Tuesday, 28 January 2014

Poisonous puffer fish or bamboozling bacteria? Who is to blame for TTX production?

Following from this Mondays lecture I thought I’d find out if there was any definite proof that bacteria are the cause of TTX in Puffer fish, and I managed to find a relatively recent paper (2011) which had ID’ed a new TTX-producing bacterial species found in puffer fish intestines.

TTX (as we learnt in Colins lecture) is seen in a wide variety of animals (from the Blue Ringed Octopus to several species of puffer fish.) The amount of TTX in puffer fish is species specific, and varies in concentration with regards to different organs and different seasons.  In general the ovary and liver are believed to be the most toxic organs as they have the highest TTX levels  - however for this species, Takifugu niphobles the toxicity of the intestines are comparable to their ovaries and liver – and is far more potent than other organs.
The origin of TTX in puffer fish has been extensively studied (see the attached Table, which shows previous studies which have documented TTX-producing bacteria isolated from puffer fish.) It is interesting to note that once again Vibrio returns! My paper mentioned that not only was Vibro alginolyticus the first identified TTX-producing bacterium, but most of the reported TTX-producing strains belong to the genus Vibrio.) We saw before that cultivating puffer fish above the sea bed/in an enclosed water system leads to the production of puffer fish who do not produce TTX – but these puffer fish do become toxic when fed with toxic puffer fish liver or returned to open water. This; alongside the fact that so many organism throughout the world produce TTX, points to the idea that TTX has an exogenous microbial origin.

In this paper
Five bacterial strains were isolated from the intestines of Takifuga niphobles (a puffer fish collected from coastal Hong Kong waters.) A mouse bioassay was used to reveal one of the strains: gutB01, caused typical symptoms of TTX intoxication (colvusions and dyspnea, with all mice bioassays killed within 10-15minutes.)
An ELISA method was also used to further demonstrate there was TTX production from the strain gutB01. Both of these methods were used to procure a toxicity value (how much TTX was produced from 24h cultivation of gutB01). This was much higher in the mouse bioassay (7.7µg/L) than the ELISA (4.3µg/L) – this could be due to the presence of some unidentified toxic components (possible TTX derivative) in the mouse bioassay, or due to the fact the cells had been in different growth phases.
Mass spectrometry (in this paper, the MALDI-TOF) was also used to confirm the chemical identity of the toxin produced by gutB01 (which matched a TTX standard.)
MIDI analysis was used to identify gutB01 as Raoultella terrigena (this method is apparently very common for microbial identification, and works via matching the fatty acid profile of an unknown species to the fatty acid profiles of various references microbial species in a database.) This ID was confirmed with the sequence obtained from a 16S-23S rDNA ITA.

In conclusion: this paper revealed a novel TTX-producing bacteria species (Raoultella terrigena) associates and lives in the intestines of the puffer fish (Takifugu niphobles.) The TTX found in puffer fish is most likely to be due to this bacterium however (as always) the papers advises caution as this study did not exclude the possibility that other bacteria in this species of puffer fish could also be contributing to TTX production.

Yu, V. C. H., Yu, P. H. F., Ho, K. C., & Lee, F. W. F. (2011). Isolation and Identification of a New Tetrodotoxin-Producing Bacterial Species, Raoultella terrigena, from Hong Kong Marine Puffer Fish Takifugu niphobles. Marine drugs,9(11), 2384-2396.


  1. So there are four classes of bacteria in that table. Do you think that horizontal gene transfer could be responsible for the transfer of TTX related genes in the free living stages or do you think that some of these results might be a bit suspicious. I know Colin mentioned that some were in doubt I wander if the current study was one of them? Having said that the distribution of TTX throughout the planet would indicate that it is very beneficial and perhaps there are just hundreds of bacterial producers. Is there some way that we could try and determine the age of the gene(s) responsible?

  2. Hi Caz, it was interesting to note that some of the bacterial species isolated in the paper I reviewed (Auawithoothij & Noomhorm, 2012) tallied with those presented in your table, but on this occasion were not found to be TTX producing. Maybe it's only under certain environmental conditions or population density (eg; quorum sensing) that upregulation of TTX production occurs?

  3. Thank you to Rachel for pointing out I had some comments on this or else I wouldn't have noticed and never replied, but better late than never!

    Rachel: the idea of TTX production occurring as a result of quorum sensing really appeals to me! Definitely an area of further research, and it would certainly explain any discrepancies between information you’re come across and the table from my paper. I didn’t see any information with regards to environmental conditions affecting TTX production in my paper, but their main focus was really isolation and identification so not surprising really.
    George: My paper appears very confident in its results (and they used a range of methods to double/triple check the species they ID’ed was the correct one and was capable of producing TTX.) However, perhaps like how Rachel suggested, the ability to produce TTX is dependent on the environment thus explaining the doubt surrounding some results we have come across.
    Having said that; I agree with the mindset that TTX related genes would be transferred via horizontal gene transfer amongst bacteria, as there seems almost a limitless amount of TTX-producing bacteria being discovered (so I’m thinking that it’s more likely for the bacteria to transfer genes rather than have all separately developed this talent.)

  4. I wonder whether the genes for TTX production are highly conserved? Also, I wonder whether phylogenetic analyses have been carried out to try and use evolutionary divergence to better understand TTX production in such a wide range of organisms? Warrants further investigation I think!