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

ResumenEl aumento de la resistencia bacteriana a los antibióticos ha causado preocupación a nivel mundial, por lo que se ha promovido la búsqueda de nuevos compuestos. Debido a su abundancia y diversidad, el fitoplancton marino constituye una importante fuente potencial de tales compuestos. La investigación sobre dinoflagelados ha llevado al descubrimiento de inhibidores de crecimiento bacteriano. El dinoflagelado marino Lingulodinium polyedrum causa proliferaciones algales en diferentes regiones del mundo, incluyendo México, y también se sabe que regula el crecimiento de otras especies en las aguas costeras. En este trabajo, se investiga la taxonomía de este dinoflagelado y se caracteriza la capacidad de sus extractos para inhibir el crecimiento de dos bacterias de importancia médica (Vibrio vulnificus y Staphylococcus aureus) en placas de cultivo de agar. La caracterización taxonómica se realizó por PCR y amplificación del gen de ITS, y se confirmó que la especie aislada en la costa del Pacífico de México fue L. polyedrum. Para demostrar el efecto inhibidor de los extractos de L. polyedrum, los cultivos se cosecharon por centrifugación. Los pellets de tres abundancias celulares se extrajeron con agua, metanol, hexano y cloroformo. Los experimentos en V. vulnificus mostraron una inhibición alta del crecimiento para los cuatro extractos, variando entre 77 y 98 %. Sorprendentemente, la inhibición del crecimiento fue menor cuando los extractos se originaron a partir de una mayor abundancia de células L. polyedrum, varía de 0 a 34 %. Para S. aureus, la inhibición del crecimiento también fue alta, pero no estadísticamente diferente para todos los extractos y abundancias de células, con un rango de 62 hasta 99 %. Esto resultados son prometedores para futuras aplicaciones farmacológicas. La cepa mexicana de L. polyedrum no produjo yesotoxinas detectables.

AbstractThe 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.

Development of an ICR mouse bioassay for toxicity evaluation in neurotoxic poisoning toxins-contaminated shellfish / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop an ICR (female) mouse bioassay (MBA) for toxicity confirmation and evaluation of neurotoxins (brevetoxins)-contaminated shellfish.</p><p><b>METHODS</b>Brevetoxins (BTX-B) as a causative agent of neurotoxic shellfish poisoning (NSP) under different shellfish matrices were intraperitoneally injected at different doses into mice to study their toxic effects and to differentiate the range of lethal and sublethal dosages. Their sensitivity and specificity were analyzed with 2 competitive ELISA kits for quantitative determination of standard BTX-B and dihydroBTX-B under different shellfish matrix-diluent combinations. Detection rates of MBA and two antibody-based assays for BTX-B from field NSP-positive shellfish samples were compared.</p><p><b>RESULTS</b>BTX-B could be detected in shellfish tissues at concentration of 50-400 μg/100 g under shellfish matrix-Tween-saline media, which were appropriate to identify toxic shellfish at or above the regulatory limit (80 μg/100 g shellfish tissues). The LD50 identified was 455 mg/kg for BTX-B under general shellfish matrices (excluding oyster matrices) dissolved in Tween-saline. The presence of shellfish matrices, of oyster matrices in particular, retarded the occurrence of death and toxicity presentation in mice. Two antibody-based assays, even in the presence of different shellfish matrix-diluent combinations, showed acceptable results in quantifying BTX-B and dihydroBTX-B well below the regulatory limit.</p><p><b>CONCLUSION</b>The two ELISA analyses agree favorably (correlation coefficient, r³⋝0.96; Student's t-tests, P>0.05) with the developed bioassay.</p>

Environmental and health effects associated with Harmful Algal Bloom and marine algal toxins in China / 生物医学与环境科学（英文）

The frequency and scale of Harmful Algal Bloom (HAB) and marine algal toxin incidents have been increasing and spreading in the past two decades, causing damages to the marine environment and threatening human life through contaminated seafood. To better understand the effect of HAB and marine algal toxins on marine environment and human health in China, this paper overviews HAB occurrence and marine algal toxin incidents, as well as their environmental and health effects in this country. HAB has been increasing rapidly along the Chinese coast since the 1970s, and at least 512 documented HAB events have occurred from 1952 to 2002 in the Chinese mainland. It has been found that PSP and DSP toxins are distributed widely along both the northern and southern Chinese coasts. The HAB and marine algal toxin events during the 1990s in China were summarized, showing that the HAB and algal toxins resulted in great damages to local fisheries, marine culture, quality of marine environment, and human health. Therefore, to protect the coastal environment and human health, attention to HAB and marine algal toxins is urgently needed from the environmental and epidemiological view.

The cytotoxicity of maitotoxin to LLC-PK(1) and its antagonism to calcium channel blocking agents / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the cytotoxicity of maitotoxin (MTX) and its protective effects on calcium-channel blocking agents, so as to provide the data for control and treatment of MTX poisoning.</p><p><b>METHODS</b>Cytotoxicity was measured by MTT detecting system, and cytoplasmic free [Ca(2+)]i was measured by F-4500 fluorometry.</p><p><b>RESULTS</b>Incubation with 8 ng/ml MTX for 3 h reduced the survival ratio of LLC-PK(1) cells. The response was found in a time- and concentration-dependent manner, with significant differences as compared with the control group. The MTX-induced increase in [Ca(2+)]i was inhibited by Verapamil and Nifedipine at 5 x 10(-5) mol/L and 1 x 10(-4) mol/L respectively. Both of them significantly reduced the death of the LLC-PK(1) cells.</p><p><b>CONCLUSIONS</b>Cytotoxicity of MTX may be caused by the elevated intracellular [Ca(2+)]i. Calcium-channel blocking agents could protect LLC-PK(1) cells from injury by MTX.</p>