Vol. 53, No.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1987, p. 1714-1715

0099-2240/87/071714-02$02.00/0
Copyright 1987, American Society for Microbiology

Marine Bacteria Which Produce Tetrodotoxin

USIO SIMIDU,1* TAMAO NOGUCHI,2 DENG-FWU HWANG,2 YASUO SHIDA,3 AND KANEHISA HASHIMOTO2
Ocean Research Institute, University of Tokyo, Minamidai, Nakano-ku, Tokyo 164,1 Laboratory of Marine Biochemistry,
Faculty of Agriculture, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113,2 and Tokyo College of Pharmacy, Horinouchi,

Hachioji, Tokyo 192-03,3 Japan

Received 2 February 1987/Accepted 20 April 1987

A number of type strains of marine bacteria, including members of the family Vibrionaceae, were cultured
and examined for tetrodotoxin productivity by high-performance liquid chromatography and gas chromatography-mass spectrometry. Most of the Vibrionaceae strains produced tetrodotoxin, anhydrotetrodotoxin, or
both.

were incubated in a seawater medium (ORI medium [8]

without thiosulfate and sulfite) which contained 0.2%
proteose peptone no. 3 (Difco), 0.2% yeast extract (Difco),
and 0.1% Phytone (BBL Microbiology Systems). The pH of
the medium was 7.8. For three strains, Aeromonas
hydrophyla, Aeromonas salmonicida, and E. coli, the medium was also prepared with distilled water. After incubation at 20C for 24 to 30 h, cells were harvested by centrifugation. Tetrodotoxin and anhydrotetrodotoxin were then

Tetrodotoxin is a strong neurotoxin and is known as the

cause of pufferfish poisoning. Since 1964, when tarichatoxin
in the eggs of the California newt, Taricha torosa, was
identified as tetrodotoxin (3), the toxin has been isolated
from several marine and land animals. Recently, bacteria
associated with these animals have intensively been examined for the origin of the toxin (4, 6, 9). We report here that
tetrodotoxin is produced by a series of marine bacteria,
including members of the Vibrionaceae, which are frequently encountered in seawater and associated with marine
animals.
Tetrodotoxin is accumulated, most typically, in the ovaries and liver of many species of Tetraodontidae fish. This
toxin affects the nervous system, causing paralysis when
administered by oral, intraperitoneal, intramuscular, or subcutaneous route. The origin of tetrodotoxin in marine animals has been the subject of a number of recent investiga-

TABLE 1. Analyses of tetrodotoxin and anhydrotetrodotoxin in

bacterial cells
Toxin detected bya:
Bacterial strain

tions.
Noguchi et al. (6) reported the isolation of a tetrodotoxinproducing bacterium from the intestines of a crab, Atergatis
floridus, which is known to have the toxin. Another
tetrodptoxin-producing bacterium has also been found in a
calcareous red alga, Jania sp. (9). Since then tetrodotoxinproducing bacteria have been isolated from various marine
organisms, including a starfish, Astropecten polyacanthus
(4), a horseshoe crab, Carcinoscorpius rotundicauda (A.
Kungsuwan, T. Noguchi, U. Simidu, K. Tsukamoto, Y.
Shida, and K. Hashimoto, Toxicon, in press), and the
blue-ringed octopus, Octopus maculosus (U. Simidu et al.,
unpublished data). The number of bacterial strains claimed
to produce the toxin has been expanding quickly. Most
strains have been identified as members of the genus Vibrio.
A striking point is that some of the strains which produced
tetrodotoxin most distinctly were identified as Vibrio
alginolyticus (4), a species that is widely distributed in
seawater and marine animals of temperate and tropical
latitudes.
To confirm the involvement of marine bacteria in tetrodotoxin production, screening experiments with some typical
marine bacterial strains were carried out. The bacteria used
were from culture collections, mostly the American Type
Culture Collection (Rockville, Md.) and the National Collection of Marine Bacteria (Aberdeen, Scotland), and included
15 strains of the family Vibrionaceae, 5 strains of the genus
Alteromonas, and 1 strain of Escherichia coli. The strains
*

Vibrio alginolyticus ATCC 17749

V. alginolyticus NCMB 1903
V. anguillarum NCMB 829
V. anguillarum NCMB 1291
V. costicola (V. costicolus) NCMB
701
V. fischeri NCMB 1281
V. fischeri (Photobacterium fischeri)
NCMB 1381
V. harveyi (Aeromonas harveyi)
NCMB 2
V. marinus Ps 207
V. parahaemolyticus NCMB 1902
V. parahaemolyticus ATCC 17802
Photobacterium phosphoreum
NCMB 844
Aeromonas hydrophila NCMB 89
A. hydrophila NCMB 89b
A. salmonicida ATCC 14174
A. salmonicida ATCC 14174b
Plesiomonas shigelloides ATCC
14029
Escherichia coli IAM 1268
E. coli IAM 1268b
Alteromonas communis IAM 12914
A. haloplanktis IAM 12918
A. nigrifaciens 1AM 13010
A. undina IAM 12922
A. vaga IAM 12923

HPLC
TTX
Anh-TTX

GC-MS

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aHPLC, High-performance liquid chromatography; GC-MS, gas chromatography-mass spectrometry. TTX, Tetrodotoxin; Anh-TTX, anhydrotetrodotoxin. +, Clearly detected; , difficult to detect; -, not detected.
bCultivated in a freshwater medium.

Corresponding author.
1714

VOL. 53, 1987

NOTES

:1.+^.
v. aiginauyiuu

..U

ATCC 17749

V. alninolyticus
MCMB 1903

__

~~~~77~~~~1
-21"" 11--Iizmd

E=

t-

AnhydratetroIotoxin

tetrodotoxinl
I

,,

1715

tetrodotoxins. None of the five Alteromonas strains tested

here produced tetrodotoxin or the anhydrated form. The
present results, coupled with our observations on marine
bacteria associated with the toxin-containing animals, suggest that tetrodotoxins found in animal organs are the
products of marine bacteria. If the animals accumulate the
bacterial products for their own purpose, this would suggest
a symbiotic relationship. Although the role of the toxin in
animals is still not entirely clear, the fact that scared animals
exude the toxin into surrounding seawater suggests that the
toxin has a protective function (7). There are a few established cases where the toxin has a distinct function; e.g., the
blue-ringed octopus, 0. maculosus, which lives in tropical
and subtropical seas, stores the toxin in large, second
salivary glands and injects it when it attacks other animals,
thus poisoning them (1).
The role of tetrodotoxin in the bacteria themselves is not
yet clear. The fact that tetrodotoxin regulates the transfer of
sodium ions through biological membranes may have some
relevance to the function of the toxin in marine bacterial
cells. Further research should be aimed at elucidating the
mechanisms of toxin production and function.

12
4
16
8
Retention time (min)

20

FIG. 1. High-performance liquid chromatography of the tetrodotoxin fraction from two V. alginolyticus strains (top, middle),
compared with that of authentic tetrodotoxins (bottom).

extracted from the cells and purified to permit confirmation.

The confirmation of these compounds was carried out in two
steps, first by high-performance liquid chromatography (Y.
Nagashima, J. Maruyama, T. Noguchi, and K. Hashimoto,
Nippon Suisan Gakkaishi, in press) and then by characterization using gas chromatography-mass spectrometry (5, 6).
Of 15 strains of the family Vibrionaceae, 10 strains,
including those of V. alginolyticus, Vibrio parahaemolyticus, and Vibrio anguillarum, along with Photobacterium
phosphoreum, clearly showed the ability to produce the
anhydrated form of tetrodotoxin (Table 1 and Fig. 1).
Anhydrotetrodotoxin is only slightly toxic but is easily
converted into tetrodotoxin in solution, particularly at lower
pH values. Conversely, tetrodotoxin also changes into the
anhydrated form in solution, the equilibrium being approximately 4 parts tetrodotoxin to 1 part anhydrotetrodotoxin.
V. alginolyticus ATCC 17749 killed five mice when cultivated in 400 ml of ORI medium for 24 h and injected
intraperitoneally (2). Most of the Vibrio and Photobacterium
species are indigenous marine bacteria. Some of them,
including the three Vibrio species listed above, are also
known as human and animal pathogens.
A. salmonicida, which is a typical fish pathogen, and
Plesiomonas shigelloides, a group of intestinal flora of
freshwater fishes, produced anhydrotetrodotoxin (Table 1).
E. coli, which is a typical terrestrial form, did not produce

This work was supported in part by a grant from the Research

Council, Ministry of Agriculture, Forestry and Fisheries Japan for
creative research projects in biotechnology, by a grant from Nihon
Life Insurance Foundation, and also by a research fund from the
Institute of Physical and Chemical Research of Japan.
LITERATURE CITED
1. Hashimoto, Y. 1979. Marine toxins and other bioactive marine
metabolites. Japan Scientific Societies Press, Tokyo.
2. Kawabata, T. 1978. Assay method for tetrodotoxin, p. 232-240.
In Environmental Health Bureau, Ministry of Health and Welfare
(ed.), Food hygiene examination manual, vol. 2. Japan Food

Hygiene Association, Tokyo.

3. Mosher, H. S., F. A. Fuhrman, H. D. Buchwald, and H. G.
Fischer. 1964. Tarichatoxin-tetrodotoxin: a potent neurotoxin.
Science 144:1100-1110.
4. Narita, H., S. Matsubara, N. Miwa, S. Akahane, M. Murakami,
T. Goto, M. Nara, T. Noguchi, T. Saito, Y. Shida, and K.
Hashimoto. 1987. Vibrio alginolyticus, a tetrodotoxin-producing
bacterium isolated from the starfish Astropecten polyacanthus.

Nippon Suisan Gakkaishi 53:617-621.

5. Narita, H., T. Noguchi, J. Maruyama, Y. Ueda, K. Hashimoto, Y.
Watanabe, and K. Hida. 1981. Occurrence of tetrodotoxin in a
trumpet shell, "boshubora" Charonia sauliae. Nippon Suisan
Gakkaishi 47:935-941.
6. Noguchi, T., J.-K. Jeon, 0. Arakawa, H. Sugita, Y. Deguchi, Y.
Shida, and K. Hashimoto. 1986. Occurrence of tetrodotoxin and
anhydrotetrodotoxin in Vibrio sp. isolated from the intestines of
a xanthid crab, Atergatisfloridus. J. Biochem. 99:311-314.
7. Saito, T., T. Noguchi, T. Harada, 0. Murata, and K. Hashimoto.
1985. Tetrodotoxin as a biological defense agent for puffers.
Nippon Suisan Gakkaishi 51:1175-1180.
8. Simidu, U., and K. Tsukamoto. 1985. Habitat segregation and
biochemical activities of marine members of the family
Vibrionaceae. Appl. Environ. Microbiol. 50:781-790.
9. Yasumoto, T., D. Yasumura, M. Yotsu, T. Michishita, A. Endo,
and Y. Kotaki. 1986. Bacterial production of tetrodotoxin and
anhydrotetrodotoxin. Agric. Biol. Chem. 50:793-795.