INTRODUCTION:

Candidiasis is one of the common fungal infections, and nowadays, it is ranked third as the most frequent nosocomial or hospital-borne infection^1,2. C. albicans has an ability to form biofilm, making it one of the. Candidiasis is frequently associated with biofilm formation that complicates the treatment process in the future³.

C.albicans has an ability to form biofilm, making it one of the virulence factors that causes candidiasis; a common infection occurring around the world⁴.

Every year, among patients infected with C. albicans, 50% of adults and 30% of children die because of candidiasis, and most of them are biofilm-related^5,6(Pfraller et al.,1998; Negri et al., 2012). Biofilms always consist of several types of microbes and some are very antibiotic-resistant^7,8.

Candida species in general and C. albicans, in particular, are common fungi that are related to biofilm-related infection⁹. Approximately 65-80% of C.albicans infection in human is biofilm-related⁴. Over 10 years ago, the seminal report from National Institute of Health (NIH) stated that 80% of soft and hard tissues infections in human are caused by biofilm microbes¹⁰. Bacterial and fungal infections caused by biofilm are very difficult to treat. In order to kill the bacteria and fungi in biofilm form, it takes 1000 times more than the normal dose of antimicrobials needed to reach the same result as the planktonic cell¹¹. C. albicans ability to form biofilm makes it one of the virulence factors that causes the candidiasis; a common infection occurred on the world⁴.

Every year, 50% of adults and 30% of children die because of candidiasis, most of them are biofilm-related^5,6 (Pfraller et al.,1998; Negri et al., 2012). Biofilm always consisted of some types of microbes and very antibiotic-resistant⁷. Candida species in general and C. albicans, in particular, are common fungi that related to biofilm-related infection¹².

Approximately 65-80% of C. albicans infections in human are biofilm-related⁸. The novel report from National Institute of Health (NIH) stated that 80% of soft and hard tissues infections in human are caused by biofilm^10,13. Bacterial and fungal infections that caused by biofilm are very difficult to treat, in order to kill the bacteria and fungi in biofilm form, it takes 1000 times than normal dose of antimicrobial needed to reach the same result as the planktonic cell^11,14,15.

Biofilm is controllable by using a chemical compound that can be obtained from nature¹⁶. Nowadays, there are plenty of natural products known that contain active biological components, which are beneficial in medication especially in antimicrobial therapy¹⁷. Biofilm control can be performed in a chemical manner by adding some chemical substances such as detergents that contain enzymes, or in a physical manner by escalating the temperature and biological manner by using bacteriophages and relying on microbiological interactions^18.

In recent years, there has been a growing interest in the discovery of treatments called generally regarded as safe (GRAS) compounds from natural substances to combat new and existing diseases. Several plants are attractive species for this application because they contain an abundance of potentially active secondary metabolites¹⁹.

Demethoxycurcumin belongs to the curcumin group (Curcuma longa Linn.) that possess a high antimicrobial activity^20,21.Nevertheless, the biofilm activity of demetoxin is not yet known. Addressing that problem, our study aimed to examine the effectiveness of the demetoxin compound on C. albicans biofilm in order to find a new strategy to fight against biofilm infection caused by C. albicans.

MATERIALS AND METHODS:

Materials:

Materials used were Demethoxycurcumin compound from isolation of (Curcuma longa Linn.), Standard biofilm-forming C. albicans isolate (C. albicans ATCC 10231) from the Microbiology Laboratory, Faculty of Pharmacy, UGM, Nystatin, DMSO 1%, NaCl, McFarland 0.5 standard, sterile aquadest, Sabaroud Dextrose Agar (Merck, Germany), RPMI 1640 (Merck, Germany), PBS (Phosphate Buffer Saline) solution, and crystal violet 1% (Merck, Germany).

Equipment:

Some equipment’s used in this research were Laminar Air Flow, incubator (IF-2B) (Sakura, Japan), micropipettepipetman (Gilson, France), multichannel micropipette (Socorex, Swiss), microplate flat-bottom polystyrene 96 well (Iwaki, Japan), microtiter plate reader (Optic Ivymen System 2100-C, Spain), spectrophotometry(Genesys 10 UV Scanning, 335903) (Thermo Scientific Spectronic, USA), autoclave (Sakura, Japan),and analytic scales (AB204 -5, Switzerland).

Fungal strains and inoculum preparation:

Isolates tested in this study were C. albicans (ATCC 10231). The first step performed was inoculating several fungi colonies from SDA media to 15 mL YPD medium, later the colonies were incubated at room temperature and were put in a shaker overnight. Later, the substances were centrifuged for 15 minutes at 300 rpm speed and washed by PBS. Pellets obtained were re-suspended with RPMI medium and a 1x10⁸ fungi suspension CFU/mL was made in 5mL RPMI medium (per the MacFarland standard 0.5). Next, we took 1 mL of this suspension and added it to the 9mL of RPMI medium, so that the 1x10⁶ CFU/mL fungal suspension colony can be obtained as a stock solution. The tested solution was used by 1:10 dilution so the 1x10^6
CFU/mL was obtained²² (CLSI, 2008).

Minimum biofilm inhibitory concentration (MBIC):

Demethoxycurcumin effect was assessed on strain C. albicans ATCC 10231 biofilm. Biofilm was inoculated in a microtiter plate polystyrene flat bottom 96-well. A 200 uL C. albicans suspension (10⁶CFU/mL) was inserted in each pit on the microtiter plate, then the suspension was incubated at ± 37°C for 90 minutes²³. After the incubation phase, the plate was washed with PBS. There was 200 uL media that contained pure isolate with concentration series (1% b/v - 0125% b/v), was added to all washed-pits. Media that contained DMSO 1% was used as solvent control, and microbe suspension was used as negative control. A microbe suspension that was used an antifungal (nystatin 1% b/v) previously was used as positive control, while a media with no microbial growth was used as media control⁹. The plate was then incubated at 37°C for 24 hours for mid-phase biofilm forming and 48 hours for maturing phase²⁴. Then, the plate was washed with PBS. Next, 125uL crystal violet 1% solution was added to each pit, next they were incubated at room temperature for 15 minutes. After the incubation, the microplate was washed with PBS and added with 200uL of ethanol 96% in each pit to dilute the formed biofilm. An Optical Density (OD) examination was performed with a microplate reader at 595 nm wavelength.

Minimum biofilm eradication concentration (MBEC):

Demethoxycurcumin effect was also assessed on strain C. albicans ATCC 10231 using the previous published methods²⁵. Biofilm was inoculated inside a microtiter plate with the same manner as explained above. After incubated at 37°C for 48 hours, the cultures from each pit were decantated, and planktonic cells were diminished by washing it with PBS. The biofilm cells were exposed by Demethoxycurcumin at several concentrations, started from 1% b/v and up to 0.125% b/v, later on they were incubated at 37^oC for 48 hours. Nystatin 1% b/v was used as positive control. After incubated, plates were washed three times with 200 mL of sterile PBS in order to diminish any attached cells. Biofilm degradation was quantified by 125 uL of crystal violet 1% solution in each pit; then incubated at room temperature for 15 minutes. After incubation, microplates were washed with PBS and ethanol 96% was added inside each pit to dilute the biofilm formed. An Optical Density (OD) examination was performed with microplate reader at 595 nm wavelength^9,26.

Scanning electron microscopy (SEM):

For SEM observations, C. albicans biofilms were formed on the sterile polyvinyl chloride coverslips (with 0.13—17 mm thickness and 22 mm diameter) inside a 12-well microtiter plates (Corning1 Costar1, Sigma—Aldrich, Missouri, USA) in the presence of 0.25 % b/v of Demethoxycurcumin for 72 h at 35°C, as described in the previous section. A biofilm grown in the absence of the test drug served as a control. The biofilms were assayed according to the method of Atshan et al, (2012)²⁷, with slight modifications. Briefly, the coverslips were removed, washedtwice with sterile PBS (0.1 M and pH 7.2) and placed in a primary fixative solution [glutaraldehyde 0.15 M 2.5 % (vol/vol) in PBS] at 4 8C for 60 min. The coverslips were subsequently rinsed 2 times with PBS for 5 min, then treated with the secondary fixative (osmium tetroxide OsO₄ 1% w/v) for1 h. The samples were subsequently washed with distilled water, dehydrated in an ethanol series (70 % for 10 min, 95 %for 10 min and 100 % for 20 min) and air-dried overnight in a desiccator. The coverslip was coated twice with platinum vanadium using a sputter ion (Bal-Tec SCD 005), followed by bonding to carbon double-side tape for examination by SEM (JEOL JSM T-300, Japan).

Statistical analysis:

Statistical analysis was started by normality test and homogeneity test of the data. From the normality test, if the result was p<0.05 which means data were not distributed in a normal manner, then, the data was analyzed using One Way Anova, followed by post-hoc Bonferroni test to compare the control group with the treatment group, using the significance level of p<0.005.

RESULT:

Demethoxycurcumin Effect on Mid-phase (24 h) Mono-species C. albicans biofilm:

In this study, we evaluated the Demethoxycurcumin potential in the inhibition of mono-species biofilms C. albicans. The results showed that Demethoxycurcumin could inhibit 50% of biofilm mono-species C. albicans (Figure 1).

Figure 1. Effect of Demethoxycurcumin againts mono-spesies C. albicans. Blue = mid-phase (24h), Yellow = maturation phase(48h).

The inhibition activity of Demethoxy curcuminon mid-phase C. albicans 82.70 % ± 0.02 is superior compared to nystatin medication control 70.67 ± 0.01. The MBIC₅₀ activity of Demethoxycurcumin compound reached 0.123% b/v level, demonstrating that Demethoxycurcumin compound’s activity is beneficial in inhibiting the antibiofilm development (Table 1).

This result also shown that Demethoxycurcumin compound activity is faster than biofilm forming, making the biofilm not able to form a complex structure and community which would trigger the more severe and chronic infection.

Demethoxycurcumin Effect on Maturing Phase (48 h) Mono-species C. albicans biofilm:

Demethoxycurcumin can account to 50% of the damage in the maturing phase from C.albicans monospecies with inhibition activity of 62.94 ± 0.01, while nystatin 1% (the control) shown a lower inhibition activity compared to Demethoxycurcumin (55.64% ±1.24) (Figure 1). MBIC₅₀ value of Demethoxycurcumin in the maturing phase is placed on 0.5% b/v concentration.

Effect Demethoxycurcumin on Eradication Phase Mono-species C. albicans biofilm:

Eradication phase is a long phase in biofilm forming, so the biofilm formed is also stronger and able to work in a synergy with one another. The biofilm defense Eradication phase is stronger than the maturing phase, when EPS composition is more complete, caused by the longer time the biofilm growth takes, resulting in the more nutrition it gets. We report that the Demethoxycurcumin 1% b/v compound can degrade up to 50% of the biofilm with inhibition activity of 57.25%± 0.01 (Figure 2).

Figure 2. Effect of Demethoxycurcumin againts mono-spesies C. albicans degradation phase.

This result shown that Demethoxycurcumin1% compound can damage the defenses in biofilm forming of C.albicans. This compound can cut off the communication cell pathways so that the number of biofilms grown can be diminished. With the control drug nystatin, the inhibition activity that was given in C.albicans eradication phase of the biofilm was lower compared to Demethoxycurcumin compound with inhibition activity of 50.21% ±1.24. This result matched with a previous study from Atik (2018)²⁸, showing that nystatin had a lower effectiveness compared to pure C-10 massoialactone isolate.

Result of Scanning Electron Microscopy of C.albicans biofilm with No Treatment

C.albicans biofilm that received no treatment shown a dense, complex cell walls with high cell density and the presence of EPS matrix that protected C.albicans (Figure 3).

Figure 3. Biofilm C. albicans without Treatment, use Scanning electron microscopy with (X 10, 000)

This finding supports the theory from Pierce et al, (2017)⁴, that stated the biofilm matrixacted as the connecting of the protective binding and cohesive interaction that provided a mechanic stability of the biofilm, controlling cell dispersion from the biofilm, and acted as the nutrition provider for cell communication. Biofilm matrices of C. albicans also protected the biofilm cells and acted as the main barrier that protect biofilm cells from the response of the immune system and antifungal medication attacks during infection.

Results of the Scanning Electron Microscopy of C.albicans biofilm with Demethoxycurcumin 0.25 % administration b/v.

The administration of Demethoxycurcumin compound 0.25%b/v (Figure 4)

Figure 4. Biofilm C. albicans by giving tannin compounds 0.25 % b/v use Scanning electron microscopy (X 10, 000)

On C.albicans biofilm indicated that there were damaged cells caused by the integrity of the host cells, preventing C.albicans to form hyphae. On the other hand, Demethoxycurcumin 0.25% b/v also caused a decline of the cells’ attachment and density, and there was also an inhibition on biofilm forming that was shown by lysis; this was caused by the active compound that attacked and damaged the EPS matrices that protected the C.albicans biofilm. Aside from that, the administration of Demethoxycurcumin also was able to change the morphological structure of the C.albicans biofilm and cut off the intra-cellular communication pathway in C.albicans biofilm. This compound can penetrate through the biofilm’s polysaccharide matrices, and dissolve the lipids inside the biofilm matrices as stated by Cowan (1999)²⁹.

DISCUSSION:

Biofilm is a collection of microbial cells that become attached in an irreversible manner on a surface and wrapped inside the extracellular polymeric substance matrix (EPS) that is produced by itself. Biofilms show a phenotype change such as a change in the growing stage growth and gene transcription from planktonic cells or free cells^26,30,31,32.

This evidence supports an early conclusion that provides some preliminary explanation, that microbe can live as a single free cells (planktonic), in nature, while some microbes tend to live together forming a colony, attaching and growing on a surface, and forming a layered-structure known as biofilm. Biofilms always consist of different types of microbes and some are very resistant towards antibiotics.

The findings of Drake et al, (2018) ³¹explaining that bacteria on the biofilm are different than planktonic cells, according to their growing style. One of the consequences from the differences is the bacteria inside the biofilm are proven more resistant to antibiotic and antimicrobes³².

Demethoxycurcumin undergoes a decline in activity compared to the cell in mid-phase. This is due to the growth in this phase, where C. albicans biofilm is made perfectly so that it has obtained enough protection. This can be seen from the mucous layers produced by the biofilm on the maturing phase are more abundant compared to the mid-phase, in addition to that, the EPS matrix produced in this phase are also more abundant compared to the mid-phase, demonstrating the fact that EPS acts as a protector and provider of nutrition for C.albicans biofilm survival, so that new colonies are formed. This result match the study from Hamzah et al, (2018)⁹, showing that in the maturing phase, antimicrobial agents will face more challenge to go through the biofilm defense system. Adult biofilms consist of yeast with hyphae that form complex tissues that are covered in an EPS and exist away from surface^33,34,35.

The trial inhibition process for the growth of mature phase biofilms by inhibiting microbial adhesion of the surface increases biofilm development, causing the development of disturbed biofilms, and this will affect the structure of biofilms to increase resistance to antimicrobials. In addition, test compilation also damages EPS biofilms, so that cell and microbial nutritional communication pathways are cut off so that microbes aimed to make more biofilms will become lysis or die, because they require nutrients to be made up from biofilm production.

The inhibition activity performed by the Demethoxycurcumin compound was lower compared to the mid-phase and maturing phase. This finding can be caused by the complex microbes that had been formed, and they are attached deeper, in addition to the EPS produced which was more abundant compared to the two previous phases.

Microbes in the degradation phase can be harder to damage compared to the microbes in mid-phase and inhibition phase. This occurs due to the longer time, allowing greater cooperation among the bacteria in the community so that antibiotics could not get in through the EPS matrix easily⁹, C. albicans and S.aureus form a complex polymicrobial inside the serum. Although S.aureus forms a small amount of monospecies inside the serum, it can build a bigger polymicrobial biofilm by the presence of C.albican³³. The MBIC₅₀ values in this phase reach the 0.50 % b/v (table 1). This result demonstrates that Demethoxycurcumin can be developed and used as one of the antibiofilm medication candidates.

CONCLUSION:

Demethoxycurcumin compound has the ability to inhibit and degrade C.albicans biofilm and shown a better activity compared to nystatin medication control. Accordingly, this compound has the potential to be developed as one of the candidates of the new antibiofilm agents against C. albicans.

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Received on 18.07.2019 Modified on 21.08.2019

Research J. Pharm. and Tech. 2020; 13(1): 377-382.

DOI: 10.5958/0974-360X.2020.00075.X