The inhibitory effect of a non-yessotoxin-producing dinoflagellate , Lingulodinium polyedrum ( Stein ) Dodge , towards Vibrio vulnificus and Staphylococcus aureus

The increased bacterial resistance to antibiotics has caused global concern, prompting the search for new compounds. Because of their abundance and diversity, marine phytoplankton are an important potential source of such compounds. Research on dinoflagellates has led to the discovery of inhibitors of bacterial growth. The marine dinoflagellate Lingulodinium polyedrum blooms in different regions of the world, including Mexico, and is also known to regulate the growth of other species in coastal waters. Here, we investigated the taxonomy of this dinoflagellate and characterized the ability of its extracts to inhibit the growth of two bacteria of medical importance (Vibrio vulnificus and Staphylococcus aureus). Taxonomic characterization was performed by PCR and gene amplification of ITS, and confirmed that the species isolated off the Pacific coast of Mexico was L. polyedrum. To prove the inhibitory effect of L. polyedrum extracts, cultures were harvested by centrifugation. Pellets from three cellular abundances were extracted with water, methanol, hexane and chloroform. The experiments on V. vulnificus showed a high growth inhibition for the four extracts, ranging from 77 to 98 %. Surprisingly, the growth inhibition was lower when the extracts originated from a higher L. polyedrum cell abundance, ranging from 0 to 34 %. For S. aureus, the growth inhibition was also high, but not statistically different for all extracts and cell abundances, ranging from 62 to 99 %. This study obtained promising results for future pharmacological applications. Our Mexican strain of L. polyedrum did not produce any detectable yessotoxins. Rev. Biol. Trop. 64 (2): 805-816. Epub 2016 June 01. Palabras clave: proliferaciones algales, antibiótico, resistencia bacteriana, Lingulodinium polyedrum, fitoplancton, Staphylococcus aureus, Vibrio vulnificus, yessotoxina. Since the discovery of antibiotics in the early 1940s, their clinical use has resulted in greatly improved health care. Human deaths from bacterial infections have been reduced and life expectancy has increased. After the emergence of antibiotic resistance in the 1970s, research has been focused principally on modifications to semisynthetic compounds that were already clinically proven (Stach, 2010). Currently, the resistance of pathogenic bacteria to multiple drugs is a global problem, causing increased concern in health care institutions (Klevens et al., 2007; Fischbach & Walsh, 2009). One possible solution is to discover and introduce new antibacterial medications. Marine eukaryotic microalgae are a potential

The inhibitory effect of a non-yessotoxin-producing dinoflagellate, Lingulodinium polyedrum (Stein) Dodge, towards Vibrio vulnificus and Staphylococcus aureus Sonia Quijano-Scheggia 1 , Maribel Barajas-Gonzalez 1 , Hong Chang Lim 2 , Chui Pin Leaw 2 , Aramis Olivos-Ortiz 1 , Juan Gaviño-Rodriguez 1 , Juan Blanco Pérez 3 & Stephen S. Bates 4 Since the discovery of antibiotics in the early 1940s, their clinical use has resulted in greatly improved health care.Human deaths from bacterial infections have been reduced and life expectancy has increased.After the emergence of antibiotic resistance in the 1970s, research has been focused principally on modifications to semisynthetic compounds that were already clinically proven (Stach, 2010).Currently, the resistance of pathogenic bacteria to multiple drugs is a global problem, causing increased concern in health care institutions (Klevens et al., 2007;Fischbach & Walsh, 2009).One possible solution is to discover and introduce new antibacterial medications.
Marine eukaryotic microalgae are a potential source of these new compounds; they offer a high genetic diversity and constitute an untapped resource of novel natural products.Their ability to withstand environmental stress and outcompete other marine organisms is related to their capacity to produce a vast array of secondary metabolites, which have considerable value in the biotechnology, aquaculture, health and food industries (Anderson, 1996).Likewise, several studies have reported antibacterial activity in the cell lysates or extracts of various microalgal species (Desbois et al., 2009;Blunt et al., 2011;de Jesus Raposo et al., 2013).
Dinoflagellates are a large group of flagellate protists, belonging mostly to marine phytoplankton, some of which are known to form harmful algal blooms that are an important source of marine biotoxins (Tomas, 1997;Gallardo-Rodríguez et al., 2012).Bioactive compounds from dinoflagellates have received increased attention because of their impact on the safety of seafood and their potential uses in biomedical, toxicological and pharmacological research.Despite the many interesting bioactive compounds isolated from dinoflagellates (Konishi et al., 2004;Camacho et al., 2007;Blunt et al., 2011), only a few have led to commercial products.This is partly due to the limited amount of dinoflagellate toxin that can be amassed for detailed clinical evaluation (Gallardo-Rodríguez et al., 2012).Nevertheless, recent patents and patent applications related to dinoflagellate toxins have been published (Selander & Pavia, 2008;Paul, 2011).One of the few examples of medical uses is the dinoflagellate toxin, gonyautoxin (Garrido et al., 2005).Lingulodinium polyedrum (Stein) Dodge is a dinoflagellate that blooms in coastal waters of Colima, Mexico (Quijano-Scheggia et al., 2013).It is easy to isolate and maintain in culture.Here, we probe its potential as source of antibacterial compounds against two bacteria of medical importance: Vibrio vulnificus and Staphylococcus aureus.Vibrio vulnificus is a Gram-negative, oxidase-positive, facultative anaerobe.In warm coastal waters, it causes vomiting, diarrhea and abdominal pain, when raw or undercooked shellfish, especially oysters, are consumed (Strom & Paranjpye, 2000;Tortora Funke & Case, 2007;Horseman & Surani, 2011).It has become resistant to various antibiotics (Kim et al., 2011;Shaw et al., 2014).Staphylococcus aureus is a Grampositive facultative anaerobe, which is widely distributed and capable of producing a broad range of diseases, from skin infections to life-threatening illnesses.It, also, has become resistant to several antibiotics (Lowy, 2003), resulting, for example, in Methicillin-resistant Staphylococcus aureus (MRSA) infection (Appelbaum, 2007).
The present study was carried out to discover if cell extracts of L. polyedrum may contain antibacterial compound(s) that may inhibit the growth of the pathogenic bacteria V. vulnificus and S. aureus.

Sampling and culturing:
Phytoplankton samples were collected with a plastic bottle during a bloom of L. polyedrum in May 2012, off the coast of Manzanillo, Colima, Mexico (19°7'3.00"N -104°22'23.52"W), and maintained at 4 °C.Single cells were isolated under a Motic AE31 inverted microscope (Ted Pella, Inc., Redding, California, USA) with a glass Pasteur pipette.They were then transferred to tissue culture plates (Assay Plate 96 Well Flat Bottom, Corning, NY, USA) containing 0.4 mL of L1 medium (Guillard, 1975) at a salinity of 30, modified by addition of 10 -8 M H 2 SeO 3 and by reducing the concentration of CuSO 4 to 10 -8 M (Band-Schmidt et al., 2005).After reaching a density of 10 4 cells/L, the non-axenic cultures were transferred successively to 50, 250 and 1 000-mL flasks.All cultures were maintained at 21 °C under cool-white fluorescent tubes (Phillips F96T12/TL865/EW, 60 W, USA) with an irradiance of 90 μmol photons/m 2 .sand a 12:12 h light:dark photoperiod.Cell abundance was determined at 200-400 x magnification using a Motic AE31 inverted bright-field light microscope, according to Utermöhl (1931) and Throndsen (1995).
Crude extracts: Exponential-phase L. polyedrum cells were harvested by centrifugation for 10 min each at 2 500 × g and then at 20 000 × g, at 4 °C, with the same centrifuge as above.Three cell abundances of L. polyedrum were used for each test of antibacterial activity (see below).The pellets were lyophilized using a FreeZone 6 lyophilizer (Labconco, Missouri, USA).Four separate extractions were performed on the pellets by adding 1.5 mL of water, methanol, hexane or chloroform for 10 min.Silicon pearls were added to each solvent and then cells were disrupted using a Mini-BeadBeater (Biospec, Oklahoma, USA) for 10 s.Each supernatant was then placed in an Eppendorf tube and allowed to evaporate at room temperature.The extracts were stored in the evaporated state at 4 °C and then resuspended in 500 µL of sterile distilled water just prior to use.

Experiments to test for antibacterial activity:
To determine if there was antibacterial activity in the compounds extracted from L. polyedrum, two bacteria were used: Vibrio vulnificus (ATCC 27562, Gram-negative) and Staphylococcus aureus (ATCC 29213, Gram-positive).The former was incubated in tryptone soy broth and the latter in Brain Heart Infusion (BHI) broth, each for 24 h at 35-37 °C.The bacterial abundance in the broth was standardized to 0.05 absorbance units (at 400 nm) with a Jenway 6 500 spectrophotometer (Staffordshire, UK).One µL of each broth was then diluted into 999 µL of Phosphate Buffered Saline (PBS) and labeled as standardized bacterial dilution (SBD).
Inhibition experiments were carried out in triplicate by adding 10 µL of SBD to 10, 25 and 50 µL of each extract and completing to 100 µL with PBS.As a control, 10 µL of SBD was added to 90 µL of distilled water.Each tube was incubated for 2 h at 35-36 °C, after which three drops of each solution were placed onto single Petri plates made with tryptone soy agar for V. vulnificus and BHI agar for S. aureus.As a control, three drops of SBD solution were placed onto another Petri plate.The Petri plates were incubated for 24 h at 35-37 °C.Cell colonies were counted in the control plates and compared to those numbers in each cell extract and cell abundance treatment, and the percent inhibition for each treatment was then calculated.The control ranged from 15 to 30 Colony Forming Units (CFU).The Student's t-test was used to determine whether the results were statistically significant.
Toxin extraction and analysis: The L. polyedrum extracts were examined for the possible presence of yessotoxins (YTXs) and some analogs and derivatives (Table 2), as this dinoflagellate is a known producer of YTXs (Paz et al., 2008).YTXs were extracted from 50 mL of late-exponential phase L. polyedrum culture, centrifuged for 10 min each at 2 500 × g and then at 20 000 × g, as above.The pellets were extracted with 10 mL of methanol and then frozen at -20 °C prior to being sent to the Centro de Investigación Médica Aplicada (CIMA; Galicia, Spain) for further processing, as follows.The extract was concentrated in a Thermo SpeedVac rotary evaporator and filtered through a Macherey-Nagel 0.22-µm PES syringe filter.A 10-µL aliquot was then injected into a LC-MS/MS system composed of a Thermo TSQ Quantum Access Max coupled to an Accela UHPLC system through an HESI-II electrospray interface.The method of Regueiro Martín-Morales, Álvarez & Blanco (2011) was used, but modified by the use of a 50-mm Gemini NX C18 3 µm, 2.1 × 50 mm column (Phenomenex), and a different elution gradient, as detailed below.The chromatographic phases were 6.7 mM ammonium hydroxide (phase A) and acetonitrile in 6.7 mM ammonium hydroxide (9:1, v:v) (phase B).For the on-line solid-phase extraction (SPE), a Security Guard with a Gemini NX C18 4 × 2 mm cartridge (Phenomenex), and a mixture of phases A and B (90:10, v/v) as loading phase, were used.
The chromatographic conditions started at 90 % phase A, held until min 1.50, after which the flow through the SPE column was diverted to the chromatographic column.The percentage of this phase was then reduced to 20 %, 15 % and 5 % at min 3.85, 4.00 and 4.75, respectively, before being held at 5 % for two additional min.Finally, the conditions were reverted to the initial proportion in order to equilibrate the column for the next injection.The mass spectrometer conditions were as follows: ionization mode: negative spray; voltage: 3 000 V; sheet gas: 40 (nominal); auxiliary gas 10 (nominal); vaporizer temp: 105 ºC; capillary temp: 360 ºC; collision cell gas pressure: 1.5 Torr; collision energy: 30.The transitions used to identify YTXs are given in Table 2.The limit of detection was 0.13 ng/mL of extract.

Phylogenetic inference:
The phylogenetic analysis using ITS nucleotide sequences yielded similar tree topologies by NJ, ML and BI.Aligned sequences produced a total of 593 characters (including gaps), of which 489 characters were constant, and 218 variable characters were parsimony informative.Our strain of L. polyedrum from Manzanillo clustered with other strains from different regions of the world in a moderate to strongly supported clade, giving bootstraps values of 99 %, 64 % and 96 %, for NJ, ML and BI, respectively (Fig. 2).
In general, the ITS2 transcript of L. polyedrum showed four universal helices.The secondary structure of ITS2 of L. polyedrum from Manzanillo showed some genetic divergence with strains from America, Europe and Asia, giving eight Single Nucleotide Polymorphisms (SNPs), four Hemi-Compensatory Base Change (HCBCs) and one deletion (Fig. 3).
There were significant differences in the growth inhibition of V. vulnificus between the different extracts in each group with the 2 × 10 6 and 1 × 10 7 cell abundances (Table 3).With the 2 × 10 6 and 5 × 10 6 cell abundances, the difference was significant only for the aqueous  extract.For the cells abundances of 1 × 10 7 and 5 × 10 6 , there were significant differences for most combinations of the different extracts, with the exceptions of the following combinations: water-methanol, hexane-chloroform, and methanol-hexane-chloroform.Results for the experiments with S. aureus did not show any significant differences in growth inhibition, for any of the four extracts and three cell concentrations.

Toxin analysis:
No YTXs, nor analogs or derivatives, were detected in the L. polyedrum isolates from Manzanillo.

DISCUSSION
Marine microorganisms remain an abundant source of novel and biologically active metabolites, with 273 new compounds reported up to 2009 (Blunt et al., 2011).Thus, dinoflagellates can also provide numerous and potentially useful bioactive products, although relatively few have been investigated because of a limited supply from nature (Gallardo-Rodríguez et al., 2012).
We confirmed that this non-axenic L. polyedrum strain inhibits the growth of V. vulnificus and S. aureus.The inhibition of V. vulnificus growth by extracts in the four solvents was high, with no statistical differences between the L. polyedrum cell abundances of 2 × 10 6 and 5 × 10 6 , except for the aqueous extract.In contrast, the inhibition was lower when higher cell abundance (1 × 10 7 ) was used, and no inhibition was observed for the aqueous extract.We hypothesize that the bioactive compounds trigger the growth inhibition at low concentrations because the compounds can penetrate the cell wall.At higher concentrations, however, they may saturate the receptors on the cell wall, thus not penetrating the cell and allowing bacterial growth.The production of inhibitory compounds may also be higher at low cell abundances.For example, Pérez, Band, Ortíz & Sobrino.(2014) found that less toxic compounds were generated at higher cell concentrations of Chattonella spp.(Raphidophyceae).
Our results showed that the growth of S. aureus is also inhibited by extracts from the four solvents and three cell abundances of L. polyedrum, with no statistically significant differences found among these treatments.Likewise, methanolic extracts from the diatom Chaetoceros muelleri showed effective inhibition of S. aureus growth (del Pilar Sánchez, Licea & Bernáldez 2010).The antibacterial activity from this diatom species has been associated with several fatty acids, which induced lysis in bacterial protoplasts by disrupting the cell membrane.These compounds can penetrate the meshwork of peptidoglycan polymers in the cell wall and reach the bacterial membrane, which leads to the disintegration of Enterobacteriaceae such as E. coli (Shanmugapriya & Ramanathan, 2011).
These results showed that L. polyedrum contains useful secondary metabolites against V. vulnificus and S. aureus, and that it could be a source of biologically active compounds with potential application in the pharmaceutical industry.Although many compounds isolated from dinoflagellates have shown bioactivities of interest (Camacho et al., 2007;Blunt et al., 2011), only a few have led to commercial products (Gallardo-Rodríguez et al., 2012).
The morphological and molecular characterization of the isolate from Manzanillo, on the Mexican west coast, confirmed it as L. polyedrum.Cell extracts from this dinoflagellate showed a high percentage of growth inhibition against both a Gram-negative (V.vulnificus) and a Gram-positive (S. aureus) pathogenic bacterium.This is the first phase in the quest to obtain antimicrobial agents with possible pharmaceutical uses from L. polyedrum.It is now necessary to identify and evaluate the antibacterial activity of individual compounds, a step that is underway.

ACKNOWLEDGMENTS
We thank the University of Colima and Terminal KMS de GNL, S. de R.L. de C.V. for the funding provided for this research, Maria Rivera-Vilarelle for her onsite effort and invaluable lab work, and the Centro de Investigación en Alimentación y Desarrollo (Mazatlán, Sinaloa, Mexico) for providing the Vibrio vulnificus strain.

Fig. 2 .
Fig. 2. Neighbor-joining (NJ) phylogenetic tree of Lingulodinium polyedrum based on ITS1, 5.8S, and ITS2 sequences.Bootstrap values from NJ, maximum likelihood (ML) and Bayesian are shown.Only bootstrap values > 50 % are indicated.Sequence in box indicates strain obtained for the present study (from Manzanillo, Mexico); sequences of the other strains were obtained from GenBank.

Fig. 4 .
Fig. 4. Percent growth inhibition of Vibrio vulnificus with four extracts and three cell abundances of Lingulodinium polyedrum.

Fig. 5 .
Fig. 5. Percent growth inhibition of Staphylococcus aureus with four extracts and three cell abundances of Lingulodinium polyedrum.

TABLE 3
Student's t-test analysis of the growth inhibition of Vibrio vulnificus, with three cell abundances and four extraction solvents