Bacterial diversity and tetrodotoxin analysis in the viscera of the gastropods from Portuguese coast

• Bacterial diversity study in the viscera of the gastropods.
• UPLC/MS/MS analysis of Tetrodotoxin.
• PCR screening of biosynthesis genes (PKS, NRPS and AMT) for TTX production.
• Phylogenetic analysis of 16S rRNA.
• Five classes of bacteria were isolated.

To trace the pathway of tetrodotoxin (TTX) producing microorganism in the Atlantic coast of Portugal, culture-dependent evaluation of the bacterial isolates from the viscera of the gastropods Monodonta lineata, Gibbula umbilicalis, Nucella lapillus and Patella intermedia, and from the environmental samples (biofilm and surrounding sea water) was carried out. Samples were collected from eight different coastal locations of Northern Portugal. A total of 311 isolates were identified. The observed bacterial diversity was distributed over five different classes (Gammaproteobacteria, Alphaproteobacteria, Flavobacteria, Bacilli and Actinobacteria) with the greatest number of 16S rRNA gene sequence derived from the Gammaproteobacteria (75%). Phylogenetic analysis based on the 16S rRNA gene showed that bacterial isolates were highly diverse and most of which were found in other marine environment. Among the different species isolated, Vibrio was found abundant. Eventhough TTX was not detected (UPLC-MS/MS) in the isolates from this study, PCR screening identified some natural product biosynthesis genes (PKS and NRPS) involved in its assembly. Further PCR screening of the TTX producing two ATCC Vibrio sp. reveals that NRPS might be involved in the biosynthesis of TTX through the incorporation of arginine.