Effect of multiple antibiotic treatments on a paralytic shellfish toxin-producing culture of the dinoflagellate Alexandrium minutum

Abstract

Alexandrium minutum is one of several dinoflagellate species capable of producing paralytic shellfish toxins. Previous work suggests that toxin levels are influenced by a number of parameters, including dinoflagellate-associated bacteria. In the present study, a toxin-producing culture of A. minutum isolated from Anakoha Bay in the Marlborough Sounds of New Zealand was subjected to an antibiotic treatment regimen designed to eliminate the associated bacteria. Antibiotics used included penicillin, streptomycin, kanamycin and tetracycline (Treatment 1); ciprofloxacin and gentamicin (Treatment 2); and penicillin, streptomycin and ciprofloxacin (Treatment 3). Enzyme immunoassay showed that saxitoxin levels in the A. minutum culture fell significantly following the first round of antibiotic treatment, and this coincided with a large reduction in the associated copiotrophic bacterial population. HPLC data indicated that there was also a reduction in gonyautoxins (GTX 1-3 ). The oligotrophic population was more difficult to eliminate and required 2 additional rounds of antibiotic treatment, but saxitoxin levels did not change any further. Scanning laser confocal microscopy following acridine orange staining was used to observe intracellular bacteria-like particles, which were considerably reduced by the end of the treatments, probably due to the inclusion of antibiotics that penetrate eukaryotic cells. Algal mean generation times were not significantly affected by the antibiotic treatments. Qualitative and quantitative changes in toxin production coincided with a reduction in the culturable, copiotrophic and/or intracellular bacteria in the A. minutum Anakoha A culture. The premise that bacteria can exert a strong influence on algal toxicity was supported by this study, although the mechanisms remain unknown.

Is photosynthesis a requirement for paralytic shellfish toxin production in the dinoflagellate Alexandrium minutum algal–bacterial consortium?

“…The bacteria may alternate between planktonic (free-living) and sessile forms attached to the dinoflagellate cells (Doucette 1995;Hold et al 2001). Intracellular bacteria have also been observed in toxic dinoflagellates, including A. minutum Anakoha A (Lu et al 2000;Maas et al 2007). Thus, any investigation into the toxicity of a dinoflagellate culture should take into account the accompanying bacteria.…”

“…Prior to the experiment, A. minutum was tested for photosynthetic cyanobacteria by light microscopy and culture on ASN III agar (Gerhardt et al 1981). On days 0 and 22 of the incubation period, total STX, Neo-STX, gonyautoxins (GTX-1-4) levels were measured by HPLC (Maas et al 2007). The day 22 samples were chosen for analysis as there was a marked loss of algal viability after this time.…”

“…One set of bottles was wrapped in four layers of aluminum foil to exclude light. All cultures were incubated in a Shearer-controlled Environment Chamber (Model CEL-37-14) for 32 days under the following conditions; 20°C±2°C, a 12/12 h dark-light cycle and illumination of 80-100 µmol m −2 s −1 (Maas et al 2007).…”

“…The viability of the algae in each sample was determined by assessing the appearance and motility of the cells under the light microscope. Samples (1 mL) for bacterial culture were serially diluted 10-fold in sterile seawater and spread-plated in duplicate on Marine Agar ZoBell 2216 (MA; Difco, BD, North Ryde, NSW, Australia), Seawater complete agar (SWC) and GP medium B modified with 0.01% yeast extract (GPLY; Maas et al 2007). MA was included to obtain an estimate of the copiotrophic bacteria, which require relatively high concentrations of nutrients, whereas the other media contained low concentrations of nutrients to support growth of oligotrophic bacteria.…”

“…Researchers have found that the presence or absence of bacteria in an algal culture can profoundly influence toxin levels and it seems that many species of marine bacteria are capable of either autonomously producing or actively metabolizing PST (Gallacher and Smith 1999;Smith et al 2002). Previous work on A. minutum isolated from the Anokoha Bay bloom event indicates there is a link between the presence of high nutrient requiring copiotrophic and/or intracellular bacteria and PST production (Maas et al 2007). Most researchers have sought to investigate the relationship between PSTproducing algae and their associated bacteria by eliminating the latter, mainly through the addition of antibacterial compounds (Ho et al 2006;Hold et al 2001;Lu et al 2000;Uribe and Espejo 2003).…”