Antimicrobial Activity of Brown Alga Eisenia bicyclis against Methicillin-resistant Staphylococcus aureus

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    We screened for antibacterial substances against methicillin-resistant Staphylococcus aureus (MRSA). Methanolic extract of Eise-nia bicyclis exhibited anti-MRSA activity according to a disk diffusion assay. To identify the active compound(s), the methanolic extract was further fractionated using hexane, dichloromethane, ethyl acetate, and n-butanol. The ethyl acetate-soluble fraction showed both the greatest anti-MRSA activity and the highest polyphenol content. The minimum inhibitory concentrations of the ethyl acetate fraction ranged from 32 to 64 μg per mL against methicillin-susceptible S. aureus and MRSA strains. High-perfor-mance liquid chromatography analysis revealed that both the methanolic extract and the ethyl acetate soluble fraction contained sizeable quantities of dieckol, which is a known anti-MRSA compound. Thus, these data strongly suggest that the anti-MRSA activity of E. bicyclis may be mediated by phlorotannins such as dieckol.


    Anti-MRSA activity , Eisenia bicyclis , Methicillin-resistant Staphylococcus aureus

  • Introduction

    Since methicillin was introduced in 1959 to resolve infec-tions caused by penicillin-resistant Staphylococcus aureus, the incidence of methicillin resistance in staphylococci has increased rapidly (Kaplan, 2005). As a result of widespread methicillin use, methicillin-resistant S. aureus (MRSA) has become a major problem globally (Lee et al., 2008). MRSA infections are difficult to treat because of the multidrug-re-sistance properties of MRSA, which is resistant to β-lactams as well as several other classes of antibiotics (Bramley et al., 1989; Hiramatsu et al., 1997). Vancomycin is commonly used for the treatment of MRSA-related infection. Vancomy-cin resistance was discovered first in enterococci and later in staphylococci (Isnansetyo and Kamei, 2009). The emergence of vancomycin-resistant S. aureus has recently been recog-nized and leaves physicians with few options available to treat MRSA infections. Therefore, much attention has been given to the search for new antimicrobial agents (Lee et al., 2008).

    In an effort to decrease usage of vancomycin and discover an alternative therapeutic agent for treating MRSA infection, we have screened marine algae for anti-MRSA compounds. Eisenia bicyclis is a common perennial phaeophyceae (brown alga) that generally inhabits the coast of Ulleung Island in the East Sea of Korea. This seaweed is used in various dishes, including appetizers, casseroles, muffins, pilafs, and soups (Eom et al., 2011; Kim et al., 2011). The antioxidant activity of E. bicyclis phlorotannins such as eckol (a trimer), phlorofu-cofuroeckol A (a pentamer), dieckol, and 8,8′-bieckol (hexam-ers) has been reported (Okada et al., 2004). This brown algae also exhibits activity against tumors (Noda et al., 1989), Al-zheimer’s disease (Jung et al., 2010), atherosclerosis (Kang et al., 2003), inflammatory diseases (Shibata et al., 2003), aller-gic disease, and cancer (Shibata et al., 2002). However, to our knowledge, no study has reported on the antimicrobial activ-ity of E. bicyclis. Here, we examined the antibacterial activity of E. bicyclis against several pathogenic bacteria, including MRSA.

    Materials and Methods

      >  Raw materials and extraction

    In late September 2010, the brown seaweed E. bicyclis was gathered from Ulleung Island, Korea. Dried E. bicyclis was ground and then finely powdered using a food mixer (HMF-1000A; Hanil Electronics, Seoul, Korea). The dried powder was stored at -20℃ until required. Powdered E. bicyclis (1 kg) was extracted with methanol (3 times × 10 L) for 3 h. The combined filtrate was concentrated by rotary evaporation at 40℃. After suspending in water (1 L), the methanol extract was partitioned with n-hexane (hexane), dichloromethane (DCM), ethyl acetate (EtOAc), and n-butanol (BuOH), in se-quence.

      >  Microorganisms and culture

    Standard bacterial strains were obtained from the Ko-rean Collection of Type Cultures (KCTC; Daejeon, Korea) and the Korean Culture Center of Microorganisms (KCCM; Seoul, Korea). The bacterial strains used were methicillin-susceptible S. aureus (MSSA; KCTC 1927), two MRSA strains (MRSA; KCCM 40510 and KCCM 40511), Bacillus cereus (KCTC 3624), B. subtilis (KCTC 1028), Enterococcus faecalis (KCTC 3206), Escherichia coli (KCTC 1682), Lis-teria monocytogenes (KCTC 3710), Salmonella typhimurium (KCTC 1925), and Vibrio parahaemolyticus (KCTC 2729). All strains were grown aerobically at 37℃ in Mueller-Hinton broth (MHB; Difco, Detroit, MI, USA) or tryptic soy broth (TSB; Difco) for determination of the minimum inhibitory concentration (MIC) and in Mueller-Hinton agar (MHA; Dif-co) for disk diffusion and minimum bactericidal concentration (MBC) assays.

      >  Disk diffusion assays

    Antibacterial activity was evaluated by a disk diffusion assay, as described by the National Committee for Clinical Labo-ratory Standards (National Committee for Clinical Laboratory Standards, 2004). In brief, bacterial strains were cul-tured in TSB at 37℃ until an OD600 of 0.5. Bacterial culture (1 mL), containing approximately 104 CFU, was spread on a MHA plate, and a paper disk (6 mm diameter) containing 1 mg extract was then placed on the agar surface. After incuba-tion for 24 h at 37℃, the diameter of the inhibition zone was measured. The determination was performed three times and the mean values presented.

      >  Minimum inhibitory concentrations (MIC)

    The MIC is the lowest concentration of antimicrobials that will inhibit the visible growth of microorganisms after over-night incubation (Grierson and Afolayan, 1999). MICs of the extracts and vancomycin were determined by the twofold seri-al dilution method in MHB (NCCLS, 2003). MIC was defined as the lowest concentration of crude extract that inhibited vi-sual growth after incubation at 37℃ for 20-24 h, and was per-formed in triplicate (Grierson and Afolayan, 1999).

      >  Minimum bactericidal concentrations (MBC)

    The MBC value was defined as the lowest concentration of E. bicyclis extracts required for a 99.9% reduction in the viable cell population (Shen et al., 2002; NCCLS, 2003). For determining MBC values, an aliquot (0.1 mL) of MIC mix-tures that showed no growth was inoculated onto MHA plates and incubated at 35℃ for 48 h (Syu et al., 2004).

      >  High-performance liquid chromatography

    To identify the active compound(s) in E. bicyclis extracts, high-performance liquid chromatography (HPLC) was per-formed using a Hitachi 2000 series HPLC system (Hitachi Tech, Tokyo, Japan) equipped with Shiseido C18 reverse-phase column (250 mm × 4.6 mm, I.D. 5 ㎛; Shiseido Co., Tokyo, Japan). For detection of the bioactive substance, a linear gradi-ent elution of 90% water with 10 to 100% (v/v) methanol was used at a flow rate of 1.0 mL per min for 45 min. Eluates were monitored at 230 nm.

    Standard phlorotannins (dieckol, eckol, and eckstolonol) were used to allow quantification. Standard compound con-centrations were calculated in the range of 0.01 to 0.1 mg by linear regression from the respective calibration curves. Eckol (Y = 12.973X - 0.4261; -r2 = 0.9991), dieckol (Y = 17.065X + 1.3499; -r2 = 0.9993), and eckstolonol (Y = 10.192X + 0.7048; -r2 = 0.9963) standard curves were obtained (data not shown).

    Results and Discussion

      >  Anti-MRSA activity of E. bicyclis extract

    E. bicyclis methanolic extract exhibited anti-MRSA activi-ty, suggesting the presence of an antibacterial substance (Table 1). Also, the extract exhibited similar activity against MSSA (Table 1). To identify the antimicrobial substance, the extract was further fractionated using organic solvents. Lyophilized E. bicyclis extract (1.0 kg) was percolated in methanol (3 times × 1,000 mL), followed by fractionation with organic sol-vents to yield hexane- (0.05 g), DCM- (0.05 g), EtOAc- (0.11 g), BuOH ion- (0.39 g), and water-soluble (0.57 g) fractions. The anti-MRSA activity of the hexane, DCM, EtOAc, BuOH, and water-soluble fractions was evaluated by measuring the inhibition zones. Of these, the EtOAc-soluble fraction showed the strongest anti-MRSA activity, followed by DCM, BuOH, and hexane, in that order (Table 1). No anti-MRSA activity was detected in the water-soluble fraction. These results were consistent with the reports of Lee et al. (2008) and Choi et al. (2010) stating that the EtOAc-soluble fraction of Ecklonia stolonifera and Ecklonia cava exhibited the strongest anti-MRSA activity. The antibacterial substance was subsequently identified.

      >  Determination of the MIC and MBC of E. bicyclis extract

    The current study focused on the antibacterial activity of E. bicyclis extracts against MRSA. To quantitatively evaluate this antibacterial activity, we investigated the MIC and MBC values of the extract (Table 2). The highest anti-MRSA and MSSA activities were present in the EtOAc-soluble fraction at 32-64 μg/mL. These results were consistent with those of the disk diffusion assay. The MICs and MBCs of the methanolic extract against MSSA and MRSA ranged from 64 to 256 μg/mL; those of the other fractions (hexane, DCM and BuOH) were between 32 and 64 μg/mL for MICs and 64 and 256 μg/mL for MBCs. However, no antibacterial activity was detected in the water-soluble fraction (Table 2). These results strongly suggest the presence of an anti-MRSA compound in the EtO-Ac-soluble fraction of the E. bicyclis methanolic extract.

    Kim et al. (2002) reported that the EtOAc extract of this seaweed showed the highest antibacterial activity against the cavity-causing Gram-positive bacterium Streptococcus mu-tans. Therefore, we investigated the antibacterial activity of E.bicyclis extract against several pathogenic and spoilage bac-teria (Table 2). The EtOAc fraction showed the highest anti-bacterial activity against these bacterial strains. The MICs and MBCs of the EtOAc fraction were 32-256 μg/mL.

    Vancomycin, a tricyclic glycopeptide antibiotic, is used to treat MRSA infections (Lee et al., 2008). Vancomycin in-terferes with bacterial cell wall synthesis, as does penicillin, eventually leading to cell lysis (Barna and Williams, 1984). Most Gram-negative bacteria are less sensitive to vancomycin than Gram-positives (Totsuka et al., 1999; Lee et al., 2008). As expected, the MICs of vancomycin against Gram-neg-ative bacteria were over 512 μg/mL compared to 0.5-4 μg/mL against Gram-positives (Table 2). Unlike vancomycin, the EtOAc fraction exhibited strong antibacterial activity (MICs 32-128 μg/mL; MBCs 32-256 μg/mL) against Gram-negative bacteria. These data suggest the existence of different mecha-nisms of inhibiting cell growth and bacterial cell wall synthe-sis.

      >  Identification of an anti-MRSA substance from E. bicyclis

    The biological activities of plant materials are related to their phenolic compound content (Kim et al., 2006; Lin et al., 2008). Such activity is based on the physiological functions of polyphenol polymers (McDougall et al., 2005). Seaweed polyphenol (phlorotannin) is the predominant EtOAc-soluble compound in brown algae (Lee et al., 2008; Choi et al., 2010). Phloroglucinols, such as eckol, phlorofucofuroeckol-A, diek-

    col, and 8,8′-bieckol, also exhibit antibacterial activity (Isnan-setyo et al., 2001; Nagayama et al., 2002).

    We detected anti-MRSA activity in E. bicyclis methanolic extract. Additionally, the ethyl acetate-soluble fraction exhib-ited the highest antibacterial activity against other pathogenic bacteria, suggesting that the antibacterial compound is abun-dant in the EtOAc fraction (Table 2). To identify the active compound, we conducted HPLC analysis (Figs. 1 and 2). Pu-rified phloroglucinol compounds (dieckol, eckol, and ecksto-lonol) were used as controls. Identification of unknown phlo-roglucinol compounds was achieved by comparing retention time with those of control compounds. The retention times of dieckol, eckol, and eckstolonol were 12.98 ± 0.30, 10.87 ± 0.29, and 21.52 ± 0.45 min, respectively (Fig. 1). HPLC analy-sis showed that only the methanolic extract and the EtOAc-

    soluble fraction contained sizeable quantities of dieckol (76.9 mg/g in the EtOAc-soluble fraction) (Table 2).

    Dieckol is a known antibacterial substance with activity against MRSA (Lee et al., 2008). Of the soluble fractions, di-eckol was present only in the EtOAc fraction, which exhibited the highest anti-MRSA activity. Our previous report showed that of the pholorotannins tested, dieckol showed the highest anti-MRSA activity (Lee et al., 2008). Considering these data, we suggest that the anti-MRSA activity of E. bicyclis is likely mediated by phlorotannins such as dieckol.

  • 1. Barna JCJ, Williams DH 1984 The structure and mode of action of glycopeptide antibiotics of the vancomycin group. [Annu Rev Microbiol] Vol.38 P.339-357 google doi
  • 2. Bramley AJ, Patel AH, O’Reilly M, Foster R, Foster TJ 1989 Roles of alpha-toxin and beta-toxin in virulence of Staphylococcus au-reus for the mouse mammary gland. [Infect Immun] Vol.57 P.2489-2494 google
  • 3. Choi JG, Kang OH, Brice OO, Lee YS, Chae HS, Oh YC, Sohn DH, Park H, Choi HG, Kim SG, Shin DW, Kwon DY 2010 An-tibacterial activity of Ecklonia cava against methicillin-resistant Staphylococcus aureus and Salmonella spp. [Foodborne Pathog Dis] Vol.7 P.435-441 google doi
  • 4. Eom SH, Kang YM, Park JH, Yu DU, Jeong ET, Lee MS, Kim YM 2011 Enhancement of polyphenol content and antioxidant activity of Eisenia bicyclis extract by microbial fermentation. [Fish Aquat Sci] Vol.14 P.192-197 google doi
  • 5. Grierson DS, Afolayan AJ 1999 Antibacterial activity of some in-digenous plants used for the treatment of wounds in the Eastern Cape South Africa. [J Ethnopharmacol] Vol.66 P.103-106 google doi
  • 6. Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T, Tenover FC 1997 Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. [J Antimicrob Chemother] Vol.40 P.135-136 google doi
  • 7. Isnansetyo A, Kamei Y 2009 Anti-methicillin-resistant Staphy-lococcus aureus (MRSA) activity of MC21-B an antibacterial compound produced by the marine bacterium Pseudoalteromonas phenolica O-BC30T. [Int J Antimicrob Agents] Vol.34 P.131-135 google doi
  • 8. Isnansetyo A, Horikawa M, Kamei Y 2001 In vitro anti-methi-cillin-resistant Staphylococcus aureus activity of 24-diacetylphlo-roglucinol produced by Pseudomonas sp. AMSN isolated from a marine alga. [J Antimicrob Chemother] Vol.47 P.724-725 google doi
  • 9. Jung HA, Oh SH, Choi JS 2010 Molecular docking studies of phlo-rotannins from Eisenia bicyclis with BACE1 inhibitory activity. [Bioorg Med Chem Lett] Vol.20 P.3211-3215 google doi
  • 10. Kang K, Park Y, Hwang HJ, Kim SH, Lee JG, Shin HC 2003 An-tioxidative properties of brown algae polyphenolics and their per-spectives as chemopreventive agent against vascular risk factors. [Arch Pharm Res] Vol.26 P.286-293 google doi
  • 11. Kaplan SL 2005 Implications of methicillin-resistant Staphylococcus aureus as a community-acquired pathogen in pediatric patients. [Infect Dis Clin N Am] Vol.19 P.747-757 google doi
  • 12. Kim JH, Lee DS, Lim CW, Park HY, Park JH 2002 Antibacte-rial activity of sea-mustard Laminaria japonica extracts on the cariogenic bacteria Streptococcus mutans. [J Korean Fish Soc] Vol.35 P.191-195 google
  • 13. Kim SM, Shang YF, Um BH 2011 A preparative method for isola-tion of fucoxanthin from Eisenia bicyclis by centrifugal partition chromatography. [Phytochem Anal] Vol.22 P.322-329 google doi
  • 14. Kim MM, Ta QV, Mendis E, Rajapakse N, Jung WK, Byun HG, Jeon YJ, Kim SK 2006 Phlorotannins in Ecklonia cava extract in-hibit matrix metalloproteinase activity. [Life Sci] Vol.79 P.1436-1443 google doi
  • 15. Lee DS, Kang MS, Hwang HJ, Eom SH, Yang JY, Lee MS, Lee WJ, Jeon YJ, Choi JS, Kim YM 2008 Synergistic effect between dieckol from Ecklonia stolonifera and β-lactams against methicil-lin-resistant Staphylococcus aureus. [Biotechnol Bioprocess Eng] Vol.13 P.758-764 google doi
  • 16. Lin RD, Chin YP, Hou WC, Lee MH 2008 The effects of antibiot-ics combined with natural polyphenols against clinical methicillin-resistant Staphylococcus aureus (MRSA). [Planta Med] Vol.74 P.840-846 google doi
  • 17. McDougall GJ, Shpiro F, Dobson P, Smith P, Blake A, Stewart D 2005 Different polyphenolic components of soft fruits inhibit al-pha-amylase and alpha-glucosidase. [J Agric Food Chem] Vol.53 P.2760-2766 google doi
  • 18. Nagayama K, Iwamura Y, Shibata T, Hirayama I, Nakamura T 2002 Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome. [J Antimicrob Chemother] Vol.50 P.889-893 google doi
  • 19. 2003 Methods for Dilution Antimicrobial Susceptibility Tests for Bac-teria That Grow Aerobically: Approved Standard. google
  • 20. 2004 Performance Standards for Antimicrobial Disk Susceptibility Test-ing. 14th Informational Supplement. google
  • 21. Noda H, Amano H, Arashima K, Hashimoto S, Nisizawa K 1989 Studies on the antitumour activity of marine algae. [Nippon Suisan Gakkaishi] Vol.55 P.1259-1264 google doi
  • 22. Okada Y, Ishimaru A, Suzuki R, Okuyama T 2004 A new phloro-glucinol derivative from the brown alga Eisenia bicyclis: potential for the effective treatment of diabetic complications. [J Nat Prod] Vol.67 P.103-105 google doi
  • 23. Shen CC, Syu WJ, Li SY, Lin CH, Lee GH, Sun CM 2002 Antimi-crobial activities of Naphthazarins from Arnebia euchroma. [J Nat Prod] Vol.65 P.1857-1862 google doi
  • 24. Shibata T, Fujimoto K, Nagayama K, Yamaguchi K, Nakamura T 2002 Inhibitory activity of brown algal phlorotannins against hy-aluronidase. [Int J Food Sci Technol] Vol.37 P.703-709 google doi
  • 25. Shibata T, Nagayama K, Tanaka R, Yamaguchi K, Nakamura T 2003 Inhibitory effects of brown algal phlorotannins on secretory phospholipase A2s lipoxygenases and cyclooxygenases. [J Appl Phycol] Vol.15 P.61-66 google doi
  • 26. Syu WJ, Shen CC, Lu JJ, Lee GH, Sun CM 2004 Antimicrobial and cytotoxic activities of neolignans from Magnolia officinalis. [Chem Biodivers] Vol.1 P.530-537 google doi
  • 27. Totsuka K, Shiseki M, Kikuchi K, Matsui Y 1999 Combined ef-fects of vancomycin and imipenem against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo. [J Antimicrob Chemother] Vol.44 P.455-460 google doi
  • [Table 1.] Growth inhibitory of Eisenia bicyclis extracts against methicillin-resistant Staphylococcus aureus (MRSA) and other strains
    Growth inhibitory of Eisenia bicyclis extracts against methicillin-resistant Staphylococcus aureus (MRSA) and other strains
  • [Table 2.] Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of Eisenia bicyclis extracts against methicillin-resistant Staphylococcus aureus (MRSA) and other strains
    Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of Eisenia bicyclis extracts against methicillin-resistant Staphylococcus aureus (MRSA) and other strains
  • [Fig. 1.] High-performance liquid chromatography (HPLC) profile of standard phlorotannins. HPLC analysis was performed as described in Materials and Methods. EK 100 μg per mL of eckol; DK 100 μg per mL of dieckol; ES 100 μg per mL of eckstolonol.
    High-performance liquid chromatography (HPLC) profile of standard phlorotannins. HPLC analysis was performed as described in Materials and Methods. EK 100 μg per mL of eckol; DK 100 μg per mL of dieckol; ES 100 μg per mL of eckstolonol.
  • [Fig. 2.] HPLC profiles of Eisenia bicyclis extracts after 5 days of fermentation. A B C D E and F are the HPLC profiles for methanol extract n-hexane-soluble extract dichloromethane-soluble extract ethyl acetate-soluble extract n-butanol-soluble extract water-soluble extract. The injection amount was 1000 ppm.
    HPLC profiles of Eisenia bicyclis extracts after 5 days of fermentation. A B C D E and F are the HPLC profiles for methanol extract n-hexane-soluble extract dichloromethane-soluble extract ethyl acetate-soluble extract n-butanol-soluble extract water-soluble extract. The injection amount was 1000 ppm.