Tetrodotoxins (TTXs) and Vibrio alginolyticus in Mussels from Central Adriatic Sea (Italy): Are They Closely Related?

Abstract

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg^-1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018-2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8-26 µg kg^-1) and 3 (2%) contained levels above the EFSA safety threshold (61-76 µg kg^-1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.

Keywords: Adriatic Sea; HILIC-MS/MS; NRPS gene; PKS gene; Vibrio alginolyticus; bacterial pellets; mussels; tetrodotoxins (TTXs).

Figure 1

HILIC-MS/MS chromatograms of the (a) TTX matrix-matched standard (65 ng mL⁻¹); (b) digestive gland of the field sample SN190702W (196 µg kg⁻¹). 320.1 > 302.1, multiple reaction monitoring (MRM) transition for TTX quantification. 320.1 > 162.1, MRM transition for TTX confirmation.

Figure 2

TTX concentrations and Vibrio alginolyticus in mussels collected from Pesaro and Ancona natural beds during 2019. X = not analyzed for TTXs.

Figure 3

Agarose gel electrophoresis of PCR products of (a) gyr B (560 bp), (b) NRPS (300 bp), and (c) PKS (300 bp) genes in Vibrio alginolyticus strains isolated from mussels collected from the coast of the Marche region. Lane 1–6 in (a), 1–3 in (b), and 1–2 in (c) represent field samples; lane 7 in (a) represents the positive control of V. alginolyticus ATCC 33787(CTRL⁺). Lanes 4 in (b) and 3 in (c) represent the positive controls of V. parahaemolyticus ATCC 17802 (CTRL⁺); lane 5 in (b) represents the negative control (CTRL⁻). M = molecular weight marker.

Figure 4

Mytilus galloprovincialis sampling sites along the Marche coast. Nine breeding sites: PS (PS), FN (Fano), SG (Senigallia), AN1 (Ancona 1), AN2 (Ancona 2), MC1 (Macerata 1), and MC2 (Macerata 2), SB1 (San Benedetto del Tronto 1), SB2 (San Benedetto del Tronto 2). Seven natural beds: Mississipi, Vallugola, Sottolacroce in the Pesaro area, An nord, An sud, Sir nord, and Sir sud in the Ancona area.

Figure 5

Molecular structure of TTX analogues monitored via the HILIC-MS/MS method.