Studies on the toxification of puffer fish - I. Production to tetrodotoxin by the intestinal bacteria of a puffer fish Takifugu niphobles.

Abstract

We surveyed tetrodotoxin (TTX) production on 15 bactria strains isolated from the intestine of a puffer fish caught at Hakatajima, Ehime Prefecture, where the puffer fish is highly toxic. The mouse bioassay revealed that all culture broths were none toxic or very weakly toxic with the exception of one strain (code-named SA-4). The SA-4 strain was confirmed for its toxicity by the mouse bioassay, and mice died after showing the characteristic symptoms for TTX intoxication. The amount of toxin was 15 MU in 250ml of culture broth. After bioassays, all the remained solutions were purified through a Toyopearl HW-40 column, an Amberlite CG-50 column (NH+ form), and a charcoal column. Chromatography of purified toxins demonstrated that 2 strains in addition to SA-4 strain contained TTX and its derivatives in their broths. The cells of SA-4 strain were proved to be lethal to mice, and the existence of TTX was confirmed. The purified toxins and authentic TTX were hydrolyzed to 2-amino-6-hydroxyl-methyl-8-hydroxyquinazoline (C9-base), and converted into its TMS-C9-bases to be analyzed by a GC-MS analyzer. Mass fragment ions m/z 407 and 392, which were specific for TMS-C9-base, appeared in the same retention time. The results from detail examinations done on SA-4 strain led us to the conclusion that SA-4 strain was Schewanella putrefaciens.