Hedera rhombea inhibits the biofilm formation of Candida, thereby increases the susceptibility to antifungal agent, and reduces infection

Candida is an opportunistic pathogen and a common cause of fungal infections worldwide. Anti-fungal use against Candida infections has resulted in the appearance of resistant strains. The limited choice of anti-fungal therapy means alternative strategies are needed to control fungal infectious diseases. The aim of this study was to evaluate the inhibition of Candida biofilm formation by Hedera rhombea (Korean name: songak) extract. Biofilm formation was assessed using the crystal violet assay which showed a dose dependent reduction in the presence of extract with the biofilm formation inhibitory concentration of C. albicans (IC50 = 12.5μg/ml), C. tropicalis var. tropicalis (IC50 = 25μg/ml), C. parapsilosis var. parapsilosis (IC50 = 6.25μg/ml), C. glabrata (IC50 = 6.25μg/ml), C. tropicalis (IC50 = 12.5μg/ml), and C. parapsilosis (IC50 = 12.5μg/ml) without directly reducing Candida growth. Treatment with 6.25μg/mL of extract increased the antifungal susceptibility to miconazole from 32% decreasing of fungal growth to 98.8% of that based on the fungal growth assay. Treatment of extract dose-dependently reduced the dimorphic transition of Candida based on the dimorphic transition assay and treatment of 3.125μg/mL of extract completely blocked the adherence of Candida to the HaCaT cells. To know the molecular mechanisms of biofilm formation inhibition by extract, qRT-PCR analysis was done, and the extract was found to dose dependently reduce the expression of hyphal-associated genes (ALS3, ECE1, HWP1, PGA50, and PBR1), extracellular matrix genes (GSC1, ZAP1, ADH5, and CSH1), Ras1-cAMP-PKA pathway genes (CYR1, EFG1, and RAS1), Cph2-Tec1 pathway gene (TEC1) and MAP kinases pathway gene (HST7). In this study, Hedera rhombea extract showed inhibition of fungal biofilm formation, activation of antifungal susceptibility, and reduction of infection. These results suggest that fungal biofilm formation is good screen for developing the antifungal adjuvant and Hedera rhombea extract should be a good candidate against biofilm-related fungal infection.

Introduction Candida albicans is an opportunistic pathogen which is responsible for systemic infections in immunocompromised patients. C. albicans can persist inside the host and can be aided by drug resistance traits which often lead to failure of therapeutic strategies [1]. One of the features of Candida species pathogenesis is their ability to form biofilms, and nosocomial infections are often related to the ability to produce biofilm on mucosal surfaces and implanted medical devices [2][3][4][5].
The formation of biofilms involves multiple interconnected signaling pathways [6][7][8][9][10][11][12][13][14], and is a finely controlled process that involves attachment to surface and embedment in the exopolymer extracellular matrix [15][16][17][18]. The biofilm matrix acts to structure microbial communities and includes sessile cells that are frequently much more resistant to antifungal agents. In fact, adherent C. albicans cells without specific drug resistant gene expression are up to 1,000 times more resistant to common antifungal agents than planktonic cells [19]. Therefore, the biofilm of C. albicans is a reservoir of viable fungal cells that can potentially cause systemic infections, with a mortality rate of around 40-60% [20,21]. Efforts are being made to develop alternative strategies to eradicate biofilm-related infections [22,23]. Medicinal plants are used for diverse traditional methods to treat cancer, infection, fever, asthma, and many other diseases. Herbal medicines usually have fewer side effects compared to over-the-counter medicines. Accordingly, medicinal plants should be a new provenance of replacement remedies to treat Candida infectious diseases [24][25][26].
Hedera rhombea is a species of ivy (genus Hedera) that is native to the coast and some islands of East Asia [27,28]. In oriental medicine, H. rhombea is mainly used for arthritis, low back pain, hepatitis, high blood pressure, hemostasis, anti-rheumatism, facial paralysis, jaundice anti-inflammatory action, hypertension, and antitumor [27][28][29].
In this study, H. rhombea extract showed anti-biofilm formation activity against several fungi including C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Interestingly, the activity of the extract also increased susceptibility to antibiotics. These results suggest that H. rhombea extract can be used as a potential anti-fungal adjuvant to control the biofilm-related infection.

Strains
The Candida strains used in this study are listed in Table 1. All strains were stored in 20% glycerol at −70˚C and cultured in YPD plates [peptone 20 g/L (BD Difco, Belgium), yeast extract 10 g/L (BD Difco, Belgium) and 2% glucose (w/v) (Daejung, Korea)].

H. rhombea extraction
The leaf of H. rhombea was obtained from Jeju island. The extracts were produced using distilled water in 3L containing 300g of the sample at 80˚C for 8 h, concentrate at 40˚C using a rotary evaporator, and freeze-dried. 10mg of plant extract powder was dissolved in 1mL dimethyl sulfoxide for the experiments [15,[24][25][26][27][28]30].

Combinatorial antifungal effects of H. rhombea extract with antifungal agents
C. albicans were grown overnight in YPD diluted to 1 × 10 6 cells/mL. The induction of biofilm formation was performed as described above. Miconazole (3.125μg/mL), magnoflorine (3.125μg/mL) and dioscin (3.125μg/mL) alone or with the indicated concentration of H. rhombea extract were added and incubated at 37˚C for 24 h [26,[30][31][32][33][34]. The experiments were performed in triplicate. Dimorphic transition of C. albicans C. albicans were grown overnight in YPD medium. 1 × 10 6 cells/mL of Candida with or without extracts were incubated in RPMI 1640 medium and YPD media supplemented with 10% fetal bovine serum medium to induce dimorphic transition at 37˚C for 4 h. RPMI without sodium barcarbonate and with glutamine buffered with MOPS [3-(N-morpholino) propanesulfonic acid] to pH 7. Inhibition quantification of the yeast-to-hyphal-form transition was accomplished by counting the number of hyphae cells in the population as previously described [26,[32][33][34][35][36][37][38][39][40]. More than 1,000 cells were counted for each well in duplicate, and all assays were repeated five times. Representative results of images were obtained using a fluorescence microscope (EVOS1 FL, ThermoFisher Scientific, Waltham, MA, USA). The experiments were performed in triplicate.

Candida adherence test
A human epithelial keratinocyte HaCaT cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37˚C in a humidified atmosphere of 5% CO 2 .
HaCaT cells (0.5×10 6 cells per wall) were grown to confluence on 24 well plates for 24 h. DMEM were drained and then plates were cautiously washed three times with PBS to remove nonadherent cell. 1×10 6 cell/mL C. albicans mixed with H. rhombea extract ranged 1.56-25μg/ mL concentration was treated into each well. The 24 well plates were incubated at 37˚C for 24 h. Representative results of images were obtained using a microscope [32,34]. The experiments were performed in triplicate.

qRT-PCR analysis
C. albicans was grown overnight in YPD and diluted to 1 × 10 6 cells/mL. The diluted suspension with 6.25-100μg/mL of H. rhombea extract was incubated RPMI 1640 at 37˚C for 24 h with shaking. Total RNA was isolated using TRIzol reagent (Life Technology, Thermo Fisher Scientific, USA) according to the manufacturer's instruction and the reverse transcriptase (NanoHelix, Korea) reaction was prepared using 1 μg of RNA to obtain cDNA. qRT-PCR was carried out with the 2X SybrGreen qPCR Mater Mix (CellSafe, Korea). The transcript level of detected genes was calculated using the formula 2-ΔΔCT . Primer sequences used are listed in Table 2. ACT1 was used as internal control [26,[30][31][32][33][34][38][39][40]. The experiments were performed in triplicate.

MTT assay
The cytotoxicity of H. rhombea extract against HaCaT and THP-1 were tested by a slightly modified MTT assay [26,[32][33][34]. Briefly, 1×10 4 HaCaT and THP-1 in DMEM and RPMI1640 medium, respectively were added to each well containing the indicated concentration of extract and incubated for 24 h. Cell viability was calculated by optical density (OD 540 ) values measured using a microplate reader (BioTek Instruments, Korea) and is reported as the percentage of the vehicle control [32]. The experiments were performed in triplicate.

Statistical analysis
All experiments were performed at least three times and data were presented as the ± mean S.D.

Inhibition of Candida biofilm formation by the treatment of H. rhombea extract
Biofilm is especially important for the fungi to survive and infect. H. rhombea was used to test whether it blocked fungal biofilm formation or not. H. rhombea extract dose-dependently

H. rhombea extract increased the susceptibility to an antifungal agent against C. albicans
H. rhombea extract increased the susceptibility of antifungal agents against C. albicans. Treatment of 3.125μg/mL of extract increased the susceptibility to miconazole with 99% of fungal growth inhibition from 38% inhibition by miconazole treatment. The extract also increased the susceptibility to plant-derived antifungal candidates including magnoflorine (99% growth inhibition by 6.25μg/mL of extract compared with 29% growth inhibition by 3.125ug/ml of only magnoflorine treatment) and dioscin (99% growth inhibition by 6.25μg/mL of extract compared with 39% growth inhibition by 3.125ug/ml of only dioscin treatment) (Fig 2A, 2B  and 2C).

H. rhombea extract blocked dimorphic transition from yeast to hyphae form
The yeast-to-hyphae conversion is an important virulence property of C. albicans. The formation of hyphae aids the subsequent invasive growth of C. albicans to penetrate host tissues and lead to the establishment of systemic infection [7]. Extract tested whether the dimorphic transition was influenced and found 1.56μg/mL of the extract significantly inhibited hyphae formation in RPMI 1640 or a 10% FBS YPD medium, and extract with higher than 6.25μg/mL completely blocked the hyphae formation (Fig 3A, 3B, 3C and 3D).

H. rhombea extract reduced fungal adherence to the HaCaT cells
Fungal biofilm formation on device-associated infection is an important medical problem. H. rhombea extract showed dose-dependently reduced adhesion of the fungi to the human HaCaT cells with 90% reduction by treatment of 1.56μg/mL of extract (Fig 4).
Total RNA was extracted from C. albicans treated with the indicated concentration of H. rhombea extract using RNA extraction kit, converted to cDNA, and analyzed by qPCR with the respective primers.

H. rhombea extract did not affect the growth of human originated cell
The cytotoxic effects of H. rhombea extract on HaCaT cells and macrophage THP-1 were checked using an MTT assay. H. rhombea extract has no significant cytotoxic effect on both macrophage THP-1 and HaCaT cells (Fig 6A and 6B).

H. rhombea extract did not inhibit the Candida growth
Several antifungal agents inhibit fungal biofilm formation because they can kill the fungi and indirectly decrease biofilm formation. Extract of H. rhombea have no effect on the growth of C. albicans after 24h of incubation and that suggested the biofilm formation inhibition of extract is not because of the reduced growth of Candida (Fig 7).

Discussion
Many fungi including Candida live in and on the human body, but when Candida begins to grow uncontrollably, it can cause an infection known as candidiasis. In fact, Candida is the most common cause of fungal infections in humans [41]. Antifungal drugs can cause side effects and resistance, and there have been multiple recent reports of resistance including in the emerging problematic organism Candida auris [42][43][44]. Outbreak response is complicated by the limited treatment options and inadequate disinfection strategies. So new approaches with a new target for the anti-fungal agents are required.
In the present study, H. rhombea extract was tested for inhibition of Candida biofilm formation and showed strong anti-biofilm formation activity against all tested Candida species including C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis (Fig 1).
Even though the mechanism of Candida biofilm formation is diverse depending on the species, H. rhombea extract inhibited Candida biofilm formation that suggests there should be a common mechanism to induce the biofilm among the Candida species, but further studies must be conducted. Moreover, H. rhombea increased the antifungal activity of miconazole, magnoflorine, and dioscin (Fig 2), and reduced Candida infection (Figs 3 and 4), these results confirmed that biofilm formation is related to the susceptibility of antifungal agents and fungal infection, but further studies should be undertaken. In the case of the H. rhombea we tested, no inhibition of growth was observed. However, in the experiments in the reference literature [29], H. rhombea extract showed inhibition of growth. The reason might be the differences in the method of extracting plants.
Based on the gene expression analysis, the expression of genes related to biofilm formation, hyphae growth, and cell adhesion was significantly reduced by treatment with H. rhombea extract. The biofilm formation is tightly related to adherence to Candida and the early step is the attachment mechanism. C. albicans biofilm formation is determined by various

PLOS ONE
transcription factors including BCR1, EFG1, TEC1, and NDT80 that function as components in several pathways and influence adherence of Candida, suggesting that even though biofilm formation was initially tested, adherence or infection factors could be also regulated by treatment of H. rhombea extract. Further studies, including the precise target determination of H. rhombea extract, effector components, and in vivo testing must be carried out.

PLOS ONE
In conclusion, H. rhombea extract inhibited C. albicans biofilm formation, increased the antifungal activity of antibiotics and putative antifungal agents, and decreased fungal adherence to the host cell. Therefore, H. rhombea extract could be a good treatment option for biofilm-forming fungal infections.