Malaysian Journal of Microscopy 10: 47-53 (2014) ISSN: 1823-7010 BACTERIAL MEMBRANE DISRUPTION IN BACILLUS SUBTILIS, STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLIBY CYNODON DACTYLON EXTRACT Syahriel A’, Jamuarius G? and Khim-Phit ‘Sustainable Palm Oil Research Unit (SPOR), Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Malay *Paculty of Sustainable Agriculturest) ‘Campus, Mile 10, Sg. Batang, Jalan UMS, 88400, Kota Kinabalu, siti Malaysia Sabah, Sandakan 90000, Sandakan, Malaysia Cynodon dactylon is a type of perennial grass that possesses great medicinal values. It has been reported to possess great antibacterial activity against many Gram positive and negative bacteria. It is believed that most antibacterial compounds show great antibacterial mechanism through membrane toxicity effect. Electron microscope (EM) is a powerful tool in observatory analysis which offers capability to observe extremely small sample or structure up 10 ‘micron size. In present study the effect 1 of C. dactylon extract on Bacillus subtilis, Staphylococcus aureus and Escherichia coli cellular membrane was investigated using Scanning Electron Microscope (SEM). SEM observation revealed the disruption of bacterial membrane and caused complete lyse in B. subtilis and E. coli and S. aureus after which led to bacterial cell death. treated with C. dactylon SPE extract Keywords: Cynodon dactylon, membrane disintegration, antibacterial, SPE INTRODUCTION ‘The bacterial cytoplasmic membrane is a very delicate organelle and is highly active metabolically. It acts mainly as a selective permeability barrier between the cytosol and the cell's external environment. Any membrane active agent can induce damage by action upon cither the membrane potentials, ionic interaction, bound enzymes or permeability. Most antibiotic compounds exert toxicity effect on bacterial cellular membrane which eventually lead to the death of cell, Therefore, researchers tum their interest to cellular membrane toxicity effect and investigate the mode of action of most of the antibacterial agents. Meanwhile, natural products derived from natural resources including plant, bacteria, fungi and actinomycetes are known to exhibit great biological activity [1-4]. Some of antibiotic compounds such as_peni cephalosporin and carbapenems are derived from these sources [5]. In recent years medicinal plants have gained most interest to be investigated for their potential against bacterial pathogens. It is believed that medicinal plants contain some potent antimicrobial compounds more than any other types of plants [6]. Cynodon dactylon is a type of perennial grass that possesses ‘great medicinal values. It is traditionally used as a rejuvenator, for wound healing and believed able to cure many diseases and * Corresponding author: E-mail: chongkp@ums.xiamySyabriel A etal, infections [7]. Previously C. dactylon has been studied for its potential agains many Gram positive and negative bacterial pathogens [8-12]. Meanwhile, previous study had also shown C. dactylon ethanol Solid Phase Extraction (SPE) extract exhibit ‘greater antibacterial activity compared to the plant crude extracts, suggesting that C. dactylon SPE. extracts might contain the Potent antibacterial compounds against bacterial pathogens [13]. Therefore, study aims to investigate the mode of action of C. dactylon ethanol SPE extract against selected bacterial such as Bacillus subtilis, Escherichia coli, and Staphylococcus aureus by understanding the effect of the extract on the bacterial cellular membrane. Effect on the bacterial cellular membrane was observed under Scanning Electron Microscope (SEM) for bacterial membrane degradation MATERIALS AND METHODS Plant Collection Wild ecotype of Cynodon dactylon was collected in the area of Kota Kinabalu (Lat: 6.034826, Long: 116.12316), . Sabah, Malaysia. Voucher (jgobilik 1090/2011) was kept in Faculty of Sustainable Agriculture, Universiti Malaysia Sabah (UMS) and a duplicate was deposited at BORH Herbarium, Institute of Tropical Biology and Conservation, UMS for future reference. Cynodon dactylon Ethanol Crude Extraction . The whole part of C. dactylon was thoroughly cleaned using distilled water to remove soil and dirt and then dried for 24-72 hours in a drying chamber at 40-50°C to remove the water content and moisture from the plant, To optimize and enhance the extraction yield, the dried plant was homogenized using a mechanical blender (Waring® Commercial Blender). Approximately 100g of the plant powder later was soaked into 200mL of ethanol and ater shaken on an orbital shaker (LabCompanion™) at 150 rpm at temperature of 25°C. The soaking process ‘was repeated three times for each extraction to obtain a complete extraction. The extracts obtained were then evaporated and concentrated under reduced pressure (768mmhg to Tmmhg) using Rota Vapor™ (BUCH to achieve final concentration of 1g of extract per mL of solvent. The Aliquot was then kept in -20°C refrigerator until further ‘Solid Phase Extraction (SPE) Solid Phase Extraction (SPE) protocol as described previously with slight ‘modification was used. Methanol absolute (1 mL) was used to activate the sorbent and further equilibrated with sterile deionized distilled water (1 mL). Sample from C. daciylon ethanol crude extract was. then loaded into the cartridges and remained in the SPE sorbent matrix for few seconds. The loaded sample was then washed with 1% methanol (I mL). The resulted fraction yielded from wash procedure was collected and labelled as ‘flush fraction’. Finally, the remaining sample in the SPE sorbent matrix was eluted = with mL methanokacetonitrile (1:1; viv), collected and labelled as ‘elute fraction’. The flush fraction from SPE, protocol for C. daciylon ethanol crude extract later was used to study the effect on bacterial cellular membrane (4). Bacterial Cultures Pure cultures of bacterial pathogens: Bacillus subtili, Escherichia coli, and Staphylococcus aureus were obiained from Queen Elizabeth Hospital, Kota Kinabalu, Malaysia. The pathogens were sub-cultured outo Netrient. Agar (NA) prior to Preservation. The bacterial cultures were ea preserved in 30% glycerol stock soletion at 85°C temperature. Prior to mode Malaysian Journal Vel. 10 (2014) 48 of MiconBacterial Membrane Disruption ia Bacillus subiils, Staphylococcus aureus and Escherichia coli by (Cymoddon dactylon Extract ofaction study, the bacterial pathogens were sub-cultured onto NA, incubated at 37°C for 24 hours before the inocula of the test pathogens were grown in Nutrient Broth (NB) and adjusted according to Mac Farland Standard to achieve approximately 1x10* CFU/mL before being treated with C. dactylon ethanol SPE. flush fraction extract (1). ‘Scanning Electron Microscope (SEM) Observation In the present study, all bacterial pathogens of the mid-exponential growth phase were diluted with NB medium to the cell den approximately 1x10 CFU/mL according to Mac Farland standard and further treated accordingly at SOmgmL.” of flush fraction of C. dactyion ethanol SPE extract for 7 hours at 37°C. Untreated controls were prepared in [NB medium. After 2 hours of incubation, the bacterial cells were collected via centrifugation at 10,000g and then the pellet formed was washed with PBS for 3 times. Fixation was done by suspending the bacterial cells into 0.25% of glutaraldehyde solution (in PBS, pHi 7.0) and then incubated at room temperature for 1 hour. After the incubation, the bacterial cells were washed with PBS for three times and then the fixed bacterial pellet was collected by centrifugation at 10,000g. Dehydrolysis of the bacterial cells were done by washing the pellets with different ethanol concentration starting with 30%, 50%, 70%, 80%, 90% and 100%, and for each ethanol treatment was incubated for 10 minutes. Incubation with 100% ethanol was done up o 1 hour. Prior to observation under SEM, the bacterial cells ‘was mounted on carbon formvar and then coated with gold-palladium in Emitech K5S0x carbon coater for 1 minute. Microscopic observation was performed with a Zeiss Supra SSVP (Oberkochen, Germany) microscope. Secondary electron images were taken at low electron energies between 2 keV and 2.5 keV. RESULTS AND DISCUSSION The present study revealed remarkable membrane disruption effect on the tested bacterial pathogens. Figure 1 provides morphological evidence for B. subtilis, S. ‘aureus and F. coli using Scanning Electron Microscope (SEM). Based on the SEM observation, it is suggested that the flush fraction of C. dactyton ethanol SPE extract is able to cause membrane damage as observed “bn both E. coli and B. subtilis. After treated with the extract, serious membrane disruption, indicated by a large hole was observed on B. subtilis which most probably causing the bacterial death. Meanwhi flatten shape observed on E. coli might a ‘200d indicator that the bacterial cell had burst out after it was treated with the plant extract. However, no clear morphological evidence for membrane disruption was observed on S. aureus after 7 hours treated with the flush fraction of C. dactylon ethanol SPE extract. In this study, itis suggested that time of incubation for treatments on S. ‘aureus may play important role to provide this morphological evidence. It is believed that the longer the time of incubation, the higher number of cells .will experience complete cellular membrane disruption, and thus burst out and dissolved in the mixture. Previous study had shown how different incubation time of antibacterial treatment on Methycillin Resistant Staphylococcus aureus (MRSA) affected the number of cell that completely lyse [15]. Meanwhile, another study provided important information on how antibacterial treatment can completely lyse bacterial pathogens after long period of incubation [16]. Therefore, itis reasonable to conclude that S. aureus had experienced complete lysis after 7 hours treated by C. dactylon ethanol SPE flush fraction extract. This resulted in reduction of bacterial cellular number since most of the lysed cells were dissolved in the treatment mixture. Previously study [13] has reported flush fraction of C. dactylon possess very strong, antibacterial activity against Gram positive Malaysian Journal af Micraseapy Vol. 10 (2014) 9Syahriel A etal. Fig. 1: Effect of C. dactylon ethanol SPE-extract (flush fraction) on B. subtilis (A: Untreated: B: Treated), S. aureus (C: Untreated; D: Treated) and £. coli (E: Untreated: F: Treated) after approximately 7 hours of treatment, Note the membrane damage due to the flush fraction of C. dactylon ethanol SPE- extract on B. subtilis and E: coli (red arrowed). Large hole was observed on B. subtilis cellular surface after treated By the plant extract. Flatten shape and protruding structure on E. coli surface resulted after the treatment. Treatment on S. aureus shows reduction on the cell number but no clear morphological evidence on membrane disruption. Malaysian Journal of Microscopy Vol. 10 (2014) 50Bacterial Membrane Disruption im Bacillus subtilis, Staphylococcus aureus and Escherichia coli by (Cymodom dactylon Extract and negative bacteria. It is believed that Cdactylon contains potent antibacterial agents broad spectrum of bacterial pathogens. Lot of studies has been conducted previously to understand the membrane disintegration action on biological cell membrane. Antimicrobial agents that exert ‘membrane disintegration effects can happen through cationic and anionic interaction or via hydrophobicity interaction with membrane hydrophobic region {17-20}. Cationic and anionic interaction between antimicrobial agents and the bacterial membrane cell wall had been thoroughly discussed previously by many reseachers 20, 21}. Previously, flush fraction of C.dactylon ethanol SPE extract was reported to contain cardiac glycosides, terpenoids and steroids [14]. Cardiac glycoside consist of alycone, which is the sugar component and aglycone, the non-sugar components which can be either C-23 or C-24 steroidal compounds. This compound exerts the amphiphilic characteristic which the hydrophilic region exerts by the glycone component while the hydrophobic region exerts by the steroidal component. Both steroids and terpenoids are well known to exhibit hydrophobie characteristic due to the presence of C-skeleton in the compounds structure thus making them permeable into biological cell membrane. Some hydrophobic compounds are known to interact with bacterial membrane lipids and it bactericidal are linearly related to their hydrophobicity [18, 19]. In this study, the disintegration of bacterial cell membrane in B. subtilis, E. coli and possible complete lyse of S. aureus might due to the compounds hydrophobicity characteristic that give the compounds capability to insert themselves into the bacterial phospholipids bilayer. Due to long or heavy C-skeleton structure, the insertion of steroidal and terpenoidal compounds from the plant extract into the bacterial inner membrane caused it to become more fluid and permeable [17, 22- 24), ‘The increased permeability of the membrane by the insertion of hydrophobic area and the C-skeleton structure can allow internal contents to leak from the cell and eventually burst, which can cause growth inhibition or even death [17, 22, 24, 25]. If membrane fluidity increases excessively, the ‘membrane can become unstable and the cell will ultimately lyse [24]. CONCLUSION ‘The present study suggests the flush fraction of C. dactylon ethanol SPE extract contains antibacterial agent that can disrupt B. subtilis, E, coli cellular membrane while caused complete lyse on S. aureus which might the causal of the bacterial death. This study also suggests C. dactylon might become one of the potential sources for antimicrobial agent against many critical bacterial pathogens by exerting critical and spectacular antibacterial action against the bacterial pathogens. ACKNOWLEDGMENTS ‘The authors acknowledge their profound gratitude to Ministry of Education Malaysia for financially supporting the research through Fundamental Research Grant Scheme (FRGS), the Sustainable Palm Oil Research Unit (SPOR) and Faculty of Science and Natural Resources (FSNR), Universiti Malaysia Sabah for providing the facilities for the research work. REFERENCES, [1] Baltz, R. H. (2007). Antimicrobials from Actinomycetes: Back to the Future Microbe, 2(3) 125-131. [2] Yin, N. 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