Anti-candida biofilm properties of Cameroonian plant extracts

Candida infections can be superficial, invasive or disseminating. The virulence of Candida species has been attributed to several factors, including the promotion of hyphae and biofilm formation, adherence to host tissues, and response to environmental changes and morphogenesis. Resistance to many clinically used antifungal agents has led to the need to identify new compounds and drugs for therapeutic use. Therefore, the objective of this study was to evaluate the anti-candida and anti-biofilm activities of some Cameroonian plant extracts against Candida albicans and Candida glabrata. The biofilm biomass of C. albicans and C. glabrata was quantified using the violet crystal protocol. A microbroth dilution method was used to determine the minimum inhibitory concentrations (MICs), and a biofilm enumeration assay was employed to determine the minimum biofilm inhibition concentrations (MBICs) and minimum biofilm eradication concentrations (MBECs) of the extracts. The absorbance value of the biofilm biomass of C. albicans was 0.14±0.01 and that of C. glabrata was 0.51±0.06. Eugenia uniflora and Terminalia mantaly aqueous leaf extracts showed MICs of 0.3125 and 0.625 mg/mL for C. glabrata, while the MICs for C. albicans were 10 and 0.625 mg/mL, respectively. The MBIC and MBEC of C. glabrata of E. uniflora aqueous leaf extracts were 0.125 and 0.5 mg/mL, respectively, and 0.45 and >1.8 mg/mL, respectively for T. mantaly. The results of this study demonstrated the in vitro anti-biofilm potential of T. mantaly and E. uniflora aqueous leaf extracts against Candida biofilm. Nonetheless, further analyses of a larger number of Candida isolates and plant extracts are needed to validate these findings.

. The incidence of infections is increasing among compromised patient groups such as cancer patients on chemotherapy, patients receiving broad-spectrum antibiotic treatment, and HIV-infected individuals (Neeta and Uttamkumar 2011;Ye et al., 2004).Vaginal candidiasis is quite common in women and approximately 75% present this infection once in their lifetime.C. albicans is the most prevalent fungal pathogen in humans.Mucosal infections of Candida albicans are often benign, but systemic infections are usually fatal (Al-Ahmadey and Mohamed 2014; Foxman et al., 2013).Although C. albicans is the most frequent cause of infection, non-albicans species infections are on the rise (Mohandas and Ballal, 2011).Thus, Candida glabrata was reported to be the second most common agent of vaginal candidiasis; however, the increasing incidence of cases of vaginal candidiasis caused by non-c.albicans species has not yet been well established (Al-Ahmadey and Mohamed, 2014;Esmaeilzadeh et al., 2009).In the Littoral Region of Cameroon (Nylon District Hospital), the prevalence of oral and vaginal candidiasis in 2012 was 52.6 and 29.7%, respectively (Njunda et al., 2012).The prevalence of oral candidiasis among HIV patients in the study population of the Mutengene Baptist Hospital in the South West Region in 2013 was 66.7% (Njunda et al., 2013).It has been reported that the mortality rate of invasive infections is 40% (Klevay et al., 2009;Pfaller and Diekema, 2007;Bertagnolio et al., 2004) and C. albicans is estimated to be responsible for 50-60% of the cases of invasive candidiasis (Perlroth et al., 2007;Pfaller and Diekema, 2007).
One of the factors contributing to the virulence of Candida is the formation of surface attached microbial communities known as biofilms (Seneviratne et al., 2008).Biofilm formation helps the microorganisms evade host defenses, exist as a persistent source of infection and develop resistance against antifungal agents.The resistance of biofilm forming Candida spp. to antifungal agents represents a major challenge especially in the design of therapeutic and prophylactic strategies (Golia et al., 2011).Aside from increasing the resistance to the available antifungal compounds, the toxicity of some of these compounds is high (Shreaz et al., 2011;Georgopapadakou and Walsh, 1994).Some major antifungals are limited to a few chemical classes such as Amphotericin B, a polyene fungicidal agent that has been implicated in hepatotoxicity and nephrotoxicity, coupled with decreasing efficacy (Pan et al., 2009;Dismukes, 2000;Arthington-Skaggs et al., 2000).Hence, the need for inexpensive, effective and less toxic antifungals is imperative.
Medicinal plants have been the major health care measure of resource-poor populations worldwide (Duraipandiyan and Ignacimuthu, 2011;Tharkar et al., 2010).According to the WHO, 80% of the world's population uses natural remedies and traditional medicines (WHO, 2001(WHO, , 2003)).This is particularly common in Africa, as well as in most low-income countries, where a high proportion of the population still resorts to traditional medicine for primary health care.Cameroon has a rich biodiversity, with ~8,620 plant species (Mbatchou, 2004;Earth Trends, 2003), some of which are commonly used in the treatment of several microbial infections (Kuete and Efferth, 2010).Some plant extracts have demonstrated positive response during pharmacological investigations (Suresh et al., 2010;Patel and Coogan, 2008).
Therefore, the main objective of the present study was to evaluate the anti-candida biofilm properties of several plant extracts by determining the minimum inhibitory concentrations (MIC), minimum biofilm inhibition and minimum biofilm eradication concentrations (MBEC).

Plant material and extraction
Leaves, twigs, stem bark and stems of different plants were collected at Mount Kalla in Yaoundé (Central region) and Dschang (West region) Cameroon on the 11th of September 2011 and 2014, and voucher specimens were deposited at the National Herbarium of Cameroon, Yaoundé.The plant parts were individually dried at room temperature and then ground to fine powder.Five hundred grams (500 g) of each sample were macerated with regular stirring in 2 L of 95% ethanol or distilled water for 72 h.The filtrate was evaporated using a rotary evaporator (Rotavapor BÜCHI 011).The plant residues were dried and macerated in distilled water for 72 h and the filtrate dried at room temperature (25-28°C) using a fan.The extraction yields were calculated as percentage relative to the starting plant material.

Biofilm quantification
The biofilm forming ability was assessed by quantification of total biomass by violet crystal (VC) staining.Thus, after washing, biofilms were fixed with 200 μl of methanol 99%, which was removed after 15 min.The microtitre plates were allowed to dry at room temperature, and 200 μl of VC (1% v/v) were added to each well and incubated for 5 min.The wells were then gently washed with sterile, ultra-pure water and 200 μl of acetic acid (33% v/v) were added to release and dissolve the stain.The absorbance of the solution obtained was read in triplicate in a microtitre plate reader (Bio-Tek Synergy HT, Izasa, Lisbon, Portugal) at 590 nm.The experiment was repeated three times (Silva et al., 2009).

Screening of plants extracts for MICs
Two clinical Candida isolates (C.albicans and C. glabrata) were collected from patients with vaginal candidiasis in the Hospital Clinic of Barcelona.The inoculum of each yeast isolate and strain was prepared from a 2-day-old culture on Sabouraud Dextrose Agar (SDA) at 37°C.The suspension was adjusted to 1 x 10 3 cells/mL using yeast nitrogen base (YNB) medium from 0.5 McFarland standards.The broth micro-dilution method was used to assess yeast susceptibility to extracts using YNB medium supplemented with 5% glucose.
Briefly, each extract (200 mg/mL in 5% DMSO) was serially diluted in YNB supplemented with 5% glucose in 96-well plates.Eighty microlitres of inoculum standardized at 1×10 3 colony forming units (CFU)/mL was added to each well to achieve a final volume of 230 μL.The final concentrations tested ranged between 0.039 and 40 mg/mL for the crude extracts.The positive control consisted of microorganisms growing without extract.After 48 h of incubation at 37°C, the MIC was determined as the lowest concentration of the crude extract in the broth medium that inhibited visible growth of the microorganisms tested.All tests were performed in duplicate.Wells without inoculum or extract were included in each plate to control background sterility and growth.The extracts with the greatest activity were chosen to continue the experimental part of the work.

Determination of the MBIC and MBEC using the Calgary protocol
The isolates were cultured overnight in SDA medium.After preparation of 0.5 McFarland in broth medium, 200 µL were added to each well of a flat-bottom 96-well microtitre plate (MBEC TM Biofilm Inoculator Innovotech product panel P and G panel lot: 14040004).
For the MBIC, flat-bottom microtitre plates containing two-fold dilutions of plant extract in 150 µl of YNB per well (antibiotic challenge plate) were used.The plant extracts included Eugenia uniflora aqueous leaf extract (1-0.125 mg/mL) and Terminalia mantaly aqueous leaf extracts (1.8-0.225mg/mL) in C. glabrata, and (3.6-0.45 mg/mL) in C. albicans.Eighty microlitres of a subculture adjusted to 1x10 3 CFU/mL was added to all the wells, except for those of the negative control, covered with the pegs lid inthe biofilm growth plate, and incubated for 18-20 h at 37°C.
For the MBEC, Candida biofilms were formed by immersing the pegs of the cover lid into this biofilm growth plate, followed by incubation at 37°C for 20 h-24 h without shaking.The peg lids were rinsed three times in sterile water, placed onto new flat-bottom microtitre plates containing two-fold dilutions of plant extract in 150 µl of YNB per well (antibiotic challenge plate), and incubated for 18-20 hours at 37°C.The plant extracts included E. uniflora aqueous leaf extract (1-0.125 mg/mL) and T. mantaly aqueous leaf extracts (1.8-0.225mg/mL) in C. glabrata, and (3.6-0.45mg/mL) in C. albicans.
After antibiotic incubation, the peg lids were washed three times with sterile water and placed into extract-free YNB fresh medium in a new flat-bottom microtitre plate (biofilm recovery plate).To transfer the biofilms from the pegs to the wells, each plate was sonicated at room temperature for 20 min (using a Bransonic 220; BransonCo., Shelton, Conn.).The peg lid was discarded and replaced by a standard lid.The sonicated culture media of each well of the microtitre plate was spread on YNB agar plates and incubated at 37°C for 24 h.Adequate biofilm growth for the positive control wells was defined as the number of colonies obtained after 24 h of incubation.The positive control contained microorganisms and culture medium, and the negative control included only medium.The results were expressed as the number of CFU counted in each extract concentration and per strain.

Phytochemical screening of E. uniflora and T. mantaly aqueous leaf extracts
Phytochemical analysis was done to identify the different components responsible for the activities observed according to the protocols described by Igwe (2004), Trease and Evans (1996) and Sofowora (1982).Zeuko´o et al. 605

Plant extracts
The plant extracts used in the experiments were obtained as defined in the materials and methods section.Table 1 describes the plant collection site and date, and the extraction solvent used.

Biofilm quantification
The average value of C. albicans and C. glabrata biofilm was 0.14±0.01 and 0.51±0.06,respectively, and 0.13±0.02for the negative control.Therefore, C. albicans was not considered in the biofilm inhibition studies.

Determination of the MIC
The aqueous leaf extracts of E. uniflora and T. mantaly showed the best MIC in C. glabrata with values ranging from 0.3-0.5 to 0.625-1 mg/mL, respectively.However, only the aqueous leaf extract of T. mantaly revealed the best activity in C. albicans, showing a MIC of 0.625 to 1.8 mg/mL.These extracts were selected for the determination of the MBIC and MBEC of the strains (Table 2).

Effect of E. uniflora and T. mantaly aqueous leaf extracts on biofilm inhibition and eradication in C. glabrata
Figure 1 shows the inhibition of biofilm formation of both extracts.T. mantaly aqueous leaf extract inhibited biofilm formation of C. glabrata at a concentration of 0.45 mg/mL, while E. uniflora aqueous leaf extract presented inhibition at a concentration of 0.125 mg/mL.In C. glabrata, the MBEC of the E. uniflora aqueous leaf extracts ranged from 0.5-1 mg/mL.However, the eradication activity of the aqueous T. mantaly leaf extract was detected at concentrations >1.8 mg/mL (Figure 2).

Phytochemical studies
The different components presented in both extracts were flavonoids, saponins, tannins, glucosides, phenol, steroids, triterpenes and anthraquinones, among others.However, contrary to what was expected, anthocyanin was absent (Table 3).pathogens due to the increasing occurrence of infections by these fungi, especially in patients with cancer, diabetes and HIV (Hamza et al., 2006).However, the antifungal agents used in the treatment of Candida infections and in biofilms can select drug-resistant microbes (Agarwal et al., 2008).The ability of these microorganisms to form biofilm together with the acquisition of new antimicrobial resistance, has led to new problems in treating infections caused by this pathogen.Thus, the WHO has recommended the evaluation of the effectiveness of plants against resistant pathogens (Eisenberg et al., 1993).In this regard, new agents affecting the growth of biofilm-associated C. albicans and C. glabrata are greatly needed (Alviano et al., 2005).The present study was, therefore, carried out in order to evaluate the anti-biofilm activity of some Cameroonian plant extracts.Moreover, the exploration of additional natural resources for new antifungal agents with anti-biofilm activity could possibly reveal new antifungal agents with different modes of action or which affect different sites in Candida cells.This study summarizes the activity of the different extracts against C. albicans and C. glabrata in both the planktonic and biofilm state.This study's results show that the MIC of all the extracts ranged from 0.3 to >40 mg/mL, with the aqueous leaf extracts of E. uniflora and T. mantaly showing the best activity.Few studies have evaluated the antimicrobial activity of these extracts.The ethanoic extract of E. uniflora has antimicrobial activity against Staphylococcus epidermidis and Staphylococcus aureus, with MICs of 52 and 250 µg/mL, respectively (Bernardo et al., 2015).However, no assays using Candida species have been carried out.Other species within the genera Eugenia, such as Eugenia dysenterica, have shown antimicrobial activity against several Candida species with MICs ranging between 125 and 500 µg/mL (Correia et al., 2016).These values are similar to those found in the present study using E. uniflora.Plants are used in local communities worldwide for the treatment of various diseases.E. uniflora has been used in the traditional medicine of some African countries to treat various ailments such as wounds, skin diseases, dysentery and fever.In Brazil, E. uniflora leaf infusion is used as an antipyretic, astringent and also for treating several stomach problems.In Surinam, the E. uniflora leaf decoction is drank as a cold remedy and as an antipyretic in combination with lemongrass (Auricchio and Bacchi , 2003;Wagner et al., 1999;Morton, 1987;Stone, 1970).Likewise, T. mantaly leaf is taken as a decoction and infusion in the treatment of many ailments such as gastroenteritis, arterial hypertension, diabetes, dental affections and cutaneous and genital infections (Coulibaly, 2006).The MBICs and MBECs of these extracts were also determined.The results obtained showed that T. mantaly aqueous leaf extract inhibited biofilm formation of C. glabrata at a concentration of 0.45 mg/mL and was able to eradicate this biofilm at a concentration >1.8 mg/mL.On the other hand, E. uniflora aqueous leaf extract  inhibited biofilm formation of C. glabrata at a concentration of 0.125 mg/mL and eradicated mature biofilm at a concentration of 0.5 mg/mL.To the authors' knowledge, there is no previous study on the anti-biofilm activity of these plants.These activities could be due to the presence of tannins, steroids, triterpenes, flavonoid glucosides, saponins and anthraquinones in the E. uniflora extracts as has been suggested previously (Fiúza et al., 2008;Lorenzi and Matos, 2002).The presence of these components could act individually or in combination to produce the effects observed at the respective concentrations.Indeed, each of these constituents has a specific mode of action on the microbial strain.Thus, for example, tannins can act as antiseptic and antimicrobial agents and have antihaemorrhagic, antidiarrhoeic and wound-healing properties (Simões et al., 2004).On the other hand, terpenoids have been reported to have the ability to interfere with biofilm formation without disrupting cellular growth (Hertiani et al., 2010;Skindersoe et al., 2008).

Conclusion
The results of this study show that the studied extracts have antimicrobial activity and inhibit biofilm formation at the concentrations tested, suggesting that the bioactive compounds of these extracts are responsible for these activities.However, further studies are needed to verify which protein is inhibited and what chemical compounds in the extract are responsible for the activity observed.These compounds could be good candidates for the development of new anti-candida antibiotics, and tests with these compounds against other pathogenic microorganisms would also be of interest.

Figure 1 .
Figure 1.Biofilm inhibition concentration of E. uniflora and T. mantaly aqueous leaf extract in C. glabrata.

Figure 2 .
Figure 2. Biofilm eradication concentration of E. uniflora and T. mantaly aqueous leaf extract in C. glabrata.

Table 1 .
Plant collection site and date, and extraction solvent.

Table 3 .
Phytochemical screening of aqueous leaf extracts of T. mantaly and E. uniflora.