| Polymicrobial biofilms represent as an understudied and clinically relevant health problem with the potential to serve as an infectious reservoir for a variety of microorganisms, including bacteria and fungi. As a natural member of the mucosal microbiota, C. albicans readily forms biofilms on a wide variety of polymers such as indwelling medical devices. C. albicans is the4th leading cause of bloodstream infections and the3rd most common organism isolated from intravascular catheters and is associated with the highest incidence of mortality. It is estimated that27%of nosocomial C. albicans bloodstream infections are actually polymicrobial, and Staphylococcus aureus was the third most common organism isolated in conjunction with C. albicans. Since the therapies against polymicrobial infections are not well characterized, combination therapy was consented to be one of the feasible methods for alternative treatment. Previous work from our laboratory demonstrated that the combination of fluconazole (FLC) and minocycline (MINO) has synergistic antifungal effects against plantonic and sessile C.albicans. Whether the combinations of FLC and antibacterial agents have synergistic effctes against the mixed strains were explored in this study, and also the potential mechanisms.Objective:In order to find new therapies against mixed bioflims, the combined effects of antibacterial agents and FLC against plantonic and sessile C.albicans mixed with Staphylococcus aureus was studied, including vancomycin(VAN), Minocycline (MINO), Rifampin (RIF), Ofloxacin (OFLX), Azithromycin(AMZ) and Fosfomycin (FOM). And also the effects of calcium regulation agents on the combinations were explored. The potential mechanisms was investigated by evaluating the roles of drugs on the two strains and testing quantitative changes of extracellular matrix, as well as the expression of the gene Hwpl(encoding hypha of Candida albicans). MethodsAccording to the method described in CLSI M27-A3, the microdilution method was used to investigate the combined antimicrobial effects of fluconazole with antibacterial agents (rifampin, minocycline, ofloxacin, fosfomycin, vancomycin and azithromycin) against Candida albicans mixed with Staphylococcus aureus both in plantonic and in biofilm.The mixed suspension was separated by transwell membrane to respectively evalueate drug effects against Candida albicans and Staphylococcus Both sensitive and resistant C. albicans and S. aureus were tested in mixed planktonic cells and mixed biofilms; the results were evaluated both by visual observation and XTT (OD value) method, and two non-parametric methods (FICI and ΔE) were used to analyze the results.We tested the quantitative changes of extracellular matrix after givn drugs by weighting the bioflim dry weight, which was carried out by the palts-glass slide model; the cell counting method was used to evaluate the combined effect against the two strains respectively after dividing the two strains in the mixed bioflim by transwell membrane; the "sandwich" method was used in order to find out the effect of antibacterial agents on FLC penetrating the mixed biofilms of C. albicans and S.aureus; the expression of the gene Hwpl (encoding hypha of Candida albicans) was studied by PCR. All of these studies were to investigate the mechanism of synergistic drug-interaction and of biofilm resistance.ResultsThe combination of FLC and antibacterial agents had synergistic effects against plantonic C.albicans mixed with S. aureus, and also the mixed bioflims.MiNO had the strongest synergistic effects with FLC, and this combination can destroy the mixed BF in low concentrations which were lower than the MIC of the strains when disposed alone. There were no significant difference between plantonic and2h-BF,4h-BF or8h-BF when disposed with drug combinations, but the MIC of the combinations were higher when against12h-BF. There was no significant antibacterial effects when against24-BF.Compared to the growth group, the FLC inhibited the fungal growth slightly. There were no significant differences between BEN or EDTA disposing alone group and the control group. No significant antifungal effect was found when MINO used alone or combined with those calcium regulators. When FLC group was taken as control, the addition of MINO enhanced the antifungal effect of FLC. When calcium channel blocker BEN and calcium chelator EDTA were combined with FLC separately, slight antifungal effect was observed, while when BEN/EDTA was combined with FLC and MINO, a obvious enhancement was displayed, especially when EDTA was combined with FLC and MINO, with the MIC of BEN and EDTA4ug/ml and0.25mM, respectively.The quantity extracellular matrix was decreased in the combination group which suggests that the combination therapy can inhibit the extracellular matrix; Transwell membranes can divide the two strains in the mixed biofilm and after counting those two strains, we found that the SA was related to the concentrations of the antibacterial agents. Only when MINO combined with FLC can cause a significant larger inhibition zone against CA than using FLC alone, but there were no such changes when against SA; the polymerase chain reaction (PCR) evaluated the impact of drug combinations on mycelium encoded protein HWP1expression, there were no great disparities between drug combination group, drug alone group and growth control.Conclusion1. In this study, microdilution method was used to study the combiantion effects of the antibacterial agents and FLC, and the resualts were evaluated in FICI and ΔE method. We found that the combinations of FLC and MINO, VAN, FOM, OLFX, RIF, or AMZ have synergistc antibacterial effects against plantonic and mixed bioflim. The time of forming mixed biofilm can impact the antibacterial effects greatly, there were no significant difference between plantonic cells and2h-BF,4h-BF or8h-BF when disposed with drug combinations, but the MIC of the combinations were higher than that against12h-BF. There were no significant antibacterial effects when against24-BF.The calcium regulation agents could enhance combination effects of FLC and MINO, and the EDTA have stronger effects than BEN.2. The mechanism study found that combination drug can reduce the amount of the mixed biofilm cells, mainly due to the suppression of Candida albicans. Although drug combinations can decrease the dry weights of the extracellular matrix, the drug combination can not enlarge the amount of FLC which can penatrate the biofilm, and not reduce the expression of the Candida albicans hypha protein encoded by HWP1, which suggest that the combined drugs can inhibit the growth of the mixed strains not by reducing the formation of the biofilm, and that C. albicans play the role of anti-mixed biofilm growth.In short, the drug combinations of FLC and antibacterial agents showed synergistic effects against the mixed strains of the different sensitivity of Candida albicans and Staphylococcus aureus. Minocycline and FLC have the strongest synergy. The calcium channel blocker benidipine and calcium chelator EDTA can enhance the combined effects. C. albicans play the role of anti-mixed biofilm growth.Combined drugs can inhibit the growth of the mixed strans not by reducing the formation of the biofilm. |
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