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Applied and Environmental Microbiology, May 2001, p. 2393-2394, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.8.3.2393-2394.2001
LETTERS TO THE EDITOR
No Ability To Produce Tetrodotoxin in Bacteria
 |
LETTER |
Bacterial production of tetrodotoxin (TTX) was first reported by
Yasumoto et al. (8). Detection of bacterially produced TTX
has been primarily by high-performance liquid chromatography (HPLC) and
gas chromatography-mass spectrometry (GC-MS). In September 1995, Applied and Environmental Microbiology published a paper indicating that both the HPLC and GC-MS methods lacked specificity for
TTX (3). In that paper, I pointed out that the previously reported production of TTX by bacteria should be revised or reexamined.
In April 2000, Applied and Environmental Microbiology
published data of Lee et al. (2) reporting that
Vibrio strains isolated from the intestines of puffer fish
produce TTX and its derivatives. Among the detection methods used by
Lee et al. (2), thin-layer chromatography and
electrophoresis had been commonly used through the 1970s. These methods
have been little used since the 1980s because the separation of TTX is
better accomplished by HPLC and GC-MS. Also, while Lee et al. also used
HPLC and GC-MS, the detection of TTX by all four of these methods
relies on alkali hydrolysis (2). Therefore, the analytical
methods used by Lee et al. are nonspecific for TTX.
In the article of Lee et al. (2), a large TTX peak was
shown in Fig. 1 while no toxicity was reported in Table 1. It is, however, well known that a good correlation is observed between TTX
concentrations calculated by mouse bioassay and HPLC when TTXs from
puffer fish and newt are used (7). Also, the apparent lower limit for TTX detection by HPLC is 10 ng 10 µl
1
(= 1,000 ng ml1) (7) and that by mouse
bioassay is 220 ng ml1 (1), indicating that
TTX detected by HPLC can be easily detected by mouse bioassay.
Therefore, the results of Lee et al.'s Fig. 1 and Table 1 may indicate
false-positive results by the analytical methods. This possibility had
been pointed out in the 1995 paper (3).
There has been no description of the determination of the structures of
the toxins obtained from bacteria, although a large amount of TTX
should easily be obtained by their cultivation. The chemical structures
of TTXs from puffer fish, newt, and blue-ringed octopus have already
been reported (6). It has, therefore, been thought even by
Japanese scientists that the chemical structures of the substances
obtained from bacteria were TTX, although no one has shown these
structures. Hence, a structural determination should not be a hard task
and is indispensable for confirmation of production of TTX by bacteria.
Production of TTX by bacteria has been presented to support the food
chain origin of TTX in puffer fish (8). The food chain origin of TTX was first hypothesized from the observation that cultured
puffer fish have no TTX, which suggested that puffer fish have no
ability to produce TTX. It has, however, been demonstrated recently
that cultured puffer fish have detectable TTX (4) and that
these fish can produce TTX (5). The progress in TTX research, including the 1995 paper (3), is not referenced
in the article of Lee et al. (2).
As the methods used to detect TTX lack specificity, I cannot agree with
the conclusions of Lee et al. (2) that Vibrio
strains produce TTX.
| |
REFERENCES |
| 1.
|
Kawabata, T.
1978.
Assay method for tetrodotoxin, p. 232-240.
In
Food hygiene examination manual, vol. 2. Japan Hygiene Association, Tokyo, Japan.
|
| 2.
|
Lee, M. J.,
D. Y. Jeong,
W. S. Kim,
H. D. Kim,
C. H. Kim,
W. W. Park,
Y. H. Park,
Y. S. Kim,
H. M. Kim, and D. S. Kim.
2000.
A tetrodotoxin-producing Vibrio strain, LM-1, from the puffer fish Fugu vermicularis radiatus.
Appl. Environ. Microbiol.
66:1698-1701[Abstract/Free Full Text].
|
| 3.
|
Matsumura, K.
1995.
Reexamination of tetrodotoxin production by bacteria.
Appl. Environ. Microbiol.
61:3468-3470[Abstract].
|
| 4.
|
Matsumura, K.
1996.
Tetrodotoxin concentrations in cultured puffer fish, Fugu rubripes.
J. Agric. Food Chem.
44:1-2[CrossRef].
|
| 5.
|
Matsumura, K.
1998.
Production of tetrodotoxin in puffer fish embryos.
Environ. Toxicol. Pharmacol.
6:217-219[CrossRef].
|
| 6.
|
Mosher, H. S.
1986.
The chemistry of tetrodotoxin.
Ann. N. Y. Acad. Sci.
479:32-43[Medline].
|
| 7.
|
Yasumoto, T., and T. Michishita.
1985.
Fluorometric determination of tetrodotoxin by high performance liquid chromatography.
Agric. Biol. Chem.
50:793-795.
|
| 8.
|
Yasumoto, T.,
D. Yasumura,
M. Yotsu,
T. Michishita,
A. Endo, and T. Kotaki.
1986.
Bacterial production of tetrodotoxin and anhydrotetrodotoxin.
Agric. Biol. Chem.
50:793-795.
|
| | | | |
Kendo Matsumura
Yamaguchi Prefectural Research Institute of Public Health
2-5-67 Aoi
Yamaguchi 753-0821, Japan
Phone: 083-922-7630
Fax: 083-922-7632
E-mail:
aat43203{at}par.odn.ne.jp
|
| |
AUTHOR'S REPLY |
In the letter of Dr. K. Matusmura in response to our recent paper
(1), he pointed out that contaminants from a bacterial culture medium may confound the identification of tetrodotoxin (TTX),
which puffer fish produce. The contamination issue was possibly based
on the results of his previous study of TTX production by bacteria
(2).
Our conclusive response to his criticism is that bacterial production
of TTX is still a valid proposition and that exogeneous production
should be regarded as a theory based on this production of the toxin.
Our conclusive and persuasive claims are based on the following
reasons. (i) Our Vibrio sp. strain was isolated as a single
colony, and the single colony was separately cultured in medium. This
indicates that although high-performance liquid chromatography (HPLC)
or gas chromatography-mass spectrometry might detect the same peaks as
those obtained from the bacterial medium, positive toxicity in a mouse
bioassay would not be detected. However, our TTX and its derivatives
purified from the bacterial culture cells showed a strong toxicity by
mouse bioassay. (ii) Only one Vibrio sp. strain from three
Vibrio species tested produced TTX and its derivatives
during bacterial cultures. Therefore, efforts to examine whether
intestinal Vibrio species are responsible for the production
of TTX or not should be carefully performed. Only a few
Vibrio sp. strains produce TTX, and thus isolation and
characterization of bacterial strains may be key points. It is,
therefore, assumed that Dr. Matsumura's strain may not be a
TTX-producing Vibrio, although he cultured Vibrio
species. Please note that there are many kinds of Vibrio
species in Fugu's intestinal tissues. (iii) Our study used
both preparative thin-layer chromatography and electrophoretic
resolution for isolation of TTX from bacterial cells. (iv) How could a
single transfer of a bacterial colony be contaminated by so much TTX
before it was used in the next analytical procedure? (v) When
Fugu fish were experimentally cultivated in an artificial
aquarium, the toxicity (MU of TTX) of the Fugu fish was
severely reduced with a reduction in intestinal bacterial flora.
Dr. Matsumura's criticism may be an insignificant result derived from
the difficulties described above. Especially with HPLC data, similar
peaks are sometimes confused, and his claims may be the results of
technical problems during HPLC. Furthermore, one more possibility for
his obtaining opposing results is that he may have cultured
Vibrio spp. which do not produce any TTX. I would like to
suggest that he isolate a TTX-positive bacterium, further characterize
it, and then discuss his results with us.
Interestingly, the criticism made by Dr. Matsumura was also subjected
to evaluation in a Japanese journal named Chemistry and
Biology (3), which is published monthly by the Japan
Society for Bioscience, Biotechnology, and Agrochemistry (formerly the Agricultural Chemical Society of Japan). It was concluded that bacterial production of TTX is still a valid supposition, and thus his
conclusion was ignored by Dr. T. Noguchi, Nagasaki University Faculty
of Fisheries, Nagasaki, Japan, and Dr. T. Yasumoto, Tohoku University
Faculty of Agriculture, Sendai, Japan. We immediately contacted Dr.
Noguchi again to discuss his conclusions more carefully, resulting in a
conclusive determination that TTX is produced by marine bacteria.
In our scientific and principal elucidation of the exogenous and
indogenous production of TTX in bacteria, we had not cited Dr.
Matsumura's paper (2), in which he insists that bacteria do not have the ability to produce TTX, that contaminants from the
bacterial culture medium may confound the identification of the TTX,
and that puffer fish do produce the toxin. I would like to respectfully
ask him to consider carefully reexamining TTX production by bacteria.
As we mentioned in our paper, a final resolution of this contradiction
would be obtained by an elucidation of genes for TTX synthesis at the
molecular level.
| |
REFERENCES |
| 1.
|
Lee, M.-J.,
D.-Y. Jeong,
W.-S. Kim,
H.-D. Kim,
C.-H. Kim,
W.-W. Park,
Y.-A. Park,
K.-S. Kim,
H.-M. Kim, and D.-S. Kim.
2000.
A tetrodotoxin-producing Vibrio strain, LM-1, from the puffer fish Fugu verimcularis radiatus.
Appl. Environ. Microbiol.
66:1698-1701.
|
| 2.
|
Matsumura, K.
1995.
Reexamination of tetrodotoxin production by bacteria.
Appl. Environ. Microbiol.
61:3468-3470.
|
| 3.
|
Yasumoto, T.
1996.
There are still bacteria which produce tetrodotoxin in Fugu.
Chem. Biol. (Kagak to Seibutsu)
34:837-839. (In Japanese.)
|
| | | | |
Dong-Soo Kim
Department of Food Science and Technology
Kyungsung University
Nam-ku
Pusan 608-736, Korea
|
| | | | |
Cheorl-Ho Kim
Department of Biochemistry and Molecular Biology
College of Oriental Medicine
Research Center for Intractable Diseases
Dongguk University
Kyung-Ju
Kyungpook 780-714, Korea
Phone: 82-54-770-2663
Fax: 82-54-770-2281
E-mail: chkimbio{at}dongguk.ac.kr
|
Applied and Environmental Microbiology, May 2001, p. 2393-2394, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.8.3.2393-2394.2001
