1. RESULTS AND DISCUSSIONS ORIGINALITY OF THE WORK
Ocimum Sanctum Extract Coating on Biomaterial Surfaces to prevent Bacterial Adhesion and Biofilm Growth
Sivaramakrishnan R1, Guruprakash S1*, Gnanaprakash D2, Subashini R1
1 Department of Biomedical Engineering, SSN College of Engineering, Kalavakkam, Tamil Nadu, India
2 Department of Chemical Engineering, SSN College of Engineering, Kalavakkam, Tamil Nadu, India
INTRODUCTION
Unadsorbed extracts removed from the wells.
1 ml of bacterial suspension - added to the wells & allowed to adhere & grow aerobically at 37°C for different time points 1 h, 3 h and 24 h. Bacterial adhesion in the absence of extract coating, considered as control. Wells, washed with sterile phosphate buffer saline (10 mM potassium phosphate, 0.15M NaCl, pH 7.0) to remove unbound bacteria. Images taken using phase contrast microscopy & number of adherent bacteria per cm2 determined using ImageJ® software.
Holes of 6 mm diameter - punched into the nutrient agar plates. Holes filled with 100 µl of OS extract & incubated at 37°C for 24 h. Antibacterial activity assessed by measuring the Zone of inhibition.
Extended use of biomaterials - limited due to Biomaterials-Associated Infections (BAI) [1]. Microorganisms involved in BAI - protected from antibiotics & host immune system due to their biofilm mode of growth.
Modification of biomaterial surface - prevents microbial adhesion and biofilm formation. Several biomaterial surface modifications [2-3] developed to reduce bacterial adhesion & biofilm formation, but microbial adhesion was reduced by one or two log units & not fully eliminated.
Medicinal plants and its phytochemical compounds - effective against a wide array of diseases - attracts the interest in using extract of medicinal plants as a coating for biomaterials.
RESULTS AND DISCUSSIONS
ORIGINALITY OF THE WORK
The water contact angles of bare PMMA and PS are 73.6±1.5 & 68.5±1.2 respectively, whereas the OS extract coated surfaces were hydrophilic compared to bare surfaces (Figure 2).
A significant reduction (p<0.01) in the number of adherent bacteria on OS extract coated surfaces compared to bare surfaces was observed. A similar trend was observed in all the three bacteria (S.aureus, E.coli and P.aeruginosa) used (Figure 3, 4).
Zone of inhibition of OS extract –OS extract showed maximum inhibition for P.aeruginosa (30 mm diameter) compared to S.aureus (25 mm diameter) and E.coli (28 mm diameter) (Figure 5).
Figure 4. Number of adherent bacteria ([A] S. aureus, [B] P. aeruginosa and [C] E. coli) after 3 h of growth on different biomaterial surfaces in the absence and presence of OS extract coating. * denotes significant difference at p<0.01 compared to control.
Ocimum sanctum (OS), also known as Ocimum tenuiflorum, Tulsi or Holy basil, is one of the widely used medicinal plants, an aromatic kind that belongs to the family Lamiaceae.
Extracts from OS were identified to be extremely effective against both gram negative and gram positive bacteria [4].
OS extract demonstrates antibacterial, antioxidant, antidiabetic, anti-cancer and immuno-modulatory properties [5].
In this work, an attempt has been made to evaluate the performance of Ocimum sanctum extract as a coating on biomaterial surfaces in preventing bacterial adhesion and biofilm growth, as an effective measure to combat BAI (Figure 1).
Figure 5. Antibacterial activity of OS extract. A) Images represent the zone of inhibition of bacterial growth and B) table shows the diameter of inhibition zone in mm
Figure 2. Water contact angle of different biomaterial surfaces in the absence and presence of OS extract coating
CONCLUSIONS
This study demonstrates the effectiveness of OS extract as a coating on biomaterial surfaces to prevent bacterial adhesion and biofilm growth to combat BAI. OS coated biomaterial surfaces showed significant reduction in bacterial adhesion and biofilm growth up to 24 h. The antibacterial mechanisms may be attributed to several targets in the cell. The strategy of essential oil extract coating on biomaterial surfaces could significantly reduce BAI.
Figure 1. Schematic diagram of the experimental methodology presenting OS extract as a coating to implant surfaces to prevent bacterial adhesion and biofilm growth
REFERENCES
MATERIALS AND METHODS
1. Hermansson M. The DLVO theory in microbial adhesion. Coll Surf B-Biointerf 1999;14:105–19.
2. Maddikeri et al. Reduced medical infection related bacterial strains adhesion on bioactive RGD modified titanium surfaces: A first step toward cell selective surfaces. J Biomed Mater Res 2008; 84:425–35.
3. Boks et al. Mobile and immobile adhesion of staphylococcal strains to hydrophilic and hydrophobic surfaces. J. Colloid Interface Sci. 2009; 331:60–4.
4. Chaterjee et al. Nematicidal principles from two species of Lamiaceae. J. Nematol. 1982; 14:118–20.
5. Singh et al. Ocimum Sanctum (tulsi): Bio-pharmacological activities. WebmedCentral PHARMACOLOGY 2010; 1:WMC
Ocimum sanctum (OS) essential oil along with Gas Chromatography – Mass Spectrometry (GC/MS) report – obtained from Aromatics International, USA and stored 4°C. Poly methyl methacrylate (PMMA), Polystyrene (PS) - used as substratum surfaces.
Tissue Culture Polystyrene (TCPS) well plates - Used as control surface. Wettability of the surfaces - determined by water contact angle measurements using the sessile drop technique.
Bacteria - cultured aerobically overnight at 37°C on blood agar from a frozen stock. For each experiment, one colony inoculated in 10 ml of tryptone soy broth & cultured for 16 h.
Wells containing PMMA & PS, filled with 500 µl of OS extract & allowed to adsorb to the surface at 37°C for 10 min.
Figure 3. Number of adherent bacteria ([A] S. aureus, inset images shows the adherent S. aureus on control (bare PMMA) & OS extract coated PMMA, [B] P. aeruginosa & [C] E. coli) after 1 h growth on biomaterial surfaces in absence & presence of OS extract. * denotes notable difference at p<0.01 than with control.