ABSTRACT
Tetrodotoxin (TTX) is a potent neurotoxin frequently occurring in marine organisms along with its numerous analogues. To determine the total TTX content, we developed an enzyme-linked immunosorbent assay (ELISA) technique utilizing polyclonal antibodies against TTX. The technique was tested using extracts of marine worms of the phylum Nemertea and confirmed by HPLC-MS/MS. It proved to be suitable for a preliminary assessment of the toxicity of marine organisms.
Subject(s)
Aquatic Organisms/chemistry , Tetrodotoxin/toxicity , Animals , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay/methods , Tandem Mass Spectrometry/methodsABSTRACT
For the first time search for tetrodotoxin (TTX) and its analogues in the extracts of nemerteans using HPLC-MS/MS was performed. TTX analogues were detected in two nemertean species in addition to TTX: 7 analogues were detected in the extract of Cephalothrix simula, 3 analogues - in the extract 11-norTTX of Kulikovia manchenkoi. Presence of 5-deoxyTTX, 11-deoxyTTX, 5,6,11-trideoxyTTX and -6(R)-ol in nemerteans was shown for the first time.
Subject(s)
Aquatic Organisms/chemistry , Tetrodotoxin/analogs & derivatives , Tetrodotoxin/analysis , Animals , Aquatic Organisms/metabolism , Chromatography, High Pressure Liquid/methods , Invertebrates/chemistry , Invertebrates/metabolism , Japan , Pacific Ocean , Tandem Mass Spectrometry/methods , Tetrodotoxin/chemistry , Tetrodotoxin/metabolismABSTRACT
The taxonomic composition of bacteria associated with the Cephalotrix simula proboscis worm was studied and screening of the tetrodotoxin (TTX)-producing bacteria was carried out using confocal laser scanning microscopy and polyclonal antibodies. Bacterial isolates were identified using the 16S rRNA gene sequencing and phenotypic characteristics. A Bacillus species was found to be responsible for tetrodotoxin production in C. simula proboscis worms. Vibrio spp. predominated in the associated microflora (68.18% of the total number of isolates). Analysis of the sensitivity of 16 strains to antibiotics of various classes revealed multiple resistance to three or more antibiotics in all studied isolates. Poor growth of most of the isolates on all laboratory media was an indirect confirmation of the symbiotic relationships between the micro- and macroorganisms.
Subject(s)
Bacillus/metabolism , Invertebrates/microbiology , Tetrodotoxin/metabolism , Vibrio/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Aquatic Organisms , Bacillus/genetics , Bacillus/isolation & purification , Drug Resistance, Multiple, Bacterial , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Symbiosis , Vibrio/drug effects , Vibrio/genetics , Vibrio/isolation & purificationABSTRACT
Pathogenic properties of the natural isolate of Shewanella algae from the coelomic fluid of the sea cucumber Apostichopus japonicus (Peter the Great Bay, Sea of Japan) were investigated. The isolate had oxydative metabolism, was positive for ornithine decarboxylase, cytochrome oxidase, catalase, DNase and gelatinase, hemolytically active, did not produce acid from carbohydrates, and did not hydrolyze urea and esculin. The strain was resistant to penicillin, amoxicillin, and ampicillin and susceptible to tetracycline and carbenicillin. Among cellular fatty acids, 13:0-i, 15:0-i, 16:0, 16:1(n-7), 17:0-i, and 17:0-ai dominated. These biochemical properties made it possible to attribute the isolated bacteria to the genus Shewanella and identified as S. algae. The cells of this bacterium were introduced into the coelomic cavity of another echinoderm, the sea urchin Strongylocentrotus nudus. As a result, in about 24h the animals became slow and 3-8days after the inoculation died. Dividing bacteria were being found during the experiment in the coelomic fluid as well as in the phagosomes of amoebocytes, i.e. cells acting as phagocytes in the coelomic fluid. The studies of the invasive properties of strain 156 showed that bacterial cells entered the subcuticular space of S. nudus and A. japonicus through the cuticle and stayed there for a long time without penetrating epithelium and exerting toxic effect upon the organisms of the laboratory animals. Pathogenic effect of S. algae can be manifested only if the cutaneous epithelium is destroyed permitting it to penetrate the lower tissue layers. The toxicity of S. algae is confirmed by in vitro experiments. The inoculation of the embryonic cells of S. nudus with samples of this bacterium caused the death of 10% of cells within an hour and 100% of cells within 12h after inoculation. The results of the investigations demonstrate that S. algae could produce opportunistic infection in the sea cucumber A. japonicus and the sea urchin S. nudus, which may be natural reservoirs of this human pathogen.
