Antibiofilm Activity of Essential Fatty Acids Against Candida albicans from Vulvovaginal Candidiasis and Bloodstream Infections

Introduction

Candida species are the most common human fungal pathogens.^1,2 Although clinical infections caused by non-albicans Candida spp. (eg, C. tropicalis, C. glabrata, and C. parapsilosis) have increased in recent years, C. albicans remains the most predominant Candida spp. causing infectious diseases in both adult and pediatric populations.³ Most recurrent and severe infections by C. albicans are associated with the formation of biofilms on the surface of biological or artificial surfaces.⁴ Candida biofilms are complex communities of yeast and filamentous cells surrounded by an extracellular matrix.⁵ The biofilm formation of C. albicans is an essential virulence factor because it can increase tolerance to conventional antifungal drugs and the host immune system.⁶

Candida spp. can invade various tissues and organs of the human body and are responsible for superficial (mucosal and cutaneous) and systemic infection. Vulvovaginal candidiasis (VVC) is a common infection of the vaginal mucosa caused by Candida spp.⁷ It is estimated that 70%-75% of women experience VVC at least once in their lifetime, with 40%-50% of these women experiencing recurrent VVC (RVVC).⁸ The ability of Candida spp. to form biofilms is crucial in recurrent VVC.⁹ Candida bloodstream infection (candidemia) is a severe systemic infection, with a crude mortality rate of 25%-50%.^10,11 Recent studies have shown that the biofilm formation of Candida spp. is associated with higher mortality in patients with candidemia.^12–15

Due to the increasing occurrence of Candida biofilm-related infections and their immense impact on the health care system, there is an urgent need to find novel efficient antifungal agents. Fatty acids, which are promising to be considered as the next generation of antimicrobial agents to treat bacterial infections,¹⁶ are also found to exhibit significant inhibitory effects against Candida spp. and have recently been considered potential alternative antifungal agents.^17–22 Nevertheless, few published studies have investigated the activities of essential fatty acids (EFAs) on biofilms in the formation and mature stages of clinical C. albicans isolates. EFAs, which are types of polyunsaturated fatty acids (PUFAs), cannot be synthesized by the human body and must be obtained in the diet. It has been suggested that some PUFAs act as antibiofilm agents through functioning as molecular signals, as well as influencing PUFA metabolism and other metabolic activities.^17,19

The EFAs are classified into two series, the omega-3 (ω-3) and the omega-6 (ω-6).²³ The ω-3 series is derived from α-linolenic acid (ALA, 18:3). The ω-6 series is derived from linoleic acid (LOA, 18:2). ALA is metabolized to eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6); and LOA is metabolized to γ-linolenic acid (GLA; 18:3) and arachidonic acid (AA, 20:4) (Figure 1). This study aimed to investigate the effect of six EFAs against biofilm formation and mature biofilms of C. albicans strains, which were isolated from VVC and candidemia.

Figure 1 Chemical structure of EFAs. (A) ALA, α-linolenic acid. (B) EPA, eicosapentaenoic acid. (C) DHA, docosahexaenoic acid. (D) LOA, linoleic acid. (E) GLA, γ-linolenic acid. (F) AA, arachidonic acid.

Materials and Methods

Chemicals

The EFAs, namely α-linolenic acid (ALA; 18:3 ω-3), eicosapentaenoic acid (EPA; 20:5 ω-3), docosahexaenoic acid (DHA; 22:6 ω-3), linoleic acid (LOA; 18:2 ω-6), γ-linolenic acid (GLA; 18:3 ω-6), and arachidonic acid (AA; 20:4 ω-6) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluconazole was purchased from Solarbio (Beijing, China). Dimethyl sulfoxide (DMSO) was used as a solvent to dissolve the EFAs and fluconazole. Stock solutions were kept at −20°C for a maximum of 4 weeks and were further diluted in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) before use. DMSO at <0.1% did not affect microbial growth or biofilm formation.

Strains and Culture Conditions

A total of 135 clinical non-duplicate C. albicans isolates were collected from the Department of Infectious Diseases and Clinical Microbiology, Beijing Chao-Yang Hospital, Capital Medical University (Beijing, China) [vaginal isolates (n=70) from patients diagnosed with VVC and blood isolates (n=65) from patients diagnosed with candidemia]. C. albicans ATCC 90028 was used as the reference control. All C. albicans isolates were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) (VITEK-MS; bioMérieux, France; IVD version 3.0), and further confirmed by PCR sequencing of the ITS region of rDNA. C. albicans strains were routinely refreshed from the frozen stocks at −20°C and inoculated at least twice onto Sabouraud Dextrose Agar (SDA) at 35°C for 24 h before all experiments.

Determination of Minimum Inhibitory Concentration of Fluconazole

To determine the minimum inhibitory concentration (MIC) of fluconazole against C. albicans strains, a broth microdilution method was used as described by CLSI M27-A4.²⁴ The MIC was defined as the lowest concentration at which there is a 50% decrease in the growth of planktonic C. albicans. Briefly, the initial concentration of the C. albicans suspension was adjusted to 1×10³ CFU/mL in RPMI 1640 medium. The final concentrations of fluconazole were 0.125–64 μg/mL. The MICs were determined spectrophotometrically at OD_{630 nm} following a 24h incubation period at 35°C. C. albicans ATCC 90028 was used as a quality control strain. All isolates were measured in triplicate.

Biofilm Formation by Crystal Violet Method

To measure the ability of C. albicans to form a biofilm, the crystal violet assay in sterile 96-well round-bottomed polystyrene plates (Corning, NY, USA), was performed as described previously.²⁵ Briefly, C. albicans cells were harvested from overnight cultures and inoculated into RPMI 1640 medium at a dilution of 1:100. Then, 200 µL of suspension (1×10⁵ CFU/mL) was added to each well of the microtiter plate and incubated at 35°C in an atmosphere of 5% CO₂ for 24 h.

To determine the biofilm mass, the wells were washed three times with pH 7.2 phosphate-buffered saline (PBS) to remove planktonic cells. The remaining attached cells were fixed with 200 μL methanol for 15 min, stained with 200 μL crystal violet (0.2%) for 20 min, and washed with distilled water to remove the excess crystal violet. Then, the bound crystal violet was extracted with 200 μL of 33% acetic acid. The OD values were measured at 630 nm using a Multiskan EX microplate photometer (Thermo Fisher Scientific, Waltham, MA, United States). Experiments were independently repeated three times. Wells with RPMI 1640 medium were used as negative controls. The OD cut-off value (ODc) for biofilm formation was defined as three standard deviations above the mean OD value of the negative control.

The isolates were categorized as strong biofilm producers (OD_{630 nm} ˃ 4×ODc), moderate biofilm producers (2×ODc ˂ OD_{630 nm} ≤ 4×ODc), weak biofilm producers (ODc ˂ OD_{630 nm} ≤ 2×ODc), and non-biofilm producers (OD_{630 nm} ≤ ODc).²⁶

Inhibition of Biofilm Formation

To detect the effects of fluconazole or EFAs treatment on biofilm formation of C. albicans, the cell suspensions were co-cultured with different concentrations of fluconazole (0.125–256 μg/mL) or EFAs (0.01, 0.1, and 1 mM) in microtiter plates. RPMI 1640 medium without fluconazole or EFAs was added to the wells as the control for biofilm formation. Then, the plates were incubated at 35°C in 5% CO₂ for 24 h. Blank solvent control (RPMI 1640 medium supplemented with 0.1% DMSO) and blank medium control (RPMI 1640 medium) were also performed. The biofilm mass was detected using the crystal violet method as described above. EFAs at ≤ 1 mM did not affect the biofilm detection.

The minimum biofilm inhibitory concentration (MBIC) was defined as the minimum concentration of antifungal agents, leading to a 50% reduction of biofilm formation compared to the control group. The biofilm inhibition rate (%) was calculated according to the formula:²⁷

Eradication of Mature Biofilm

To measure the biofilm eradication ability of fluconazole or EFAs against C. albicans, the mature biofilms of C. albicans were pre-formed by adding only cell suspensions and culturing for 24h. After incubation, the non-adherent cells were removed by washing with PBS twice. Then, RPMI 1640 medium containing different concentrations of fluconazole (0.125–256 μg/mL) or EFAs (0.01, 0.1, and 1 mM) were added to the wells. The mature biofilm treated with fresh RPMI 1640 medium served as biofilm control. The microtiter plates were incubated at 35°C for an additional 24 h. Blank solvent control (RPMI 1640 medium supplemented with 0.1% DMSO) and blank medium control (RPMI 1640 medium) were also performed. The biofilm mass was detected using the crystal violet method as described above. EFAs at ≤ 1 mM did not affect the biofilm detection.

The minimum biofilm eradication concentration (MBEC) was defined as the minimum concentration of antifungal agents that eradicated mature biofilm by 50% compared to the control group. The biofilm eradication rate (%) was calculated according to the formula:²⁷

Evaluation of the Effect of Essential Fatty Acids on Fluconazole Susceptibility

To evaluate the effect of EFAs on fluconazole susceptibility, the MICs, MBICs, and MBECs of fluconazole (0.125–256 μg/mL) combined with different EFAs (0.1 mM) on C. albicans strains were tested as described above. Growth and blank controls were also included. The MICs were determined spectrophotometrically at OD_{630 nm.} The MBICs and MBECs were detected using the crystal violet method.

Growth Curves of C. albicans

Growth curves for C. albicans strains were established in the absence and presence of EFAs (0.01, 0.1, and 1 mM). The assay was performed using the HB&L system (Alifax, Polverara, Italy) as previously described.²⁸ Briefly, C. albicans cells were inoculated into 2 mL of RPMI 1640 medium (1:100) with an initial concentration of 1×10⁵ CFU/mL. The inoculated culture was grown at 35 °C under gentle agitation, and the McF was determined every hour until 14 h.

Statistical Analysis

Statistical analysis was performed using SPSS 20.0 statistical software (IBM, Armonk, NY, USA). Data were expressed as means ± standard deviation (SD) of at least three independent experiments. The chi-squared test was used to compare qualitative variables. Statistical differences between biofilms biomass in treated and untreated groups were analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett-t test. A comparison of the MIC, MBIC, and MBEC values of fluconazole alone and combined EFAs was performed using the Wilcoxon Signed Rank Test. A P-value < 0.05 was considered statistically significant.

Results

Biofilm Formation Ability of C. albicans

Of the total 135 C. albicans isolates studied, 70 were from women diagnosed with VVC, and 65 were obtained from the blood cultures of patients with candidemia. The biofilm formation ability of C. albicans was determined using the crystal violet method (Figure 2). A total of 94.1% (127/135) of C. albicans isolates exhibited biofilm formation capacity, out of which 65.2%, 20.0%, and 8.9% showed strong, moderate, and weak biofilm formation capacity, respectively. 94.3% (66/70) and 93.8% (61/65) were biofilm producers among VVC-derived strains and candidemia-derived strains, respectively. Moreover, strong biofilm producers were significantly more frequent among VVC-derived strains (57/70, 81.4%) than among candidemia-derived strains (31/65, 47.7%) (χ² = 16.903, P < 0.05).

Figure 2 The biofilm formation ability of C. albicans strains from vulvovaginal candidiasis (VVC) and candidemia.

Minimum Inhibitory Concentration of Fluconazole for Planktonic C. albicans

As shown in Figure 3, the minimum inhibitory concentration (MIC) values of fluconazole for 135 isolates of C. albicans ranged from ≤ 0.125 to 8 µg/mL. The distribution of the MICs was similar between the VVC-derived and candidemia-derived strains. Most strains obtained MIC values that were ≤ 0.125 µg/mL (118/135, 87.4%). According to the CLSI M27-A4 guideline,²⁴ 98.5% (133/135) of C. albicans were susceptible to fluconazole (MIC ≤ 2 µg/mL). Only one C. albicans strain (Y65) isolated from VVC appeared resistant to fluconazole with a MIC = 8 µg/mL.

Figure 3 Distribution of the minimum inhibitory concentrations (MICs) of fluconazole among C. albicans strains from vulvovaginal candidiasis (VVC) and candidemia.

Antibiofilm Effect of Fluconazole

The minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) values of fluconazole were tested against 12 clinical strains with a strong biofilm-forming ability and MIC > 0.125 µg/mL (Table 1). The MBIC values, which ranged from 0.25 to 8 µg/mL for biofilms, were similar to the corresponding strains’ MIC values for planktonic cells. However, the MBEC values increased drastically compared with the MIC values, ranging 64- to 1024- fold. The biofilms of C. albicans appeared to be highly resistant to fluconazole with MBEC values above 64 µg/mL.

Table 1 The MICs, MBICs, and MBECs of Fluconazole Against C. albicans Strains

Inhibitory Effect of Essential Fatty Acids on Biofilm Formation

To determine whether EFAs inhibited the biofilm formation of 12 strains of C. albicans, the biofilm biomass of EFAs treated and control groups were compared. As shown in Figure 4, after 24 h of treatment with EFAs at a concentration of 0.01 mM, none of the EFAs had inhibitory effects on biofilm formation; whereas in the presence of the highest concentration (1 mM) tested, except for LOA, other EFAs had inhibitory effects on biofilm formation (P < 0.05), and the EFAs attenuated biofilm formation in a dose-dependent manner. In addition, the most pronounced inhibitory effect on biofilm formation was observed for GLA, with the biofilm inhibition rates (%) at 0.01 mM, 0.1 mM, and 1 mM being 13.35%, 54.36%, and 80.99%, respectively (Table S1).

Figure 4 Inhibitory effect of essential fatty acids (EFAs) on biofilm formation of C. albicans. (A) 0.01 mM EFAs. (B) 0.1 mM EFAs. (C) 1 mM EFAs. The values shown are means ± standard deviations. An asterisk denotes a statistically significant difference compared to the untreated control (P < 0.05).

Eradication Effect of Essential Fatty Acids on Mature Biofilm

The eradication biofilm activity of EFAs was evaluated against preformed 24 h biofilms of 12 C. albicans isolates (Figure 5). At concentrations of 0.01 mM and 0.1 mM, there were no significant effects on the mature biofilms of C. albicans compared with the control group (P > 0.05). Nevertheless, at the increased concentration (1 mM), the biofilm of C. albicans was significantly eradicated by EPA and AA (P < 0.05), and the biofilm eradication rates (%) were 29.95% and 24.14%, respectively (Table S2). Notably, EPA and AA at 1 mM had both eradication and inhibitory activities on the biofilms of C. albicans (Figures 4 and 5).

Figure 5 Eradication effect of essential fatty acids (EFAs) on mature biofilm of C. albicans. (A) 0.01 mM EFAs. (B) 0.1 mM EFAs. (C) 1 mM EFAs. The values shown are means ± standard deviations. An asterisk denotes a statistically significant difference compared to the untreated control (P < 0.05).

Effects of EFAs on Fluconazole Susceptibility

To evaluate the impact of EFAs on fluconazole susceptibility, the MICs, MBICs, and MBECs of fluconazole combined with different EFAs (0.1 mM) on clinical C. albicans strains were determined. It was observed that under the planktonic condition the MIC values were increased after fluconazole combined with EFAs (P < 0.05) (Table 2). Under the biofilm condition no significant differences (P > 0.05) were found between the combination treatment and fluconazole alone treatment (Tables 3 and 4).

Table 2 The MICs of Fluconazole in Combination with EFAs Against C. albicans Strains

Table 3 The MBICs of Fluconazole in Combination with EFAs Against C. albicans Strains

Table 4 The MBECs of Fluconazole in Combination with EFAs Against C. albicans Strains

Effects of EFAs on C. albicans Growth

To further understand whether EFAs affect the growth rate of C. albicans and thus play an antibiofilm role, the growth curves of four C. albicans strains (Y26, Y38, Y65, and X52) were measured. These four strains were relatively sensitive to EFAs under biofilm conditions. As shown in Figure 6, None of the EFAs suppressed the growth of C. albicans at the low concentrations (0.01 mM and 0.1 mM), while EPA, DHA, GLA, and AA at 1 mM had inhibitory effects on growth.

Figure 6 Growth curves of C. albicans strains in the absence (control) or the presence of different concentrations of essential fatty acids (EFAs). (A–C) C. albicans Y26, (D–F) C. albicans Y38, (G–I) C. albicans Y65, and (J–L) C. albicans X52. The McF values were determined every hour during 14 h.

Discussion

C. albicans has been associated with many human infections and causes considerable morbidity and mortality.²⁹ One of the most potent pathogenic traits of C. albicans is its capacity to form a biofilm. Biofilms allow for enhanced adhesion to body surfaces, protection from environmental stressors and the host immune system, and often result in increased tolerance to antifungal agents.⁶ According to these properties, the biofilm formation of C. albicans is an important vital determinant during candidiasis.⁶ As traditional antifungal drugs do not work well against the biofilms, the present study intended to search for new antifungal agents to control C. albicans biofilms.

In this study, a total of 135 clinical isolates of C. albicans (vaginal isolates, n=70; blood isolates, n=65) were determined for the biofilm formation ability. The vast majority of C. albicans showed biofilm formation ability (94.1%, 127/135) under the conditions of our study. This result is similar to the previously reported data by Monfredini et al,³⁰ Gharaghani et al,³¹ and Hacioglu et al.³² However, different from our results, lower biofilm formation rates (51.2% and 23.9%) of C. albicans were reported by Marak et al³³ and Tulasidas et al.³⁴ This may be due to differences in culture conditions during biofilm formation. Weerasekera et al³⁵ compared the effect of three culture media on the biofilm formation of C. albicans. They found biofilm biomass was higher in RPMI 1640 followed by sabouraud dextrose broth (SDB) and yeast nitrogen base (YNB). Therefore, RPMI 1640 medium was used in the biofilm assays, which could induce high biofilm biomass and is beneficial for evaluating anti-biofilm agents. With the increasing attention to biofilm-related infections, there is a need to develop a standardized culture condition for the C. albicans biofilm to facilitate cross-comparison between laboratories.

Fluconazole is currently one of the most widely used antifungal drugs to treat C. albicans infections. Although C. albicans is not intrinsically resistant to fluconazole in the planktonic state, the biofilms of C. albicans exhibit inherent antifungal drug resistance.³⁶ Our investigation also confirmed that C. albicans biofilms exhibit enhanced resistance against fluconazole. The MIC values of fluconazole for most tested strains were ≤ 0.125 µg/mL. For the C. albicans strains with strong biofilm formation ability, fluconazole could inhibit biofilm formation of most strains, the MBIC values were 0.25–8 µg/mL; but when biofilms were established, resistance to fluconazole was greatly enhanced, and the MBEC values were ≥ 64 µg/mL, increased from 64- to 1024- fold compared with MIC values. The mechanisms of fluconazole resistance in Candida biofilms include the increased metabolic activity occurring in the early development of biofilm, the presence of extracellular matrix, and alterations in gene expression (such as the upregulation of CDR and MDR genes encoding azole resistance transporters).³⁷ Therefore, it is critical to search for novel antifungal agents which can efficiently inhibit biofilm formation and eradicate mature biofilm of C. albicans.

EFAs are polyunsaturated fatty acids that play crucial roles in regulating body homeostasis, such as regulating the antioxidant signaling pathway, modulating inflammatory processes, and influencing hepatic lipid metabolism and physiological responses of other organs.^23,38 Because of their nature, fatty acids are easily used as food additives. EPAs can reduce the risk of various diseases, including cardiovascular disease,³⁹ cancer,⁴⁰ and Alzheimer’s disease,⁴¹ among others.^42,43 Several studies demonstrated that EFAs (eg, LOA, EPA, GLA, and AA) exerted antifungal properties against Candida spp.^17–20 Nonetheless, few published studies have investigated the activities of EFAs on mature biofilms of C. albicans. Therefore, this study investigated the effect of EFAs, including ALA, EPA, DHA, LOA, GLA, and AA, against biofilm formation and mature biofilms of C. albicans.

Our results showed that other EFAs at 1 mM had inhibitory effects on biofilm formation except for LOA. The biofilm inhibition rates (%) ranged from 20.52% to 80.99% (LOA < ALA < AA <EPA < DHA < GLA) (Table S1). A recent study by Jamiu et al²⁰ also demonstrated LOA and GLA displayed significant inhibition of C. krusei biofilms. Both increased the susceptibility of C. krusei biofilm to fluconazole via induction of oxidative stress, cell membrane damage, and disruption of efflux pump activity. In this study, GLA exhibited a remarkable inhibitory effect against biofilm formation of C. albicans, indicating that GLA could be used as an alternative antifungal agent to prevent biofilm formation.

Furthermore, our research is the first to show that EPA and AA at 1 mM effectively eradicated mature biofilms of C. albicans, and the biofilm eradication rates were 29.95% and 24.14%. The biofilm eradication abilities of EPA and AA at 1 mM were similar to that of fluconazole at 64 μg/mL (26.15%). Although EFAs may not be as adequate as the high concentrations of fluconazole, pathogenic fungi are less likely to become resistant to antifungal EFAs.⁴⁴ Thus, EPA and AA have the potential to be used as antifungal agents for treating biofilm-associated infections.

In this study, our data also showed no synergistic effect between EFAs and fluconazole. EFAs at the low concentration (0.01 mM) even slightly increased MIC values of fluconazole. It might be due to low concentrations of EFAs promoting the growth of C. albicans. It should be noted that EPA, DHA, and GLA at 0.1 mM did not inhibit the growth rate of C. albicans, but did inhibit the biofilm formation of C. albicans. The underlying mechanisms for the antibiofilm potential of EFAs might involve affecting the adhering surface, changing cell-membrane fluidity, reducing extracellular polysaccharide or hyphae formation, and modulating quorum-sensing systems.⁴⁵

For the ω-3 fatty acids, the European Food Safety Authority suggests that long-term intake of EPA and DHA supplements up to about 5 g/day is safe;⁴⁶ and for ω-6 fatty acids, the United States Department of Agriculture indicates that the average adult intake of LOA is 17–20 g/day for men and 12–13 g/day for women.⁴⁷ Therefore, the maximum concentration of 1 mM (0.28–0.33 g/L) of EFAs used in the study is safe for patients with C. albicans infection.

Overall, this study indicates EPA and AA could serve as potential new antifungal agents for treating clinical infections caused by C. albicans biofilms.

Conclusion

In summary, the present study evaluated the antibiofilm activities of EFAs on C. albicans strains in vitro. GLA was found to inhibit biofilm formation by C. albicans markedly. Besides, EPA and AA exhibited better eradication effects on mature biofilms of C. albicans. These findings indicate GLA, EPA, and AA may be promising antifungal agents in managing C. albicans-related infections. Nevertheless, future studies are warranted to investigate the antibiofilm activity of EFAs on C. albicans in vivo, as well as the molecular mechanism of EFAs against the biofilm.