In vitro antimicrobial activities of methanolic extract from marine alga Enteromorpha intestinalis

Ibrahim Darah， Sheh-Hong Lim

Industrial Biotechnology Research Laboratory （IBRL）， School of Biological Sciences， Universiti Sains Malaysia， 11800 Minden， Penang， Malaysia

In vitro antimicrobial activities of methanolic extract from marine alga Enteromorpha intestinalis

Ibrahim Darah， Sheh-Hong Lim*

Industrial Biotechnology Research Laboratory （IBRL）， School of Biological Sciences， Universiti Sains Malaysia， 11800 Minden， Penang， Malaysia

ARTICLE INFO

Article history:

in revised form 1 Jun 2015

Accepted 15 Jun 2015

Available online 5 Aug 2015

Antibacterial activity

Enteromorpha intestinalis

Marine algae

Methicillin-resistant Staphylococcus aureus

Objective: To extract the bioactive compound from Enteromorpha intestinalis （E. intestinalis）and determine its in vitro antimicrobial activity.

Methods: E. intestinalis was extracted by methanol and subjected to antimicrobial screening. The antimicrobial activity was studied by using disc diffusion and broth dilution method. The effect of the extract on the growth profile of the bacterial was also examined via time-kill assay. Microscopy observations using SEM was done to determine the major alterations in the microstructure of methicillin-resistant Staphylococcus aureus （MRSA）.

Results: The results showed methanolic extract of E. intestinalis exhibited a favourable antimicrobial activity against tested bacteria with produced inhibition zone ranging from 8.0-19.0 mm. However， all the tested fungi and yeast were resistant to the extract treatment. Time kill assay suggested that methanolic extract of E. intestinalis had completely inhibited MRSA growth and also exhibited prolonged antibacterial activity. The main abnormalities noted from the microscopic observations were the structural deterioration in the normal morphology and complete collapsed of the bacteria cells after 36 h of treatment.

Conclusions: The significant antibacterial activity shown by crude extract suggested its potential against MRSA infection. The extract may have potential to develop as antibacterial agent in pharmaceutical use.

Short communication doi: 10.1016/j.apjtb.2015.07.012 ©2015 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.

1. Introduction

Various therapeutic benefits available in natural products interest researchers to search for alternatives to combat the rising prevalence of global antimicrobial resistance problems. Moreover， concerns on the safety of some chemical in drugs have prompted an increased great interest in natural additives.

Marine organisms such as algae offer unlimited source of new agents for pharmaceutical industries. Recently， the ability of marine algae to produce secondary metabolite has been extensively documented［1，2］. Today， marine algae have been shown to possess substances with therapeutic values［3，4］. Algae extracts have been investigated， which contains antimicrobial， cytotoxic activity，antitumor and anti-inflammatory properties［5，6］. The antimicrobialagents obtained from marine algae such as chlorellin derivatives，acrylin acid， halogenated aliphatic compounds， phenolic inhibitors were observed to inhibit the growth of microorganisms［7］. Therefore，enhanced screening method for detection of antimicrobial activity and advanced technology in oceanography is necessary to help in promoting the studies in this area. The continuous research for new antimicrobial agent from marine algae is considered an alternative to overcome the emergence of multi-drug resistance organisms and the higher risk of infectious diseases caused by these organisms.

Enteromorpha intestinalis （E. intestinalis） is a marine alga and has a wide distribution around Peninsular Malaysia. This alga belongs to the family of Ulvaceae. E. intestinalis is often one of the first macroalgae to colonize newly cleared surface on rocky shores， tide pools， in estuaries， and on the hauls of ships passing from salt water to freshwater［8］. In this communication， we report the antibacterial activity of the preparations of methanolic crude extract of E. intestinalis and the effect of this extract on the alteration of MRSA cells by microscopy study.

2. Materials and methods

2.1. Preparation of the extract

E. intestinalis was collected from the dock at Gelugor fishing village， Penang， Malaysia， during low tide period. The algal samples were washed thoroughly with running tap water and left to dry for 4-7 days at ambient temperature. The whole parts of the algae were cut into small pieces approximately 1.0 cm and use for extraction. Extraction was carried out according to the method described by Lim and Darah［9］. The 100 g of small pieces of algae were soaked in 1 000 mL of methanol for 3 days at room temperature （30 ± 2）°C. Stirring the mixture to ensure homogeneity during the extraction process. The mixture was filtered by using filtered paper （Whatman No.1， United Kingdom）. The filtrate was obtained then evaporated to obtain a dark oily paste of concentrated extracts.

2.2. Microorganisms and cultural maintenance

All the microorganisms used in this study were obtained from the stock cultures of Industrial Biotechnology Research Laboratory，School of Biological Sciences， Universiti Sains Malaysia. The bacterial strains were grown and maintained on nutrient agar slants（Oxoid， United Kingdom） at 37 °C. However， fungi and yeast were maintained on potato dextrose slants （Oxoid， United Kingdom） at 30 °C. All the stock cultures were stored at 4 °C for further use.

2.3. Antimicrobial activity test

Disc diffusion method was applied in this test. Inoculum containing 1 × 108cells/mL were added to molten nutrient agar and distributed uniformly. When the agar was solidified， sterilized antibiotic disc（Whatman， United Kingdom） were placed on the agar and 20 µL of each extract at concentration of 50 mg/mL were pipetted onto it. Similarly， each plate was placed with sterile antibiotic disc by using chloramphenicol （30 µg/mL） or ketoconazole （30 µg/mL） as positive control and solvent as negative control. The inoculated plates were then incubated at 37 °C for bacteria and 30 °C for fungi and yeast. Clear zones of growth inhibition indicated antimicrobial activities were present.

2.4. Determination of minimal inhibitory concentration （MIC）

The MIC values were studied for the microorganisms that were susceptible in the antimicrobial activity test. MIC value of the methanolic E. intestinalis extract was determined by broth dilution assay method［10］. Briefly， extracts were subjected to a serial dilution to give final concentrations between 0.39 to 50.00 mg/per mL. Extracts with different concentration were added aseptically into different labelled test tubes containing 1.5 mL sterile nutrient broth. Then， 0.5 mL of the bacteria suspension with 1 × 108cells/ mL were inoculated into the respective test tubes. The inoculated test tubes were incubated at 37 °C overnight. The lowest dilution of the tube that showed no visual turbidity was determined as the MIC value.

2.5. Time kill assay

The effect of E. intestinalis methanolic extract was observed on MRSA. This experiment was performed using the modified method of Lim and Darah［9］. A total of 250 mL Elenmeyer flasks containing 50 mL of extracts and nutrient broth were prepared with final concentration of half MIC， MIC and two times MIC. One millilitre of inoculums containing 1 × 108cells/mL was added， and incubated at 37 °C， with shaking at 150 r/min for 48 h. Every 4 h intervals， about 4 mL of the mixture was withdrawn and the optical density （OD）was read at 540 nm.

2.6. Morphological study of the bacteria cell after exposure to the extract

Scanning electron microscope observations were carried out on MRSA cells. One mililiter of the MRSA cell suspension at a concentration of 1 × 108cells/mL was inoculated on a nutrient agar plates and then incubated at 37 °C for 4 h. The extract （2 mL）， at a concentration of 6.25 mg/mL （MIC）， was then dropped onto the inoculated agar and was further incubated for another 36 h at the same incubation temperature. A 1% dimethyl sulfoxide （v/v） treated culture was used as control. A small block of bacteria containing agar was withdrawn from the inoculated plates at various time intervals（0， 12， 24 and 36 h） and were fixed for scanning electron microscopy（FESEM LEO Supra 50 VP， Carl Zeiss， Germany） study［11］.

2.7. Statistical analysis

The triplicate data obtained were subjected to an analysis of a completely random design using SPSS （version 12.0） software （SPSS，Chicago， IL， USA）. Statistical significance was assumed at the 0.05 levels （P ＜ 0.05）.

3. Results

The antimicrobial activity of E. intestinalis methanolic extract was shown in Table 1. Among the tested microorganisms only Bacillus cereus （B. cereus）， Bacillus subtilis （B. subtilis） and methicillin-resistant Staphylococcus aureus （S. aureus） （MRSA） were susceptible to the extract. The inhibition zone produced by the commercial antibiotic disk was larger than that produced by the extract disk （P ＜ 0.05）.

Table 1 Antimicrobial activity of methanolic extract of E. intestinalis.

The MIC values of E. intestinalis extract against three species of bacteria showed positive results in antimicrobial screening test. The results revealed that the lowest MIC value on MRSA was about 6.25 mg/mL. However， the MIC values on B. cereus and B. subtilis were 12.5 mg/ mL. Thus， further study was concentrated on MRSA since it showed lower MIC value at 6.25 mg/mL and also known as a pathogen on humans， which causes urinary tract infections， open wounds infection， respiratory system infections and a variety of systemic infections.

The growth profile of the selected test microorganism， MRSA after treated with the methanolic extract of E. intestinalis at different concentrations， was shown in Figure 1. This study was carried-out over a period of 48 h with the bacteria cells exposed to MIC value，half of the MIC value and twice of the MIC value of the extract. At ½MIC （3.13 mg/mL） value， the methanolic extract of E. intestinalis exhibited a drop in OD of the growth profile of MRSA after 36 h of exposure， which led to the stationary phase of the bacteria growth compared to the control. However， at the MIC （6.25 mg/mL） and 2MIC （12.5 mg/mL） values， the methanolic extract of E. intestinalis produced absolute bacteria eradication after 28 h of exposure. The timekill curves described that the methanolic extract of E. intestinalis has potency to inhibit MRSA cells （bacteriostatic） at lower concentration with ½MIC value of 3.13 mg/mL and to kill the cells （bacteriocidal） at concentrations of MIC （6.25 mg/mL） and above.

The effect of methanolic extract of E. intestinalis of 6.25 mg/ mL （MIC） on MRSA cells were clearly observed under SEM. Figure 2A shows the control bacterial cells which were not exposed to the extract. The MRSA cells in the control were tightly packed smooth cocci undergoing cell division. A progressively increasing number of cells were seen to be degenerating over 12 h of exposure， progression of changes occurred was characterized by the appearance of discrete blebs on the cell surface （Figure 2B）. After 24 h of treatment the formation of pores on the corrugated cells was seen （Figure 2C）. As for the final incubation period （Figure 2D）， dramatic lysis was apparent on the cell. Almost complete destructions of the cells were observed. At this point， the cells have lost its metabolic functions completely.

4. Discussion

In this study methanolic extract of E. intestinalis exhibited inhibition zones on some tested bacterial and none on tested fungi and yeast. These findings demonstrated that antibacterial compounds presence in that extract concentration when using methanol as an extraction solvent. The results were agreeable with the findings reported by Sasidharan et al.［12］. Moreover， Manivannan et al.［13］have reported solvents extraction systems were important in obtaining significant antibacterial activity from plants including algae. According to Imbs et al.［14］， antimicrobial activities of extracts that varied considerably between assay microorganisms also suggest that the microbial growth inhibition was mediated by a variety of antimicrobial compounds.

In present study， the MIC values ranged from 6.25-12.5 mg/mL. Sahgal et al.［15］ attributed the differences of the MIC value could be due to the morphological structure of the bacterial cells and their composition in the cells. The results also showed that the higher concentrations of extract， the greater the effect on the bacterial growth inhibition occurred. The reduction in growth possibly occurred due to interference by active compounds in the extract［16］. Similarly， Lim et al.［10］ and Darah et al.［17］ reported that the higher concentration of the extract was needed to kill the microorganismscells than to inhibit the growth of these cells on time-kill profile study.

According to Darah et al.［18］， many studies were reported to investigate the mechanism of actions involved in bacterial killing process. Among them are the interactions of antimicrobial compound with the cell membrane. The ultrastructural changes in the bacterial cell produced by antibiotics are the expression of profound biochemical alterations［19］. According to Braga and Ricci［20］， the integrity of cell wall and bacterial structure are imperative in maintaining the vitality and the virulence of bacteria. Based on SEM findings， it can be suggested that the cells had undergone some distinct morphological and cytological alterations. The same phenomenon was also observed on S. aureus after exposed to fosfomycin［21］. MRSA cells after treated with the extract for 36 h were seen to be a conglomerate of structurally collapsed and lysed. The cells were deformed evidently and lost their metabolic function would lose their virulence at this stage［21］. In fact with micrograph from SEM， crude extract of E. intestinalis were capable of collapsing the MRSA cells with the antimicrobial compounds it borne.

In this work， conclusively demonstrates the antibacterial potential of methanolic extract from E. intestinalis. This extract is proven to be bacteriostatic and bactericidal agent for MRSA. Furthermore， it may have potential for clinical treatment of MRSA infection disease although this use will require additional investigation. Further purification of active compound（s） and its individual antibacterial activity study can be suggested on the basis of the present study.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

The authors acknowledge The Ministry of Science， Technology and Innovation and Universiti Sains Malaysia for awarding the RUI research grant （RUT1001/pkimia/855006） to support this study.

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31 Mar 2015

Lim Sheh-Hong， Industrial Biotechnology Research Laboratory （IBRL）， School of Biological Sciences， Universiti Sains Malaysia， 11800 Minden， Penang， Malaysia.

Tel: +6012-4629487

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