Synergistic Antibacterial Activity of Ecklonia cava (Phaeophyceae: Laminariales) against Listeria monocytogenes (Bacillales: Listeriaceae)

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    In an effort to discover alternative antimicrobials against Listeria monocytogenes, several marine algae were screened. The methanolic extract of Ecklonia cava exhibited the highest antibacterial activity against L. monocytogenes, with the ethyl acetate (EtOAc) soluble fraction of E. cava methanolic extract having a MIC value of 256 μg/mL and a MBC value of 512 μg/mL. The MIC values of streptomycin in combination with the EtOAc fraction were markedly reduced up to 64-fold, suggesting that the antibacterial activity of the antibiotic was restored when combined with the EtOAc fraction. The interaction between streptomycin and the EtOAc fraction was assessed by fractional inhibitory concentration (FIC) indices. The combination of streptomycin and the EtOAc fraction against L. monocytogenes resulted in ΣFICmin range of 0.141 to 0.266 and ΣFICmax of 0.531 for all strains. The median ΣFIC against L. monocytogenes strains ranged from 0.172 to 0.344. Thus, synergistic ranges of FIC <1 were observed for all combinations of streptomycin and the EtOAc fraction against L. monocytogenes strains. Indeed, the median ΣFIC of streptomycin–EtOAc fraction ranged from 0.172 to 0.344, suggesting a marked synergy.


    Antibacterial activity , Ecklonia cava , Listeria monocytogenes , Synergistic effect

  • Introduction

    Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a severe invasive infection in humans with a particularly high case-fatality rate. Listeriosis is a major public health concern in all regions of the world due to the severity of manifestations (i.e., septicemia, meningitis and fetal death), with a case-fatality rate ranging from 20% to 50% (Denny and McLauchlin, 2008). Despite efficient antibiotic therapy, listeriosis represents a public health problem because it is frequently fatal. To overcome these problems, a wide range of synthetic antimicrobial agents (sodium benzoate, calcium benzoate, sorbate) has been used as food preservatives (Sherwin, 1990). However, antibiotic resistance has been described among Listeria spp., particularly L. monocytogenes strains isolated from food, the environment, or sporadic human listeriosis cases (Poyart-Salmeron, et al., 1990; Facinelli et al., 1991; Charpentier et al., 1995).

    Recently, consumers have begun demanding foods that are fresh, natural, and minimally processed, along with the requirement for enhanced safety and quality. This perspective has put pressure on the food industry for progressive removal of chemical preservatives, and has fuelled research into alternative natural antimicrobials (Lanciotti et al., 2004). Additionally, increased public awareness of the negative effects caused by synthetic chemicals has led to the search for “green solutions,” such as organic and synthetic chemical-free food products (Abutbul, 2004).

    In that regard, seaweeds have become an important source of pharmacologically active metabolites, with a broad spectrum of biological activities with possible applications in food production to prevent bacterial and fungal growth. In fact, compounds with antioxidant, antiviral, antifungal, and antimicrobial activities have been detected in brown, red, and green algae (Eom et al., 2011). Ecklonia cava, of the family Laminariaceae, ranges along the eastern coast of Korea and Japan. It is utilized as a food ingredient, animal feed, fertilizer, and medicine. Additionally, E. cava was reported to harbor compounds with various biological activities, including carotenoids, fucoidan, and phlorotannins (Kang et al., 2012). E. cava extracts exhibit various biological functions, including antioxidant, antibacterial, protease inhibitory, immunomodulatory, antiasthmatic, and tyrosinase inhibitory activity (Kim et al., 2006; Kang et al., 2012). However, the antibacterial activity of E. cava against L. monocytogenes has not been reported to date. The present study aimed to investigate the antibacterial activity of E. cava against L. monocytogenes.

    Materials and Methods

      >  Samples and extraction

    Dried E. cava powder (1.0 kg) was extracted and fractionated using organic solvents as described in Lee et al. (2014). The methanolic extract of E. cava (101.3 g) was partitioned in turn with n-hexane (Hexane; 1.0 L × 3), dichloromethane (DCM; 1.0 L × 3), ethyl acetate (EtOAc; 1.0 L × 3), and n-butanol (n-BuOH; 1.0 L × 4). The water fraction was obtained by filtering the remaining materials from the n-BuOH partition. Finally, each extract was evaporated using a rotary evaporator (Eyela, Tokyo, Japan) under vacuum at 45℃. Another 12 methanol extracts from various marine algae, previously prepared and stored under appropriate conditions (refrigerated at -70℃), were used for the same purpose.

      >  Total phenolic contents

    The contents of total phenolic (TP) compounds in the fractionated E. cava extracts were evaluated using the modified Folin–Ciocalteu method (Eom et al., 2013), taking phloroglucinol as a standard and with results expressed as phloroglucinol equivalents (PGE). The concentration of TP compounds was 241.0 ± 1.6 mg PEG/g in the MeOH extract, 26.8 ± 6.9 mg PEG/g in the hexane fraction, 83.3 ± 1.0 mg PEG/g in the DCM fraction, 556.8 ± 2.7 mg PEG/g in the EtOAc fraction, 416.3 ± 18.4 mg PEG/g in the BuOH fraction, and 34.3 ± 4.1 mg PEG/g in the water fraction.