Pufferfish (Tetraodon cutcutia) Sampled from a Freshwater River Serves as an Intermediate Reservoir of a Sucrose Nonfermenting Variant of Vibrio cholerae PS-4.

We describe the genomic characteristics of Vibrio cholerae strain PS-4 that is unable to ferment sucrose on a thiosulfate citrate bile salt sucrose (TCBS) agar medium. This bacterium was isolated from the skin mucus of a freshwater pufferfish. The genome of strain PS-4 was sequenced to understand the sucrose nonfermenting phenotype. The gene encoding the sucrose-specific phosphotransferase system IIB (sucR) was absent, resulting in the defective sucrose fermenting phenotype. In contrast, genes encoding the glucose-specific transport system IIB (ptsG) and fructose-specific transport system IIB (fruA) showed acid production while growing with respective sugars. The overall genome relatedness indices (OGRI), such as in silico DNA-DNA hybridization (isDDH), average nucleotide identity (ANI), and average amino acid identity (AAI), were above the threshold value, that is, 70% and 95 to 96%, respectively. Phylogenomic analysis based on genome-wide core genes and the nonrecombinant core genes showed that strain PS-4 clustered with Vibrio cholerae ATCC 14035T. Further, genes encoding cholera toxin (ctx), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), toxin-coregulated pilus (tcp), and lipopolysaccharide biosynthesis (rfb) were absent. PS-4 showed hemolytic activity and reacted strongly to the R antibody. Therefore, the Vibrio cholerae from the pufferfish adds a new ecological niche of this bacterium. IMPORTANCE Vibrio cholerae is native of aquatic environments. In general, V. cholerae ferments sucrose on thiosulfate citrate bile salt sucrose (TCBS) agar and produces yellow colonies. V. cholerae strain PS-4 described in this study is a sucrose nonfermenting variant associated with pufferfish skin and does not produce yellow colonies on TCBS agar. Genes encoding sucrose-specific phosphotransferase system IIB (sucR) were absent. The observed phenotype in the distinct metabolic pathway indicates niche-specific adaptive evolution for this bacterium. Our study suggests that the nonfermenting phenotype of V. cholerae strains on TCBS agar may not always be considered for species delineation.

The flagellin of Vibrio parahaemolyticus induces the inflammatory response of Tetraodon nigroviridis through TLR5M.

Vibrio parahaemolyticus is an important marine pathogen that cause inflammation even death in teleost. It has brought huge economic losses to aquaculture and serious threats to the sustainable development of marine fisheries. Here, we isolated the DNA, RNA, and total flagellin from V. parahaemolyticus, and obtained the primary spleen and head kidney cells (including leukocytes) from Tetraodon nigroviridis. V. parahaemolyticus DNA, RNA, and total flagellin were used to treat the T. nigroviridis primary cells described above. The results show that the nitric oxide (NO) production and respiratory burst response were significantly induced after stimulation with V. parahaemolyticus total flagellin in T. nigroviridis head kidney and spleen cells. And total flagellin could promote the gene expression and protein production of IL-1ß in T. nigroviridis leukocytes. T. nigroviridis TLR5M (TnTLR5M) and TLR5S (TnTLR5S) ORF sequences were obtained as the main recognition receptor for flagellin. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the expression of pattern recognition receptor TnTLR5M and TnTLR5S, the important signal molecule of inflammatory system TnMyD88 and TnTRAF6, and inflammatory cytokines TnIL-1ß and TnIFN-γ2. The results show that there were a significant upregulation after challenge with V. parahaemolyticus total flagellin. We further demonstrated that the total flagellin of V. parahaemolyticus could activate the luciferase activity of the NF-κB reporter gene mediated by TnTLR5M. Overall, our results suggest that V. parahaemolyticus total flagellin activated the NO production, respiratory burst response, and inflammatory cytokines expressions, such as TnIL-1ß and TnIFN-γ2, through the TnTLR5M-NF-κB signaling pathway in T. nigroviridis.

Description of Acinetobacter kanungonis sp. nov., based on phylogenomic analysis.

A novel strain of a member of the genus Acinetobacter, strain PS-1T, was isolated from the skin of fresh water pufferfish (Tetraodon cutcutia) collected from Mahanadi River, India. Cells were Gram-stain-negative, aerobic, coccoid and non-motile. The predominant polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phospholipid (PL) and the cell wall sugars were glucose, galactose and ribose. The major cellular fatty acids of PS-1T were C18 : 1ω9c (30.67 %), C16 : 1ω7c (19.54 %), C16 : 0 (15.87 %), C12 : 0 (7.35 %) and C12 : 0 3-OH (6.77 %). The genome size was 3.5 Mbp and the DNA G+C content was 41.97 %. Gene ontology study revealed that the major fraction of genes were associated with biological processes (53.99 %) followed by molecular function (30.42 %) and cellular components (15.58 %). Comparisons of 16S rRNA gene sequences revealed 97.94-97.05 % sequence similarity with the closely related type strains of species of the genus Acinetobacter. The average nucleotide identity (ANI) and average amino acid identity (AAI) of PS-1T with reference strains of species of the genus Acinetobacter with validly published names were bellow 95-96 and the corresponding in-silico DNA-DNA hybridization (DDH) values were below 70 %. A phylogenomic tree based on core genome analysis supported these results. Genotypic and phenotypic characteristics of PS-1T indicate that the strain represents a novel species of the genus Acinetobacter and the name Acinetobacter kanungonis sp. nov. is proposed. The type strain is PS-1T (=JCM 34131T=NCIMB 15260T).

Composition and functional characterization of the gut microbiome of freshwater pufferfish (Tetraodon cutcutia).

This study describes the community composition and functions of the gut microbiome of the freshwater omnivorous pufferfish based on metagenomic approach. Metagenome sequence data showed a dominance of the class Gammaproteobacteria followed by Fusobacteria, Actinobacteria, Anerolineae, Betaproteobacteria, Deinococci, Clostridia and Deltaproteobacteria. At the order level, the most abundant groups were Aeromonadales, Fusobacteriales, Enterobacterales, Synechococcales. The genus Aeromonas was the most predominant followed by Plesiomonas and Cetobacterium. Additionally, within the domain Archaea, class Methanomicrobia was most abundant followed by Hadesarchaea, Thermoplasmata, Candidatus Altiarchaeales, Candidatus Bathyarchaeota and Thermoprotei. The metabolic profile of the bacterial community exhibited a high prevalence of genes associated with core housekeeping functions, such as synthesis of cofactors, vitamins, prosthetic groups, pigments, amino acids and its derivatives, carbohydrate and protein metabolism. Comparative analysis with other fish gut microbiome showing similarity in protein metabolism with carnivorous Salmon and carbohydrate metabolism with herbivorous grass carp respectively. This study describes the bacterial community compositions are influenced by the trophic level.

New tetrodotoxin analogs in Brazilian pufferfishes tissues and microbiome.

While tetrodotoxin (TTX) is commonly found in pufferfish tissues, it is unclear if bacterial symbionts isolated from pufferfish tissues can produce TTX. In this investigation, UPLC qTOF-MS/MS analysis of tissue extracts obtained from Sphoeroides spengleri and Canthigaster figuereidoi identified TTX in their composition, indicating their consumption is unsafe. UPLC qTOF-MS/MS analysis coupled with Molecular Networking indicated new TTX analogs (methyl-TTX, TTX-acetate, hydroxypropyl-TTX and glycerol-TTX). Bacterial extracts from sixteen strains revealed a compound with a [M+H]+ ion at m/z 320.1088, identical to TTX. However, TTX itself was not detected in these cultures by UPLC-MS/MS. Neurotoxicity of Vibrio A665 purified fraction 2 (with precursor [M+H]+ ion at m/z 320.1088) was significant in human neural stem cells (hNSCs), but the Nav blockage activity was not confirmed by the veratridine/ouabain essays, indicating a possible difference in the mechanism of action between the bacterium A665 purified fraction 2 and TTX. Vibrios symbionts of pufferfish point out involving in the production of TTX precursors.

Enterovibrio baiacu sp. nov.

We report here the novel species to encompass the isolate A649T (=CBAS 716T = CBRVS P1061T) obtained from viscera of the healthy pufferfish Sphoeroides spengleri (Family Tetraodontidae). Genomic taxonomy analysis demonstrates that the novel strain A649T had < 95% average amino acid identity/average nucleotide identity (AAI/ANI) and < 70% similarity of genome-to-genome distance (GGDH) towards its closest neighbors which places A649T into a new Enterovibrio species (Enterovibrio baiacu sp nov.). In silico phenotyping disclosed several features that may be used to differentiate related Enterovibrio species. The nearly complete genome assembly of strain A649T consisted of 5.4 Mbp and 4826 coding genes.

Histopathological Features of Mycobacterium chelonae Infection in Two Farmed Japanese Pufferfish (Takifugu rubripes).

Granulomatous lesions were observed in the swim bladder, kidney, spleen and gills of two farmed Japanese pufferfish (Takifugu rubripes) infected with Mycobacterium chelonae. Three types of lesions were noted: unencapsulated clusters of epithelioid cells without central necrosis (type 1), encapsulated granulomas without central necrosis (type 2) and encapsulated granulomas with central necrosis (type 3). Type 3 lesions occurred most frequently in the swim bladder, while type 1 and type 2 lesions occurred frequently in the kidney and spleen, and the gills exhibited mostly type 1 lesions. This suggests that the lesions in the swim bladder were more fully developed than those occurring elsewhere and that the swim bladder may be more susceptible to infection with M. chelonae. This is the first report describing the histopathological features of M. chelonae infection in Tetraodontidae.

Two distinct interferon-γ genes in Tetraodon nigroviridis: Functional analysis during Vibrio parahaemolyticus infection.

Interferon gamma (IFNγ) is a Th1 cytokine that plays a very important role in almost all phases of immune and inflammatory responses. In this study, we explored the functions of IFNγ1 and IFNγ2 of Tetraodon nigroviridis. Treating T. nigroviridis spleen and head kidney cells in vitro with recombinant T. nigroviridis IFNγ1 protein (rTn IFNγ1) or recombinant T. nigroviridis IFNγ2 protein (rTn IFNγ2) enhanced their nitric oxide responses. Both rTn IFNγ1 and rTn IFNγ2 also induced the expression of interferon-stimulated gene 15 (ISG15), a common anti-viral gene, although the expression of the interferon-inducible Mx gene was markedly inhibited by rTn IFNγ1 and was induced by rTn IFNγ2. The in vivo effects of rTn IFNγ1 and rTn IFNγ2 on Vibrio parahaemolyticus (V. parahaemolyticus) infection were assessed by intraperitoneally injecting rTn IFNγ1 or rTn IFNγ2 (100 ng) and V. parahaemolyticus (8 × 10(10)CFU/mL) into T. nigroviridis. A comparison of the group treated only with V. parahaemolyticus and those also treated with rTn IFNγ1 or rTn IFNγ2 showed that neither of these IFNγs protected T. nigroviridis from V. parahaemolyticus infection. However, rTn IFNγ1 more rapidly and robustly promoted inflammatory responses compared with rTn IFNγ2, whereas rTn IFNγ2 was involved in inducing the host to develop a more effective response earlier during the later stage of a V. parahaemolyticus infection. Moreover, microRNA-29b (miR-29b) expression is inversely correlated with IFNγ2 expression in T. nigroviridis.

Biological Activities of Tetrodotoxin-Producing Enterococcus faecium AD1 Isolated from Puffer Fishes.

Puffer fishes were collected from the central sea in Vietnam from spring to summer season. The eggs were incubated in MRS broth that was used to test the toxicity in mice and isolate the lactic acid bacteria community that could produce tetrodotoxin (TTX). Thin layer chromatography (TLC) and high performance lipid chromatography (HPLC) were used to detect and quantify TTX. As a result, Enterococcus faecium AD1 which was identified by biochemical test and 16S rRNA analysis could produce TTX 0.3 mg/mL when cultured in MRS broth. The bacterium was optimized for TTX production and gave 0.18 mg/mL, 0.07 mg/mL, and 0.15 mg/mL in media prepared from the meat-washing water of freshwater fishes (Pangasius bocourti, Oreochromis sp.) and sea fish (Auxis thazard), respectively, that are also hopeful to answer some poisoning cases related to eating fishes. Enterococcus faecium also showed the wide antimicrobial activities on yeast, Gram-negative and -positive bacteria. Extracted exopolysaccharide (EPS) that reacted with 2,2-diphenyl-1-picrylhydrazyl to give IC50 at 5 mg/mL equaled 11 mg/mL ascorbic acid which could show effects on Hela-6 and Hep G2 using sulforhodamine B test. Enterococcus faecium can be claimed as a promising source in tetrodotoxin and biological compounds.

Detection of tetrodotoxin-producing Providencia rettgeri T892 in Lagocephalus pufferfish.

Tetrodotoxin (TTX) is a potent toxin but it could be used in pharmaceutical field. Identification of TTX producing bacteria in pufferfish is necessary for TTX yield and the pufferfish conservation. In this study, Lagocephalus was collected from Cam Ranh Sea, a central part of Vietnam during spring season. The liver and intestine were incubated in 0.9 % NaCl for TTX detection in pufferfish. To be benefited from the isolation of new TTX producing bacteria, the liver and intestine were incubated in 6.5 % NaCl. The cultures were used to test the toxin and to isolate the bacterial community that could yield TTX. Surprisingly, Providencia rettgeri T892 in intestine could produce TTX identified by biochemical test and 16S rRNA sequencing. This strain was used to test the production of TTX, based on thin layer chromatography (TLC), mouse bioassay and high performance liquid chromatography (HPLC) analysis. The bacterium was optimized for TTX production in media prepared from the meat-washing water of Auxis thazard, Megalaspis cordyla and Decapterus maruadsi. Interestingly, the TTX obtained 0.106 mg/mL and 0.055 mg/mL in medium prepared from A. thazard and M. cordyla, respectively while there was no TTX production detected in medium prepared from D. maruadsi. This paper could contribute to warn to the human health care system about a possible TTX poisoning in some cases related to eating fishes.

Shewanella putrefaciens, a major microbial species related to tetrodotoxin (TTX)-accumulation of puffer fish Lagocephalus lunaris.

AIMS: To investigate the major micro-organisms, which were isolated from internal organs, related to tetrodotoxin (TTX)-accumulation of puffer fish Lagocephalus lunaris. METHODS AND RESULTS: Puffer fish Lagocephalus lunaris around Chang Island in the Gulf of Thailand were collected to examine TTX-accumulation and microbial load in internal organs. The nine predominant micro-organisms isolated from the internal organs were determined TTX and studied in relation to the TTX-accumulation of the puffer fish. Shewanella putrefaciens, a predominant bacterium and related to the TTX-accumulation of the puffer fish, was examined growth and TTX-production after culture in modified Zobell medium. The results revealed that the average TTX-accumulation of the puffer fish directly varied to the bacterium load periodically, year-round. Furthermore, it coincided with the growth and the TTX-production of the bacterium, which grew slowly but produced high TTX at low temperature. CONCLUSIONS: Shewanella putrefaciens was a major bacterium relating to TTX-accumulation of puffer fish L. lunaris. It resulted in high TTX-accumulation of the puffer fish at low temperatures of seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: Temperature affected growth and TTX-production of S. putrefaciens that resulted in TTX accumulated in puffer fish L. lunaris.

Isolation and identification of a new tetrodotoxin-producing bacterial species, Raoultella terrigena, from Hong Kong marine puffer fish Takifugu niphobles.

Puffer fish, Takifugu niphobles, collected from the Hong Kong coastal waters were screened for tetrodotoxin-producing bacteria. A Gram-negative, non-acid-fast, non-sporing and rod shaped bacterial strain (designated as gutB01) was isolated from the intestine of the puffer fish and was shown to produce tetrodotoxin (TTX). Based on the Microbial Identification (MIDI) and 16S-23S rDNA internal transcribed spacer (ITS) phylogenetic analysis, the strain was identified as Raoultella terrigena. The TTX production ability of the strain was confirmed by mouse bioassay, ELISA and mass spectrometry (MALDI-TOF). Our results reiterate that the TTX found in puffer fish was likely produced by the associated bacteria and TTX are widely produced amongst a diversity of bacterial species.

A novel TTX-producing Aeromonas isolated from the ovary of Takifugu obscurus.

Puffer fish (Takifugu obscurus) from the Yangtze River of China were screened for tetrodotoxin-producing bacteria. An Aeromonas strain was isolated from the ovary of the puffer fish and was shown to produce tetrodotoxin; this strain was denoted Ne-1. The identity of tetrodotoxin produced by strain Ne-1 was confirmed by mouse bioassay, high performance liquid chromatography coupled with mass chromatography (LC-MS), and ELISA. Strain Ne-1 was characterized morphologically, biochemically, and by 16S rDNA phylogenetic analysis; these analyses suggested that strain Ne-1 is closely related to Aeromonas molluscorum. Given that strain Ne-1 was isolated from the ovary of T. obscurus, we propose that the TTX-producing Aeromonas sp. is a parasite or symbiotic bacterium rather than a sample contaminant. Collectively, our studies suggest that Aeromonas sp. strain Ne-1 produces tetrodotoxin in T. obscurus.

Bacterial production of tetrodotoxin in the pufferfish Arothron hispidus.

This study evaluates the presence, location and production source of tetrodotoxin (TTX) in two species of pufferfish, Diodon histrix and Arothron hispidus, common to Hawaiian waters. Organs from each fish were analysed for TTX and used to isolate bacteria for evaluation of possible TTX production. Comparative analyses of extracts of fish and bacterial culture media were performed with liquid chromatography-mass spectrometry (LC-MS) and a sodium channel specific bioassay. Bacterial cultivation experiments were performed in two different growth media and bacteria were identified through sequence homology of the 16S rRNA gene. Forty-two and forty-seven distinct strains were cultivated from D. histrix and A. hispidus, respectively. However, no commonality was found between the populations of bacteria isolated from the two fish. TTX was detected only in A. hispidus and was present in the flesh, pectoral fin and kidneys, as well as the skin slime. Sixteen of the forty-seven bacterial species isolated from A. hispidus were cultivated for further evaluation of TTX production. Among these sixteen bacterial species, Vibrio harveyi strains isolated from the skin slime and kidneys of A. hispidus were found to produce TTX, being the source of TTX produced in the pufferfish.

Morphological and genetical description of Loma psittaca sp. n. isolated from the Amazonian fish species Colomesus psittacus.

A previously unrecognised fish-infecting microsporidia (Loma psittaca n. sp.), found adherent to the intestinal mucosa of the freshwater puffer fish Colomesus psittacus (Teleostei, Tetraodontidae) from lower Amazon River, was described based on light and transmission electron microscope and phylogenetic analysis. The whitish xenoma was completely filled by numerous spores, including several developmental stages of the parasite. In all of these stages, the nuclei were monokaryotic. The merogonial plasmodium divided by binary fission and the sporont gave rise to disporoblastic ovoid spores measuring 4.2 +/- 0.4 x 2.8 +/- 0.4 microm. In mature spores, the polar filament was arranged in 10-11 (rarely 12) coils in one row in turn of posterior vacuole. The polaroplast had two distinct regions around the manubrium. The polyribosomes were organised in coiled tapes. The small subunit rRNA gene was sequenced and maximum parsimony analysis placed the microsporidian described here in the clade that includes the genera Ichthyosporidium, Loma and Pseudoloma. Based on differences from previously described microsporidians, such as ultrastructural characteristics of the xenoma, developmental stages including the spore and phylogenetic analysis supported the recognition of a new species, herein named L. psittaca n. sp.

Chryseobacterium arothri sp. nov., isolated from the kidneys of a pufferfish.

A Gram-negative, non-flagellated, rod-shaped bacterium, designated strain P2K6(T), was isolated from the kidneys of a pufferfish (Arothron hispidus) caught off the coast of Kaneohe Bay, O'ahu, Hawai'i. The strain formed yellowish colonies when grown on marine agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain P2K6(T) was related most closely to members of the genus Chryseobacterium. Levels of 16S rRNA gene sequence similarity between strain P2K6(T) and the type strains of recognized species of the genus Chryseobacterium were 94-96.6%, suggesting that the strain represents a novel species within this genus. The DNA G+C content of strain P2K6(T) was 36.5 mol%, the dominant fatty acids were iso-C(15:0) (35.3%) and iso-C(17:0) 3-OH (14.9%), and the most abundant quinone was menaquinone MK6. On the basis of the data from this polyphasic study, it is suggested that strain P2K6(T) represents a novel species of the genus Chryseobacterium, for which the name Chryseobacterium arothri sp. nov. is proposed. The type strain is P2K6(T) (=CIP 109575(T)=DSM 19326(T)).

Isolation of halotolerant Lactococcus lactis subsp. lactis from intestinal tract of coastal fish.

We isolated lactic acid bacteria from the intestinal tract of the pufferfish Takifugu niphobles caught in Shimoda, Shizuoka, Japan by using MRS broth prepared with 50% seawater. Additional screening was carried out using phenotypic tests such as Gram staining, cell morphology, catalase, oxidase and fermentation of glucose. Subsequently 227 isolates screened by the phenotypic tests were subjected to species-specific PCR for Lactococcus lactis, resulting in four positive isolates. The 16S rRNA gene sequences from three isolates were highly similar to that of L. lactis subsp. lactis (DNA database accession number M58837), while that of one isolate was identical to that of Leuconostoc mesenteroides (AB023246). These isolates were characterized by API 50 CH for carbohydrate fermentation and other phenotypic criteria for salt tolerance, and the characteristics were compared with those of L. lactis subsp. lactis from a cheese starter culture. The carbohydrate fermentation profiles of these isolates were characteristic of L. lactis subsp. lactis strains, whereas the tolerance of these isolates to salt was higher than that of L. lactis subsp. lactis from the cheese starter culture: the new L. lactis isolates showed high salt tolerance in MRS-agar plates containing 200% seawater or 6% sodium chloride. This is the first report of the isolation of halotolerant strains of L. lactis subsp. lactis from a marine environment.

Ferrimonas senticii sp. nov., a novel gammaproteobacterium isolated from the mucus of a puffer fish caught in Kaneohe Bay, Hawai'i.

A novel species, strain P2S11T, was isolated from the mucus of a puffer fish caught off the coast of Kaneohe Bay, O'ahu, Hawai'i. A phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was most closely related to Ferrimonas marina DSM 16917T and Ferrimonas balearica DSM 9799T with 93.5% and 82.9% sequence similarities, respectively, which established the novel strain as belonging to the genus Ferrimonas. The strain formed off-white coloured colonies on marine agar and cells were Gram-negative, non-motile rods. H2S was produced when strain P2S11T was grown on TSI medium with added salt. Strain P2S11T had a DNA G+C content of 54.9 mol% and the dominant fatty acids were C16:1omega9c, C16:0 and C17:1omega8c. On the basis of this polyphasic study, strain P2S11T (=ATCC BAA-1480T=DSM 18821T) represents a novel species of the genus Ferrimonas, for which the name Ferrimonas senticii sp. nov. is proposed.

Two novel species of tetrodotoxin-producing bacteria isolated from toxic marine puffer fishes.

Out of eight dominant discrete bacterial colonies isolated and purified from the toxic marine puffer fishes collected in Hong Kong waters, two novel species of non-sporing, non-acid-fast and chemoorganotrophic bacteria capable of producing tetrodotoxin (TTX, a potent non-protein neurotoxin), as well as one previously reported and confirmed TTX-producing bacterium. They were identified as Microbacterium arabinogalactanolyticum, Serratia marcescens and Vibrio alginolyticus, respectively, all of which are widely distributed in soils, sewage or marine environments. Each bacterial isolate (500 ml broth medium cultured in darkness without aeration for 10 days at 25 degrees C) could produce an amount of toxicity, after extraction and purification, ranging from 78.3 to 105.3 mouse units (MU) in 500 ml of broth medium by mouse bioassay. The principal toxic component in the bacterial cultures was determined to be TTX by thin layer chromatography and mass spectrometry.