MOLECULAR CHARACTERIZATION OF RHAMNOLIPID AND ITS EFFECT ON CANDIDA BIOFILM

Abstract

Biosurfactants are amphipathic compounds possessing both hydrophilic and hydrophobic moieties that partition preferentially at the interface between two different phases in a heterogeneous system. In past two decades, these surface active compounds have gained significant commercial importance. as compared to their synthetic chemical counterpart due to their tailor-made multifaceted diversity, eco-friendly nature, higher biodegradable ability, low toxicity, effectiveness at extreme pH and temperature suitability for large scale production and selectivity. Among various types of biosurfactant available rhamnolipid, glycolipid-type biosurfactant, specifically produced by Pseudomonas aeruginosa are most effective surfactants which have great potential for biomedical and industrial applications. Rhamnolipid have also been found to have antagonistic effects on economically important zoosporic plant pathogens, thus opening their avenues as biocontrol agents. Recently, rhamnolipid has emerged as a promising multipurpose ingredient, which exhibit emulsifying, anti-adhesive and antimicrobial activities simultaneously and are consequently suitable for many food applications. However, despite their potential and biological origin, their role in commercialized usage as an effective therapeutic agent to combat infectious diseases is still to be established. This lacuna of their usage in the biomedical field prompted us to undertake the present investigation on in vitro studies on rhamnolipid medicated biofilm inhibition using Candida albicans as a model system. C. albicans is an opportunistic dimorphic pathogen which commonly affect immuno-compromised individual and is capable of causing life threatening infection. In fact, C. albicans is ranked fourth in causing nosocomial infections, third in catheter-related infections, second highest in colonization to infection rate and highest overall in crude mortality. It is believed that C. albicans in its biofilm mode consists of a structured communities of cells embedded in biomatrix. The process of biofilm 111 formation has been divided into different stages involving initial attachment to surface, formation of microcolonies on the surfaces and finally differentiation of microcolonies into exopolysaccharide-encased mature biofilm. Such biofilm can cause potentially enormous damage specially when formed on medical devices which can result in the failure of these devices and serve as a reservoir or source for infections. Thus, it becomes important to control primary colonization of fungal growth as an effective therapeutic solution not only to removal such fungal biofilm but also to eradicate them from surfaces. The objective of present work was thus outlaid to isolate, screen, purify and assess the ability of rhamnolipid as pointed below, as potential alternative to the available conventional therapies for the inhibition of C. albicans biofilm. From the petroleum oil contaminated sites, soil samples were taken and screened for native bacterial population for biosurfactant production. Through, screening on minimal media supplemented with different hydrocarbons as sole organic source, 25 isolates (NSVP1-NSVP10, DSVPl1- DSVP20 and SSVP21-SSVP25) were selected. These 25 bacterial isolates were then tested for haemolytic ability, drop collapse assay, emulsification assay and surface tension reduction as an index for surface active agent production. Of these four isolates namely NSVP2, DSVP11, DSVP17 and DSVP20 were found to pose biosurfactant producing ability and among them DSVP20 was found to be. potential producer of biosurfactant. Through 16S rRNA sequencing and taxonomic, characterization, it was found out that DSVP20 exhibited 97-99% similarity with Pseudomonas sp.. Further, biochemical tests and cultural characteristics established DSVP20 as Pseudomonas aeruginosa (GenBank accession no. GQ865644). Extraction of biosurfactant was done from the cell free broth through acidification followed by organic extraction with ethyl acetate. The extracted biosurfactant was further purified through absorption chromatography. The FTIR spectrum of bisourfactant was recorded in the spectral region iv of 4000-500 cm' which exhibited specific absorption bands at 3381 cm 1, 2927 cm', 2859 cm 1, 1735cm 1, 1635 cm', 1300-1100 cm 1, 1013 cm 1, 906 cm' and 840 cm 1 indicating the chemical structure to be di-rhamnolipid (RL2). The weak broad band located at 3364 cm 1 can be attributed to the O—H stretching vibrations of hydroxyl groups, while the strong absorption peaks at 2927 cm' and 2859 cm' are assigned to the C—H stretching vibrations of the hydrocarbon chain positions. Stretching band of C=O at 1730 cm' is characteristic of ester bonds and carboxylic acid groups. The carbonyl stretching peak was observed at 1640 cm', which is characteristic of ester compounds. The C—O stretching bands in the range of 1457-1100 cm' related to the. bonds -formed between carbon atoms and hydroxyl groups in the chemical structures of the rhamnose rings and the ester carbonyl group was also confirmed from the peak at 1057 cm', which corresponds to C-0 stretching vibration. Relatively strong sorption bands of pyranyl at 906 cm-1 and 840 cm' suggest it to be RL2 in nature. MALDI-TOF mass spectrum analysis of the sample gave intense signals at m/z 672.81 corresponding to the sodium adduct [M + Na]' of the RL2 which is reported to be m/z 649. The characteristic chemical shifts observed from 'H NMR analysis were 0.895 ppm (for —CH3), 1.262 ppm (for — (CH2)6), 2.552 ppm (for —CH2— COO ), 4.878 ppm (for —O—CH—), and 5.243 ppm (for — COO—CH ). The 13C NMR also displayed chemical shifts of 102.317. and 94.450 ppm which is a characteristic peak of RL2. The RL2 present in the cell free broth of Pseudomonas sp. showed thermal, pH and salt stability when tested at different time intervals. The surface tension and critical micelle dilution (CMD) values remained stable after exposure to high temperatures (100°C) even after 4 h. The surface activities were retained over a pH range of 4-12 with minimal deviation in surface tension and CMD' values, whereas CMD-2 showed a slight and gradual increase in surface tension with increasing pH values. No appreciable changes were observed on surface tension and v CMD values with the addition of NaCl up to 10% in cell free broth. Scanning electron microscopy, confocal laser scanning microscopy and atomic force microscopy were used in the study to explore the structural integrity and developmental characteristics of C. albicans biofilm. SEM observations provided useful information on the different cellular morphologies present in the biofilm structure. The mature biofilm consist of a mixture of yeast and filamentous forms embedded within exopolymeric material. Confocal laser scanning microscopy of C. albicans biofilm combined with fluorescent stains, FITC-ConA and PI showed presence of EPS in cell bound as well as secreted form. It-demonstrated that C. albicans possess structural heterogeneity and displays architectural similarity to that of bacterial biofilm. Atomic force microscopy provided a surface view of the C. albicans biofilm providing resolution at the atomic level. AFM was shown to be sensitive enough to dissect the effect of subtle changes in overall C. albicans cell surface composition on the initial interaction with biomaterials. XTT (2, 3-bis [2-Methoxy-4- nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay data showed maximum adherence with 48 h grown culture. Data also suggested that biofilm were highly metabolically active in its first 24 h. However, as the Candida biofilm matures and become more complex (48 to 72 h), the metabolic activity reached its plateau, reflecting high number of cells that constitute the mature biofilm. The minimum inhibitory concentrations (MICs) of RL2 were evaluated against C. albicans by standard broth dilution assay on different stages of C. albicans biofilm formation formed on 96 well microtitre plates. In vitro studies showed that anti-adhesive activity of RL2 to inhibit the C. albicans adhesion to be concentration dependent. Our observation showed about 50% of candidal cells attached to 96-well plate after 2 h of treatment with 0.16 mg mL`1 RL2. RL2 (5.0 mg mL-1) isolated from P. aeruginosa DSVP20 was able to reduce 96% of pre-biofilm formed on 96 well microtitre plate. Cell surface hydrophobicity data recorded vi also indicated that with increase in RL2 concentration, there is a marked reduction in cell surface hydrophobicity of C. albicans biofilm. C. albicans is the most common human fungal pathogen associated with device related infections mainly in its biofilm mode of growth. As biofilm residing Candida cells achieved much more resistance against antimicrobials than their planktonic counterparts, the fact that the RL2 was able to inhibit C. albicans biofilm adhesion as well as formation asserts on the possible use of RL2 as an alternative antifungal agent.