. epidermidis, repectively.In order to evaluat the effect of the combined drugs against C.albicans and S. epidermidis, repectively, TTC-sabouraud’s medium and manitol salt agar were selected to separate the C.albicans and S. epidermidis from polymicrobial planktonic cells and biofilms, and colony counting method was used to determine the quantitative change of the isolates. We found that the main reasons for the changes in the number of C.albicans were synergism of the combined drugs and nutrient limitation, and the reason for the changes in the number of S. epidermidis is nutrient limitation and doxycyline, which suggested that the synergistic effect of fluconazole and doxycycline mainly acted on C.albicans instead of S. epidermidis, just as the result of the last chapter.5. The effect of the combined drugs on resistance gene expression of C.albicans in polymicrobial biofilms.The expression of resistance genes CDR1, CDR2, and MDR1of C.albicans in mono and poly biofilms were measured by reverse transcription PCR and real-time fluorescence quantitative PCR, and the result of real-time fluorescence quantitative PCR was analysed by2-△△Ct method. We found that the expression of resistance genes CDR1, CDR2, and MDR1of C.albicans in mono and poly biofilms were similar. For CDR1and MDR1genes, no significant differences of control group and doxycyline alone group were observed. Overexpression was caused by fluconazole used alone, and then the expression was significantly reduced by combination of fluconazole and doxycyline. For CDR2gene, compared with the control group, a significant decrease was observed in doxycycline alone group, however, inconsistent results were found in fluconazole group against mono and poly biofilms (overexpression in mono biofilms, no change in poly biofilms), and the expression was significantly reduced by combination of fluconazole and doxycyline. It can be speculated that the synergism is associated with blocking the expression of efflux pump genes CDR1, CDR2, and MDR1.6. The effect of the combined drugs on intrcellular calcium of C.albicans in polymicrobial biofilms.Firstly, the impact of calcium channel blockers (benidipine and nifedipine) on the synergism of combined drugs was observed by plate streaking. We were surprised to find that addition of calcium channel blocker to drug combination of doxycycline and fluconazole enhanced the effect dramatically, as a result, it is possible that calcium homeostasis may be account for the synergism. A new generation of calcium-seneitive fluorescent probe Fluo-3AM was selected, and the effect of combined drugs on incellular calcium concentration was observed by flow cytometry. We found that no significant change of incellular calcium concentration was observed in control and doxycycline alone group. After fluconazole using alone for4h, the incellular calcium concentration rised quickly, and a highest value was reached by8h. A sharply increasment was observed after addition of combined drugs for4h, and a slow rise after8h. We found that the incellular calcium concentration of C.albicans in mono and poly biofilms were similar. In a short, the synergism of combined drugs is associated with disordering the intracellular calcium.7. The effect of combined drugs on cell cycle of C.albicans in polymicrobial biofilms.The effect of combined drugs on cell cycle of C.albicans in mono and poly biofilms was observed by flow cytometry. For mono biofilm, the cell cycle was arrested at the G2/M phase in fluconazole alone and combined drugs groups, and the effect of fluconazole alone was stronger than combined drugs. For poly biofilm, no significant difference was observed in control, doxycycline alone, fluconazole alone, and combined drugs groups. It is possible that the cell cycle was arrested by fluconazole alone instead of the combined drugs. At the same time, we found that in control groups, the G2/M phrase was arrested in poly biofilms compared with that in mono biofilms, which suggested that the growth state of C.albicans was weakened in poly biofilms.To sum up, the synergism of fluconazole and doxycycline against polymicrobial biofilm of C. albicans and S. epidermidis was observed. The mechanism of the synergistic effect was associated with the expression inhibition of Candida drug efflux pump genes CDR1, CDR2and MDR1, and intracellular calcium disorder in C. albicans. |
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