Antimicrobial Efficacy of Some Marine Macroalgae of Red Sea

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1 International Journal of Microbiology and Immunology Research Vol.3(3), pp , June, 2014 Available online at ISSN Apex Journal International Full Length Research Antimicrobial Efficacy of Some Marine Macroalgae of Red Sea Mostafa M. El-Sheekh 1 *, Mohamed M. Gharieb 2, Sabha M. El-Sabbagh 2, Walaa T. Hamza 3 1 Botany Department, Faculty of Science, Tanta University, Egypt. 2 Botany Department, Faculty of Science, Menoufia University, Egypt. 3 National Institute of Oceanography and Fisheries, Egypt. Accepted 14 May, 2014 In this investigation, four macroalgae (Dictyota sp., Sarcodiotheca furcata, Cystoseira myrica and Sargassum ramifolium) were tested for their antimicrobial activities. Algal extracts were more effective against than and fungi. The highest antibacterial activity was recorded by S. furcata extract mostly against the tested. The extract was more effective in the growth inhibition of Shigella flexneri (33 mm) followed by Escherichia coli, Salmonella typhi, Klebsiella pneumonia and Proteus mirabilis. The antifungal activity was only observed against Candida albicans. were affected by MICs ranged from mg/ml of the tested algal extracts. The effective MICs of mg/ml were recorded against the tested. Candida species were affected by some algal extracts at MIC of 200 mg/ml. Key words: Macroalgae, antimicrobial, multi-drug resistant, MICs. INTRODUCTION Many seaweeds or macroalgae are known to synthesize bioactive secondary metabolites which have antimicrobial activities. There are numerous reports of compounds derived from macroalgae with a broad range of antibacterial activities (Magallanes et al., 2003; Freile-Pelegrìn and Morales, 2004; Oranday et al., 2004, Manilal et al. 2010). Numerous substances were identified as antimicrobial agents from algae: chlorellin derivatives, acrylic acid, halogenated aliphatic compounds, terpenes, sulphur containing heterocyclic compounds, phenolic inhibitors, etc. (Espeche et al., 1984). Sandsdalen et al. (2003) isolated an antibacterial compound from the brown alga Fucus vesiculosus and they identified it as polyhydroxylated fucophlorethol. Antibacterial activity of methanolic extracts from 32 macroalgae (13 Chlorophyta and 19 Phaeophyta) from the Atlantic and Mediterranean coast of Morocco were evaluated for the production of antibacterial compounds *Corresponding author: mostafaelsheekh@yahoo.com against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Klebsiella pnomeuniae ATCC and E. faecalis ATCC (Ibtissam et al., 2009). Kappaphycus (red algae) and Padina (brown algae) from the coast of Tamilnadu, India were tested in vitro for their antibacterial activities against different types of bacteria such as Pseudomonas flouresences, S. aureus, Vibrio chloera and Proteus mirabilis using disc diffusion method (Rajasulochana et al., 2009). Antimicrobial activity of ethanolic extract of Sargassum thunbergii was determined by paper disc assay. The ethanol extract was effective against Serratia liquefaciens, Salmonella typhimurium, Pseudomonas aerogenosa and all of the tested gram-positive bacteria at 4 mg/ml. Also, Bacillus subtilis, Clostridium perfringens and Listeria monocytogenes were susceptible to the algal extract (Lee et al., 2009). Recently, Silva et al. (2013) observed inhibitory effect of ethanol extracts of the macroalgae Padina gymnospora, Hypnea musciformes and Ulva fasciata against virulent antibiotic-resistant bacteria suggests these macroalgal species constitute a

2 022 Int. J. Microbiol. Immunol. Res. potential source of bioactive compounds. The objective of the current work was to evaluate the antimicrobial activities of four different macroalgal extracts against some multi-drug resistant bacteria and fungi. MATERIALS AND METHODS Microorganisms The organisms used in this study were obtained from clinical specimens at the Diagnostic Microbiology Laboratory of Medical Sciences College, Hodeidah University, Yemen (provided by Prof. Dr. W.A. El- Shouny, Faculty of Sciences, Tanta University, Egypt). Some bacteria were obtained from the culture collection of the Microbiology Department, Faculty of Pharmacy, Tanta University, Egypt. The tested were Staphylococcus aureus 1, Staphylococcus aureus 2, Streptococcus viridians, Streptococcus pneumoniae, Lactobacillus acidophilus and a diphtheroid (Corynebacterium sp.). included Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Shigella flexneri, Pseudomonas aeruginosa and Proteus mirabilis. The fungi; Candida albicans, Candida tropicalis, Saccharomyces servisiae and Aspergillus niger were also used for antimicrobial testing. All stock bacterial cultures were maintained on nutrient agar slants at 4 C with monthly transfers (Cheesbrough, 2000). Whereas, fungi were maintained on Sabouroud s agar slant. The purity of the identified bacteria and fungi was tested continuously by the morphological and biochemical tests according to Collee et al. (1996). Culture media used The composition of the media used for the growth of bacteria was mentioned by Cheesbrough (2000). Nutrient agar The medium composed of (g/l);beef extract 3, peptone 5, sodium chloride 5 and agar 15 and distilled water 1 litre. The medium was adjusted at ph 7.2.Nutrient broth mediumcontainedthe same components except the agar. Müller Hinton agar Beef infusion 300 ml, casein hydrolysate 17.5 g, starch 1.5 g, agar 15 g and distilled water 1 litre. The medium was adjusted at ph 7.4. Sabouraud agar This medium was used for the maintenance of fungi. The medium composed of (g/l); glucose 20, peptone 10, agar 15 and distilled water 1 litre. The medium was adjusted at ph 5.4 (Collee et al., 1996). Preparation of algal extracts The marine red and brown algae were collected from the red sea coast to study their bioactivity. The taxonomic identification of species was done by experts in this field, using standard literature and taxonomic keys: A Field Guide to the British Seaweeds Specimens of all species tested (Dictyota sp., Sarcodiotheca furcata, Cystoseira myrica and Sargassum ramifolium) are deposited in the herbarium of the Laboratory of Phycology, Microbiology Section, Department of Botany, Faculty of Science at Tanta University, Egypt. The collected algae were rinsed with sterile water to remove any associated debris and were kept under sunshade for 7 days. Then, algae were dried in an oven at 80 C for 24 h. The dry seaweeds were crushed in an electric mill until a fine powder. Three grams of dried matters were extracted in 10 ml either of boiling water for 30 min, or in a mixture of chloroform: methanol (2:1 v/v) for 72 hours (Rajasulochana et al., 2009). The mixture was stirred every 12 hours using sterile glass rods and filtered through Whatman filter paper No. 2. All extracts were collected in dark bottles and stored at 4 C until used for the estimation of the antimicrobial activity. In vitro Antimicrobial activity test The antimicrobial activity of algal extracts was assayed by agar wells diffusion. Agar plates were prepared using Müller Hinton agar. The plates were seeded with 0.1 ml of test culture corresponding to 10 6 cfu/ml. Wells of 6 mm in diameter were made using a sterile cork borer in solidified agar. The tested agents were added as 50 µl in each well. The (chloroform: methanol 2:1 v/v) algal extracts (300, 200, 100 and 50 mg/ml) were added to the wells. The extracting agent was tested as controls (Tuney et al., 2006). Plates were left for one hour at 4 C and then incubated for 24 h at 37 C (except for Aspergillus niger which was incubated at 28 C for 72 h). Inhibition zones were measured in mm and three replicates were averaged

3 El-Sheekh et al 023 Table 1. Antimicrobial activity of Dictyota sp. extract against some multi-drug resistant bacteria and fungi grown on Müller-Hinton agar. Dictyota sp. Conc. (mg/ml) Inhibition zone (mm) Staphylococcus aureus 1 17±2 14±2 0±0 0±0 Staphylococcus aureus 2 8±1 0±0 0±0 0±0 Streptococcus viridans 9±1 7±1 0±0 0±0 Streptococcus pneumoniae 12±2 10±2 0±0 0±0 Corynebacterium sp. 0±0 0±0 0±0 0±0 Escherichia coli 11±2 10±2 8±1 7±1 Klebsiella pneumoniae 15±2 12±2 10±2 9±1 Salmonella typhi 21±3 18±2 16±2 0±0 Shigella flexneri 32±4 30±3 24±3 0±0 Pseudomonas aeruginosa 0±0 0±0 0±0 0±0 Proteus mirabilis 9±1 8±1 0±0 0±0 Candida albicans 15±2 9±1 0±0 0±0 Candida tropicalis 9±1 7±1 0±0 0±0 Saccharomyces servisiae 0±0 0±0 0±0 0±0 LSD 1.08x x10-15 Each value is the mean of three readings ± standard deviation. ND: not detected. (Lis-Balchin et al., 1995). Statistical analysis One way analysis of variance (ANOVA) is used according to SPSS (1999). Each value presented in the tables is the mean of three readings ± the standard deviation (SD). The least significant difference is abbreviated as LSD and measured at P RESULTS Antimicrobial activity of different algal extracts Dictyota sp. extract The results presented in Table 1 revealed a relatively low antibacterial activity of Dictyotasp. extract against the tested. The inhibition zone (17 mm) of Staphylococcus aureus 1 followed by lower growth inhibition of S. pneumoniae, Staphylococcus aureus 2 and Streptococcus viridians. Dictyotasp. extract was more effective against Gram negative bacteria than. The highest inhibition of growth was attained against Shigella flexneri (32 mm) followed by S. typhi, K. pneumoniae, Escherichia coli and Proteus mirabilis. The highest antifungal effect of Dictyota sp. extract was recorded against Candida albicans as an inhibition zone of 15 mm. Whereas, the tested Corynebacterium sp., Pseudomonas aeruginosa and Saccharomyces servisiae were resistant. Sarcodiotheca furcata extract Table 2 showed the antimicrobial effects of Sarcodiotheca furcata extract against the tested microorganisms. Concerning the Gram positive bacteria, the tested algal extract inhibited the growth of Staphylococcus aureus 1 (17 mm) followed by Streptococcus pneumoniae (15 mm) and it showed very weak effect against Staphylococcus aureus 2 and S. viridians, while the other tested organisms were not

4 024 Int. J. Microbiol. Immunol. Res. Table 2. Antimicrobial activity of Sarcodiotheca furcata extract against some multi-drug resistant bacteria and fungi grown on Müller-Hinton agar. S. furcata conc. (mg/ml) Inhibition zone (mm) Staphylococcus aureus 1 17±2 15±2 0±0 0±0 Staphylococcus aureus 2 7±1 0±0 0±0 0±0 Streptococcus viridans 7±1 7±1 0±0 0±0 Streptococcus pneumoniae 15±2 13±2 0±0 0±0 Corynebacterium sp. 0±0 0±0 0±0 0±0 Escherichia coli 21±2 17±2 15±2 8±1 Klebsiella pneumoniae 15±2 9±1 8±1 10±2 Salmonella typhi 14±2 14±2 12±2 0±0 Shigella flexneri 33±4 31±3 28±3 0±0 Pseudomonas aeruginosa 0±0 0±0 0±0 0±0 Proteus mirabilis 8±1 9±1 0±0 0±0 Candida albicans 10±1 13±2 0±0 0±0 Candida tropicalis 8±1 8±1 0±0 0±0 Saccharomyces servisiae 0±0 0±0 0±0 0±0 LSD 2.47x x10-14 Each value is the mean of three readings ± standard deviation. ND: not detected. affected. The antibacterial activity of S. furcata extract was also tested against some. The extract was more effective in the growth inhibition of Shigella flexneri (33 mm) followed by Escherichia coli, Salmonella typhi, K. pneumoniae and P. mirabilis. The weak antifungal activity was only observed against Candida albicans and Candida tropicalis (10 &8 mm, respectively). Cystoseira myrica extract The results presented in Table 3 revealed a relatively low antibacterial activity of Cystoseira myrica extract against the tested. The inhibition zone of Staphylococcus aureus 1 (14 mm) was followed by growth inhibition of Staphylococcus aureus 2 and Streptococcus viridians. On the other hand, Streptococcus pneumoniae and Corynebacterium sp. were resistant. The highest antibacterial effect of C. myrica extract on was recorded against the growth of S. flexneri (31 mm). Lower inhibition zones were observed against E. coli, Salmonella typhi, K. pneumoniae and P. mirabilis. Poor antifungal activity was only observed against Candida species. Sargassum ramifolium extract In Table 4, Staphylococcus aureus 1 and 2 were the only susceptible Gram positive organisms to Sargassum ramifolium extract recording inhibition zones of 15 and 12 mm, respectively. The other tested organisms were resistant. The maximum susceptibility of Gram negative organisms were represented by S. flexneri, Salmonella typhi, E. coli. A considerable antifungal activity was only detected against Candida albicans (15 mm). Four algal extracts were tested for MIC determination. The data in Table 5 revealed that Gram positive bacteria were affected by MICs ranged from mg/ml of the tested algal extracts. The effective MICs of mg/ml were recorded against the tested. Candida species were affected by some algal extracts at MIC of 200 mg/ml.

5 El-Sheekh et al 025 Table 3. Antimicrobial activity of Cystoseira myrica extract against some multi-drug resistant bacteria and fungi grown on Müller-Hinton agar. C. myrica conc. (mg/ml) Inhibition zone (mm) Staphylococcus aureus 1 14±2 11±2 7±1 0±0 Staphylococcus aureus 2 10±2 9±1 0±0 0±0 Streptococcus viridans 7±1 7±1 0±0 0±0 Streptococcus pneumoniae 0±0 0±0 0±0 0±0 Corynebacterium sp. 0±0 0±0 0±0 0±0 Escherichia coli 22±3 18±2 15±2 12±2 Klebsiella pneumoniae 17±2 15±2 10±2 14±2 Salmonella typhi 16±2 15±2 13±2 0±0 Shigella flexneri 31±3 31±3 30±3 0±0 Pseudomonas aeruginosa 0±0 0±0 0±0 0±0 Proteus mirabilis 9±1 9±1 0±0 0±0 Candida albicans 9±1 8±1 0±0 0±0 Candida tropicalis 8±1 8±1 0±0 0±0 Saccharomyces servisiae 0±0 0±0 0±0 0±0 LSD x10-14 Each value is the mean of three readings ± standard deviation. ND: not detected. DISCUSSION The production of antimicrobial active compounds was considered to be an indicator of the capacity of the seaweeds to synthesize bioactive secondary metabolites (Espeche et al., 1984; Magallanes et al., 2003; Freile- Pelegrìn and Morales, 2004; Oranday et al., 2004). In this study, the antimicrobial activities of four algal extracts prepared by mixture of chloroform and methanol (2:1 v/v), were tested against some microorganisms. All types of algal extracts recorded variable inhibition levels of the tested microbial growth. The results revealed a relatively low antibacterial activity of the algal extracts against the tested. Staphylococcus aureus 1, Staphylococcus aureus 2, Streptococcus viridans and Streptococcus pneumoniae, recorded inhibition zones of 17 mm. The highest antibacterial effect on the was recorded by S. furcata extract against the growth of S. flexneri (33 mm) followed by E. coli, Salmonella typhi, K. pneumoniae and P. mirabilis. On the other hand, Corynebacterium sp. and P. aeruginosa were resistant. The antifungal activity was only observed against Candida albicans as an inhibition zone of 15 mm. It was reported that the Gram-positive bacterial strains were more susceptible to seaweeds extract than Gramnegative bacterial strains (Pesando and Caram, 1984). The extracts of Enteromorpha ramulosa and Dictyopteris membranacea were active against Gram positive and (González et al., 2001). Also, Sandsdalen et al. (2003) isolated antibacterial compound (polyhydroxylated fucophlorethol) from the brown alga Fucus vesiculosus and they found that it was active against both the Gram positive and the. Organic solvent provides a higher efficiency in extracting compounds for antibacterial activities compared to water based methods (Tuney et al., 2006). Rajasulochana et al. (2009) found that chloroform: methonal is the best solution for extracting the effective antibacterial materials from the brown algae species. Ibtissam et al. (2009) indicated that the methanol extracts of Sargassum vulgare did not show antibacterial activity

6 026 Int. J. Microbiol. Immunol. Res. Table 4. Antimicrobial activity of Sargassum ramifolium extract against some multi-drug resistant bacteria and fungi grown on Müller-Hinton agar. S. ramifolium conc. (mg/ml) Inhibition zone (mm) Staphylococcus aureus 1 15±2 13±2 7±1 0±0 Staphylococcus aureus 2 12±2 9±1 0±0 0±0 Streptococcus viridans 0±0 0±0 0±0 0±0 Streptococcus pneumoniae 0±0 0±0 0±0 0±0 Corynebacterium sp. 0±0 0±0 0±0 0±0 Escherichia coli 18±2 15±2 15±2 15±2 Klebsiella pneumoniae 16±2 14±2 0±0 14±2 Salmonella typhi 16±2 14±2 13±2 0±0 Shigella flexneri 31±3 31±3 30±3 0±0 Pseudomonas aeruginosa 0±0 0±0 0±0 0±0 Proteus mirabilis 7±1 7±1 0±0 0±0 Candida albicans 15±2 9±1 0±0 0±0 Candida tropicalis 7±1 7±1 0±0 0±0 Saccharomyces servisiae 0±0 0±0 0±0 0±0 LSD 6.31x x x10-15 Each value is the mean of three readings ± standard deviation. ND: not detected. against E. coli and S. aureus growth. Silva et al. (2013) observed that E. coli and P. aeruginosa were only susceptible to ethanol extracts of the brown macroalga Padina gymnospora. The purification of the chloroform extract from the brown macroalga Sargassum muticum, through a series of chromatographic separations resulted in a compound; galactoglycerolipids, active against the growth of two of the four bacterial species (Shewanella putrefaciens and Polaribacter irgensii) and all tested fungi (Plouguerné, et al., 2009). The antimicrobial activity of red and green seaweed extracts significantly increased when ethanol and acetone were used as extraction solvents (Cox et al., 2010). The herein obtained data revealed that Gram positive bacteria were affected by MICs ranged from mg/ml of the tested algal extracts. The effective MICs of mg/ml were recorded against the tested. Candida species were affected by some algal extracts at MIC of 200 mg/ml. In this concern, Oranday et al. (2004) showed that non polar extracts of Sargassum fluitans, and polar extracts of Gracilaria tikvahiae inhibited the growth of more than four microorganisms. Extracts were separated using chromatography column and fractions were tested against S. aureus and C. albicans. The eighty fraction of petroleum ether of S. fluitans exhibited high activity against C. albicans, MIC 0.16 µg/ml. Lee et al. (2009) determined the antimicrobial activity of S. thunbergii by paper disc assay and minimum concentration inhibitor (MIC) test. They proved that a water extract of S. thunbergii did not show the antimicrobial activity, but an ethanol extract of S. thunbergii inhibited Serratia liquefaciens, S. typhimurium, P. aeruginosa and all of the tested gram positive bacteria at 4 mg/ml. As the results of MIC test, S. thunbergii extract inhibited the growth of B. subtilis, S. aureus and Listeria monocytogenes at concentration of 0.1~0.3%, and inhibited C. perfringens at 0.01%. Christobel et al. (2011) tested the aqueous extract of seven species of marine macroalgae for their antimicrobial potency against ten pathogenic bacterial strains. Ulva fasciata, Gracilaria corticata, Sargassum wightii and Padina tetrastromatica showed significantly higher activity against 70% of the tested bacterial isolates. The maximum zone of inhibition was noted for the red alga G. corticata against P. mirabilis (17mm) and

7 El-Sheekh et al 027 Table 5. Minimum inhibitory concentrations of algal extracts against some multi-drug resistant bacteria and fungi grown on Müller-Hinton agar. Dictyota sp. Sarcodiotheca furcata MICs (mg/ml) Cystoseira myrica Sargassum ramifolium Staphylococcus aureus Staphylococcus aureus R Streptococcus viridans 300 R R R Streptococcus pneumoniae R R Corynebacterium sp. R R R R Escherichia coli Klebsiella pneumoniae Salmonella typhi Shigella flexneri Pseudomonas aeruginosa R R R R Proteus mirabilis R Candida albicans Candida tropicalis R Saccharomyces servisiae R R R R R: resistant ; ND: not detected brown alga P. tetrastromatica against the pathogens S. aureus and Vibrio harveyi (15mm). The general trend of inhibitory activity was higher towards Gram negative bacteria. The results of the present study has brought to light that Gram negative organisms were more susceptible to the aqueous extract of the algae used. In contrast, Taskin et al. (2001) and Tuney et al. (2006) reported that Gram positive bacteria were more effectively controlled by the extracts of the algae used in their study compared to. The more susceptibility of a particular group of bacteria was due to the difference in their cell wall structure and their composition (Paz et al., 1995). Although the outer membrane of Gram negative bacteria acts as a barrier to many environmental substances including antibiotics (Tortora et al., 2007), the higher susceptibility noticed against the algal extracts gives a promising indication of developing a potent drug from these marine natural sources to be used in combating the infections due to such pathogens. The high sensitivity observed in Gram negative strains need to be revealed through further experiments. The bioactive compounds derived from macroalgae may be of phenolic nature, which solubilized the lipopolysaccharide layer of bacterial cell wall, leading to the entry of the inhibitory molecules. It is well known that, are characterized by a thick peptidoglycan layer in its outer cell wall and this might have resisted the entry of the inhibitory active molecules, hence attributed to the less susceptibility. REFERENCES Cheesbrough, M. (2000). District laboratory practice in tropical countries. Part 2 Cambridge University Press, UK. Christobel, G.J., Lipton, A.P., Aishwarya, M.S., Sarika, A.R. Udayakumar A. (2011). Antibacterial activity of aqueous extract from selected macroalgae of southwest coast of India. Seaweed Res. Utiln., 33 (1&2): Collee, J.G., Fraser, A.G., Marmion, B.P., Simmons, A. (1996). Bacteria and related organisms, Mackie and McCartney, Sec. B Pseudomonas, Stenotrophomonas, Burkholderia Practical Medical Microbiology 4 th Ed., Churchill Livingstone, New York. Pp Cox S., Abu-Ghannam, N., Gupta S. (2010). An assessment of the antioxidant and antimicrobial activity

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