Inhibition of Biofilm Formation in <em>Candida albicans</em> and <em>Candida krusei</em> by <em>Combretum zeyheri</em> Leaf Extracts

Research Article

J Bacteriol Mycol. 2018; 5(3): 1068.

Inhibition of Biofilm Formation in Candida albicans and Candida krusei by Combretum zeyheri Leaf Extracts

Mtisi B1, Sithole S2, Mombeshora M2 and Mukanganyama S2*

¹School of Pharmacy, University of Zimbabwe, Mt. Pleasant, Harare, Zimbabwe

²Department of Biochemistry, University of Zimbabwe, Mt. Pleasant, Harare, Zimbabwe

*Corresponding author: Stanley Mukanganyama, Biomolecular Interactions Analyses Group, Department of Biochemistry, and University of Zimbabwe, Zimbabwe

Received: April 13, 2018; Accepted: May 16, 2018; Published: May 23, 2018


Fungal infections are currently on the increase due to use of invasive devices such as indwelling catheters, antibiotic misuse and HIV/AIDS infections. Antifungal drug resistance may be due to biofilm formation, polymorphism or phenotypic switching. Combretum zeyheri is used for the treatment of various ailments such as snakebite, diarrhea, hypertension and eye infections. The aim of this study was to identify C. zeyherileaf extracts that inhibit biofilm formation in Candida species. Eight extracts were screened to for antifungal activity using broth micro dilution assay. The screening for inhibition of biofilm by the extracts was determined using biofilms formed on 1cm2 PVC square disks. The amount of biofilm formed was determined spectrophotometrically after staining with crystal violet. The ethanol extract, ethyl acetate extract, acetone extract showed potent antifungal activity against C. albicans whilst in C. krusei, it was the methanol, ethanol, ethyl acetate and hexane extracts. The methanol extract, hexane extract, DCM extract and DCM-methanol extract showed potent inhibition of biofilm formation in C. albicans whilst in C. krusei only the water extract and ethanol extract showed inhibition of biofilm formation. The DCM extract was the most effective in inhibition of biofilms. The minimum inhibitory concentration for biofilm formation for the DCM extract was 62.5μg/ml in C. krusei and 50μg/ml in C. albicans. In addition to inhibition of growth; C. zeyheri extracts inhibit biofilm formation in both C. albicans and C. krusei. The isolation of active components that exhibit both antifungal and anti-biofilm formation from C. zeyheri is required.

Keywords: Combretum zeyheri; Candida albicans; Candida krusei; Biofilm Formation; Candidiasis


The biggest challenge to all modern day clinicians is appearance of new or resistant strains of any disease causing microorganism [1]. Microbial resistance is on the rise and it is a cause for concern and a need arises for new pathways to be explored and to discover possible answers to nature’s new challenges which are presented through microbial evolution [2]. The use of herbal or plant remedies has been part of the primary health care system in Zimbabwe for a long time and they are now commonly integrated as complements to conventional medicines [3]. Literature documents claims about the antimicrobial properties of many plants all over the world [4]. The Combretaceae family is one of the plant species extensively explored with a wide array of antimicrobial activity and plays a large part in African ethno medicine [5].

The candida genus is a heterogeneous group microorganism and 17 different species are known to cause human infections. Candida albicans, Candida krusei, Candida parapsilosis and Candida glabrata are the common species which result in over 90% of all invasive infections [6]. C. albicans is a commensal microorganism found on human skin, mucosal oral cavity, gastrointestinal tract and vagina [7]. Its presence in a healthy individual does not cause any problems, but this may change however in immune-compromised individuals. Virulence factors are genetically determined, but expressed by microorganisms when subjected to certain conditions [8]. Virulence characteristics specialized and developed by Candida spp. are involved in invasion and adhesion of host tissues, biofilm formation and evasion of the immune system and the infection [9]. Virulence factors are expressed when a suitable environment persists and these include polymorphism [10], production of adhesins and invasins [11].

Biofilms are communities found within a matrix of extracellular material. The matrix is a three–dimensional, gel-like hydrated and charged environment in which the microorganisms are immobilised [12]. Within a matrix there are channels that provide a mechanism for nutrient circulation. Failure by antimicrobial agents to penetrate is due to the biofilm matrix. The drugs that by-pass are assisted by water channels within the biofilm. The microbial biofilm matrix components could also retard access to drug via adsorption or neutralisation [13]. In general, the biofilm matrix comprises of carbohydrates, proteins, phosphorus and hexosamines. Environmental conditions such as medium composition, pH and oxygen as well as the fungal strain and species affect biofilm formation and matrix composition [14]. Biofilms can grow on any biotic or a biotic moist surface. Formation of biofilms by microorganism is also a defense mechanism for their own survival in a hostile environment [15].

Plants have been shown to contain more compounds than chemists can synthesize and have served as a source of new drugs, pharmaceutical products and provide starting materials for synthesis of many known drugs [16]. There are at least twenty four known species of Combretum family commonly used in African traditional medicine and these form the basis of most traditional health care in the management of a variety of ailments and diseases [17]. Traditionally Combretum zeyheri is used for the treatment of various ailments such as snake bite, diarrhea and hypertension. In Zimbabwe, the plant is used for diarrhea and eye infections [18]. Combretaceae and Terminalia spp have been used for the management of fevers, cough, respiratory infections, urinary tract infections, piles and worms and also useful in treating chronic diarrhea and dysentery, flatulence, vomiting, colic and enlarged spleen and liver [19].

For in vitro studies using antibacterial agents, susceptibility is commonly quantified using minimum inhibitory concentrations (MICs) to determine the lowest concentration required to exert growth inhibition. Extracts of C. zeyheri have been reported to have antimicrobial activity C. albicans, C. krusei and C. parapsilosis [20]. Isolated phytochemicals showed antifungal activity on laboratory strains and the clinical fungal isolates [21]. In a study by Mangoyi et al, [22], C. zeyheri extracts showed to be effective antifungal and drug efflux inhibitors. Mutasa et al [23], showed that C. zeyheri ethanolic extract produced a dose-dependent effect on ergosterol synthesis in C. albicans by disrupting membrane integrity. Mangoyi, et al, [24] isolated and from C. zeyheri that showed antifungal activity in both C. krusei and C. albicans. The flavonoids also showed effective inhibition of ergosterol synthesis in C. albicans. From these studies, C. zeyheri extracts has established antifungal properties, but there are few studies on its effects on virulence factors such as the ability to affect biofilm formation. The aim of this study was to determine the effects C. zeyheri extracts on biofilm formation in C. albicans and C. krusei.

Methods and Materials

Fungal strains, reagents

The following reagents and chemicals used were obtained from Sigma-Aldrich (Taufkirchen, Germany): MTT (3-(4, 5-dimethylthiazol-2-yl_-2, 5-diphenyltetrazolium bromide), DMSO, miconazole, Sabouraud’s dextrose broth (SDB), Sabouraud’s dextrose agar (SDA), crystal violet, and barium chloride. C. albicans ATCC 10231 and Candida krusei clinical strain was obtained from Parirenyatwa Group of Hospitals, Harare, Zimbabwe.

Plant collection

Leaves of C. zeyheri were collected from Norton (170 53' 0'' S, 300 42' 0'' E) Mashonaland West Province of Zimbabwe. The plant identity was authenticated and classified by Mr. C. Chapano, a taxonomist at the National Herbarium and Botanical Gardens (Harare, Zimbabwe). The herbarium samples (voucher specimen N6E7) were kept in the Department of Biochemistry, University of Zimbabwe.

Preparation of plant extracts

Leaves were dried for five days in a Labcon orbital incubator (Labotec Co., Cape Town, South Africa) at 40°C. The leaves were ground by a manual / traditional pestle and mortar to a fine powder. C. zeyheri extract was prepared by weighing 50g of powder and adding 500ml of solvent. A mixture of 50:50 DCM: Methanol was used. The mixture was left to stir for 24 hours on a magnetic stirrer. After 24 hours to prepare the total extract the mixture was first prefiltered with cotton wool. The resultant filtrate was re-filtered with a Whatman™ no.1 into a pre-weighed plastic beaker. The resultant filtrate was then air dried under a fan. After drying the residue, the second solvent was added. This serial extraction was done using solvents in the following order of increasing polarity: Hexane, DCM, ethyl acetate, acetone, ethanol, methanol and water.

Growth of Candida species

C. albicans or C. krusei from glycerol stocks were resuscitated by subculturing in SDB for 24h at 370C in a Lab Companion incubator (Jeio Tech, Seoul, South Korea) shaking at 120rpm. The resulting inoculum was streaked on SDA plates and colonies from these plates were once more cultured in SDB. The optical density of C. albicans or C. krusei was adjusted with SDB to match that of 0.5 McFaland’s standard at 600nm. Appropriate dilutions were made to get a final inoculum concentration of 2×106cfu/ml.

Antifungal susceptibility tests using C. zeyheri leaf extracts

The effect of miconazole and the eight C. zeyheri leaf extracts obtained from the serial exhaustive and total extraction was investigated using the microbroth dilution method designed by Scorzoniet al, [26]. A stock solution of miconazole or the plant extracts were prepared by dissolving 0.005g in 1.25ml absolute DMSO and made up to 25ml by SDB media to a concentration of 200μg/ml. Two-fold serial dilutions were made to end up with concentrations of 100, 50, 25, 12.5, 6.3, 3.2, 1.6, 0.8 and 0 ug/ml. Aliquots of 100μl of solutions and 100μl of standardized cells culture were added to each well on a 96-well microplate. The negative control contained 0μg/ml of the extract and the antibiotic miconazole was the positive control. For the plant extracts the positive control included wells containing miconazole and cells as well as wells containing cells only. The final concentration of DMSO in each well was 2.5%. Absorbance of the wells was read at 590nm using a Genios Pro microplate reader (Tecan, Grödig, Austria) and the plate were incubated for 24h at 370C. After incubation absorbance of the plate was read again at 590nm in the ELISA reader to determine the change in cell density. MTT was added to determine cell viability. Cells were incubated for 2 hours after which 25μL of DMSO were added to dissolve the formazan crystals.

Screening of plant extracts for inhibition of biofilm formation by Candida species

A stock solution of extracts was prepared by dissolving 5mg of extract and dissolved in 1.25ml of absolute DMSO and made up to 25ml with SDB media. An overnight culture of cells was prepared. The cells were centrifuged in a Hettich Rotofix 32 (Tuttlingen, Germany) and washed once with 0.9% saline solution. The cells were standardized to a final count of 1.5 x108 cfu/ml. The method used for quantification of biofilm was a modification of one described by Borucki et al., [27]. Squares of 1cm x 1cm polyvinylchloride (PVC) were prepared and sterilized in 70% ethanol for overnight. Four disks were placed in each well of a six-well plate. An aliquot of 1.5ml of SDB media was added to each well and 1.5ml the standardized cell suspension was added to each well. The plates were incubated for 3h at 370C shaking at 80rpm in a Lab Doctor Mini Incubated Shaker (MidSci, California, USA). After 3h the cells disks were washed once with phosphate bicarbonate saline buffer. The disks were transferred into 12-well plates. Each well of the 12 well plates had 1.5ml aliquot of extract and 1.5ml of DMSO diluted in SDB media. Each extract was in duplicate, the third well was a control with extract, DMSO in media and a disk with no cells on it. The controls were disks in cells only, disks in cells + diluted DMSO, disks in miconazole at MIC and disks in media only. The plates were incubated at 370C shaking at 80rpm for 72h. Biofilm formation was determined in triplicate for each sample.

After 72h the well the free cells were decanted and the squares were washed three times with PBS. The wells were filled with 3ml 0.1% crystal violet and left to stand at room temperature for 45min. The crystal violet solution was removed and the wells and disks washed with PBS buffer five times. The plates were left to dry under a fan. After drying the crystal violet was dissolved in 95% ethanol, by adding 3 ml of the ethanol to each well. The crystal violet was left to solubilize for 30min. The dissolved crystal violet was transferred to 96-well plate. Each well from the 12-well plate contributed to 3 wells on the 96 well plates. The absorbance of crystal violet which was directly proportional to biofilm formation was read at 590nm in a Genios Pro ELISA reader (Tecan, Grödig, Austria).

Determination of MIC for the extract with most potent biofilm formation inhibiting capacity

A stock solution of 1000μg/ml was prepared by dissolving 25mg of extract in 1.25ml of DMSO and made up to 25ml with SDB media. Serial broth dilutions of the stock solution were made to 500, 250, 125, 62.5, 31.5, 15.8, 7.9 and 0 μg/ml. The disks of 1cm 2 were prepared and after incubating, the disks were placed in wells of the 12 well plates with 1.5ml aliquot of extract and 1.5ml of DMSO diluted in SDB media. Each concentration had a duplicate and a 3rd well was a control with extract, DMSO in media and a disk with no cells on it. The controls were disks in cells only, disks in cells + diluted DMSO, disks in miconazole at MIC and disks in media only. The plates were incubated at 370C shaking at 80rpm for 72 hours. After 72 hours, the extract suspensions were removed and the wells washed with PBS buffer three times. The wells were then filled with 0.1% crystal violet, 3ml each well and left to stand at room temperature for 45min. The crystal violet solution was removed and the wells and disks washed with PBS buffer five times. The plates were left to dry under a fan and ethanol was added to solubilize crystal violet after which absorbance of the dye was determined at 590nm.

The effects of combining potent anti-biofilm extract with a standard antifungal drug

A stock solution of 1000μg/ml was prepared by dissolving 25mg of extract in 1.25ml of DMSO and made up to 25ml with SDB media. Two-fold serial broth dilutions of the stock solution to give the following concentrations: 1000, 500 and 250 μg/ml. An overnight culture of cells was prepared. The cells were centrifuged in a Hettich Rotofix 32 (Tuttlingen, Germany), washed once with 0.9% saline solution and resuspended in PBS. The optical density of the cells were adjusted to McFarland standard to give a final count of 1.5 x108 cfu/ml. Biofilm formation was measured as described above but now including a combination of the extract and miconazole. A stock solution of 200μg/ml miconazole was prepared and diluted to concentrations of 25, 12.5 and 6.3 μg/ml for C. albicans and 12.5, 6.3 and 3.2 for C. krusei. A modification of checker board assay described by [28] was done. The square disks were transferred to 12 well plates. Each of the nine combinations was done in duplicate on the 12-well plate. Controls with combination concentrations and a disk with no cells were prepared for each combination. Other controls were miconazole only at MIC, cells with DMSO, media only and cells only. Each well contained 1.5ml of media with DMSO and 1.5ml of combination concentration in 50:50 ratio. The plates were incubated at 370C shaking at 80rpm for 72h in a Lab Doctor Mini Incubated Shaker (Mid Sci, USA). After 72h, the extract suspensions were washed off three times using PBS. The wells were then filled with 0.1% crystal violet, 3ml each well and left to stand at room temperature for 45min. The crystal violet solution was removed and the wells and disks washed with PBS buffer five times. The absorbance of the crystal violet from the disks was similarly determined as described above.

Statistical analyses

Statistically significant differences between the mean of the controls and the tests were analysed using one way ANOVA with Dunnett’s multiple comparison post-test using Graph Pad Prism 5 (Version 5.03 Graph Pad Software Inc. San Diego, California USA).


Extraction of phytochemicals from C. zeyheri leaves

Solvent extraction is a method used to separate phytochemicals from plants. Solvents used separate them according to solubility in the solvent used or polarity. The yield is an indicator of the types of constituents in the leaves. After extraction and drying the water extract had the largest yield (12%), followed by DCM/Methanol (9%), ethanol (3.8%), acetone (3.7%), methanol (3.2%), hexane (2.3%), DCM (1.65%) and ethyl acetate extract (1.63%).

The effects of DMSO on cell viability

DMSO was the solvent of choice used to dissolve the extracts. All the extracts were soluble in DMSO. This assay was done to determine the possible toxic effects of the solvent to the fungal cells. C. albicans showed greater sensitivity to DMSO compared to C. krusei. Concentration of up to 5% DMSO had no effect on cell viability in C. krusei unlike in C. albicans where sensitivity began at concentrations above 2.5%.

Determination of MIC of Miconazole in C. albicans and C. krusei

Miconazole was used as the positive control in all the assays. The minimum inhibitory concentration was determined too as it was used as the reference standard. The results in showed that Candida krusei (MIC =3.2μg/ml) was more sensitive to the effects of miconazole when compared to C. albicans (MIC= 6.3μg/ml).

The effects of C. zeyheri leaf extracts on Candida growth

The ethanol extract was found to be the most effective at inhibiting the growth of C. albicans with an MIC of 3.2μg/ml, followed by methanol with an MIC of 25μg/ml (Figure 1). The ethyl acetate extract and acetone extract also had inhibitory activity, but MIC was higher than the highest concentration of 100μg/ml. The DCM-methanol extract showed no growth inhibitory activity in C. albicans for the range of concentrations used in the assay. For C. krusei the ethanol extract again had the most potent inhibitory activity with an MIC of 50μg/ml (Figure 2). The ethyl acetate extract, hexane extract and methanol extract both had an MIC of 100μg/ml. The DCM, DCMmethanol and acetone extracts had inhibitory activity but, however, the MICs were above the highest concentration of 100μg/ml used in the study. To compare the potency of the extracts, the percentage growth inhibition at the maximum concentration of 100μg/ml was determined and results are shown in Table 1.