In vitro synergism between berberine and miconazole against planktonic and biofilm Candida cultures.

OBJECTIVES: To investigate the antimycotic activity of the plant alkaloid berberine (BBR), alone and in combination with antifungal azoles, against planktonic and biofilm Candida cultures. DESIGN: The minimum inhibitory concentrations (MICs) of BBR, miconazole (MCZ), and fluconazole (FLC) towards Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida parapsilosis, and Candida tropicalis were determined by a microdilution method. For C. albicans, the synergistic effects of BBR combined with MCZ or FLC were examined in a paper disc agar diffusion assay and checkerboard microdilution assay. The effect of the BBR/MCZ combination was further investigated in a C. albicans biofilm formation model with a dual-chamber flow cell. The effect on metabolic activity of biofilm cells was established using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)/menadione. RESULTS: Berberine inhibited the growth of various Candida species (MICs 0.98-31.25mg/L) in the following order of susceptibility: C. krusei > C. kefyr > C. glabrata > C. tropicalis > C. parapsilosis and C. albicans. Synergism between BBR and MCZ or FLC was observed in the disc diffusion assay as well as in suspension showing an FIC index <0.5 (∑FIC=0.19). Whilst neither BBR (16 mg/L) nor MCZ (0.8 mg/L) alone significantly inhibited biofilm formation of C. albicans, their combination reduced biofilm formation by >91% after 24 h, as established from the reduction in surface area coverage (P<0.01). The BBR/MCZ combination also exhibited synergy against the metabolic activity of pre-formed C. albicans biofilms in polystyrene microtiter plates (∑FIC=0.25). CONCLUSION: Berberine exhibits synergistic effects with commonly used antimycotic drugs against C. albicans, either in planktonic or in biofilm growth phases.

Factors affecting antimicrobial activity of MUC7 12-mer, a human salivary mucin-derived peptide.

BACKGROUND: MUC7 12-mer (RKSYKCLHKRCR), a cationic antimicrobial peptide derived from the human low-molecular-weight salivary mucin MUC7, possesses potent antimicrobial activity in vitro. In order to evaluate the potential therapeutic application of the MUC7 12-mer, we examined the effects of mono- and divalent cations, EDTA, pH, and temperature on its antimicrobial activity. METHODS: Minimal Inhibitory Concentrations (MICs) were determined using a liquid growth inhibition assay in 96-well microtiter plates. MUC7 12-mer was added at concentrations of 1.56-50 microM. MICs were determined at three endpoints: MIC-0, MIC-1, and MIC-2 (the lowest drug concentration showing 10%, 25% and 50% of growth, respectively). To examine the effect of salts or EDTA, a checkerboard microdilution technique was used. Fractional inhibitory concentration index (FICi) was calculated on the basis of MIC-0. The viability of microbial cells treated with MUC7 12-mer in the presence of sodium or potassium was also determined by killing assay or flow cytometry. RESULTS: The MICs of MUC7 12-mer against organisms tested ranged from 6.25-50 microM. For C. albicans, antagonism (FICi 4.5) was observed for the combination of MUC7 12-mer and calcium; however, there was synergism (FICi 0.22) between MUC7 12-mer and EDTA, and the synergism was retained in the presence of calcium at its physiological concentration (1-2 mM). No antagonism but additivity or indifference (FICi 0.55-2.5) was observed for the combination of MUC7 12-mer and each K+, Na+, Mg2+, or Zn2+. MUC7 12-mer peptide (at 25 microM) also exerted killing activity in the presence of NaCl, (up to 25 mM for C. albicans and up to 150 mM for E. coli, a physiological concentration of sodium in the oral cavity and serum, respectively) and retained candidacidal activity in the presence of KCl (up to 40 mM). The peptide exhibited higher inhibitory activity against C. albicans at pH 7, 8, and 9 than at pH 5 and 6, and temperature up to 60 degrees C did not affect the activity. CONCLUSION: MUC7 12-mer peptide is effective anticandidal agent at physiological concentrations of variety of ions in the oral cavity. These results suggest that, especially in combination with EDTA, it could potentially be applied as an alternative therapeutic agent for the treatment of human oral candidiasis.

Effect of MUC7 peptides on the growth of bacteria and on Streptococcus mutans biofilm.

OBJECTIVES: To investigate the susceptibility of selected bacteria as well as Streptococcus mutans biofilm to MUC7 peptides and compare the activities with those of other known antimicrobial peptides. METHODS: MIC and MBC of peptides for S. mutans, Escherichia coli, Streptococcus gordonii, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Pseudomonas aeruginosa were determined using the microdilution method. For S. mutans, the effects of the peptides on the kinetics of growth inhibition, time-killing, and on biofilm formation and reduction were also examined. For biofilm studies, polystyrene microtitre plates, Calgary Biofilm Device (CBD) and hydroxylapatite (HA) discs, along with Crystal Violet and Alamar Blue dyes, and/or EM observations, were employed. RESULTS: S. mutans was the most susceptible to all peptides tested (MICs of 9.4-25.0 microM), compared with the other species (MICs of 3.1->100 microM). MUC7 peptides (except MUC7-12-mer-L4) exerted 2-fold higher activity against S. mutans than Hsn5-12-mer and magainin-II, and faster killing of S. mutans than Hsn5-12-mer. The MUC7 peptides also had an effect on S. mutans biofilm. On the polystyrene plates, they suppressed the biofilm formation, with MBIC(50) of 6.25-12.5 microM, and reduced the 1 day developed biofilm in a batch culture, with MBRC(50) of 25-50 microM. On the CBD pegs, the viabilities of the biofilm were suppressed by >95% in the presence of MUC7 peptides at 4x MIC (50 microM). One day developed biofilm viabilities were inhibited by 49-75%. On HA, the formation of biofilm (as observed by EM) was also considerably reduced. CONCLUSIONS: MUC7 peptides present somewhat preferential antimicrobial activity against S. mutans. They also have an effect on in vitro formation and reduction of the preformed S. mutans biofilm.

Human salivary mucin MUC7 12-mer-L and 12-mer-D peptides: antifungal activity in saliva, enhancement of activity with protease inhibitor cocktail or EDTA, and cytotoxicity to human cells.

MUC7 12-mer-L exhibits potent in vitro antifungal activity in low-ionic-strength buffers. In this study, we investigated the anticandidal activity and stability of MUC7 12-mer-L and its all-D-amino-acid isomer, along with Hsn5 12-mer (P113) and magainin-II, in human clarified and unclarified saliva in the absence or presence of protease inhibitor cocktail (PIC, which includes EDTA) or EDTA alone. In the absence of PIC or EDTA in saliva, only MUC7 peptides showed significant candidacidal activity. At a 100 microM concentration in clarified saliva and unclarified saliva, MUC7 12-mer-D demonstrated 94 versus 64% killing, respectively; MUC7 12-mer-L showed 57 versus 32% killing; Hsn5 12-mer showed 16 versus 0% killing; and magainin-II showed no killing. Addition of PIC or EDTA to either saliva caused the enhancement of antifungal activities of all peptides, although to different degrees. Taken together, the results suggest that EDTA (a metal-dependent protease inhibitor and/or divalent cation chelator) enhanced the antifungal activity of all four peptides mainly by chelation of divalent cations present in saliva (known to inhibit peptide antifungal activity), and PIC enhanced the activity of the three L peptides above that achievable by EDTA alone through inhibition of all classes of proteases. Peptide stability in saliva monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no degradation of MUC7 12-mer-D and 23, 60, and 75% degradation of MUC7 12-mer-L, Hsn5 12-mer, and magainin-II, respectively. Cytotoxicity assays determined that, at 100 microM peptide concentrations, MUC7 12-mer-D and 12-mer-L caused 3.5 and 4.3% hemolysis in phosphate-buffered saline and no toxicity to the HOK-16B cell line (derived from normal human oral keratinocytes). In summary, MUC7 12-mer peptides appear to be excellent candidates for investigation of antifungal activity in in vivo models of oral candidiasis.

In vitro synergic antifungal effect of MUC7 12-mer with histatin-5 12-mer or miconazole.

OBJECTIVES: MUC7 12-mer (RKSYKCLHKRCR), a cationic peptide derived from human salivary MUC7 mucin, exhibits potent in vitro antifungal activity, as determined by killing assays in phosphate buffer. In this study we examined the MUC7 12-mer antifungal activity alone or in combination with other antifungal agents in LYM medium (modified RPMI 1640). METHODS: Antifungal activities of MUC7 12-mer and other compounds against several fungal strains were first measured by MIC and minimum fungicidal concentration (MFC) tests using broth microdilution assay. The viability of Candida albicans and Cryptococcus neoformans were also determined by killing assays and time kinetics of peptide-mediated killing. Antifungal activities of MUC7 12-mer in combination with other compounds [histatin-5 (Hsn5) 12-mer: AKRHHGYKRKFH, amphotericin B or miconazole] against C. albicans and C. neoformans were determined by chequerboard assays and confirmed by killing assays. Toxicities of individual compounds were determined by haemolytic assays. RESULTS: MICs and MFCs of MUC7 12-mer ranged from 3.13 to 6.25 mg/L for most of the strains tested, and were, in most cases, comparable to those of amphotericin B and miconazole (0.78-6.25 mg/L). ED(50) values of MUC7 12-mer and Hsn5 12-mer were 7.1 and 7.4 micro M (or 11.2 and 11.6 mg/L), respectively, for C. albicans; and 1.2 and 1.1 micro M (or 1.9 and 1.7 mg/L), respectively, for C. neoformans. The killing of C. albicans and C. neoformans was achieved after 30 and 10 min exposure to the peptides, respectively. Combinations of MUC7 12-mer and Hsn5 12-mer, and of MUC7 12-mer and miconazole have a synergic antifungal effect on C. neoformans, with a fractional inhibitory concentration index (FICI) of 0.37 and 0.25, respectively; and a slightly lower than synergic effect on C. albicans, with a FICI of 0.63 and 0.56, respectively. In addition, using human erythrocytes, the two salivary peptides showed low levels of haemolytic activity. CONCLUSIONS: This study suggests that MUC7 12-mer and Hsn5 12-mer peptides may be suitable candidates for use in combination antifungal therapy.