Effects of Euterpe oleracea Mart. extract on Candida spp. biofilms

Abstract

Problem of research

Candida spp. biofilms are complex microbial communities that have been associated with increasing resistance to clinically available antifungal drugs. Hence, novel pharmacological approaches with ability to inhibit biofilm formation have been investigated.

Aim of study

The aim was to analyze in vitro antifungal activity of Euterpe oleracea Mart. (açaí berry) extract on biofilm strains of Candida albicans, C. parapsilosis, and C. tropicalis that were formed on abiotic surfaces.

Remarkable methodology

Biofilms of C. albicans, C. parapsilosis, and C. tropicalis were grown in vitro. They were then treated with E. oleracea Mart. extract at different concentrations (7.8, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 μg/mL) for evaluation of both biofilm removal and anti-biofilm activity.

Remarkable results

All Candida species analyzed formed biofilms on abiotic surfaces. Yet, increased biofilm formation was displayed for C. tropicalis in comparison with the other two species. E. oleracea Mart. extract was shown to inhibit biofilm formation at all concentrations used when compared to no treatment (p < 0.05).

Significance of the study

In the current study, the extract of E. oleracea Mart. demonstrated antifungal activity against Candida albicans, C. parapsilosis, and C. tropicalis biofilms, regardless of the dose utilized. These results are important to evaluate a natural product as antifungal for Candida species.

Introduction

Infections by Candida species, such as Candida albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis, are increasingly frequent [1–3]. They often occur due to host immunosuppression and/or virulence factors expressed by these yeasts, which contribute to their ability to colonize, penetrate, and invade tissues [4]. Such strains have also the ability to form biofilm.

According to Costerton (1999), biofilm consists of cells that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms [5]. Importantly, biofilms may hinder the penetration of most antifungal agents, thereby leading to a poor response to treatment and drug resistance [6–9]. The formation and development of distinct phases of biofilm (adhesion/colonization, maturation, and dispersion) are mediated by regulatory genetic alterations and complex molecular events [10].

Because they consist in a matrix of microorganisms, biofilms can be considered a defense strategy of pathogens, affecting either biotic or abiotic surfaces such as medical devices (e.g., catheters, bladder probes) [11–13].

Moreover, biofilms have been increasingly linked to both mucosal infections such as candidiasis, which is facilitated by virulent factors from Candida species, including their capacity to form biofilms and the transition to filamentous or hyphal form [14].

In this context, natural products with ability to inhibit or disrupt biofilms have been investigated as a potential source of novel antifungals [15, 16]. Al-Sokari and Sheikha (2015) evidenced that crude extracts of Ruta graveolens L have good inhibition zone against Escherichia coli and Pseudomonas aeruginosa. Latex of Ficus carica Linn plants showed that it is a good inhibitor for Candida albicans [17].

Khan and Ahmad had studied the inhibitory effect of essential oil of Cymbopogan citratus and Syzygium aromaticum on the biofilm of drug-resistant Candida from clinical origin. C. citratus was capable to inhibit the biofilm formation of approximately 88% in C. albicans 04 and 82% in C. albicans SC5314 and S. aromaticum inhibited 52% and 57% biofilm in above-mentioned test strains at the same concentration [18].

Açaí (Euterpe oleracea Mart.) is a native plant from Amazonian region, and it has been used despite of its high antioxidant activity as antimicrobial. Euterpe genus includes over 28 species distributed throughout the Amazon region in Latin America, where E. oleracea, E. precatoria, and E. edulis are the most frequent species.

The phytochemical composition of the fruit known as “açaí berry” has been well characterized. It includes phenolic acids, anthocyanins (e.g., cyanidin-3-rutinoside and cyanidin-O-glucoside), proanthocyanidins, lignans (e.g., aryltetrahydronaphthalene, dihydrobenzofuran, furofuran, 8-O-4′-neolignan, and tetrahydrofuran), and polyphenolic constituents (e.g., epicatechin, the catechin homoorientin, orientin, isovitexin, and taxifolin deoxyhexose) [19].

Hence, the present study was aimed at investigating in vitro whether the treatment with E. oleracea Mart. extract would have the ability to inhibit or disrupt biofilms from C. albicans, C. parapsilosis, and C. tropicalis formed on abiotic surfaces.

It is important to study natural products as antifungal agents, considering the remarkable resistance of Candida species to imidazoles.

This is relevant to have alternative treatments because the increasing incidence of drug-resistant pathogens and the toxicity of existing antifungal compounds have increased interest in the antifungal properties of natural products. Furthermore, most of the available antifungals are either ineffective against Candida biofilms or exhibit their inhibitory activity at high concentrations.

Methods

Microbial strain identification

Commercially available strains of C. albicans (ATCC 10,231), C. tropicalis (ATCC 1369), and C. parapsilosis (ATCC 22,019) were obtained from Plast Labor (Rio de Janeiro, RJ, Brazil) and kept under refrigeration until use. Field experiments have been approved by the Brazilian Ministry of the Environment, Instituto Chico Mendes de Conservação da Biodiversidade—ICMBio (approval number: 57805–1).

The strains were kept on Sabouraud Dextrose Agar with 4% chloramphenicol. The fungal suspension for the experiments was prepared from 24 h colonies diluted in 0.85% saline according to the 0.5 MacFarland scale (1–5 × 10⁶ cells/mL).

Preparation of E. oleracea Mart. extract

The fruits of E. oleracea Mart. used in this study were collected from Juçara Park, located in São Luís, MA, Brazil (latitude: 02° 31′ 47″ S, longitude: 44° 18′ 10″ W, altitude: 24 m). Approval was obtained through the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen, protocol A91B0BA). The extract was prepared using a protocol previously utilized with some adaptations [20]. Briefly, fruits were thawed and washed three times with distilled water, then soaked in warm water for 1 h. Subsequently, 365 g of whole fruit extract was grinded and mixed with 400 mL of ethyl alcohol p.a. For 10 days, the mix was shaken for 2 h/day, followed by vacuum filtration. The solvent was removed by rotary evaporation, lyophilized, aliquoted, and then refrigerated until use. After the maceration period, the extract was concentrated in rotoevaporator, lyophilized, and sent for identification of chemical compounds through mass spectrometry analysis.

Mass spectrometry analysis

Mass spectrometry analysis was performed using an electrospray ionization (ESI) source and a cyclotron analyzer coupled to a Fourier transform (ESI-FT-ICR MS). Samples were diluted with 0.1% acetic acid for positive analysis, and the resulting solution was then infused directly into the SOLARIX 9.4 T mass spectrometer (Bruker Daltonics, Bremen, Germany), operating in a range of 100–1000 m/z. The general conditions for EIS analysis were gas pressure of 0.3 psi, capillary voltage of 4.5 kV, and 220 °C for the ion transfer capillary temperature. The ESI ( +)—FT—ICR MS spectra were acquired and processed using the Compass Data Analysis software (Bruker Daltonics).

Evaluation of biofilm formation

Biofilm formation was evaluated in 96-well polystyrene microplates as previously described [21]. First, Candida spp. strains were cultivated in Sabouraud Dextrose Agar and incubated at 37 °C for 24 h. Next, isolates were diluted in saline solution to match the 0.5 McFarland turbidity standard, corresponding to 1 × 10⁶ to 5 × 10⁶ cells per mL [22]. The wells of microplates were filled sequentially in triplicates. In the negative control group, only 200 μL of BHI with 6% glucose were added. In the remaining wells, 180 μL of BHI with 6% glucose plus 20 μL of the suspension of each Candida species in saline solution was added. The microplates were incubated (37 °C, 24 h) and, subsequently, washed three times with sterile distilled water, and received 200 μL of crystal violet dye each well for 5 min. They were then washed three times with sterile distilled water, and lastly, 200 μL of sterile distilled water was added to each well for spectrophotometric analysis at 570 nm wavelength absorbance.

Adhesion and antibiofilm activity of E. oleracea Mart. extract on Candida spp.

Biofilms in 96-well polystyrene microplates were formed by inoculum of C. albicans, C. parapsilosis and C. tropicalis (100 μL), incubated for 1 h and 30 min at 37 °C, which corresponds to adhesion phase of yeasts to the abiotic surface [21].

Subsequently, the medium was aspirated, the wells were washed three times with PBS 1X and 200 μL of different concentrations (7.8, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 μg/mL) diluted in BHI with 6% glucose were added. The wells were washed with sterile PBS 1X 3 times, fixed with PA ethanol for 2 min and stained with 1% violet crystal solution for 5 min and again washed with sterile 1X PBS 5 times to remove excess dye.

After this step, 200 μL of sterile 1X PBS was added to each well and the reading was performed on the Epoch microplate reader with a wavelength of 570 nm. In columns A1, A2, and A3 the positive control was inoculated adding only the culture medium.

Biofilm removal activity of E. oleracea Mart. extract on Candida spp. biofilms

To analyze in vitro biofilm removal activity of E. oleracea Mart. extract on biofilms formed by C. albicans, C. parapsilosis, and C. tropicalis on abiotic surfaces of microplates (TPP) after 72 h, different concentrations (7.8, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 μg/mL) of E. oleracea Mart. extract (200 μL) were added in each well over the mature biofilm [21].

Subsequently, the wells were washed with sterile 1X PBS 3 times, fixed with PA ethanol for 2 min and stained with 1% violet crystal solution for 5 min and again washed 5 times with 1X PBS to remove excess dye. After this step, 200 μL of sterile 1X PBS was added to each well and the reading was performed on the Epoch microplate reader with a wavelength of 570 nm.

Antibiofilm activity of E. oleracea Mart. extract in coverslips

The biofilm evaluation in coverslips was performed using protocols previously reported with some adaptations [23–25]. C. albicans, C. tropicalis, and C. parapsilopsis yeasts were grown in a 24-well plate containing 13 cm round glass coverslip in 1-mL 0.85% saline.

The fungal samples were seeded in Sabouraud Dextrose Agar and incubated at 37 °C for 24 h. Next, isolates were diluted in saline solution to match the 0.5 McFarland turbidity standard, corresponding to 1 × 10⁶ to 5 × 10⁶ cells per mL [22]. In each well, 100 μL of the fungal suspension was added and placed to adhere for 6 h. The wells were subsequently washed three times with 0.85% saline solution and then 1 mL of each concentration of the extract (7.8, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 μg/mL) was added, and plates were incubated for 48 h.

Next, the plates were incubated at 37 °C for 48 h in a BOD oven. After that, the coverslips were gently removed, washed with PBS 1X 3 times, dried at room temperature, fixed with PA ethanol, stained with 1% violet crystal solution. The coverslips were glued with mesh onto the surface of a glass slide and analyzed under the Ninkon optical microscope.

Cells adhered to the coverslip were counted and classified according to cell arrangement patterns adhered to glass coverslips as: diffuse pattern, when yeast cells adhered to entire surface of the glass coverslip without forming cell groups; localized adhesion, when involving groups of yeast that adhered to localized regions of the coverslip; aggregative, which is characterized by yeast clumps arranged as “stacked bricks” or “grape clusters” that attached to the glass slide surface. The formation of filaments or pseudohyphae along the surface of the coverslip characterized the filamentous or pseudohyphal pattern.

Statistical analysis

Data were presented as means ± standard deviations or as medians and interquatile ranges. Normality of variables was analyzed using the Shapiro–Wilk test. Comparisons were performed using the Kruskal–Wallis or Wilcoxon tests. A p value ≤ 0.05 was considered to be statistically significant. Statistical analyzes were performed using STATA (StataCorp, Release 14; College Station, TX, USA).

Results

Figure 1 displays the chemical characterization of crude ethanolic extract of açaí berry fruit by mass spectrometry analysis using the positive method. The extract was shown to be rich in polyphenols, and the compounds identified are exhibited in Table 1.

Fig. 1.

Mass spectrometry from Euterpe oleracea Mart extract

Table 1.

Compounds isolated from Euterpe oleracea Mart extract

m/z	Compounds
291,08,652	Epicatechin
338,34,154	Erucamide
381,07,924	N-(3-methoxy-5-nitrophenyl)-2-(5-methyl-3,4-dinitro-1H-pyrazol-1-yl) acetamide
391,28,411	Not identidied
579,15,031	Procyanidin B3
633,20,235	(4-Acetoxy-5-((2-((4.5-dihyroxytetrahydro-2H-pyran-2-yl) oxy)-4.5-dilydroxytetrahydro-2H-pyran-3-yl) oxy)-3-hydroxytetrahydrofuran-2-yl) methyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate
723,19,485	Not identified

Further, Candida spp. used in this study had the ability to form biofilms on abiotic surfaces. Higher biomass formation on abiotic surfaces was observed in C. tropicalis (2.397 ± 0.23) and C. parapsilosis (1.176 ± 0.37) biofilms, whereas lower biomass was shown for C. albicans biofilm (0.53 ± 0.07).

A variation in biomass formed between Candida species was observed, where C. tropicalis was the most adherent, thereby producing more biofilm. Table 2 shows the absorbance before and after the addition of E. oleracea Mart. extract to surfaces containing biofilms formed by each Candida species analyzed. It was found a statistically significant difference between the median absorbance measured before and after treatment with E. oleracea Mart. extract for all Candida species evaluated (p < 0.001).

Table 2.

Median absorbance (570 nm) of Candida albicans, Candida parapsilosis, and Candida tropicalis species during biofilm formation, pre- and postuse of E. oleracea Mart extract

Strains	Absorbance		p value**
	Pre (n = 72)	Post (n = 72)
	Median [IQR*]	Median [IQR*]
C. albicans	0.773 [0.625–1.072]	0.058[0.049–0.081]	< 0.001
C. parapsilosis	1.504 [1.113–1.680]	0.049[0.047–0.053]	< 0.001
C. tropicalis	2.500 [2.332–2.587]	0.125 [0.056–0.333]	< 0.001

*Interquartile range (p25– p75)

**Wilcoxon test for paired samples

Since the treatment with E. oleracea Mart. extract decreased Candida spp. biomass formation, we further tested different concentrations of the extract. Table 3 displays significant differences in medians from before and after the addition of the extract at all concentrations (p < 0.05). Therefore, regardless of concentration, antibiofilm activity of açaí berry extract was maintained. Finally, when the removal activity of E. oleracea Mart. extract at the different concentrations was analyzed, there was a statistically significant difference between the values obtained for C. albicans, C. parapsilosis, and C. tropicalis (Table 4).

Table 3.

Absorbance for different concentrations of E. oleracea Mart extract, pre- and postuse, on biofilms of C. albicans, C. parapsilosis, and C. tropicalis on abiotic surfaces

Concentration (μg/mL)	Absorbance		p value**
	Pre (n = 72)	Post (n = 72)
	Median [IIQ*]	Median [IIQ]
7.8	1.341[0.765–2.538]	0.047 [0.046–0.055]	0.019
15.6	1.112 [0.812–2.489]	0.051 [0.049–0.323]	0.007
31.2	1.614 [0.964–2.379]	0.077 [0.053–0.146]	0.007
62.5	1.631[1.123–2.511]	0.085[0.049–0.253]	0.007
125	0.709 [0.518–1.969]	0.058 [0.047–0.386]	0.007
250	1.644 [1.274–1.664]	0.052 [0.049–0.066]	0.007
500	1.688[0.843–2.479]	0.053 [0.050–0.063]	0.007
1000	1.579 [1.462–2.462]	0.055 [0.053–0.086]	0.007

*Interquartile range (p25– p75)

**Wilcoxon test for paired samples

Table 4.

Evaluation of the removal activity in C. albicans, C. parapsilosis, and C. tropicalis specimens in relation to the concentration of E. oleracea Mart extract

Concentration (μg/mL)	Removal activity
	C. albicans	C. parapsilosis	C. tropicalis
	Mean ± SD	Mean ± SD	Mean ± SD
7.8	0.607 ± 0.096	0.154 ± 0.021	0.440 ± 0.204
15.6	0.504 ± 0.064	0.373 ± 0.213	0.665 ± 0.282
31.2	0.656 ± 0.058	0.196 ± 0.007	0.658 ± 0.168
62.5	0.844 ± 0.161	0.344 ± 0.254	0.805 ± 0.101
125	0.715 ± 0.140	0.497 ± 0.073	0.589 ± 0.065
250	0.962 ± 0.549	0.471 ± 0.060	0.807 ± 0.110
500	0.251 ± 0.112	0.660 ± 0.074	0.648 ± 0.068
1000	1.275 ± 0.279	1.083 ± 0.005	1.640 ± 0.093
p value*	0.016	0.010	0.034

*Kruskal–Wallis test

These findings corroborate with those obtained for the antibiofilm activity of E. oleracea Mart. extract on glass surface, which was found to prevent biofilm formation. Isolated cells (4 to 6 cells isolated per field) and 99% absence of biofilm (Fig. 2a and b) and 39 isolated cells and absence of biofilm at concentrations of 250, 500, and 1000 μg/mL (Fig. 2c) were observed, preventing biofilm development on glass coverslips. At concentrations of 31.2 to 125 μg/mL, there was also no biofilm formation; however, the presence of aggregative arrangements was observed (Fig. 2j–u). Notably, concentrations 7.8 (a–c*) and 15.6 (v–z) μg/mL were not able to prevent biofilm formation.

Fig. 2.

Antibiofilm effect of different concentration of the Euterpe oleracea ethanolic extract (μg/mL) on biofilm of C. albicans, C. parapsilosis, and C. tropicalis

Discussion

In the current study, the ability of C. albicans, C. parapsilosis, and C. tropicalis to form biofilms on abiotic surfaces was demonstrated, and the biomass produced varied according to each species. C. tropicalis adhered more easily to the abiotic material and was thus associated with greater biofilm formation.

The increasing incidence of drug-resistant pathogens and the toxicity of existing antifungal compounds have increased the interest from antifungal properties of natural products [26]. Several studies have been conducted using natural products to evaluate interference in C. albicans biofilm and anticandidal activity on planktonic and biofilm cultures of the C. parapsilosis complex [27, 28].

Most of the available antifungals are either ineffective against Candida biofilms or exhibit activity at very high concentrations [29, 30]. Plants are rich sources of bioactive molecules exhibiting various biological and pharmaceutical properties. Various phytochemicals are known to possess strong antimicrobial/antifungal activities [31]. Use of these phytochemicals against biofilms could be an excellent strategy [32, 33].

Cannas et al. (2014) evidenced a considerable activity of essential oil of Myrtus communis L. against C. albicans and C. parapsilosis after 24–48 h [34].

Borges et al. [35] demonstrated an increased adhesion of C. parapsilosis, which formed biofilm in copper fragments after 6 and 24 h of incubation, corroborating to our findings. In addition, açaí berry extract, in contact with abiotic surfaces containing biofilms formed by C. albicans, C. parapsilosis, and C. tropicalis, presented with a biofilm removal effect, whereby medians from before and after the treatment with the extract varied significantly in this study.

Several plant extracts, essential oils, and phytomolecules have been found to inhibit biofilm formation by Candida spp. [36]. Nair et al. [37] analyzed several phytochemicals and identified plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a phytochemical of Plumbago species, as a potent antifungal agent against C. albicans, with a low minimum inhibitory concentration that was effective at preventing and dispersing biofilms in catheters formed by C. albicans. Therefore, in vivo and in vivo evaluation as well as clinical trials is required to further investigate the use of phytochemicals as candidate molecules for anti-biofilm drugs.

Polyphenols and flavonoids are the main chemical compounds from açaí. Polyphenols display excellent biofilm inhibitory activities in C. albicans. Studies showed that curcumin, pyrogallol and pyrocatechol possess anti-Candida biofilm activity [38]. Epigallocatechin-3-gallate extracted from green tea prevented biofilm formation by C. albicans [39].

Epigallocatechin-3-gallate (ECGC) has antifungal activity against human-pathogenic yeasts like Candida albicans. Although the mechanistic effects of EGCG are not fully understood, there are results indicating that EGCG binds to lipid membranes and affects the folic acid metabolism of bacteria and fungi by inhibiting the cytoplasmic enzyme dihydrofolate reductase [40].

In the current study, data from before and after the addition of açaí berry extract on biofilms of C. albicans, C. parapsilosis, and C. tropicalis on abiotic surfaces demonstrated a significant difference in biofilm formation at both the lowest (7.8 μg/mL) and the highest (1000 μg/mL) concentrations of the extract. It is worth mentioning that anti-biofilm activity of E. oleracea Mart. extract was maintained at all concentrations tested, suggesting that even at low doses, açaí berry extract shows an antifungal effect against Candida spp. biofilm. Nadaf et al. [41] observed that Hymenocallis littoralis leaf extract at concentrations of up to 70 μg/mL presented with anti-biofilm properties, reducing biomass production by C. albicans through interaction with active site residues of adhesin proteins.

The pathogenicity of Candida species through various virulence factors, such as adhesion to host surfaces, formation of biofilms and secretion of hydrolytic enzymes has been demonstrated [10]. The apparent increase in the emergence of C. glabrata, C. tropicalis, and C. parapsilosis species can be attributed to better identification methods and has also been associated with clinical impairment, interventions performed, and pharmacological therapy. Although studies to identify virulence factors, particularly in C. albicans, are frequent, relatively little is known regarding non-albicans Candida species. Millot et al. [42] analyzed several lichen extracts towards identifying their potential activity against C. albicans biofilm, eleven of which were found to inhibit biofilm maturation by C. albicans.

In relation to the dosage of E. oleracea Mart. extract utilized, the greatest inhibitory action on biofilm formation in the species analyzed in this study was obtained at a concentration of 250 μg/mL. Dias-Souza et al. [43] evaluated the effects of different doses of E. oleracea Mart. against Staphylococcus aureus biofilm and found a minimum biofilm eradication concentration of 250 μg/mL. This indicates that low concentrations are required to obtain an antibiofilm activity of açaí berry extract against Candida spp. biomass production.

Conclusion

In summary, an extract obtained from E. oleracea Mart. presented with both anti-biofilm activity and removal effect against Candida albicans, C. parapsilosis, and C. tropicalis biofilms, even when low concentrations were used. These results are important for the development of a new antifungal from a natural product. Further in vitro investigation is required to determine which compounds from açaí berry extract are responsible for the actions observed in the present study before developing in vivo analysis and clinical trials in this regard.

Abbreviations

ATCC

Anatomical Therapeutic Chemical Code

BHI

Brains heart infusion

ESI

Electrospray ionization

ESI-FT-ICR MS

Cyclotron analyzer coupled to a Fourier transform

ICMBio

Instituto Chico Mendes de Conservação da Biodiversidade

Sisgen

Sistema Nacional de Gestão do Patrimônio Genético

Data Availability

All data is included in the manuscript.

Declarations

Conflict of interest

The authors declare no competing interests.