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

archives of oral biology 56 (2011) 565–572

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In vitro synergism between berberine and miconazole against planktonic and biofilm Candida cultures Guo-Xian Wei 2, Xin Xu 3, Christine D. Wu 1,* Department of Pediatric Dentistry, University of Illinois at Chicago, College of Dentistry, MC850, 801 S. Paulina Street, Chicago, IL 60612-7212, United States

article info

abstract

Article history:

Objectives: To investigate the antimycotic activity of the plant alkaloid berberine (BBR), alone

Accepted 26 November 2010

and in combination with antifungal azoles, against planktonic and biofilm Candida cultures. Design: The minimum inhibitory concentrations (MICs) of BBR, miconazole (MCZ), and

Keywords:

fluconazole (FLC) towards Candida albicans, Candida glabrata, Candida kefyr, Candida krusei,

Combination therapy

Candida parapsilosis, and Candida tropicalis were determined by a microdilution method. For C.

Natural oral antifungal

albicans, the synergistic effects of BBR combined with MCZ or FLC were examined in a paper

Hydrastis canadensis

disc agar diffusion assay and checkerboard microdilution assay. The effect of the BBR/MCZ

Antimycotics

combination was further investigated in a C. albicans biofilm formation model with a dual-

Candida biofilm

chamber flow cell. The effect on metabolic activity of biofilm cells was established using 2,3bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)/menadione. Results: Berberine inhibited the growth of various Candida species (MICs 0.98–31.25 mg/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 P 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 P 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. Published by Elsevier Ltd.

1.

Introduction

Fungal infections, including oral candidiasis, constitute a persistent public health problem.1 The high global incidence

and prevalence of oral candidiasis may be attributed to an increasing usage of broad-spectrum antibiotics, cytotoxics, and corticosteroids, and to a growing number of immuno-suppressed individuals as well as those with common endocrine

* Corresponding author at: Department of Pediatric Dentistry, University of Illinois at Chicago, College of Dentistry, MC850, 801 S. Paulina Street, Room 469J, Chicago, IL 60612-7212, United States. Tel.: +1 312 355 1990; fax: +1 312 996 1981. E-mail address: [email protected] (C.D. Wu). 1

This author has previously published under the name of ‘‘Christine D. Wu-Yuan’’. Current address: Boston University, Goldman School of Dental Medicine, Department of Periodontology and Oral Biology, 700 Albany Street, CABR W221, Boston, MA 02118, United States. 3 Current address: Department of Operative Dentistry, West China College of Stomatology, Sichuan University, Chengdu, China. 0003–9969/$ – see front matter . Published by Elsevier Ltd. doi:10.1016/j.archoralbio.2010.11.021 2

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disorders (such as diabetes mellitus) or severe nutritional deficiencies.2 Approximately 50–60% of the human population harbours Candida species as part of the normal oral microbiota.3 A variety of predisposing factors aids in the change of Candida from a commensal to a parasitic existence, resulting in the resurgence of oral candidiasis as a relatively common illness.2–4 Candida albicans has been considered the most virulent and frequently isolated Candida species associated with human oral candidiasis.2,5 However, the non-albicans Candida species, such as Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida dubliniensis, and Candida krusei, are also frequently isolated from human fungal infectious sites.6 Amongst these, C. dubliniensis, C. glabrata, and C. krusei have been found more frequently in HIV-positive patients than in non-HIV populations.5 C. albicans may colonize the host on mucosal surfaces as a biofilm.7 The formation of the Candida biofilm is an important step in the series of events leading to C. albicans infections.7 Biofilms of pathogenic fungal species such as C. albicans are frequently observed in patients treated with indwelling devices, causing chronic as well as life-threatening acute systemic infections.8,9 Of particular concern is that biofilms display increased resistance to antifungal therapy, cause failure of implant devices, and serve as a reservoir or source for future continuing infections.10,11 Therefore, the search for new antifungal agents effective against biofilms has important clinical implications that may affect the outcomes of patients suffering from these difficult-to-treat infections. Common treatments for candidiasis include antifungal agents belonging to the azoles, polyenes, and echinocandins. These agents impair fungal cell integrity by inhibiting the synthesis of ergosterol and glucan in the cell membrane and cell wall.12 However, their therapeutic applications in certain patient population are limited, due to their toxicity and the emergence of drug-resistant Candida species.13–15 The latter represents a special risk in immunocompromised patients.5 Berberine is an alkaloid present in a number of clinically important medicinal plants, including Hydrastis canadensis (goldenseal), Coptis chinensis (coptis or golden thread), Berberis aquifolium (Oregon grape), Berberis vulgaris (barberry), and Berberis aristata (tree turmeric).16 Berberine functions as a defence compound in plants by protecting them against invading micro-organisms. Studies have investigated the pharmacology and clinical efficacy of berberine, and have reported its broad antimicrobial spectrum against bacteria, fungi, protozoans, virus, helminthes, and Chlamydia.16–20 Amongst the berberine-containing phytomedicines, H. canadensis (goldenseal) is one of the top 12 remedies sold in the U.S., and its historic usage for treating oral diseases has been documented.18 Although researchers have reported the antibacterial activity of berberine against selected oral pathogens,18,19,21–23 and its effects on in vitro planktonic fungi associated with oral candidiasis,20,24–26 relatively few have examined its effects in animal models27 or other model to simulate the physiological conditions. The use of antimicrobial agents in combination has been a common practice to improve the therapeutic efficacy and to reduce the severity of adverse effects of these drugs. Combinations may also lower drug dosage, thus reducing their toxic side-effects. In vitro synergy between berberine and

fluconazole has been reported against C. albicans planktonic cells.20,26 However, whether such synergy exists against the more resistant biofilm cells is not clear at the moment. In this study, we explored the antifungal activity of berberine against medically important Candida species, and examined the synergistic effects when berberine is combined with commonly used antifungal agents. Given the antifungal resistance and clinical relevance of biofilms, our studies also addressed the effectiveness of berberine/antifungal drug combinations in biofilm formation and viability in vitro.

2.

Materials and methods

2.1.

Candida strains and antifungal agents

Candida strains used were C. albicans SC5314 (provided by Dr. H.Y. Sroussi, University of Illinois at Chicago, College of Dentistry), C. albicans ATCC10231, C. glabrata ATCC66032, Candida kefyr ATCC46467, C. krusei ATCC14213, C. parapsilosis ATCC22019, and C. tropicalis ATCC13803. All species were cultured in Sabouraud dextrose agar (SDA, Difco, Sparks, MD, USA) at 37 8C for 24 h to obtain primarily the yeast form of the fungus. Cells were harvested and suspended in 0.9% NaCl to an OD of 0.1 at 530 nm (Milton Roy Spectronic 601, Thermo Spectronic, Madison, WI, USA), corresponding to 5  106 cells/ mL. Sabouraud dextrose broth (SDB, Difco) was routinely used for growth assays. Antifungal agents used in this study included miconazole (MCZ, Fisher BioReagents, Fair Lawn, NJ, USA) and fluconazole (FLC, LKT Laboratories, Inc., St. Paul, MN, USA). Berberine (BBR), a plant alkaloid from H. canadensis (goldenseal), was purchased from Sigma–Aldrich (St. Louis, MO, USA). MCZ was dissolved in 100% dimethyl sulfoxide (DMSO), whereas FLC and BBR were prepared in sterile distilled water.

2.2. Determination of minimum inhibitory concentrations in broth The minimum inhibitory concentrations (MICs) of BBR, MCZ, or FLC against the Candida species were determined according to the microdilution method of the CLSI (formerly NCCLS) (M27-A)28 using 96-well microtiter plates with some modifications. Each well contained the Candida species at a final concentration of 2.5  103 cells/mL, SDB, and test agent. The final concentrations ranged from 1.95 to 250 mg/L for BBR; from 0.125 to 4 mg/L for MCZ; and from 0.5 to 16 mg/L for FLC. SDB without test agents was included as an agent-free control, and uninoculated SDB as medium blank. All plates were incubated in an aerobic incubator at 37 8C for 24 h, after which the growth was determined spectophometrically at 530 nm by means of a microplate reader (PowerWave 200, Bio-Tek Instruments, Winooski, VT, USA). The MIC90 was defined as the lowest concentration of test agent demonstrating more than 90% of growth inhibition compared with that of the agent-free growth control. The MIC90 of reference strain C. albicans ATCC10231 (used for quality control) was in the expected range for FLC in SDB (0.25–1.0 mg/L).28 The data are reported as the median of at least 3 independent tests.

archives of oral biology 56 (2011) 565–572

2.3. Assessment of drug synergy against C. albicans growth 2.3.1.

Paper disc agar diffusion assay

To examine the effect of BBR alone or in combination with MCZ or FLC on the growth of Candida species, we performed the paper disc-diffusion assay in agar. Candida yeast cell suspension in half-strength molten SDA (4 mL, 2.5  106 cells/ mL) was poured evenly over a pre-prepared SDA plate. After the top layer had solidified, sterile paper discs (Sterile Blanks Concentration Discs, ¼00 , Difco Bacto, Detroit, MI, USA) were placed on the agar surface, and test agents were added, i.e., 5–40 mg for BBR; 3–12 mg for MCZ or FLC; and 5–40 mg BBR and 9 mg of MCZ or FLC for combination. After incubation at 37 8C for 48 h, the size and pattern of the growth inhibition zone around the disc on agar were evaluated.

2.3.2.

to allow for initial attachment. After 2 h of initial colonization, the flow of SDB with or without test agents was started at a rate of 0.2 mL/min to mimic human saliva flow rates,32 and to generate a shear rate (9.7 s1) that was compatible with the fluid flow reported in the oral cavity (0.1–50 s1).33 For evaluation of the effects of BBR alone or in combination with MCZ on the development of C. albicans biofilm, one of the flow chambers was perfused with SDB only (agent-free control), whilst the other was perfused with SDB containing BBR (16 mg/L) or MCZ (0.8 mg/L) alone, or a combination of the two agents. Dynamic comparison of the effects of antifungal agents on biofilms between treated and untreated cells was performed in real time by dark-field microscopy (Leica DMR, Wetzlar, Germany). Micrographic images were taken at the desired time intervals and analysed with Image-ProPlus 5.1 software (Media Cybernetics, Inc., Bethesda, MD, USA), Data were statistically analysed by student t test.

Checkerboard microdilution assay

Synergistic effects of BBR in combination with MCZ or FLC on the growth of C. albicans SC5314 were quantitatively determined by a checkerboard microtiter plate method.29 Each well of the 96-well microtiter plate contained C. albicans SC5314 (final concentration of 2.5  103 cells/mL), SDB, and serially diluted test agents in combinations. The concentrations for BBR were 1–30 mg/L and 0.125–64 mg/L for MCZ or FLC. After incubation at 37 8C for 24 h, cell growth was examined at 530 nm by means of a microplate reader as previously described. All MIC data were expressed as the median values obtained from at least three independent experiments. We used the MIC values of BBR/MCZ or BBR/FLC to determine the fractional inhibitory concentration (FIC), i.e., the ratio of the MIC of an agent used in combination to the MIC of the agent P used alone. The FIC index ( FIC, the sum of individual FICs) P was calculated using the formula: FIC = MIC(Acombo)/MIC(AaP FIC < 0.5 indicates synergy; lone) + MIC(Bcombo)/MIC(Balone). 0.5–4.0, indifference; and >4, antagonism.29

2.4. Assessment of drug synergy against C. albicans biofilm 2.4.1.

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Dual-flow cell model

An in vitro dual-flow cell model (FC 271, BioSurface Technologies, Bozeman, MT, USA), modified from Leung et al.30 was used. The flow cell consisted of two compartments, each containing a polycarbonate flow chamber with two recesses to hold glass discs (polycarbonate coupons, RD 128 PC 10 mm in diameter and 2 mm in thickness) upon which biofilms were formed. Glass discs provided appropriate background that allowed for visualization of the unstained fungal biofilms by dark-field microscopy. For biofilm formation, the glass discs in flow cells were pre-conditioned with sterile artificial saliva (AS), which contained 1 g/L Lab-lemco powder (Oxoid, Basingstoke, UK), 2 g/ L yeast extract (Difco), 5 g/L proteose peptone (Bacto, Sparks, MD, USA), 2.5 g/L mucin from porcine stomach (Sigma), 0.2 g/L calcium chloride (Sigma), and 0.2 g/L potassium chloride (Sigma) in distilled water. After the mixture was autoclaved, a sterilized urea (Sigma) solution was added (final concentration, 0.5 g/L).31 The flow chambers were pre-conditioned for 1 h, and C. albicans SC5314 yeast cell suspension (1.5 mL, 1  107 cells/mL in AS) was injected into both flow chambers

2.4.2.

Microtiter plate model

The effects of BBR and MCZ on biofilm cell viability were established by measurement of cell metabolic activity of C. albicans biofilm in a 96-well microtiter plate based on the method reported by Pierce et al.34 A 100-mL aliquot of a suspension of C. albicans SC5314 yeast cells in SDB (1  106 cells/mL) was added in wells for biofilm formation. After incubation at 37 8C for 24 h, the growth medium was carefully removed by aspiration without disruption of the integrity of the biofilm, and the formed biofilms were carefully washed 3 times with phosphate-buffered saline (PBS, 50 mM, pH 6.8) to remove non-adherent cells. The plate with the seeded biofilms was used subsequently for a checkerboard microdilution assay to determine the effect of BBR/MCZ on the metabolic activity of the biofilm. The final concentrations of BBR ranged from 16 to 4000 mg/L, and for MCZ, from 9 to 1250 mg/L. After incubation at 37 8C for 24 h, the antifungal agents were removed, and the treated biofilms were washed 3 times with PBS. The metabolic activity of the biofilms was determined by the addition of 100 mL of 2,3-bis(2-methoxy-4nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)/ menadione solution (0.5 g/L XTT and 1 mM menadione at final concentrations). All plates were covered with aluminium foil and further incubated in the dark at 37 8C for 2 h. Aliquots (70 mL) of coloured cell-free supernatant in each well were then transferred to another blank microtiter plate, and absorbance was determined spectrophotometrically at 490 nm. The sessile minimum inhibitory concentration (SMIC90) was defined as the lowest concentration resulting in a 90% reduction in absorbance when compared with agentP free biofilm control. The FIC was calculated as described for the planktonic cells to determine synergism or antagonism.

3.

Results

3.1. FLC

Susceptibility of Candida species to BBR, MCZ, and

The inhibitory activities of BBR, MCZ, and FLC against a series of medically important fungi were investigated (Table 1). Berberine exhibited MIC values ranging from 1.95 to 31.25 mg/L. The

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Table 1 – Susceptibility of Candida species to berberine and antifungal agents. MIC90 (mg/L)a

C. C. C. C. C. C. C.

albicans SC5314 albicans ATCC10231 glabrata ATCC66032 kefyr ATCC46764 krusei ATCC14243 parapsilosis ATCC22019 tropicalis ATCC13803

BBR

MCZ

FLC

31.25 31.25 5.89 3.91 1.95 31.25 7.81

2.00 0.25 0.13 0.03 0.25 0.05 0.25

16.00 0.50 0.38 0.25 0.25 0.19 0.65

BBR, berberine; MCZ, miconazole; FLC, fluconazole. MIC90 defined as the lowest concentration of agent demonstrating more than 90% inhibition of the growth in comparison with that of the agent-free growth control. Each data-point represents the median of at least 3 independent tests. The MIC90 of reference strain C. albicans ATCC10231 was in the expected range for FLC in SDB (0.25–1.0 mg/L).28

a

susceptibility to BBR, ranked from low MICs to high MICs, was in the order of C. krusei > C. kefyr > C. glabrata > C. tropicalis > C. parapsilosis and C. albicans (Table 1). The MICs of MCZ and FLC against the test strains of Candida species were lower than those of BBR, ranging from 0.02 to 0.25 mg/L, and 0.06 to 16 mg/L, respectively. Amongst the two C. albicans strains tested, C. albicans SC5314 was the least susceptible to MCZ and FLC. The higher than expected FLC MIC for C. albicans SC5314 may be attributed to the usage of Sabouraud dextrose broth for growth experiments in this study. C. albicans SC5314 was selected for further studies exploring the synergistic effects of BBR/MCZ and BBR/FLC combinations.

3.2. Effects of BBR/MCZ (or FLC) combinations on C. albicans growing on agar and in broth Paper disc agar diffusion testing revealed that BBR in combination with MCZ or FLC enhanced the growth inhibition

[()TD$FIG]

Table 2 – Synergistic effect in checkerboard microdilution assay of berberine combined with miconazole or fluconazole on growth of C. albicans planktonic cells. P Agent MIC90 (mg/L) FICa

BBR MCZ BBR FLC

Alone

Combo

31.25 2.00 31.25 16.00

3.90 0.13 3.90 2.00

0.19 0.25

BBR, berberine; FLC, fluconazole; MCZ, miconazole. a P FIC = M IC ( A c o m b o ) /M IC ( A a l o n e ) + M IC ( B c o m b o ) / MIC ( B a l o n e ) . P FIC < 0.5 indicates synergy; 0.5–4.0, indifference; and >4, antagonism. Data represent the median of at least 3 repeated experiments.

of C. albicans SC5314 (Fig. 1). Three types of inhibition zone were noted: (1) a transparent area surrounding the disc, showing complete inhibition (CI); (2) an opaque area, resulting from partial inhibition (PI); and (3) a transparent area between discs, demonstrating synergistic inhibition (SI) by the two agents (Fig. 1a). BBR (40 mg) alone showed a slight PI zone (Fig. 1d). FLC and MCZ showed a dose-dependent increase in zone diameter. When a 40-mg quantity of BBR was combined with 9 mg of either FLC or MCZ, inhibition zones increased in size and changed in appearance from PI to CI (Fig. 1), clearly indicating a synergistic effect between the combined drugs. To quantify the indicated synergistic effects of BBR in combination with either of the azole agents, we evaluated BBR/MCZ and BBR/FCZ combinations in a checkerboard microdilution assay. The MICs of both FLC and MCZ were reduced 8–10 times when the individual agents were comP bined with BBR (31.25 mg/L) (Table 2). The calculated FIC was 0.25 for BBR/FLC and 0.19 for BBR/MCZ, indicating synergism and supporting the observations made in the disc diffusion assay.

Fig. 1 – Effect of berberine alone and in combination with fluconazole or miconazole on growth of C. albicans SC5314. Growth inhibition zones on SDA (panel a). SI, synergistic inhibition zone; CI, complete inhibition zone; PI, partial inhibition zone. Inhibition zones of agents at different doses (panels b–f). Panels a0 –f0 describe the images for panels a–f. B, berberine; F, fluconazole; M, miconazole; BF or BM, combination of berberine with either fluconazole or miconazole. Number represents the quantity of agent (mg) in each disc.

archives of oral biology 56 (2011) 565–572

[()TD$FIG]

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Fig. 2 – Effect of BBR alone and in combination with MCZ on C. albicans biofilm development in a dual-flow cell, as revealed by dark-field microscopy. (A) The formation of C. albicans biofilm expressed in terms of attached C. albicans cells/mm2. No significant difference was found between control (open bar) and BBR (16 mg/L) (dotted bar) or MCZ (0.8 mg/L) (hatched bar) alone (t test, P > 0.05). However, significant differences between control or each agent alone and BBR/MCZ (16/0.8 mg/L) combination (solid bar) were observed (P < 0.01). Results represent one of the 3 experiments. Error bars represent the standard deviation of the 6 observations for each time-point. (B) The continuous perfusion with BBR/MCZ medium inhibits biofilm formation. Images of untreated biofilm cells [panels (a) and (e), agent-free control], showing the development of biofilm from C. albicans adhering to artificial saliva-coated glass disc surfaces in the flow chamber perfused with agent-free medium. Images of BBR/MCZ-treated biofilm cells [panels (d) and (h)]. Side-by-side images of treated vs. untreated cells were obtained at intervals of 4 h [panels (a)–(d)], 14, 16, 18, 20, 22 h (not shown), and 24 h [panels (e)–(h)] after inoculation into the parallel chambers of the dual-flow cell. Magnification, 200T.

3.3. Effects of BBR/MCZ combinations on in vitro biofilm formation Given the importance of biofilm formation in Candida establishment and colonization, inhibition of the formation of C. albicans biofilm formation by BBR, alone and in combination with MCZ, was investigated in a flow cell model. In this model, C. albicans cells attached to glass surfaces preconditioned with artificial saliva. The area of cell coverage was 9.7  3.2  103/mm2 after 4 h, increasing to 59  25  103/

mm2 after 14 h, and 242  38  103/mm2 after 24 h (Fig. 2A). Dark-field microscopic examination of 24-h biofilm revealed the presence of abundant yeast clusters and microcolonies, without filamentation or cell elongation (Fig. 2B). In the presence of BBR (16 mg/L) or MCZ (0.8 mg/L), no significant inhibition of biofilm formation was observed, and the surface coverage areas were comparable with those of agent-free control throughout the 24-h period (t test, P > 0.05) (Fig. 2A). However, biofilm formation was virtually completely inhibited in the presence of the BBR/MCZ combination, showing

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Table 3 – Effect of berberine in combination with miconazole on metabolic activity of C. albicans biofilm. P Agent SMIC90 (mg/L)a FIC

BBR MCZ

Alone

Combo

2000 625

250 78

0.25

BBR: berberine; MCZ: miconazole. a SMIC90 defined as the lowest concentration of agent demonstrating more than 90% inhibition of the metabolic activity against C. albicans biofilm in comparison with that of the agent-free growth control. Data represent the median of at least 3 repeated experiments.

>91% inhibition at the latest tested time-point (24 h). Photomicrography of the biofilm formed under these conditions confirmed few adherent C. albicans cells or microcolonies (Fig. 2B, panel h). Inhibition of the metabolic activity of pre-formed C. albicans biofilm was evaluated in a microtiter plate model. In this model, biofilms were first grown for 24 h before being exposed to BBR or BBR/MCZ combinations. As shown in Table 3, the SMIC90, the lowest concentration of agent that showed 90% inhibition of metabolic activity against C. albicans biofilm compared with the agent-free control, was 2000 mg/L for BBR alone and 625 mg/L for MCZ alone. The relatively high doses required for an effect to be observed were consistent with the reported resistance of biofilms to cell killing.35,36 When BBR and MCZ were combined, the SMIC90 values decreased fourfold for BBR (250 mg/L) or MCZ (78 mg/L), and the calculated P FIC value was 0.25. The results demonstrated that synergistic effects can also be observed in cells that have already grown in a biofilm, which are typically the most difficult to treat.

4.

Discussion

Antifungal azoles, including fluconazole and miconazole, are commonly used to treat fungal infections in patients. The emergence of Candida strains resistant to this class of conventional antifungal drugs has posed increasing medical complications37–39 and supports the need for alternative therapeutic options. Investigations on novel classes of drugs or novel combinations of drugs are ongoing to thwart the development of drug resistance and to achieve an increase in overall drug efficacy. We explored the usefulness of BBR, an alkaloid isolated from a variety of medicinal plants, which has previously been reported to exert a broad antimicrobial spectrum against micro-organisms including fungi and oral pathogens.16,18,19,22 The present study demonstrated that BBR exhibits antifungal activity against Candida species. Most of the non-albicans Candida species tested were more susceptible to BBR than C. albicans (Table 1). Amongst the non-albicans Candida tested, C. krusei was the most sensitive to BBR. These results suggested the potential benefit of BBR to immunocompromised individuals through inhibition of both C. albicans and the non-albicans Candida species. More importantly, this study demonstrated that BBR/MCZ and BBR/FLC exert strong synergistic effects towards both the planktonic and the

biofilm phase of C. albicans. In particular, the observation against biofilm phase contributes to the need for drug therapies amenable to treat this resistant form of fungal growth, which is often associated with persistent, devicerelated fungal infections. In the natural ecosystem, most microbes exist as attached communities of cells within an organized biofilm, and approximately 80% of all human microbial infections have a biofilm aetiology.35,40 Microbial biofilm exhibits increasing levels of resistance to most antibiotics or therapeutic agents35,36 and reduced susceptibility of Candida biofilms to antifungal agents has also been well-documented.9,10,41 For example, FLC is less effective against C. albicans biofilm than against its planktonic counterparts.34 We also noted that increased concentrations of MCZ or BBR were required to reduce C. albicans biofilm metabolic activity, the SMIC90 values of BBR and MCZ being 60- and 300-fold higher, respectively, than the MICs for their planktonic counterparts (Tables 2 and 3). To maintain their niche, adherent populations need to withstand the continuous bathing action of physiological fluids (e.g., saliva and blood), which also provide water and nutrients to fungal cells. In an effort to simulate the physiological conditions encountered by biofilm microorganisms in vivo, we used flow cells to study bacterial30,42 and fungal biofilms43,44 under flowing conditions. The use of a dual-flow cell, coupled with dark-field microscopy and an image analysis technique, provides a unique method for examination of the effects of antifungal agents on biofilm formation under a constant flow of replenishing nutrients and shear forces. In this study, C. albicans yeast cell biofilm was successfully developed in a flow cell perfused with SDB. Formation of C. albicans biofilm is believed to occur in three stages: early, intermediate, and mature. The biofilms formed in this study consisted mainly of the yeast form with few hyphal cells, which were similar to the composition of early biofilms reported previously.45,46 The early stage of C. albicans biofilm, characterized by the adhesion of single cells to the surface, is the initial step towards the formation of mature biofilms.45,46 Inhibition of early-stage biofilm is crucial to prevent the development of mature C. albicans biofilm that shows an increased resistance to antimycotic drugs. The present study revealed that neither BBR (16 mg/L) nor MCZ (0.4 mg/L) inhibited biofilm development up to 24 h, but virtually complete inhibition was observed when the agents were combined (Fig. 2). The inhibition observed could be related either to inhibition of cell growth (multiplication) or to interference with adhesion of growing cells to the artificial saliva-treated glass discs. Based on the growthinhibitory effects of BBR/MCZ in the broth microdilution assay, it is reasonable to assume that the effect of the combination on biofilm formation is related to an effect on C. albicans cell growth rather than an effect on cell adhesion, although this additional desirable effect cannot be excluded at this time. In the search for the mechanism of synergism between BBR and MCZ, their individual antifungal modes of action need to be considered. The antimicrobial mechanism of BBR has been reported by investigators.12,17,47–50 Davidson et al. reported that BBR was an excellent DNA intercalator by binding

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preferentially to AT-rich sequences.47 BBR was found to inhibit key enzymes (sterol 24-methyl transferase and chitin synthase) in the pathways of ergosterol and chitin biosynthesis, leading to increased permeability of cell membranes and cell walls of C. albicans.48–50 In addition, BBR may also contribute to oxidative cell damage by increasing the generation of reactive oxygen species (ROS).51 MCZ belongs to the azole antifungals, which inhibit biosynthesis of ergosterol, a critical component of fungal cell membranes, leading to permeability changes.52,53 The inhibition of ergosterol biosynthesis also results in the accumulation of toxic methylated sterol intermediates and, subsequently, arrests fungal cell growth.54 In addition, MCZ may induce changes in the actin cytoskeleton55 and produce reactive oxygen species (ROS)56 leading to cell death. Xu et al. have recently demonstrated that BBR in combination with FLC augmented the production of endogenous ROS through enhancing the tricarboxylic acid cycle and inhibiting ATPsynthase activity.51 Although it is not clear at this time, a similar mechanism may exist in the BBR/MCZ combination against biofilm cells. In summary, analysis of the data obtained from this study demonstrates that the natural antimicrobial agent BBR displays in vitro growth inhibition of C. albicans and selected pathogenic non-albicans Candida species. The observed synergism between this natural agent and MCZ may open new avenues for exploring the potential therapeutic application of such combinations in the treatment of persistent oral candidiasis.

Acknowledgments The authors thank Dr. Herve Sroussi and Dr. Lin Tao at the College of Dentistry, University of Illinois at Chicago (UIC) for providing test Candida species; and the United States Army Dental and Trauma Research Detachment (USADTRD), Great Lakes, Illinois, for use of the Leica DMR microscope. This study was supported by the Department of Pediatric Dentistry, College of Dentistry, the University of Illinois at Chicago, Chicago, IL. Funding: This study was supported by the Department of Pediatric Dentistry, UIC College of Dentistry. Competing interests: None declared. Ethical approval: Not required.

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