Antimicrobial effects of marine algal extracts and cyanobacterial pure compounds against five foodborne pathogens

Antimicrobial effects of marine algal extracts and cyanobacterial pure compounds against five foodborne pathogens

Food Chemistry 199 (2016) 114–118 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Short...

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Food Chemistry 199 (2016) 114–118

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Short communication

Antimicrobial effects of marine algal extracts and cyanobacterial pure compounds against five foodborne pathogens Dominic Dussault a, Khanh Dang Vu a, Tifanie Vansach b,1, F. David Horgen b, Monique Lacroix a,⇑ a b

INRS-Institut Armand-Frappier, Canadian Irradiation Center, Research Laboratories in Sciences Applied to Food, 531, Blvd des Prairies, Laval, QC H7V 1B7, Canada Department of Natural Sciences, Hawaii Pacific University, 45-045 Kamehameha Hwy, Kaneohe, HI 96744, United States

a r t i c l e

i n f o

Article history: Received 4 March 2015 Received in revised form 28 October 2015 Accepted 26 November 2015 Available online 26 November 2015 Keywords: Antimicrobial Foodborne pathogens Marine algae Cyanobacteria

a b s t r a c t The marine environment is a proven source of structurally complex and biologically active compounds. In this study, the antimicrobial effects of a small collection of marine-derived extracts and isolates, were evaluated against 5 foodborne pathogens using a broth dilution assay. Results demonstrated that algal extracts from Padina and Ulva species and cyanobacterial compounds antillatoxin B, laxaphycins A, B and B3, isomalyngamide A, and malyngamides C, I and J showed antimicrobial activity against Gram positive foodborne pathogens (Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus) at low concentrations (6500 lg/ml). None of the algal extracts or cyanobacterial isolates had antibacterial activity against Gram negative bacteria (Escherichia coli and Salmonella enterica serovar Typhimurium). Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction The consumption of contaminated food can cause serious illness. In the United States it has been estimated that 76 million people, annually, are victims of food diseases, resulting in five thousand deaths each year. Known pathogenic organisms cause 9.4 million infections per year, including viruses (59%), bacteria (39%), and parasites (3%) (Scallan et al., 2011). Thus, controlling foodborne pathogens in food products is important to public health. Aquatic microorganisms and algae produce a pool of underinvestigated secondary metabolites and are potential sources of drug-like compounds to inhibit food borne pathogens. Despite the fact that a number of cyanobacterial species produce toxins that pose a significant risk to human health, cyanobacteria and algae are both photosynthetic aquatic organisms that have a history of human consumption. In addition to serving as food staples, several species of algae and cyanobacteria are used as food supplements around the world (Gantar & Svircev, 2008), which reflects the importance of many species as sources of nutrients and anti-oxidants. In fact, many potential beneficial uses have been associated with extracts and compounds from cyanobacteria and

⇑ Corresponding author. E-mail address: [email protected] (M. Lacroix). Current address: Department of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Rd, Boca Raton, FL 33431, United States. 1

http://dx.doi.org/10.1016/j.foodchem.2015.11.119 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

algae, including antioxidant, immunomodulatory, antimicrobial, and antitumor activities (Plaza, Herrero, Cifuentes, & Ibanez, 2009). As an example of antimicrobial studies, Engel and coworkers (2006) evaluated the antimicrobial effects of extracts from 49 marine algae from the tropical Atlantic against the pathogenic bacterium Pseudoaltermonas bacteriolytica, the pathogenic fungus Lindra thalassiae, the saprophytic fungus Dendryphiella salina, and the saprophytic stramenopiles Halophytophthora spinose and Schizochytrium aggregatum. The majority of extracts (90%) were active against one or more pathogens, and 77% were active against two or more of the target microorganisms. The same research group also screened 54 species of marine algae collected from Indo-Pacific reefs against the same microorganisms (Puglisi, Engel, Jensen, & Fenical, 2007). In that study, 95% of all species yielded extracts that were active against one or more of the target microorganisms, and 77% extracts were active against two or more of them. Although these studies, and others, demonstrate the potential of marine plant extracts as antimicrobial agents, few studies have specifically investigated marine algal or cyanobacterial extracts or compounds against foodborne pathogens. The aim of this study is to evaluate antibacterial activity of isolates and crude extracts from marine algae and cyanobacteria collected in Hawaii and the Caribbean against 5 foodborne pathogens. These pathogens include Escherichia coli O157:H7 EDL 933, Staphylococcus aureus ATCC 29213, Bacillus cereus LSPQ 2872, Salmonella enterica serovar Typhimurium SL 1344 (Salmonella Typhimurium SL 1344), and Listeria monocytogenes HPB 2812.

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2. Materials and methods 2.1. General experimental ESI-MS spectra were measured and performed on a Thermo Finnigan (San Jose, CA, USA) LCQ Deca XP Max ion trap mass spectrometer equipped with an electrospray ionization (ESI) probe. The NMR experiments were performed on a General Electric GN Omega 500 spectrometer operating at 500 and 125 MHz, for 1H and 13C respectively, or a Varian Mercury Plus 300 spectrometer operating at 300/75 MHz. Preparative HPLC (20 ml/min flow rate) was performed on an Agilent 1100 binary preparative system with an Agilent multi-wavelength UV detector. Semi-preparative scale HPLC (5 ml/min flow rate) was performed using an Agilent 1100 binary preparative system with a Thermo Finnigan Surveyor Plus photodiode array detector (PDA). 2.2. Collection and extraction of algae biomass Algal samples were collected in waters off Oahu, Hawaii. Algal biomass samples were collected by hand leaving holdfasts in place. Dried algal voucher specimens were held in collections at Hawaii Pacific University (Department of Natural Sciences). The freshly collected material was frozen and freeze-dried. Dried biomass was extracted exhaustively with methanol, which was then removed under vacuum. Extracts were generally eluted from a short silica vacuum liquid chromatography (VLC) column with dichloromethane/methanol mixtures to select for moderately polar metabolites, and the fractions were dried under vacuum and stored at 20 °C and with desiccant until use. The collection site and date and the VLC elution solvent for each sample are as follows: Dictyosphaeria sp. (Chlorophyta), Pearl Harbor, adjacent to Hickam Airforce Base, August 12, 1997, dichloromethane/methanol (50:50); Dictyota sp. (Phaeophyta), Kailua Beach Park, November 3, 2001, dichloromethane/methanol (50:50); Eucheuma striatum (Rhodophyta), Kaneohe Bay, April 4, 2000, not subjected to VLC; Galaxaura rugosa (Rhodophyta), Kahala Beach Park, March 23, 2002, dichloromethane/methanol (50:50); Gracilaria salicornia (Rhodophyta), Kaneohe Bay, April 4, 2000, dichloromethane/methanol (70:30); Laurencia nidifica (Rhodophyta), Kahala Beach Park, March 23, 2002, dichloromethane/methanol (50:50); Liagora sp. (Rhodophyta), Maunalua Bay, May 9, 2001, dichloromethane/methanol (50:50); Padina sp. (Phaeophyta), Ulehawa Beach Park, February 3, 2001, dichloromethane/methanol (50:50); Ulva sp. (Chlorophyta), Ulehawa Beach Park, February 3, 2001, dichloromethane/methanol (90:10). Species were identified by the late Professor I.A. Abbott (University of Hawaii). 2.3. Isolation of cyanobacterial bioactive compounds 2.3.1. General Several compounds were isolated from the following fieldcollected cyanobacteria: (1) an unidentified cyanobacterium (HPU code 071007-KAN-07) that was collected in Kaneohe Bay, Kaneohe, HI on October 7, 2007; (2) a sample of Moorea producens (syn: Lyngbya majuscula) from True Blue Bay, Grenada, West Indies, on May 12, 2001; (3) a Moorea sp. collected from the waters of Kaneohe Bay, Kaneohe, HI on June 7, 2008. Generally, as described below, organic extracts were subjected to repeated reversed-phase or both normal and reversed-phase chromatography, and compounds were identified by comparison of their MS and 1H NMR data with literature values.

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2.3.2. Isolation of compounds from an unidentified cyanobacterium (071007-KAN-07) The fresh collected materials were frozen and freeze-dried. The freeze-dried sample (9.0 g dry weight) was extracted with dichloromethane followed by dichloromethane/methanol (50:50), and the combined extracts were dried under vacuum. The crude extract (830 mg) was subjected in several portions to reversed-phase HPLC [Luna C18(2), 10 lm, Phenomenex, 21.2  250 mm, acetonitrile/ water gradient: 40:60 to 100% acetonitrile (0–25 min), 100% acetonitrile (25–30 min); 20 ml/min]. Peaks were collected and rechromatographed [Luna C18(2), Phenomenex, 10.0 x 250 mm, acetonitrile/water gradient: 50:50 to 100% acetonitrile (0–10 min), 100% acetonitrile (10–20 min); 4.6 ml/min] to yield laxaphycins A (10.4 mg), B (4.8 mg), and B3 (4.5 mg) (Fig. 1), which were identified by comparing their MS and 1H NMR data with literature data (Bonnard et al., 2007). 2.3.3. Isolation of compounds from Moorea producens The sample was air-dried and stored at 20 °C until extraction. The dried sample (250 g dry wt) was extracted with dichloromethane/isopropanol (50:50), followed by methanol. The resulting organic extracts were combined, dried, and fractionated over a C18 medium pressure liquid chromatography (MPLC) column with methanol/water mixtures (40:60, 60:40, 80:20) and finally 100% methanol. The methanol/water (70:30) fraction (600 mg) was further fractionated in several portions by reversed-phase HPLC [Luna C18(2), Phenomenex, 21.2  250 mm, acetonitrile/water gradient: 60:40 to 100% acetonitrile (0–17 min); 20 ml/min]. Repeated HPLC of resulting fractions [Luna C18(2), Phenomenex, 10.0  250 mm, acetonitrile/water gradient: 65:35 to 100% acetonitrile (0–20 min); 4.7 ml/min] yielded malyngamide C (12.0 mg), malyngamide C acetate (11.9 mg), malyngamide J (14.6 mg), and antillatoxin B (11.5 mg) (Fig. 1), which were identified by comparison of MS and 1H NMR data with reported values (Ainslie, Barchi, Kuniyoshi, Moore, & Myndersel, 1985; Nogle, Okino, & Gerwick, 2001; Wu, Milligan, & Gerwick, 1997). 2.3.4. Isolation of compounds from Moorea sp Immediately after collection the sample was frozen and freezedried. The freeze-dried sample (7.0 g dry wt) was extracted with dichloromethane then dichloromethane/methanol (50:50). The extracts were combined and dried under vacuum. The crude extract (567 mg) was subjected in multiple portions to HPLC chromatography [Luna C18(2), Phenomenex, 21.2  250 mm, methanol/water (80:20); 20.0 ml/min]. Resulting fractions were rechromatographed [Luna C18(2), Phenomenex, 10.0  250 mm, acetonitrile/water gradient: 50:50 to 100% acetonitrile (0–10 min), 100% acetonitrile (10–20 min); 4.6 ml/min] to yield isomalyngamide A (4.7 mg), majusculamide A (11.1 mg), and malyngamide I (6.2 mg) (Fig. 1), which were identified by MS and comparison of previously reported 1H NMR data (Kan, Sakamoto, Fujita, & Nagai, 2000; Marner, Moore, Hirotsu, & Clardy, 1977; Todd & Gerwick, 1995). 2.4. Microorganisms and growth condition E. coli O157:H7 EDL 933, S. aureus ATCC 29213, B. cereus LSPQ 2872, Salmonella Typhimurium SL 1344, L. monocytogenes HPB 2812 were subcultured in tryptic soy broth (1%, v/v) (TSB, Difco Laboratories, Detroit, USA) at 37 °C for 24 h from the stock culture maintained at 80 °C in TSB containing glycerol (20%, w/v). Prior to the experiment, 1 ml of culture was incubated through one cycle of 24 h at 37 °C in TSB to obtain a working culture containing

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O

O N H H N

N O

N

O O

O

O

NH2

N

O

O

O

antillatoxin B

majusculamide A O

O

R

O

O

O

N N

O

O O

O

O N H

Cl

O Cl

isomalyngamide A R=H R = Ac

malyngamide C malyngamide C acetate O

H N

N H NH

H N O

O

O

O N O Cl

O O

O

O

O

H N

N H

HO

OH

O

O

O HN

O

NH

HN

malyngamide I OH

NH

N

O

O O

HO

HO laxaphycin A

O

O

O

O

O O O

N H

N H

HO

H N

OH

O

NH

N H malyngamide J

O

NH

O

O OH HN

NH O O

O

N

O

R

N H OH HO

HN

O

O O

N H2N

N H O

H2 N laxaphycin B laxaphycin B3

R=H R = OH Fig. 1. Chemical structure of the cyanobacterial isolates.

approximately 109 CFU/ml. The bacterial culture was centrifuged at 2000g for 15 min at 4 °C, and then washed with NaCl (0.85%, w/v) and resuspended in TSB and incubated for 24 h preceding the experiment. 2.5. Minimal inhibitory concentration (MIC) assay in micro titer plates The influence of varying concentrations of extract efficacy was assessed against pathogen microorganisms (106 cells per ml) using 96-well micro titer plates (Sarstedt Inc., Montreal, Canada) according to a modified protocol of Gutierrez and coworkers (2008). On the day of the experiment, the lyophilized extracts or isolates were

dissolved in methanol at a concentration of 1 mg/ml. The first column of the microplate was filled with 200 ll of Mueller–Hinton (M–H) broth and columns 2–12 were filled with 150 ll M–H broth. Next 58 ll of extract or isolates was added to the first column. Two fold serial dilutions were performed from columns 1–11 by transferring 125 ll of mixture from the previous column. Finally, 15 ll of a bacterial suspension (107 cells per ml) was added to each well prior to 24 h incubation at 37 °C. Column 12 served as a negative control without test substances (broth + 5% methanol + bacteria). Each row from 1 to 5 contained a different bacterial strain and row 6 served as a blank without bacteria (broth + methanol + extract or isolates). For the positive control, allylisothiocyanate

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alginolyticus, Vibrio cholerae, S. aureus, Salmonella Typhimurium, and E. coli (Vijayavel & Martinez, 2010). Concerning the safety of these extracts for food preservation, it is known that some algal species of the genera included in this study – Dictyota, Eucheuma, Gracilaria, Laurencia, Padina and Ulva – are edible in Pacific Island countries (McDermid & Stuercke, 2003; Novaczek, 2001). Uses include salad, dried flakes/powders for dressings, fritters, or animal feed. (Novaczek & Athy, 2001). The history of human consumption for the algal genera under study suggests that the extracted components are likely have no or low human toxicity. Thus, it is of interest to further evaluate the antibacterial effects of the extracts from Ulva sp. and Padina sp. and their active components in a food system, since the activity of antibacterial agents may be affected by food composition (Gutierrez, Barry-Ryan, & Bourke, 2008). For cyanobacterial isolates, isomalyngamide A was active against B. cereus (MIC = 7.8 lg/ml) while malyngamide J was moderately active against B. cereus (MIC = 63 lg/ml) (Table 1). To our knowledge, there was only one previous report (Gerwick, Reyes, & Alvarado, 1987) of antibacterial activity for the extensive series of the malyngamide-class of compounds. At the same time, both antillatoxin B and malyngamide C showed antibacterial activity against B. cereus (MICs = 130 lg/ml) and laxaphycin A inhibited S. aureus (MIC = 125 lg/ml) (Table 1). Other compounds showed only moderate or no antibacterial effects (MIC P 250 lg/ml) against the tested bacteria. Concerning the safety of these compounds for food application, there is no information on their safety levels in animals or human. However, there are some studies on other properties of these compounds which may relate to their safety. For example, isomalyngamide A has antiproliferative activity against breast cancer cells MCF7 (IC50 = 4.6 lM, 2.5 lg /ml) and MDA-MB-231 (IC50 = 2.8 lM, 1.5 lg/ml) (Chang et al., 2011)l These IC50 values are similar to the MIC value of isomalyngamide A against B. cereus (7.8 lg /ml), but a general toxic effect was not observed against other pathogens in this study as the MIC’s for isomalyngamide A ranged from 7.8 to >500 lg/ml (Table 1). Malyngamide J had brine shrimp toxicity (LC50 = 18 lg/ml) and goldfish toxicity (LC50 = 40 lg/ml) (Wu et al., 1997), which may suggest a mechanism of general toxicity. However, this was not reflected in our results as malyngamide J was active against only one pathogen (Table 1). Antillatoxin B is a neurotoxic lipopeptide, it has ichthyotoxicity (LC50 = 1.0 llM, 0.57 lg/ml) (Nogle et al., 2001). Malyngamide C

was used and subjected to the same procedure as mentioned above. On the following day, bacterial growth was detected by optical density measured at 540 nm (ELISA reader, CLX800BioTek Instruments) after the addition of 20 ll of an iodonitrotetrazolium chloride (INT; 0.5 mg/ml) alcoholic solution (Sigma–Aldrich Canada Ltd., Oakville, ON, Canada). The microplates were again incubated at 37 °C for 60 min, and in those wells, where bacterial growth occurred, INT changed from clear to red. For each measurement, triplicates were tested.

3. Results and discussion Extracts of nine algae (2 Chlorophyta, 5 Rhodophyta, and 2 Phaeophyta) and 10 cyanobacterial isolates (Fig. 1) were evaluated for their antimicrobial effects against 5 common foodborne pathogens. Three strains are Gram positives (B. cereus, S. aureus and L. monocytogenes) and 2 are Gram negative (E. coli and Salmonella Typhimurium). The MIC values of the algal extracts and cyanobacterial isolates against 5 pathogenic bacteria are presented in Table 1. The positive control allylisothiocyanate showed MIC values ranging from 39 to 156 ppm, which are comparable to a previous report (Burt, 2004). None of the extracts or isolates showed antimicrobial activity against the Gram negative bacteria. This could be explained by the hydrophobicity of the compounds extracted. The Gram negative bacterial membrane contains lipopolysaccharides that create a hydrophilic barrier that may prevent the moderate to low polarity extract components that were eluted from silica with dichloromethane/methanol mixtures from entering the cell. Non-specific antimicrobial activity of hydrophobic compounds, where compounds disrupt the cell lipid membrane and create leakage of the cytoplasm, is common (Burt, 2004; Oussalah, Caillet, & Lacroix, 2006), but apparently not observed in this study. Of the 9 algal extracts tested, extracts from Ulva sp. and Padina sp. showed the highest antibacterial activity against Gram positive bacteria (Table 1). The Padina sp. extract inhibited growth of B. cereus with an MIC of 63 lg/ml and S. aureus with an MIC of 130 lg/ml. The Ulva sp. extract inhibited both B. cereus and S. aureus, each with an MIC of 130 lg/ml, while showing a moderate anti-Listerial activity at an MIC of 250 lg/ml. The activity observed for the Ulva extract is consistent with a recent study that investigated an ethanolic extract of Ulva fasciata, which demonstrated antimicrobial activity against Enterococcus faecalis, Vibrio

Table 1 Minimal inhibitory activity (lg/ml) of marine algae extracts and cyanobacterial isolates. B. cereus

S. aureus

L. monocytogenes

E. coli

Salmonella Typhimurium

Organic algal extracts

Extracts or isolates Dictyosphaeria sp. Dictyota sp. Eucheuma striatum Galaxaura rugosa Gracilaria salicornia Laurencia nidifica Liagora sp. Padina sp. Ulva sp.

>500 >500 >500 >500 >500 500 >500 63 130

>500 >500 >500 >500 >500 >500 >500 130 130

>500 >500 >500 >500 >500 >500 >500 >500 250

>500 >500 >500 >500 >500 >500 >500 >500 >500

>500 >500 >500 >500 >500 >500 >500 >500 >500

Cyanobacterial isolates

Antillatoxin B Isomalyngamide A Laxaphycin A Laxaphycin B Laxaphycin B3 Majusculamide A Malyngamide C Malyngamide C Ac Malyngamide I Malyngamide J

130 7.8 250 250 250 >500 130 >500 250 63

250 500 125 250 500 >500 >500 >500 >500 >500

250 >500 250 250 >500 >500 >500 >500 >500 >500

>500 >500 >500 >500 >500 >500 >500 >500 >500 >500

>500 >500 >500 >500 >500 >500 >500 >500 >500 >500

Positive control

Allylisothiocyanate

39

78

39

156

39

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has cytotoxic effects against colorectal cells HT29 (IC50 of 5.2 lM, 2.4 lg/ml) (Kwan, Teplitski, Gunasekera, Paul, & Luesch, 2010). These concentrations are lower than the MIC values of antillatoxin B or malyngamide C against tested bacteria (Table 1). Laxaphycin A at a concentration of 20 lM (24 lg/ml) was not active against human leukemic lymphoblasts (Bonnard, Rolland, Francisco, & Banaigs, 1997); however, Laxaphycin B and B3 were more active against these cells, with an IC50 value of less than or equal to 1.4 lM (2.0 lg/ml) (Bonnard et al., 1997, 2007) and less than or equal to 1.5 lM (2.1 lg/ml) (Bonnard et al., 2007), respectively. Again, the concentrations of the aforementioned compounds are lower than those of their respective MIC values against tested bacteria (Table 1) and do not follow the rank potency for cancer cell toxicity. Although we cannot rule out that the effects of the active cyanobacterial compounds are due to general toxicity, it is clear that the ichthyotoxicity (fish toxicity), cancer cell cytotoxicity, and brine shrimp toxicity reported in the literature for the cyanobacterial compounds tested in this study do not mirror our results against food borne pathogens. However, as the potential of these compound is further investigated, it will become necessary to evaluate their toxicity in non-cancerous cell lines and/or in animals before considering their applications in a food system. 4. Conclusions The effects of nine algal extracts and ten cyanobacterial isolates on inhibiting the growth of foodborne pathogens were investigated. Two algal extracts (Padina sp. and Ulva sp.) and several cyanobacterial compounds (antillatoxin B, laxaphycins A, B and B3, and malyngamides A, C, I and J) were found to have antimicrobial activity against the Gram positive foodborne pathogens L. monocytogenes, B. cereus and S. aureus at low concentration (6500 lg/ml). Further research to explore the in situ potential of extracts from Ulva sp and Padina sp. in preventing pathogen growth should be conducted. Moreover, it is necessary to evaluate the toxicity of cyanobacterial isolates in non-cancerous cell lines and/or in animals. Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) – Canada, and BSA Food Ingredients s.e.c/l.p. (Montreal, Qc, Canada) under a research contract agreement with INRS. We also acknowledge support from the U.S. National Institutes of Health (NIGMS P20GM 103466) to FDH and the HPU Shared Instrumentation Facility. We thank Dr. Bryan Sakamoto for providing cyanobacterial biomass. NSERC, le Fond de Recherche sur la Nature et les Technologies (FQRNT) and BSA Food Ingredients s.e.c/l.p. supported Dominic Dussault through the Industrial Innovation Scholarships BMP Innovation.

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