Abstract
Listeria monocytogenes (LM) is an important food borne pathogen responsible for listeriosis. Further, LM is an etiological agent associated with life threatening conditions like meningitis and encephalitis. Biofilm forming and drug resistant LM may potentially become difficult to treat infections and hence effective controlling measures are required to prevent LM infections. In view of this, the present study evaluated an anti-listerial potential of edible brown seaweed, Eisenia bicyclis, by disc diffusion and micro-dilution methods. The results of the present study suggested that the anti-listerial activity of various phlorotannins isolated form E. bicyclis were in the range of 16–256 µg/ml. Among the phlorotannins isolated, fucofuroeckol-A (FAA) exhibited the highest anti-listerial potential (MIC range 16–32 µg/ml) against LM strains tested. Further, in checker board synergy assays, FFA-streptomycin combination exhibited significant synergy (fractional inhibitory concentration index, ∑FIC < 0.5) against aminoglycoside resistant clinical strains of LM. The results of the present study suggested the potential use of edible seaweed E. bicyclis as a source of natural phlorotannins to control food borne pathogenic infections.
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Keywords: Antibiotic resistance, Anti-listerial agents, Eisenia bicyclis, Phlorotannins
Listeria monocytogenes (LM), an important foodborne pathogen is responsible for listeriosis, meningitis and encephalitis. The ability of LM to produce virulence factors causes listeriosis a difficult to treat infection. Also listerial biofilms formed in food processing environment are resistant to antibiotics and disinfectants [1, 2]. Increased antimicrobial resistant pathogens in the food chain [3] demands novel food pathogen control strategies. Eisenia bicyclis, edible marine algae, is popular in Asian cuisine [4] and is known to exhibit antioxidant, anti-hypertension, anti-allergic, and anti-tumor activities [5]. In search of novel food-compatible anti-listerial compounds of seaweed origin, the present study evaluated the anti-listerial property of phlorotannins purified from E. bicyclis.
All chemicals, antibiotics, and reagents used were purchased form Sigma Chemical Co (St. Louis, MO, USA) unless or otherwise mentioned. E. bicyclis (Ulleung Trading Co. Ulleung-gun, Korea) was thoroughly washed with distilled water and dried at 60 °C in a hot air oven. A reference LM strain (ATCC 19112) was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Clinical strains of LM were obtained from Gyeongsang National University Hospital (GNUH; Jinju, Korea). All strains were aerobically cultured in brain heart infusion broth (BHI; Difco Inc., Detroit, MI), and incubated at 37 °C. In case of determining antibacterial properties, Mueller–Hinton broth and agar (MHB and MHA; Difco Inc.) were used. The extraction and purification of E. bicyclis was performed according to the method previously reported [6]. The crude and isolated compounds were dissolved in dimethyl sulfoxide (DMSO). All antimicrobial assays were performed according to the guidelines of Clinical and Laboratory Standards Institute [7]. For disc diffusion assays, MHA plates seeded with test strains (104 CFU/ml) and sterile paper discs (6 mm diameter) impregnated with crude extract, fractions or pure compounds and placed on the surface of MHA and incubated at 35 °C. Untreated and solvent treated (DMSO, 10%) discs served as blank and negative controls, respectively. After 24 h, the size of inhibitory zones was determined. In growth assays, MHB was adjusted with two fold serial dilutions of either fractions or pure compounds in DMSO (1% v/v in final volume) were inoculated with test strains (104 CFU/ml) and incubated at 35 °C. After 24 h, the optical density (OD610 nm) of the culture was determined using microplate reader. DMSO (1% v/v) served as negative controls. MIC is determined as the lowest concentration of test agent which cause > 90% inhibition in the growth of the test pathogen. For synergy testing, a checker board synergy testing was employed where a serial two-fold dilution of antibiotic as well as test compound was mixed to obtain different combinations of concentrations in MHB. The plates were inoculated with the test bacteria (104 CFU/ml) and incubated stationary at 35 °C for 24 h. The fractional inhibitory concentration index (∑FIC) was determined according to the method previously reported [8]. A potential synergy was conferred when FIC index was < 0.5.
In disc diffusion assay, the methanolic extracts of E. bicyclis at 1 and 5 mg/disc exhibited a zone of inhibition (ZOI) in the range of 9.0 ± 0.5 to 14.0 ± 0.1 mm against LM strains (Table 1). These results are in agreement with the results of previous study [9] that methanolic extracts of seaweed plants were inhibitory against food borne pathogens including LM. A liquid–liquid extraction system selectively separates bioactive compounds based on their partition coefficient with different solvents. Using this approach we partitioned aqueous methanolic crude extracts of E. bicyclis with various non polar and polar solvents and the ZOI and MICs of each solvent soluble fraction was tested against LM strains. Among the solvent soluble fractions, the ethyl acetate fraction exhibited ZOI in the range of 9.5 ± 0.4 to 15.0 ± 0.1 mm against LM strains (Table 2). Further, ethyl acetate fraction exhibited significantly lower MIC values (MIC range 128–256 μg/ml) against test LM strains which is comparable to the MIC values obtained (128 µg/ml) for ethyl acetate fraction of Ecklonia cava against LM [10].
Table 1.
Strains | Conc. (mg/disc) | Zone of inhibition (mm)a | |||||
---|---|---|---|---|---|---|---|
MeOHb | Hexane | DCM | EtOAc | BuOH | H2O | ||
KCTC 19112 | 1 | 9.5 ± 0.2c | 8.0 ± 0.1 | 8.0 ± 0.3 | 9.5 ± 0.4 | ND | ND |
5 | 13.0 ± 0.5 | 10.0 ± 0.4 | 10.0 ± 0.5 | 15.0 ± 0.1 | 9.0 ± 0.2 | ND | |
Strain 2148 | 1 | 10.0 ± 0.2 | 7.5 ± 0.2 | 7.0 ± 0.3 | 10.0 ± 0.4 | ND | ND |
5 | 14.0 ± 0.1 | 9.0 ± 0.1 | 8.5 ± 0.4 | 14.5 ± 0.3 | 10.0 ± 0.1 | ND | |
Strain 2637 | 1 | 9.0 ± 0.5 | 7.5 ± 0.4 | 7.5 ± 0.5 | 9.5 ± 0.4 | ND | ND |
5 | 13.0 ± 0.1 | 8.5 ± 0.1 | 10.0 ± 0.1 | 14.0 ± 0.2 | 9.5 ± 0.2 | ND | |
Strain 2868 | 1 | 9.0 ± 0.2 | ND | ND | 9.0 ± 0.2 | ND | ND |
5 | 12.0 ± 0.5 | 8.0 ± 0.1 | 8.5 ± 0.4 | 13.5 ± 0.2 | 8.0 ± 0.3 | ND |
ND no detected antibacterial activity
a Methanol extract and its fractions from E. bicyclis were loaded onto discs (6 mm in diameter)
b MeOH methanolic extract, Hexane n-hexane-soluble fraction, DCM dichloromethane-soluble fraction, EtOAc ethyl acetate-soluble fraction, BuOH n-butanol-soluble fraction, H 2 O water-soluble fraction
c Data are the averages of triplicate experiments
Table 2.
Strains | MIC (µg/mL)a | |||||
---|---|---|---|---|---|---|
MeOHb | Hexane | DCM | EtOAc | BuOH | H2O | |
KCTC 19112 | 512 | 512 | 512 | 256 | 1024 | –c |
Strain 2148 | 128 | 256 | 512 | 128 | 512 | – |
Strain 2637 | 256 | 512 | 512 | 256 | 512 | – |
Strain 2868 | 256 | 512 | 512 | 256 | 1024 | – |
–, No activity detected
EtOAc ethyl acetate-soluble fraction, BuOH n-butanol-soluble fraction, H 2 O water fraction
a Methanol extract and its fractions from E. bicyclis were loaded onto discs (6 mm in diameter)
b MeOH methanolic extract, Hexane n-hexane-soluble fraction, DCM dichloromethane-soluble fraction
Usually ethyl acetate fractions of seaweeds were reported to contain antimicrobial phenolics, tannins and flavonoids [9]. The purification of ethyl acetate soluble fraction resulted in six phlorotannins (Fig. S1) namely eckol (EK), fucofuroeckol-A (FFA), 7-phloroeckol (7-P), dioxinodehydroeckol (DD), phlorofucofuroeckol (PFF), dieckol (DE) whose identity was confirmed in comparison with the proton and carbon NMR spectra of authentic samples [9, 11, 12]. Further, these pure phlorotannins were evaluated against LM. As shown in Table 3, it is evident that FFA exhibited potent anti-listerial activity (MIC range 16–32 µg/ml) followed by PFF (32–128 µg/ml) and DE (64–128 µg/ml). All other compounds exhibited moderate toxicity against LM strains. Phlorotannins isolated from E. kurome and E. bicyclis (phloroglucinol, EK, PFF, DE and 8,8′-bieckol) were reported to possess antibacterial activity against Gram-positive and Gram-negative pathogens [13, 14]. Although the ethyl acetate fraction of E. cava was reported to possess an anti-listerial activity (MIC, 256 µg/ml), the active compound was not reported [10]. Additionally, this study suggested a potent antibacterial activity of FFA against LM strains.
Table 3.
Strains | MIC (µg/mL) | ||||||
---|---|---|---|---|---|---|---|
EtOAc fr.a | EK | FFA | 7-P | DD | PFF | DE | |
KCTC 19112 | 256 | 128 | 16 | 64 | 64 | 32 | 64 |
Strain 2148 | 128 | 128 | 16 | 128 | 64 | 32 | 64 |
Strain 2637 | 256 | 256 | 32 | 128 | 128 | 64 | 128 |
Strain 2868 | 256 | 256 | 32 | 128 | 128 | 128 | 128 |
a EtOAc fr. ethyl acetate-soluble fraction, EK eckol, FFA fucofuroeckol-A, 7-P 7-phloroeckol, DD dioxinodehydroeckol, PFF phlorofucofuroeckol, DE dieckol
Antibiotic-resistant LM strains are frequently observed in the retail food [15]. One of the effective ways to develop new antimicrobials is to repurpose the existing antimicrobials against drug-resistant pathogens [16]. The clinical strains of LM used in the present study exhibited border line resistance to aminoglycoside antibiotics especially streptomycin (MIC > 8 µg/ml). The checkerboard assay results suggested that FFA-streptomycin combination exhibited synergy against all LM strains. The combination of streptomycin and FFA resulted in a ΣFICmin range of 0.18–0.26 and ΣFICmax range of 0.53–0.56 against all strains (Table 4). The median ΣFIC against LM strains ranged from 0.32 to 0.37 suggesting FFA-streptomycin combination exhibited marked synergy (Table 4). Earlier, β-lactam adjuvant action of phlorotannin from E. bicyclis and E. cava and against methicillin-resistant Staphylococcus aureus was reported [17, 18]. However, in our best of knowledge, the anti-listerial activity of FFA from E. bicyclis has not been investigated. The concentration of the FFA used in the present study is low and is clinically achievable. Brown algae, E. bicyclis and its phlorotannins were reported to be safe when orally administered in mice and exhibits relatively less cytotoxicity [13]. Hence, development of seaweed based therapeutic intervention is expected to reduce the listerial infections. In conclusion, the findings obtained from this study suggest that the marine brown algae E. bicyclis could be novel natural materials for the control and spread of L. monocytogenes infections in food and humans.
Table 4.
Strains | Test compound | MIC (µg/mL) | Median ∑FICa | ∑FICbmax | ∑FICcmin | Minimum conc. for observing synergy |
---|---|---|---|---|---|---|
Listeria monocytogenes (KCTC 19112) | FFA | 16 | 0.344d | 0.531 | 0.266 | 0.25 |
Streptomycin | 8 | 2.0 | ||||
L. monocytogenes isolate 2148 | FFA | 16 | 0.375 | 0.563 | 0.266 | 0.25 |
Streptomycin | 16 | 4.0 | ||||
L. monocytogenes isolate 2637 | FFA | 32 | 0.375 | 0.563 | 0.266 | 0.5 |
Streptomycin | 16 | 4.0 | ||||
L. monocytogenes isolate 2868 | FFA | 32 | 0.328 | 0.563 | 0.188 | 2.0 |
Streptomycin | 32 | 4.0 |
∑FIC = 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
a∑FIC, the sum of FICs
b∑FICmax, the maximum ∑FIC
c∑FICmin, the minimum ∑FIC
dThe FIC index indicated synergistic; < 0.5, addictive; 0.5 to < 1.0, indifferent; > 1.0 to < 2.0, antagonistic; > 2.0
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Acknowledgements
This research was supported by Marine Biotechnology Program (20150220) funded by Ministry of Oceans and Fisheries, Republic of Korea. The pathogens for this study were provided by the Gyeongsang National University Hospital Branch of National Culture Collection for Pathogens (GNUH-NCCP).
Compliance with Ethical Standards
Conflict of interest
None.
Footnotes
Electronic supplementary material
The online version of this article (10.1007/s12088-017-0693-x) contains supplementary material, which is available to authorized users.
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