Antibacterial and anti-biofilm activities of Dicranopteris linearis leaf extracts

2017-03-27T23:40:55Z (GMT) by Christina Injan Anak Mawang
Biofilms are defined as cells that are irreversibly attached to a surface and embedded in an exopolysaccharide matrix. Biofilms can grow on living tissues and indwelling medical devices, which can threaten human health. Biofilms are more resistant to antibacterial agents and are difficult to eradicate. Moreover, the presence of non-multiplying cells in biofilms further complicates antibacterial treatments as they are able to tolerate extremely high doses of antibacterials. Staphylococcus aureus is one of the most frequent causes of biofilm-associated infections. Due to the various challenges in biofilm treatments, there is a need to search for effective compounds for biofilm treatments.<br> <br>   Dicranopteris linearis or “resam” has been used in traditional medicine to treat fever, constipation and burns. The objective of this study was to determine the antibacterial and anti-biofilm activities of D. linearis against the non-multiplying cells and biofilms of S. aureus. Methanol crude extraction (MCE) and sequential solvent extraction (SSE) of D. linearis was conducted. The extracts were assessed for antibacterial and anti-biofilm activities.<br><br>   Through broth microdilution assay, antibacterial activity against <i>S. aureus </i>was observed for MCE of <i>D. linearis</i> leaves (MCE(L)), MCE of <i>D. linearis</i> roots (MCE(R)) and methanol (MeOH) fraction of SSE. The minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) for MCE(L) and MCE(R) were at 2.5 – 5.0 mg/ml while MeOH fraction had MIC and MBC values at 5 mg/ml and 10 mg/ml, respectively. Furthermore, time-kill assay against non-multiplying cells of <i>S. aureus </i>was also conducted, by testing against <i>S. aureus</i> cultures that were growth arrested through nutrient depletion, cold temperature and protein synthesis inhibition. MCE(L) demonstrated bactericidal activity at 20 mg/ml against the growth arrested cultures of <i>S. aureus</i> caused by nutrient depletion and protein synthesis inhibition, and was not effective against culture growth arrested at cold temperature.<br><br>   For anti-biofilm activity, the water (H<sub>2</sub>O) fraction and hexane (HEX) fraction was the most effective for biofilm inhibition activity and biofilm disruption activity, respectively, when tested against five <i>S. aureus</i> biofilm strains. The H<sub>2</sub>O fraction demonstrated biofilm inhibition activity at 0.31 – 2.5 mg/ml while HEX fraction showed biofilm disruption activity at 0.07 – 5 mg/ml. This is the first study to report on the anti-biofilm activity of <i>D</i>. <i>linearis</i>. Both H<sub>2</sub>O fraction and HEX fraction did not inhibit cell growth, thus the anti-biofilm effect observed was only due to the biofilm structure itself or the genes that codes for the biofilm.<br><br>   Additionally, H<sub>2</sub>O fraction was able to inhibit <i>S. aureus</i> biofilm formation on various polymer materials commonly used in medical settings: polystyrene (85-93% inhibition), polyvinyl chloride (76-91% inhibition), polyethylene (68-90% inhibition); polypropylene (52-93% inhibition), silicone rubber (68-94% inhibition). The presence of various phytochemicals such as flavonoids terpenoids, tannins, cardiac glycosides, phenols, quinones and saponins were identified in H<sub>2</sub>O fraction. However, further purification and isolation of H<sub>2</sub>O fraction was not conducted due to difficulties in identifying the specific phytochemical responsible for the biofilm inhibition effect.<br><br>   HEX fraction was able to disrupt about 42-75% of <i>S. aureus</i> biofilms. Through scanning electron microscopy, HEX fraction demonstrated destruction of the biofilm structure and scant biofilms were observed, with only few bacterial cells. Few phytochemicals were identified in HEX fraction, and thus, HEX fraction was selected for further purification and isolation process. Purification of HEX fraction had yielded Fraction A and based on nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry data, the compound from Fraction A was identified as Alpha-tocopherol.<br><br>   Alpha-tocopherol was tested for anti-biofilm activity and was found to exhibit biofilm disruption activity against <i>S.</i> <i>aureus</i> biofilms at 0.01 – 0.5 mg/ml. Currently, there has not been any study reported on the biofilm disruption effect of alpha-tocopherol. This will be the first study to report on the biofilm disruption activity of alpha-tocopherol against <i>S. aureus</i> biofilms or any other bacterial biofilms.<br><br>    Further investigation revealed that alpha-tocopherol affects the biofilm matrix and not the cells within biofilms. Alpha-tocopherol was also effective in disrupting <i>E</i>. <i>faecalis</i> biofilm (23% disruption) and <i>E. coli</i> biofilm (31% disruption), and the polymicrobial biofilms of <i>S. aureus</i> + <i>E. faecalis</i> (22-25% disruption) at 0.01 – 0.5 mg/ml. The combination of alpha-tocopherol with vancomycin had mostly showed indifferent effect towards the disruption of biofilm biomass. The combination of alpha-tocopherol and vancomycin was indifferent to the presence of each other in reducing the biofilm biomass of <i>S. aureus</i> and would not cause a greater effect in disrupting biofilm as compared to using either alpha-tocopherol and vancomycin alone. Furthermore, the combination of alpha-tocopherol and vancomycin at low concentrations (4 µg/ml of alpha-tocopherol + 0.008 µg/ml of vancomycin) was shown to affect the viability of cells within <i>S. aureus</i> biofilms.<br><br>  In conclusion, findings from this study demonstrated the antibacterial and anti-biofilm activities of <i>D. linearis</i>, with alpha-tocopherol being the active constituent for biofilm disruption activity. Further work on the biofilm disruption effect of alpha-tocopherol is necessary to explore its potential use in anti-biofilm therapies.