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.
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.
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.
The bacterial strains used in this study were L. monocytogenes KCTC 3710 from the Korean Collection for Type Cultures (KCTC; Daejeon, Korea) and three clinical isolates provided by the Gyeongsang National University Hospital (Jinju, Korea), a member of the National Biobank of Korea. All strains were cultured aerobically at 37℃ in Brain Heart Infusion broth (BHI; Difco, USA).
The antibacterial efficacy of marine algae extracts was evaluated by disk diffusion assay followed by the CLSI (2009), as described in our previous reports (Lee et al., 2014; Eom et al., 2011). In brief, bacterial strains were cultured in BHI at 37℃ until an OD at 600 nm of 0.5. One hundred microliters of bacterial culture containing approximately 104 - 105 CFU/ mL were spread on Mueller Hinton agar (MHA; Difco, USA) plates. Paper discs (6 mm in diameter) containing 1 and 5 mg of extract were placed on the MHA plate. After incubating for 24 h at 37℃, the diameter of the inhibition zone was measured. The experiment was carried out three times and the mean values were calculated.
Minimum inhibitory concentration (MIC) is defined as the lowest concentration of antimicrobial that inhibits visual growth of microorganisms after 20 - 24 h of incubation at 37℃ (Gierson and Afolayan, 1999). The MICs of marine algae extracts and some commercial antibiotics were determined by a twofold serial dilution method in Mueller–Hinton broth (MHB; Difco) (CLSI, 2008).
Minimum bactericidal concentration (MBC) is defined as the lowest concentration of an antimicrobial required for a 99.9% reduction in the viable cell population (NCCLS, 2003). For MBC determination, an aliquot of inoculums was taken from a MIC test well that did not show turbidity and was poured onto BHI agar plates. The agar plates were incubated at 37℃ until growth was seen in the growth control plates. The number of colonies on each agar plate was counted. The MIC and MBC experiments were repeated in triplicate.
Sensitivity and/or resistance of L. monocytogenes KCTC 3710 and clinical isolates was evaluated using the disk diffusion method with 16 antibiotics, either individually or in combination.
The interaction between the EtOAc fraction of E. cava and antibiotics containing streptomycin (Sigma Chemical Co., St. Louis, MO, USA) against L. monocytogenes strains was tested by the checkerboard method using fractional inhibitory concentration (FIC) (Hsieh et al., 1993; Meletiadis et al., 2010). The sum of the FICs (ΣFIC) was calculated for each well with the equation: ΣFIC = FICA + FICB = (CA/MICA) + (CB/MICB), where MICA and MICB are the MICs of drugs A and B alone, respectively, and CA and CB are the concentrations of the drugs in combination, respectively, in all of the wells corresponding to an MIC (isoeffective combinations). Among all of the ΣFICs calculated for all isoeffective combinations, we reported the minimum ΣFIC (ΣFICmin) and the maximum ΣFIC (ΣFICmax) to capture synergistic and antagonistic interactions, respectively. The synergistic effect was evaluated as a fractional inhibitory concentration (FIC) index as described by Lee et al. (2014). The interaction was defined as synergistic if the FIC index was <1.0, additive if the FIC index was 1.0, subadditive if the FIC index was between 1.0 and 2.0, indifferent if the FIC index was 2, and antagonistic if the FIC index >2.0. Synergy was further sub-classified as either marked (FIC index, ≤0.5) or weak (FIC index, between 0.5 and 1.0).
Data are reported as means ± standard errors of the mean. Differences at P<0.05 were considered statistically significant. SPSS 12.0 (SPSS Inc., Chicago, IL) was used to perform the statistical analysis.
MeOH extracts from 13 marine algae, including E. cava, were screened for their antibacterial activity against L. monocytogenes by disk diffusion assay as described in the Materials and methods. E. cava methanolic extract exhibited the greatest antibacterial activity against L. monocytogenes KCTC 3710 and all three isolates of L. monocytogenes used in this study (data not shown). The diameters of inhibition zone ranged from 7.3 mm to 13 mm for 1 mg/disk, and 9 mm to 18 mm for 5 mg/disk (Table 1). These results indicate that E. cava contains higher levels of antibacterial compounds active against L. monocytogenes than do other seaweeds. Also, these results are in line with the study by Cox et al. (2010) of the antimicrobial properties of several Irish edible brown seaweeds and reported good antibacterial activities against L. monocytogenes for MeOH extracts of the seaweed Laminaria digitata.
To further investigate the mechanism underlying the antibacterial effect against L. monocytogenes, E. cava methanolic extract was fractioned using several solvents. Of those, the EtOAc fraction exhibited the greatest antibacterial activity against L. monocytogenes (the diameter of the clear zone around the disk ranged from 13 to 23.0 mm for 5 mg/disk) as compared to other fractions (clear zone diameters ranged from 6.0 to 16.3 mm for 5 mg/disk) (Table 1).
These results suggested that a substance with antibacterial activity against L. monocytogenes was present in the EtOAc soluble fraction of the E. cava extract, therefore supporting the first hypothesis. According to Choi et al. (2010), marine-derived polyphenols (phlorotannins) are the predominant EtOAc-soluble compound in brown algae (Choi et al. 2010). In this study, it was also found that the EtOAc fraction of E. cava methanolic extract contained the highest amount of TP compounds, as described in the Materials and Methods. Thus, on the basis of the aforementioned results, E. cava was subjected to further investigation of its antibacterial activity against L. monocytogenes.
The MIC assay was carried out to quantitatively evaluate the antibacterial activity of E. cava methanolic extract and its soluble fractions. MIC values of solvent fractions against L. monocytogenes varied depending on the polarity of the solvent. In general, the MIC values were in the range 256 to 1,024 μg/mL (Table 2). Among the solvent-soluble fractions, the EtOAc-soluble fraction evinced the lowest MIC values, as it completely inhibited the growth of L. monocytogenes KCTC 3710 and three clinical isolates (P2148, P2637 and P2868) at 256 μg/mL. This indicated that the EtOAc solvent-soluble fraction had the highest antibacterial activity. No marked difference between the standard strain and L. monocytogenes clinical isolates was observed (Table 2).
The antibacterial activities of E. cava extracts were also quantitatively evaluated by MBC assay. The MBC values—the lowest concentration that killed 99.99% of the initial inoculum—were slightly higher than MIC values, but in all cases did not exceed twice the MIC value. The MBC values for the EtOAc fraction were 512 μg/mL for all strains used, while MBC values of the other fractions ranged from 512 to >1,024 μg/mL for all strains (Table 2). These results are in accordance with those reported by Rahman et al. (2013) that, at 400 μg/disc, an EtOAc extract of Poncirus trifoliata seeds showed the greatest antibacterial activity against four of five L. monocytogenes strains. A similar result was reported by Bajpai and Kang (2009); i.e., the EtOAc fraction of the plant Metasequoia glyptostroboides showed the strongest antibacterial effect against four L. monocytogenes strains.
Recent reports show an increased rate of resistance to one or more clinically relevant antibiotics in environmental L. monocytogenes isolates (Charpentier et al., 1995; Conter et al., 2009) and, less frequently, in clinical strains (Safdar and Armstrong, 2003). However, few studies have evaluated antimicrobial resistance in Listeria spp. (Hansen, 2005; Safdar and Armstrong, 2003). Thus the present study used both the disk diffusion and minimum inhibitory concentration assays to evaluate the antibacterial susceptibility of L. monocytogenes against several commercial antibiotics.
The antibiotic resistance patterns of the type strain and three clinical isolates of L. monocytogenes were assessed by a disk diffusion assay using 16 commercial antibiotic kits. In the disk diffusion assay, no clear zones were found around the disks containing either gentamycin or streptomycin for all three clinical isolates used in this study (data not shown). The failure of streptomycin and gentamycin to inhibit the growth of all three clinical L. monocytogenes isolates suggests resistance to these agents (data not shown).
The MIC value for streptomycin against L. monocytogenes KCTC 3710 was 8 μg/mL, compared to 16 μg/mL for P2148 and P2637, and 32 μg/mL for P2868. On the other hand, the MIC values of gentamycin against the L. monocytogenes strains tested in this study were 2 to 8 μg/mL (Table 3). The MIC values of streptomycin against L. monocytogenes strains were equal or higher than the generally accepted Soussy’s MIC breakpoint values, ranging from 8 to 16 μg/mL (Aureli et al., 2003). In contrast, the MIC values of gentamycin against L. monocytogenes strains, with the exception of the type strain KCTC 3710, were lower than the generally accepted Soussy’s MIC breakpoint values, ranging from 4 to 8 μg/mL (Aureli et al., 2003). These results indicate that streptomycin is no longer useful for treating Listeria infections. This adds to other reports, including Charpentier et al. (1995), Facinell et al. (1991), and Poyart-Salmeron et al., (1990), of cases of clinical isolates of L. monocytogenes resistant to multiple antibiotics, including gentamycin, streptomycin, erythromycin, kanamycin, sulfamethoxazole, rifampin, and others.
It has been demonstrated that one of the more effective strategies in developing new drugs or alternative therapies is the restoration of antibiotic activity, in combination with antibacterial materials derived from natural products and traditional medicines, against drug-resistant bacteria (Eom et al. 2014; Lee et al., 2014). In this study, the interaction between the EtOAc fraction of E. cava and the commercial antibiotic streptomycin against L. monocytogenes strains was evaluated by FIC assay. Gentamycin was not assayed since most of the L. monocytogenes strains tested failed to exhibit resistance to this agent.
As shown in Table 4, the MIC values of streptomycin in combination with the EtOAc fraction of E. cava (at concentrations of 2 to 32 μg/mL) were markedly reduced up to 64-fold for KCTC 3710, 8-fold for P2148 and P2868, and 4-fold for P2637. Thus, the antibacterial activity of a traditional antibiotic, streptomycin, was restored by combination with the EtOAc fraction, since the minimum concentrations that inhibited growth with streptomycin were 0.125 to 4 μg/mL.
The synergistic antibacterial activity between streptomycin and the EtOAc fraction of E. cava was assessed by the FIC analysis. The FIC indices of streptomycin in combination with the EtOAc fraction are presented in Table 4. The combination of streptomycin and the EtOAc fraction resulted in a ΣFICmin range of 0.141 to 0.266 and ΣFICmax of 0.531 for all strains (Table 4). The median ΣFIC against L. monocytogenes strains ranged from 0.172 to 0.344. As described by Lee et al. (2014), the 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 the streptomycin–EtOAc fraction ranged from 0.172 to 0.344, suggesting marked synergy (Table 4). These results suggest that E. cava extracts showed synergistic effects in combination with streptomycin. In addition, Choi et al. (2010) reported that the combination of E. cava extracts with ampicillin improved the inhibition of S. aureus and Salmonella spp.
In conclusion, this study evaluated the antibacterial activity of the edible marine brown alga E. cava against L. monocytogenes. The EtOAc fraction of E. cava extract showed the strongest antibacterial activity against L. monocytogenes among the solvent fractions tested, suggesting that the antibacterial activity of E. cava against L. monocytogenes may be related to the phlorotannin or marine-derived polyphenolic contents. Additionally, streptomycin in combination with the EtOAc fraction restored antibacterial activity against L. monocytogenes in a synergistic manner. Thus the results of the present investigation will contribute to the development of an alternative phytotherapeutic agent against Listeria infection.