Reevaluation of Production of Paralytic Shellfish Toxin by Bacteria Associated with Dinoflagellates of the Portuguese Coast

Abstract

Paralytic shellfish toxins (PSTs) comprise a suite of potent neurotoxins that act by blocking sodium channels in nerve axons (3). These toxins may cause severe human poisoning upon consumption of contaminated shellfish. The production of PSTs by dinoflagellates has been thoroughly documented and confirmed worldwide. Strains of the dinoflagellates Alexandrium lusitanicum Balech and Gymnodinium catenatum Graham isolated from Portuguese waters have been shown by different detection methods to produce PSTs (1,6,7,16).The potential involvement of dinoflagellate-associated bacteria in PST production, specifically the autonomous synthesis of these toxins, has been addressed using bacterial strains isolated from laboratory dinoflagellate cultures (5, 10, 15) in conjunction with high-performance liquid chromatography (HPLC) (5, 15) and in vitro assay-based toxin detection (10). Two bacterial strains isolated previously by our group from A. lusitanicum and G. catenatum and identified as Pseudomonas stutzeri and Pseudomonas diminuta, respectively, were reported to produce PSTs (5, 7). Nevertheless, the autonomous production of PSTs by bacteria remains a controversial subject, and there have been several recent reports of the incorrect identification of bacterial metabolites as PSTs by HPLC analysis (2,17,18,20,21).In the context of such findings and the continuing uncertainty regarding bacterial involvement in PST toxigenesis, the present investigation was undertaken to reevaluate our previous results on the toxin content and profile in P. stutzeri and P. diminuta. Both biological in vitro (mouse neuroblastoma [MNB] assay) and chemical (HPLC) methods were employed to account for sodium channel-blocking (SCB) activity and/or compounds with the same chromatographic behavior as PSTs produced by these two bacterial isolates. Additionally, since nutritional status has been reported to influence bacterial toxicity (4), analyses for intra-and extracellular toxins under both nutrient-replete and phosphorus-limited growth conditions were conducted.For this study, P. stutzeri and P. diminuta (5, 7) were individually inoculated into 500-ml volumes of two growth media: phosphorus-limiting artificial seawater experimental (ASWE) medium (11) containing 0.5 M Na 2 HPO 4 as well as 37.5 mM sodium succinate as the sole carbon and energy source, and a complex organic seawater complete medium (SWC) (4, 11) rich in phosphate (5 g of Bacto Tryptone, 3 g of yeast extract, and 6 ml of 50% glycerol per liter of seawater). Cultures were incubated at 19 to 21°C (150 oscillations min Ϫ1 ) under 22-E m Ϫ2 s Ϫ1 irradiance until late log phase. At that time, a cell count (in CFU per milliliter) was obtained and cells were separated from growth medium by centrifugation (10,000 ϫ g, 20 min, 8°C).For toxin extraction, cell pellets were lyophilized after centrifugation and extracted with 1 ml of 0.5 N acetic acid per 100 mg of lyophilized weight. Bacterial cells were mechanically disrupted, and the suspension was centrifuged (2,000 ϫ g, 10 min, room temper...

Diversity and distribution of epibiotic bacteria on Pseudo-nitzschia multiseries (Bacillariophyceae) in culture, and comparison with those on diatoms in native seawater

“…Reports indicate that bacteria may produce ''phycotoxins'' autonomously (Simidu et al, 1990;Córdova et al, 2003) or be major contributors to toxin production in association with other organisms (Stewart et al, 1997;Hold et al, 2001a). However, conclusive evidence has been elusive (Baker et al, 2003), and Martins et al (2003) have reevaluated previous published reports of paralytic shellfish toxin production by bacteria associated with dinoflagellates.…”

Evolution and Distribution of Saxitoxin Biosynthesis in Dinoflagellates

“…This hypothesis has been investigated by numerous studies, though the results were conflicting [30,31,32,33,34]. Additionally, some of the methods used to measure bacterial metabolites have subsequently been shown to lack specificity, with compounds thought to be STX later being identified as chemical imposters [32,34]. …”

Bacterial diversity of Gymnodinium catenatum and its relationship to dinoflagellate toxicity

“…There is no doubt that many cyanobacteria can autonomously produce PST compounds, and a plausible biosynthetic pathway has been recently described (Kellmann et al 2008), but the evidence for PST production by other heterotrophic bacterial groups is weaker. While a number of bacteria have been reported to produce toxins having chromatographic and biological activity similar to that of PST compounds (Kodama et al 1988, Gallacher et al 1997, Smith et al 2002, several studies have demonstrated that this activity can be attributed to compounds structurally unrelated to PSTs (Sato & Shimizu 1998, Baker et al 2003, Martins et al 2003. Alternatively, bacteria may be directly involved in dinoflagellate PST biosynthesis by supplying precursor compounds or synthesizing key intermediates in the biosynthetic pathway (Gallacher & Smith 1999).…”