Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00248-023-02341-4

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmicb.2023.1298681

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2965/jwet.22-095

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmicb.2023.1230548

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.rsma.2023.102984

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

DOI： 10.3390/w14071018

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jgar.2022.04.024

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DOI： 10.1264/jsme2.ME22038

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Here, we report the draft genome sequences of putative aerobic anoxygenic phototrophic bacterial strains
<italic>Jannaschia</italic>
sp. AI_61 and AI_62, isolated from seawater around a coastal aquaculture in Ainan, Ehime, Japan. These genome sequences could be useful for our understanding of the variation of aerobic anoxygenic phototrophs in the genus.

DOI： 10.1128/MRA.00491-21

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Water systems in Southeast Asia accumulate antibiotics and antibiotic resistance genes (ARGs) from multiple origins, notably including human clinics and animal farms. To ascertain the fate of antibiotics and ARGs in natural water environments, we monitored the concentrations of these items in Thailand. Here, we show high concentrations of tetracyclines (72,156.9 ng/L) and lincomycin (23,968.0 ng/L) in pig farms, followed by nalidixic acid in city canals. The city canals and rivers contained diverse distributions of antibiotics and ARGs. Assessments of targeted ARGs, including sul1, sul2, sul3, and tet(M), showed that freshwater (pig farm wastewater, rivers, and canals) consistently contained these ARGs, but these genes were less abundant in seawater. Although sulfonamides were low concentrations (<170 ng/mL), sul1 and sul2 genes were abundant in freshwater (minimum 4.4 x 10(-3) -maximum 1.0 x 10(0) copies/16S), suggesting that sul genes have disseminated over a long period, despite cessation of use of this class of antibiotics. Ubiquitous distribution of sul genes in freshwater appeared to be independent of selection pressure. In contrast, water of the coastal sea in the monitored area was not contaminated by these antibiotics or ARGs. The density of Enterobacteriales was lower in seawater than in freshwater, suggesting that the number of ARG-possessing Enterobacteriales falls after entering seawater. From the pig farms, through rivers/canals, to the coastal sea, the occurrence of tetracyclines and tet(M) exhibited some correlation, although not a strong one. However, no correlations were found between concentrations of total antibiotics and ARGs, nor between sulfonamides and sul genes. This is the first comprehensive study showing Thai features of antibiotics and ARGs contaminations. The pig farm is hot spot of antibiotics and ARGs, and sul genes ubiquitously distribute in freshwater environments, which become less abundant in seawater. (C) 2021 The Authors. Published by Elsevier B.V.

DOI： 10.1016/j.scitotenv.2021.148423

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

Background and Objectives: One major source of antibiotic contamination in the sea is from aquaculture. We monitored the concentration of commonly used antibiotic classes and antibiotic resistance genes (tet(M), sul1, sul2 and sul3) in aquaculture farms in Peninsular Malaysia. Methods: Antibiotic residues and resistance genes were quantified using high-performance liquid chromatography and real-time PCR respectively. Risk quotients in European technical guidance document on risk assessment was used to assess the potential environmental risk. Results: We detected 23 antibiotics with tetracyclines, sulfonamides and quinolones were the most frequently detected classes, indicating a wide distribution of antibiotics in Malaysian aquaculture farms. The dendrogram and heatmap revealed three groups of antibiotic concentration patterns but with no differences in the types of antibiotics usage among aquaculture farms. The ARGs (10(-3) copies/16S) were detected in >90% of the sites except for sul3. Ciprofloxacin, enrofloxacin, norfloxacin and lincomycin posed risks to cyanobacteria and algae in Kelantan, Perak and Pahang. Conclusion: Relative to Asian aquaculture farms, the residues detected here were at low or moderate levels except for quinolones. This study will be useful to develop effective management of aquaculture wastewater in order to mitigate antibiotic pollution and transmission of ARGs to humans through the food chain.

DOI： 10.1080/20964129.2021.1926337

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DOI： 10.1016/j.etap.2020.103557

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Research Foundation  

Most animals cannot digest cellulose but have symbiotic microbes that degrade the matrix polysaccharides of plant matter. Herbivorous and omnivorous marine fish are similarly expected to rely on symbiotic microbes, but reports to date on cellulase-producing bacteria in fish intestines are limited. Here, we report the isolation of new cellulase-producing bacteria from the marine omnivorous teleost, blackfish (Girella melanichthys), and the characterization of cellulase activity. Three strains of cellulase-producing bacteria sp. were isolated from the hindgut of wild G. melanichthys. The strains of cellulase-producing bacteria grew in medium with artificial seawater but not in NaCl alone. Growth was optimum at 20-35°C, but there was no growth at 40°C, suggesting adaptation in a marine environment at a low temperature. Isolates were identified to Microbulbifer sp., among which GL-2 strain produced a high enzyme activity. The GL-2 strain was further used for enzyme characterization with carboxymethyl cellulose (CMC) as the substrate. Maximum activity of the cellulase was observed at 60°C, and activity was more than 30% at 20°C, while commercial cellulase Enthiron showed an optimum activity at 50°C and 17% activity at 20°C. Hydrolytic products by GL-2 cellulase were cellobiose but not glucose, suggesting a deficiency of β-glucosidase activity. Active gel electrophoresis containing CMC showed five bands, suggesting several cellulolytic enzymes. The GL-2 strain and its enzyme are potential probiotics for aquaculture fish and the industrial production of cellobiose.

DOI： 10.2323/jgam.2020.05.001

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Microbial Ecology  

DOI： 10.1264/jsme2.me20150

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Informa {UK} Limited  

DOI： 10.1080/10643389.2019.1692611

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

<title>ABSTRACT</title>
<italic>Microbulbifer</italic> sp. strain GL-2 was isolated from the intestine of a teleost, <italic>Girella melanichthys.</italic> Here, we report the complete genome sequence of this strain, which produces cellulase(s). Twelve cellulase candidate genes were found on the chromosome.

DOI： 10.1128/MRA.00746-20

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Marine aquaculture fish and the environment are possible hot spots for the maintenance and spread of antibiotic resistance genes (ARGs). We here show the time courses of changes of six tetracycline resistance genes (tet) in fish rearing seawater and fish intestine in tank experiments. Experimental tanks were prepared as oxytetracycline (OTC) administration tanks and those without OTC. It was found that tet(B), tet(M), and tet(W) were dominant in seawater among the six tet genes. tet(B) and tet(M) abundances increased immediately after OTC administration, indicating that OTC served as a selective pressure to increase the proportion of tet-possessing bacteria. In contrast, the abundance of tet genes in the fish intestine did not differ between the with- and without-OTC administration groups, and clearly was not altered by OTC administration. Profile changing of tet in seawater and fish intestine did not synchronize. These observations suggested that the dynamics of intestinal tet-possessing bacteria do not directly reflect the environment, but reflect selection within the intestine.

DOI： 10.3389/fmicb.2020.01764

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

<title>ABSTRACT</title>Biofilms in water environments are thought to be hot spots for horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). ARGs can be spread via HGT, though mechanisms are known and have been shown to depend on the environment, bacterial communities and mobile genetic elements. Classically, HGT mechanisms include conjugation, transformation and transduction; more recently, membrane vesicles (MVs) have been reported as DNA reservoirs implicated in interspecies HGT. Here, we review the current knowledge on the HGT mechanisms with a focus on the role of MVs and the methodological innovations in the HGT research.

DOI： 10.1093/femsec/fiaa031

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Other Link： http://academic.oup.com/femsec/article-pdf/96/5/fiaa031/33144680/fiaa031.pdf

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Plankton Society of Japan/The Japanese Association of Benthology  

DOI： 10.3800/pbr.14.276

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.scitotenv.2019.06.304

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

© 2019 Elsevier B.V. The use of antibiotics in aquaculture causes selection pressure for antibiotic-resistant bacteria (ARB). Antibiotic resistance genes (ARGs) may persist in ARB and the environment for long time even after stopping drug administration. Here we show monthly differences in the occurrences of genes conferring resistance to sulfonamides (i.e. sul1, sul2, sul3), and tetracyclines (tet(M)) in Japanese aquaculture seawater accompanied by records of drug administration. sul2 was found to persist throughout the year, whereas the occurrences of sul1, sul3, and tet(M) changed month-to-month. sul3 and tet(M) were detected in natural bacterial assemblages in May and July, but not in colony-forming bacteria, thus suggesting that the sul3 was harbored by the non-culturable fraction of the bacterial community. Comparison of results from Taiwanese, Japanese, and Finnish aquaculture waters reveals that the profile of sul genes and tet(M) in Taiwan resembles that in Japan, but is distinct from that in Finland. To our knowledge, this work represents the first report to use the same method to compare the dynamics of sul genes and tet(M) in aquaculture seawater in different countries.

DOI： 10.1016/j.scitotenv.2019.03.111

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/mbo3.710

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/mbo3.710

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Microbial Ecology  

DOI： 10.1264/jsme2.me19099

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

File： Reprint Text1-s2.0-S0048969718310660-main.pdf

DOI： 10.1016/j.scitotenv.2018.03.305

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1128/genomeA.00297-18

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Language：English   Publishing type：Research paper (scientific journal)  

The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a 'non-conjugative' integrative element. To identify active MGEs in a Vibrio ponticus isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between Escherichia coli and Vibrio ponticus. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOBH group, ARGs, and a 13.8-kb integrative element Tn6283. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn6283 integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn6283 copy in the chromosome and that in the newly transferred pSEA1. Tn6283 is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer.

DOI： 10.1371/journal.pone.0198613

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media S.A.  

Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities) in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO) and 2-methoxyethanol (MTXE). The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution) DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube), protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In conclusion, the materials and method for measurements should be carefully selected in order to accurately determine the activities of microbial extracellular hydrolytic enzymes in aquatic ecosystems
especially, low protein binding materials should be chosen to use at overall processes of the measurement.

DOI： 10.3389/fmicb.2017.01952

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

Background The novel tandem genes mef(C) and mph(G) have been reported in marine bacteria in Japan. This paper aimed to characterise the extent of environmental distribution of mef(C) and mph(G) as well as their dissemination and persistence in aquatic bacterial communities. Methods Erythromycin-resistant bacteria were isolated from Japan, Taiwan and Thailand aquaculture sites. The mef(C)–mph(G) genes were detected by PCR. The size of mobile genetic elements conveying mef(C) and mph(G) was examined by Southern blotting. The conjugation rate was assessed by filter mating. Results The mef(C)–mph(G) tandem genes were distributed in erythromycin-resistant isolates from aquaculture seawater in Japan and northern Taiwan and in animal farm wastewater in Thailand. A total of 29 bacterial isolates were positive for mef(C)–mph(G). The genes were found on vectors of various sizes. Partial sequencing of the traI relaxase gene revealed homology with a pAQU1-like plasmid, an IncA/C-type plasmid and an SXT/R391 family integrative conjugative element (SRI) as vectors. Thirteen isolates (45%) were positive for traI(pAQU-IncA/C-SRI), whereas the others were negative. The traI(pAQU-IncA/C-SRI)-positive isolates exhibited a higher transfer frequency (10−4–10−5 transconjugants/donor) than traI(pAQU-IncA/C-SRI)-negative isolates (&lt
10−9). Conclusions These results suggest that mef(C)–mph(G) are coded on various vectors and are distributed among marine and wastewater bacteria in Asian countries. Vectors with traI(pAQU-IncA/C-SRI) play a role in the spread of mef(C)–mph(G).

DOI： 10.1016/j.jgar.2017.03.015

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media S.A.  

DOI： 10.3389/fmicb.2017.00014

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Microbial Ecology  

Extracellular DNA (exDNA) is released from bacterial cells through various processes. The antibiotic resistance genes (ARGs) coded on exDNA may be horizontally transferred among bacterial communities by natural transformation. We quantitated the released/leaked tetracycline resistance gene, tet(M) over time under grazing stress by ciliates and heterotrophic nanoflagellates (HNFs), and found that extracellular tet(M) (ex-tetM) increased with bacterial grazing. Separate microcosms containing tet(M)-possessing bacteria with ciliates or HNFs were prepared. The copy number of ex-tetM in seawater in the ciliate microcosm rapidly increased until 3 d after the incubation, whereas that in the HNF microcosm showed a slower increase until 20 d. The copy number of ex-tetM was stable in both cases throughout the incubation period, suggesting that extracellular ARGs are preserved in the environment, even in the presence of grazers. Additionally, ARGs in bacterial cells were constant in the presence of grazers. These results suggest that ARGs are not rapidly extinguished in a marine environment under grazing stress.

DOI： 10.1264/jsme2.ME17042

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media S.A.  

Aerobic anoxygenic phototrophic bacteria (AAnPB) rely on not only heterotrophic but also phototrophic energy gain. AAnPB are known to have high abundance in oligotrophic waters and are the major portion of the bacterial carbon stock in the environment. In a yearlong study in an aquaculture area in the Uwa Sea, Japan, AAnPB, accounted for 4.7 to 24% of the total bacteria by count. Since the cell volume of AAnPB is 2.23 � 0.674 times larger than the mean for total bacteria, AAnPB biomass is estimated to account for 10-53% of the total bacterial assemblage. By examining pufM gene sequence, a common phylogenetic AAnPB species was found in all sampling sites through the year. The common species and other season-specific species were phylogenetically close to unculturable clones recorded in the Sargasso Sea and Pacific Ocean. The present study suggests that the common species may be a cosmopolitan species with worldwide distribution that is abundant not only in the oligotrophic open ocean but also in eutrophic aquaculture areas.

DOI： 10.3389/fmicb.2016.01996

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Research Foundation  

Antimicrobials are widely used, not only for treating human infections, but also for treatment of livestock and in fish farms. Human habitats in Southeastern Asian countries are located in close proximity to aquatic environments. As such, the human populations within these regions are at risk of exposure to antimicrobial resistant bacteria, and thereby disseminating antimicrobial resistance genes (ARGs). In this study, we collected water samples from 15 sites (5 sites in Chao Phraya River, 2 sites at the mouth of Chao Phraya River, 3 sites in Ta Chin River, and 5 sites at city canals) and 12 sites (6 sites at city canals
2 sites at chicken farms
2 sites at pig farms
and 2 samples from sites at pig farms, which were subsequently treated at a biogas plant) in Thailand in 2013 and 2014, respectively. In total, 117 Aeromonas spp. were isolated from the water samples, and these organisms exhibited various antimicrobial susceptibility profiles. Notably, there was a significant correlation between the environmental concentration of tetracyclines and the rates of tetracycline resistance in the isolated Aeromonas spp.
however, both the concentration and rates of tetracycline resistance in samples derived from pig farms were higher than those of samples harvested from other aquatic environments. These findings suggest that the high concentrations of antimicrobials observed in these aquatic environments likely select for ARGs. Furthermore, they indicate that Aeromonas spp. comprise an effective marker for monitoring antimicrobial resistance in aquatic environments.

DOI： 10.3389/fmicb.2016.00710

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Microbial Ecology  

The persistence of the multi-drug resistance plasmids pAQU1 and IncFIB was examined in bacterial populations under very low selective pressure. We herein demonstrated that these plasmids stably remained not only in the original host, but also in a transconjugant, even after being in a non-culturable state. In seawater microcosms containing Photobacterium damselae 04Ya311 possessing pAQU1, no significant loss of pAQU1 was observed during a 30-d starvation period. The copy numbers of pAQU1 and IncFIB in E. coli were constant. The results of the present study suggest that these plasmids have the ability to remain among various bacteria under oligotrophic conditions with low antibiotic selection pressure.

DOI： 10.1264/jsme2.ME15122

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Verlag  

Antibiotic-resistant bacteria can be detected in pristine environments and animals. Tetracycline (TC) is frequently used for wide areas of veterinary medicine, which selects TC-resistant bacteria. The TC resistance genes are known from natural environments, and tet(M) is the broadest host range tet gene. Here, we report that TC-resistant bacteria and the TC resistance gene tet(M) were diverse in Adélie penguin intestines, even within a single penguin colony. Total bacterial counts were as high as 107 CFU g−1, and TC-resistant bacteria ranged from 1.4 × 102 to 6.6 × 103 CFU g−1 intestinal contents, which was 0–0.54 % of the total viable count. Phylogenetic affiliation of TC-resistant bacteria revealed a variety of Gram-positive and Gram-negative bacteria. The tet(M) gene was identified in 32.3 % of TC-resistant strains, and two tet(M) genotypes were identified within one penguin colony, suggesting various contamination origins of tet(M).

DOI： 10.1007/s00300-015-1732-x

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Other Link： http://link.springer.com/article/10.1007/s00300-015-1732-x/fulltext.html

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

The biodegradation of proteins in seawater requires various proteases which are commonly thought to be mainly derived from heterotrophic bacteria. We, however, found that protists showed a high protease activity and continuously produced trypsin-type enzymes. The free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium was isolated and used for microcosm incubation with different concentrations of killed bacteria as food for 10 days. The results showed that the co-existence of the ciliate with its associated bacterium produced a significant protease activity in both cell-associated and cell-free fractions while that in the associated bacterium only microcosm was negligible. The protease profiles are different between cell-associated and cell-free fractions, and a trypsin-type enzyme hydrolyzing Boc-Val-Leu-Lys-MCA was detected throughout the period in the presence of ciliates. This suggests that ciliates release proteases into the surrounding environment which could play a role in protein digestion outside cells. It has been previously suggested that bacteria are the major transformers in seawater. We here present additional data which indicates that protists, or at least ciliates with their specific enzymes, are a potential player in organic matter degradation in water columns.

DOI： 10.1016/j.marenvres.2015.06.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The aim of this study was to determine whether mef(C) and mph(G), originally found on the transferable multi-drug plasmid pAQU1 from Photobacterium damselae subsp. damselae isolated from seawater of a fish farm, are responsible for conferring macrolide resistance. Since these genes are localized head-to-tail on pAQU1 and only four nucleotides exist between them, the single- and combination-effect of these genes was examined. When mph(G) alone was introduced to Escherichia coli, the minimum inhibitory concentrations (MICs) against erythromycin, clarithromycin and azithromycin increased, whereas introduction of mef(C) alone did not influence macrolide susceptibility. Introduction of both mef(C) and mph(G) dramatically increased the MICs to the same three macrolides, i.e. &gt;512gml(-1), &gt;512gml(-1) and 128gml(-1) respectively. These results suggest that the macrolide phosphotransferase encoded by mph(G) is essential for macrolide resistance, while the efflux pump encoded by mef(C) is required for high-level macrolide resistance. The tandem-pair arrangements of the mef(C) and mph(G) genes were conserved on plasmids ranging in size from 240 to 350kb of the 22 erythromycin-resistant strains belonging to Vibrio and Photobacterium obtained from the fish farm. Sixteen of 22 plasmids ranged in size from 300 to 350kb. This is the first report of novel macrolide resistance genes originating from a marine bacterium.
Significance and Impact of the StudyIn this study, mef(C) and mph(G) were found to be novel macrolide-resistance genes, and this is the first report of macrolide-resistance genes originating from a marine bacterium. These genes may be responsible for previously reported cases of the emergence of erythromycin-resistant bacteria in aquaculture sites by an unknown mechanism. The introduction of the tandem arrangement of the mef(C) and mph(G) genes in Escherichia coli increased the MICs to erythromycin, clarithromycin and azithromycin, suggesting a novel mechanism conferring high-level macrolide resistance via combined expression of the efflux pump and macrolide phosphotransferase.

DOI： 10.1111/lam.12414

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Other Link： http://orcid.org/0000-0003-2347-616X

Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

Tributyltin (TBT) is one of the most toxic xenobiotics ubiquitous in the aquatic environment. Several reports have described the negative impact of TBT in living organisms, from bacteria to mammals. Over the world, TBT contamination has being described as a serious problem. Thus, it is imperative to decontaminate TBT polluted sites. Bioremediation strategies may constitute an alternative to conventional decontamination methods, benefiting from the microorganisms potential to metabolize xenobiotics. Several microorganisms among bacteria, fungus, and algae have been reported to possess the ability to resist and, in certain cases, degrade TBT in their simple and less toxic derivatives. Due their characteristics, some of those microorganisms have been used for bioremediation studies and to construct bioreporters to detect TBT in the environment. This review provides an overview regarding microbial TBT resistance, while focusing on TBT degradation and bioremediation. A comprehensive revision on the several applications of organotin compounds, adverse biological effects on living organisms, and information regarding the available TBT bioreporters is also included.

DOI： 10.1080/10643389.2014.924181

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Aerobic anoxygenic phototrophic bacteria (AAnPB) are bacteriochlorophyll a-containing photoheterotrophic organisms that rely on phototrophy and heterotrophy processes for energy acquisition. Here, we demonstrate the ubiquitous distribution of AAnPB in the Uwa Sea and their relatively larger cell size among members of the bacteria community. The contribution of AAnPB in terms of cell number to the total bacterial abundance ranged from 3.5 % to 7.9 %. The mean cell volume for AAnPB was 2.78 +/- A 0.72 times larger than the mean cell volume of the total bacteria. The ratio of the contribution of their biovolume to the total bacteria was estimated to be 9.7-22 %.

DOI： 10.1007/s10872-014-0267-z

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media S.A.  

Antibiotic resistant bacteria are ubiquitous in the natural environment. The introduction of e?uent derived antibiotic resistance genes (ARGs) into aquatic environments is of concern in the spreading of genetic risk. This study showed the prevalence of sulfonamide and tetracycline resistance genes, sul1, sul2, sul3, and tet(M), in the total bacterial assemblage and colony forming bacterial assemblage in river and estuarine water and sewage treatment plants (STP) in South Africa. There was no correlation between antibiotic concentrations and ARGs, suggesting the targeted ARGs are spread in a wide area without connection to selection pressure. Among sul genes, sul1 and sul2 were major genes in the total (over 10-2copies/16S) and colony forming bacteria assemblages (~10-1copies/16S). In urban waters, the sul3 gene was mostly not detectable in total and culturable assemblages, suggesting sul3 is not abundant. tet(M) was found in natural assemblages with 10-3copies/16S level in STP, but was not detected in colony forming bacteria, suggesting the non-culturable (yet-to-be cultured) bacterial community in urban surface waters and STP e?uent possess the tet(M) gene. Sulfamethoxazole (SMX) resistant (SMXr) and oxytetracycline (OTC) resistant (OTCr) bacterial communities in urban waters possessed not only sul1 and sul2 but also sul3 and tet(M) genes. These genes are widely distributed in SMXr and OTCr bacteria. In conclusion, urban river and estuarine water and STP e?uent in the Durban area were highly contaminated with ARGs, and the yet-to-be cultured bacterial community may act as a non-visible ARG reservoir in certain situations.

DOI： 10.3389/fmicb.2015.00796

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Tributyltin (TBT) is a biocide extremely toxic to a wide range of organisms, which has been used for decades in antifouling paints. Despite its global ban in 2008, TBT is still a problem of great concern due to the high levels trapped in sediments. Aeromonas molluscorum Av27 is a TBT degrading bacterium that was isolated from an estuarine system. We investigated the ability and the role of this bacterium on TBT degradation in this estuarine system, using a microcosm approach in order to mimic environmental conditions. The experiment was established and followed for 150 days. Simultaneously, changes in the indigenous bacterial community structure were also investigated. The results revealed a maximum TBT degradation rate of 28% accompanied by the detection of the degradation products over time. Additionally, it was observed that TBT degradation was significantly enhanced by the presence of Av27. In addition a significantly higher TBT degradation occurred when the concentration of Av27 was higher. TBT degradation affected the bacterial community composition as revealed by the changes in the prevalence of Proteobacteria subdivisions, namely the increase of Deltaproteobacteria and the onset of Epsilonproteobacteria. However, the addition of Av27 strain did not affect the dominant phylotypes. Total bacterial number, bacterial biomass productivity, 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analyses also indicated alterations on the bacterial community structure over time, with bacteria non-tolerant to pollutants increasing their representativeness, as, for instance, the increase of the number of Alphaproteobacteria clones from 6% in the beginning to 12% at the end of the experiment. The work herein presented confirms the potential of Av27 strain to be used in the decontamination of TBT-polluted environments. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.envres.2014.04.031

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS RESEARCH FOUNDATION  

Emergence of antibiotic-resistant bacteria in the aquaculture environment is a significant problem for disease control of cultured fish as well as in human public health. Conjugative mobile genetic elements (MGEs) are involved in dissemination of antibiotic resistance genes (ARGs) among marine bacteria. In the present study, we first designed a PCR targeting tral gene encoding essential relaxase for conjugation. By this new PCR, we demonstrated that five of 83 strains isolated from a coastal aquaculture site had tral-positive MGEs. While one of the five strains that belonged to Shewanella sp. was shown to have an integrative conjugative element of the SXT/R391 family (ICE VchMex-like), the MGEs of the other four strains of Vibrio spp. were shown to have the backbone structure similar to that of previously described in pAQU1. The backbone structure shared by the pAQU1-like plasmids in the four strains corresponded to a similar to 100-kbp highly conserved region required for replication, partition and conjugative transfer, suggesting that these plasmids constituted "pAQU group." The pAQU group plasmids were shown to be capable of conjugative transfer of tet(M) and other ARGs from the Vibrio strains to E. colt. The pAQU group plasmid in one of the examined strains was designated as pAQU2, and its complete nucleotide sequence was determined and compared with that of pAQU1. The results revealed that pAQU2 contained fewer ARGs than pAQU1 did, and most of the ARGs in both of these plasmids were located in the similar region where multiple transposases were found, suggesting that the ARGs were introduced by several events of DNA transposition into an ancestral plasmid followed by drug selection in the aquaculture site. The results of the present study indicate that the "pAQU group" plasmids may play an important role in dissemination of ARGs in the marine environment.

DOI： 10.3389/fmicb.2014.00152

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Persistence and dispersal of antibiotic resistance genes (ARGs) are important factors for assessing ARG risk in aquaculture environments. Here, we quantitatively detected ARGs for sulphonamides (sul1 and sul2) and trimethoprim (dfrA1) and an integrase gene for a class 1 integron (intI1) at aquaculture facilities in the northern Baltic Sea, Finland. The ARGs persisted in sediments below fish farms at very low antibiotic concentrations during the 6-year observation period from 2006 to 2012. Although the ARGs persisted in the farm sediments, they were less prevalent in the surrounding sediments. The copy numbers between the sul1 and intI1 genes were significantly correlated suggesting that class 1 integrons may play a role in the prevalence of sul1 in the farm sediments through horizontal gene transfer. In conclusion, the presence of ARGs may limit the effectiveness of antibiotics in treating fish illnesses, thereby causing a potential risk to the aquaculture industry. However, the restricted presence of ARGs at the farms is unlikely to cause serious effects in the northern Baltic Sea sediment environments around the farms.

DOI： 10.1371/journal.pone.0092702

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Language：English   Publisher：Oceanographic Society of Japan  

DOI： 10.1007/s10872-013-0212-6

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Language：English   Publisher：Oceanographic Society of Japan  

DOI： 10.1007/s10872-013-0216-2

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media S. A  

Proteins constitute the major portion of labile substances in the marine environment and are an important source of organic matter supporting marine ecosystems. However, previous studies have revealed that specific bacterial membrane proteins are refractory in the oceans. We here show by kinetic analyses of protease degradation activity using inactivated Pseudomonas aeruginosa (Pa) cells as a proteinaceous substrate that bacterial proteases are insufficient to completely hydrolyze proteins, which may partially cause the protein accumulation in seawater. Protease activity was monitored simultaneously in 8 microcosms subjected to differing conditions. Some Pa proteins were retained for 30 days in the presence of bacteria without protists, whereas the Pa proteins were completely disappeared in the presence of both, indicating that these proteins were substantially incorporated into protist biomass. Our result suggests that protists play an important role in the transformation of bacterial proteins in seawater. Our experiments also imply that the functional/taxonomic diversity should be taken into account when considering decomposition activity in marine environments.

DOI： 10.3389/fmars.2014.00069

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Other Link： http://orcid.org/0000-0003-4041-100X

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Aeromonas molluscorum Av27 is an estuarine bacterium highly resistant to tributyltin (TBT). Also, the strain is able to degrade TBT into the less toxic compounds dibutyltin and monobutyltin. Therefore, this bacterium has potential to be employed in bioremediation processes. In this context, defining its biological safety is crucial. With that purpose a number of intrinsic characteristics, usually present/associated with virulent strains, were investigated. Few virulence factors were detected in strain Av27. For instance, a DNase gene is present, but it is not apparently expressed in vitro. Motility, adherence factor and phospholipase activity were also detected. Additionally, cytotoxicity to Vero cells was negative. Resistance to penicillin (10 mu g ml(-1)), amoxicillin/clavulanic acid (30 mu g ml(-1)) and cephalothin (30 mu g ml(-1)) and also to the vibriostatic agent O/129 was observed. Five plasmids (4, 7, 10, 100 kb and one greater than 100 kb) were identified. No Class I and II integrons were detected. Study of the optimal growth conditions showed that Av27 easily adapts to different environmental conditions. Overall, the results suggest that A. molluscorum Av27 can be considered safe to use to bioremediate TBT in contaminated environments.

DOI： 10.1007/s10482-013-9961-x

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Language：English   Publisher：US DEPT HEALTH HUMAN SCIENCES PUBLIC HEALTH SCIENCE  

BACKGROUND: There is growing concern worldwide about the role of polluted soil and water environments in the development and dissemination of antibiotic resistance.
OBJECTIVE: Our aim in this study was to identify management options for reducing the spread of antibiotics and antibiotic-resistance determinants via environmental pathways, with the ultimate goal of extending the useful life span of antibiotics. We also examined incentives and disincentives for action.
METHODS: We focused on management options with respect to limiting agricultural sources; treatment of domestic, hospital, and industrial wastewater; and aquaculture.
DISCUSSION: We identified several options, such as nutrient management, runoff control, and infrastructure upgrades. Where appropriate, a cross-section of examples from various regions of the world is provided. The importance of monitoring and validating effectiveness of management strategies is also highlighted. Finally, we describe a case study in Sweden that illustrates the critical role of communication to engage stake-holders and promote action.
CONCLUSIONS: Environmental releases of antibiotics and antibiotic-resistant bacteria can in many cases be reduced at little or no cost. Some management options are synergistic with existing policies and goals. The anticipated benefit is an extended useful life span for current and future antibiotics. Although risk reductions are often difficult to quantify, the severity of accelerating worldwide morbidity and mortality rates associated with antibiotic resistance strongly indicate the need for action.

DOI： 10.1289/ehp.1206446

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

With an increasing need for assessing the risk of aquaculture antibiotics, there has been growing interest in their fate and effect on sedimentary bacteria. Here we show the risk assessment for oxytetracycline (OTC) use in seawater and its subsequent transfer to sediment, and illustrate that the sediment bacterial community was stable against OTC at dosed concentrations. Water-sediment microcosm experiments were conducted to simulate quiescent aquaculture conditions. The sorption coefficient (K-d) was 12.3-44.2 mL/g, which is lower than the previous reports employing vigorous mixing. In a denaturing gradient gel electrophoresis (DGGE) analysis, the addition of OTC at 50 mu g/L into the water phase had little effect on the major sediment bacterial community structure. This finding suggests that low concentrations of OTC in the water phase - such as those used within many aquaculture operations - do not pose a high risk of causing major changes in environmental sediment bacterial community structures. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.etap.2013.03.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Seven sulfonamides, trimethoprim, five macrolides, lincomycin and three tetracyclines were measured in 150 water samples of sewage, livestock and aquaculture wastewater, and river and coastal waters, in five tropical Asian countries. The sum of the concentrations of the target antibiotics in sewage and heavily sewage-impacted waters were at sub- to low-ppb levels. The most abundant antibiotic was sulfamethoxazole (SMX), followed by lincomycin and sulfathiazole. The average concentration of SMX in sewage or heavily sewage-impacted waters was 1720 ng/L in Vietnam (Hanoi, Ho Chi Minh, Can Tho; n=15), 802 ng/L in the Philippines (Manila; n=4), 538 ng/L in India (Kolkata; n=4), 282 ng/L in Indonesia (Jakarta; n=10), and 76 ng/L in Malaysia (Kuala Lumpur; n=6). These concentrations were higher than those in Japan, China, Europe, the US and Canada. A predominance of sulfonamides, especially SMX, is notable in these tropical countries. The higher average concentrations, and the predominance of SMX, can be ascribed to the lower cost of the antibiotics. Both the concentration and composition of antibiotics in livestock and aquaculture wastewater varied widely. In many cases, sulfamethazine (SMT), oxytetracycline (OTC), lincomycin, and SMX were predominant in livestock and aquaculture wastewater. Both human and animal antibiotics were widely distributed in the respective receiving waters (i.e., the Mekong River and Manila Bay). SMT/SMX ratios indicate a significant contribution from livestock wastewater to the Mekong River and nearby canals, with an estimated similar to 10% of river water SMX derived from such wastewater. Mass flow calculations estimate that 12 tons of SMX is discharged annually from the Mekong River into the South China Sea. Riverine inputs of antibiotics may significantly increase the concentration of such antibiotics in the coastal waters. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scitotenv.2013.02.027

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We analyzed polychlorinated biphenyls (PCBs), dichlorodiphenyl dichloroethane and its metabolites, hexachlorocyclohexanes (HCHs), polycyclic aromatic hydrocarbons (PAHs), and hopanes, in plastic resin pellets collected from nine locations along the Portuguese coast. Concentrations of a sum of 13 PCBs were one order of magnitude higher in two major cities (Porto: 307. ng/g-pellet
Lisboa: 273. ng/g-pellet) than in the seven rural sites. Lower chlorinated congeners were more abundant in the rural sites than in the cities, suggesting atmospheric dispersion. At most of the locations, PAH concentrations (sum of 33 PAH species) were ∼100 to ∼300. ng/g-pellet
however, three orders of magnitude higher concentrations of PAHs, with a petrogenic signature, were detected at a small city (Sines). Hopanes were detected in the pellets at all locations. This study demonstrated that multiple sample locations, including locations in both urban and remote areas, are necessary for country-scale pellet watch. © 2013 Elsevier Ltd.

DOI： 10.1016/j.marpolbul.2013.02.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Research Foundation  

DOI： 10.2323/jgam.59.47

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We developed a simple aquatic microbial ecosystem model in order to examine potential effects of zinc on microorganisms and the related degradation of dissolved organic matter (DOM). The model is a combination of both a traditional food chain and a microbial loop. The traditional food chain is mainly composed of phytoplankton and zooplankton, whilst the microbial loop is composed of DOM, bacteria and bacterivorous protozoa. We incorporated the suppressive effect of zinc on the bacterial uptake of DOM and assessed the steady state responses of the model for various zinc concentrations. The analytical and numerical results of the model implied that either zooplankton or bacterivorous protozoa might be the most vulnerable group to excessive zinc load than bacteria, depending on the grazing preference of zooplankton between phytoplankton and bacterivorous protozoa. The sensitivity analyses supported that the microbial loop solely is more sensitive to zinc than the coupled system combining both the traditional food chain and the microbial loop. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2012.09.014

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Research Foundation  

Recent evidence has shown that antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are ubiquitous in natural environments, including sites considered pristine. To understand the origin of ARGs and their dynamics, we must first define their actual presence in the natural bacterial assemblage. Here we found varying distribution profiles of sul genes in "colony forming bacterial assemblages" and "natural bacterial assemblages." Our monitoring for antibiotic contamination revealed that sulfamethoxazole (SMX) is a major contaminant in aquatic environments of Metro-Manila, which would have been derived from human and animal use, and subsequently decreased through the process of outflow from source to the sea. The SMX-resistant bacterial rate evaluated by the colony forming unit showed 10 to 86% of the total colony numbers showed higher rates from freshwater sites compared to marine sites. When sul genes were quantified by qPCR, colony-forming bacteria conveyed sul1 and sul2 genes in freshwater and seawater (10-5-10-2 copy/16S) but not sul3. Among the natural bacterial assemblage, all sul1, sul2, and sul3 were detected (10-5-10-3 copy/16S), whereas all sul genes were at an almost non-detectable level in the freshwater assemblage. This study suggests that sul1 and sul2 are main sul genes in culturable bacteria, whereas sul3 is conveyed by non-culturable bacteria in the sea. As a result marine bacteria possess sul1, sul2 and sul3 genes in the marine environment. © 2013 Suzuki, Ogo, Miller, Shimizu, Takada and Siringan.

DOI： 10.3389/fmicb.2013.00102

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INTER-RESEARCH  

Protein biodegradation in the marine environment is caused by proteases derived from various organisms, including bacteria, which are considered to be a major source of these enzymes. We investigated the succession of bacterial proteases in seawater to determine the variation in protease activity over time. The potential activities of proteolytic enzymes in stored seawater and isolated bacteria were studied using 19 different synthetic oligopeptide substrates for aminopeptidase, trypsin, chymotrypsin and elastase. In time-course experiments carried out over 112 d, aminopeptidase activity increased, whereas trypsin activity decreased over time. Amino peptidase activity was mainly found in unfiltered seawater containing bacterial cells, whereas trypsin activity was mainly found in 0.2 mu m seawater filtrates. Individual bacterial isolates showed different proteolytic properties but all exhibited aminopeptidase activity. Members of the Gammaproteobacteria and Bacteroidetes showed high trypsin and chymotrypsin activities. Based on these results, we conclude that protein degradation in seawater occurs via the combined action of various bacterial proteases.

DOI： 10.3354/ame01618

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Vanadium is a contaminant from steel additive and ship fuel in coastal and port areas, and its effect on marine microbes remains largely unknown. We showed that vanadium accelerates transfer of the tetracycline resistance gene tet(M) from Photobacterium to Escherichia coli, and found a positive correlation between the concentration of vanadium in natural marine sediment and the rate of oxytetracycline resistance. These results suggest the possibility that vanadium may play a role in the preservation and horizontal transfer of antibiotic resistance genes in the marine environment.

DOI： 10.1111/j.1574-6968.2012.02653.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The effect of tributyltin (TBT) on growth and metabolic activity of three estuarine bacteria with different TBT resistance profiles was investigated in an organic-rich culture medium (TSB) and in phosphate buffered saline (PBS) buffer. Exposure to TBT was assessed by determining its effect on growth (OD600 nm measurement), bacterial productivity (leucine incorporation), viability (CFU counts), aggregation and cell size (from Live/Dead analysis), ATP and NADH concentrations. TBT exposure resulted in decrease of bacterial density, cell size, and metabolic activity. In addition, cell aggregates were observed in the TBT-treated cultures. TBT strongly affected bacterial cell metabolism and seemed to exert an effect on its equilibrium, interfering with cell activity. Also, TBT toxicity was lower when cells were grown in TSB than in PBS, suggesting that a nutrient-rich growth medium can protect cells from TBT toxicity. This study contributes to our understanding of the TBT-resistant cell behavior reflected in its physiology and metabolic activity. This information is of utmost importance for further studies of TBT bioremediation. (C) 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

DOI： 10.1002/tox.20605

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS RESEARCH FOUNDATION  

Southeast Asia has become the center of rapid industrial development and economic growth. However, this growth has far outpaced investment in public infrastructure, leading to the unregulated release of many pollutants, including wastewater-related contaminants such as antibiotics. Antibiotics are of major concern because they can easily be released into the environment from numerous sources, and can subsequently induce development of antibiotic-resistant bacteria. Recent studies have shown that for some categories of drugs this source-to-environment antibiotic resistance relationship is more complex. This review summarizes current understanding regarding the presence of quinolones, sulfonamides, and tetracyclines in aquatic environments of Indochina and the prevalence of bacteria resistant to them. Several noteworthy findings are discussed: (1) quinolone contamination and the occurrence of quinolone resistance are not correlated; (2) occurrence of the su/ sulfonamide resistance gene varies geographically; and (3) microbial diversity might be related to the rate of oxytetracycline resistance.

DOI： 10.3389/fmicb.2012.00067

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOL RES FOUNDATION  

A tributyltin (TBT) resistance gene was isolated from the TBT-resistant marine origin bacterium Pseudomonas aeruginosa 25W. This gene was identical to PA0320 deposited in the P. aeruginosa PAO1 database (http://www.pseudomonas.com). The deduced amino acid sequence of PA0320 appears to be homologous to the YgiW proteins of Escherichia coli and Salmonella enterica. The deletion mutant of PA0320 showed a reduction of growth rate in the presence of TBT. A susceptibility test to cadmium, mercury, hydrogen peroxide and acidic pH in the deletion mutant showed an increasing susceptibility to them. PA0320 plays a certain role in stress tolerance against TBT as well as in stressors producing reactive oxygen species.

DOI： 10.2323/jgam.58.283

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The ubiquitous application and release of antibiotics to the environment can result in bacterial antibiotic resistance, which in turn can be a serious risk to humans and other animals. Southeast Asian countries commonly apply an integrated recycling farm system called VAC (Vegetable, Aquaculture and Caged animal). In the VAC environment, antibiotics are released from animal and human origins, which would cause antibiotic-resistant bacteria (ARB). This study evaluated occurrence of ARB in the VAC environment in northern Vietnam, with quantitative analysis of antibiotic pollution. We found that sulfonamides were commonly detected at all sites. In dry season, while sulfamethazine was a major contaminant in pig farm pond (475-6662 ng/l) and less common in city canal and aquaculture sites, sulfamethoxazole was a major one in city canal (612-4330 ng/l). Erythromycin (154-2246 ng/l) and clarithromycin (2.8-778 ng/ml) were the common macrolides in city canal, but very low concentrations in pig farm pond and aquaculture sites. High frequencies of sulfamethoxazole-resistant bacteria (2.14-94.44%) were found whereas the occurrence rates of erythromycin-resistant bacteria were lower (&lt;0.01-38.8%). A positive correlation was found between sulfamethoxazole concentration and occurrence of sulfamethoxazole-resistant bacteria in dry season. The sulfamethoxazole-resistant isolates were found to belong to 25 genera. Acinetobacter and Aeromonas were the major genera. Twenty three of 25 genera contained sul genes. This study showed specific contamination patterns in city and VAC environments and concluded that ARB occurred not only within contaminated sites but also those less contaminated. Various species can obtain resistance in VAC environment, which would be reservoir of drug resistance genes. Occurrence of ARB is suggested to relate with rainfall condition and horizontal gene transfer in diverse microbial community. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scitotenv.2011.04.030

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE  

Fluoroquinolone antibiotics (FQs) have been used worldwide for chemotherapy, animal husbandry, and aquaculture, and the occurrence of FQ-resistant (FQs(r)) bacteria in natural environments has been reported. Plasmid-mediated transferable quinolone resistance (PMQR) genes are suspected to originate from the chromosomes of water-dwelling bacteria. However, the occurrence of and the potential reservoir of FQs(r) bacteria and PMQR genes in aquatic environments have not been elucidated. In this study, we detected FQs(r) bacteria and PMQR genes in aquatic environments in Thailand and Vietnam, and measured FQ contamination. Levels of contamination were greater Thailand (avg. 5130, max 46100 ng L(-1)) than in Vietnam (avg. 235, max 1130 ng L(-1)); however, the occurrence of FQs(r) bacteria was higher in Vietnam (similar to 15%) than in Thailand (similar to 7.0%), suggesting that contamination by FQs is not directly linked to the development of FQs(r) bacteria. Diverse taxonomic groups of FQs(r)-bacteria were identified, and one of the PMQR genes, qnrB, was detected from bacteria of environmental origin, not enteric bacteria. This suggests that the environmental bacteria are a potential reservoir of antibiotic resistance determinants even at un-contaminated sites.

DOI： 10.1264/jsme2.ME10204

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The prophylactic and therapeutic use of tetracyclines in aquaculture has been shown to contribute to the spread of tetracycline resistance in the environment In this work, the prevalence of four different tetracycline-resistance genes, tetA, tetC, tetH, and tetM, in sediments from four aquaculture farms and their surroundings in the Baltic Sea was monitored by quantitative polymerase chain reaction (qPCR). The presence of three additional tetracycline-resistance genes (tetE, tetG, and tetW) was studied qualitatively by standard PCR, and the amount of bioavailable tetracyclines and total amounts of tetracycline and oxytetracycline in samples were also measured. None of the farms were using tetracycline at the time of the sampling and one of the farms had stopped all antibiotic use six years prior to the first sampling. Two of the farms were sampled over four successive summers and two were sampled once. Our results showed greater copy numbers of tetA, tetC, tetH, and tetM at the farms compared to pristine sites and demonstrated the presence of tetE, tetG, and tetW genes in the sediments under aquaculture farms at most sampling times. However, no resistance genes were found in samples collected 200 m from any of the farms. None of the samples contained therapeutically active concentrations of tetracyclines at any of the sampling times, suggesting that the increase in the prevalence of tetracycline resistance genes is caused by the persistence of these genes in the absence of selection pressure.

DOI： 10.1021/es102725n

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INTER-RESEARCH  

To understand the role of zinc (Zn) in the biogeochemical cycle in coastal environments, we examined the bacterial remineralisation of dissolved organic matter (DOM) with 2 composite experiments using microcosms supplemented with Zn. In Expt 1, using samples collected from 2 stations in the Seto Inland Sea, Japan, we found that a decrease in DOM due to bacterial remineralisation during a 14 d experimental period had negative responses to Zn at both sites, but we found an inhibitory effect on bacterial abundance only at a station in the western part of the Seto Inland Sea. In Expt 2, comparison of the response of the remineralisation process to Zn among 3 kinds of organic substrate showed that Zn has little effect on 2 authentic standards (laminarin and bovine serum albumin) and that remineralisation of DOM originating from natural seawater was significantly suppressed by the addition of Zn. Based on the regression curves, we estimated the potential impact of Zn on the remineralisation of DOM. At a water quality standard of Zn concentration (86 mu g Zn l(-1)), DOM concentrations at the end of the experimental period (Day 14) increased 2.4 to 6.9%, and turnover time prolonged with a timescale of weeks to months. These potential shifts induced by Zn suggest that the allochthonous input of Zn into the coastal environment leads to suppression of energy flow in the microbial loop and enhances transport of DOM from coastal to offshore areas.

DOI： 10.3354/ame01481

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Microbial degradation and utilization of proteins derived from bacterial detritus were investigated in a microcosm experiment using Pseudomonas aeruginosa detritus as a substrate. To assess the effects of natural marine microbial communities on degradation and utilization of protein derived from P. aeruginosa cells, four microcosms were prepared: natural seawater (containing the natural microbial community) with P. aeruginosa detritus (N+Pa), autoclaved seawater with P. aeruginosa detritus (A+Pa), natural seawater (N) and autoclaved seawater (A) without adding anything as a control. The numbers of total and growing bacterial cells, protease activity, and transition of P. aeruginosa proteins were monitored in the four microcosms. Changes in the numbers of total and growing bacterial cells and protease activities indicated that bacterial detritus significantly stimulated the microbial community in the microcosms. Both the surviving P. aeruginosa in A+Pa and natural microbial community in N+Pa microcosms were able to degrade and utilize P. aeruginosa detritus; however, the community in N+Pa including various microbes maintained high activity longer, indicating that diversity is an important factor in keeping the community active. Even under the very high protease activity in N+Pa, 39-kDa and 48-kDa proteins from P. aeruginosa remained in the microcosm during the entire experiment (150 days). Immunoblotting suggested the 48-kDa protein was an intact molecule of OprP, which had been detected from the dissolved fraction of natural seawater in previous studies. This result suggests that the protein molecules that had been detected from natural seawater actually had a high tolerance to microbial degradation.

DOI： 10.1007/s10872-010-0043-7

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

P&gt;Pseudomonas aeruginosa is an opportunistic pathogen, and ubiquitously found in natural environments. However, details on difference between clinical and environmental isolates have not been reported enough. In this study, we defined existence of marine specific genogroup and different drug susceptibility among isolates from clinical, river and coastal seawaters. Pseudomonas aeruginosa were isolated by using cetrimide kanamycin nalidixic acid agar media and incubation at 42 degrees C, which was specific selection method of this bacterium from the natural aquatic samples. Pulse field gel electrophoresis analysis showed that the levels of genetic variation within P. aeruginosa were different among environmental sites. Pulse field gel electrophoresis also showed a lower diversity within P. aeruginosa in the coastal waters; and coastal strains isolated different sampling points were positioned closely in the same cluster. Most of the aquatic isolates were sensitive to most of the drugs tested and 'intermediate' to panipenem on the contrast to the clinical isolates, suggesting that the clinical use of antibiotics affect significantly to the emergence of the drug-resistant P. aeruginosa.

DOI： 10.1111/j.1758-2229.2010.00178.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Samples of polyethylene pellets were collected at 30 beaches from 17 countries and analyzed for organo-chlorine compounds. PCB concentrations in the pellets were highest on US coasts, followed by western Europe and Japan, and were lower in tropical Asia, southern Africa and Australia. This spatial pattern reflected regional differences in the usage of PCBs and was positively correlated with data from Mussel Watch, another monitoring approach. DDTs; showed high concentrations on the US west coast and in Vietnam. In Vietnam, DDT was predominant over its metabolites (DDE and DDD), suggesting the principal source may be current usage of the pesticide for malaria control. High concentrations of pesticide HCHs were detected in the pellets from southern Africa, suggesting current usage of the pesticides in southern Africa. This study demonstrates the utility and feasibility of the International Pellet Watch approach to monitor POPs at a global scale. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.marpolbul.2009.06.014

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The effect of water-surface discharge on the inactivation of Bacillus subtilis ATCC6633 in water was examined by using a very short high-voltage pulse generator. The surviving number of spore cells at 10(4) CFU/ml in initial concentration exponentially decreased with increasing discharge-treatment time. The input energy into the water-surface discharge under an O-2 gas flow for reduction in the survival number to 10% was lower than that under an air flow because many oxidation agents such as ozone and OH radical were produced under the O-2 gas flow.
The input energy density for the one-tenth reduction depended not only on the spore state but also on the initial cell concentration. The input energy for the high-concentration spore cells (10(7) CFU/ml) was much higher than that for the low-concentration spore cells (10(4) CFU/ml). Cellular proteins and DNA were degraded by a 30-min discharge treatment of vegetative cells, whereas DNA of the high-concentration spore cells was relatively resistant.

DOI： 10.1271/bbb.90153

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10(-7) to 10(-3); however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10(-6) to 10(-5); however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids, while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner.

DOI： 10.1007/s11274-009-0004-8

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOLOGICAL SOCIETY KOREA  

Distribution of marine type of Aquabirnavirus (MABV) was examined in shellfish and fish from Okinawa and Ishigaki Islands, Japan, where water temperature is higher than 25A degrees C through the year. Genome detection and virus isolation were performed for shellfish and fish samples, and the results revealed the prevalent distribution of MABV in diverse species in the area, although isolation was not frequently. Detection rate of MABV genome in bivalves was higher than gastropods, which was similar result to former report in mainland of Japan. Furthermore, the unique five-nucleotide deletion was found with a high rate of occurrence in the MABV genome from shellfish and fish. This study showed distribution status of MABV in organisms in subtropical waters by wide monitoring, and discovered new genome variation in VP2/NS region of this virus.

DOI： 10.1007/s12275-008-0250-8

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To assess the presence and distribution of the sul genes (sul1, sul2, and sul3) and plasmids in human-mediated environments of north Vietnam, we examined a total of 127 sulfonamide-resistant (SR) bacterial isolates from four shrimp ponds (HNAQs), a city canal (HNCs) and three fish ponds that received wastewater directly from swine farms (HNPs). Results from the SR isolates revealed that sul genes were most frequently detected in the HNPs (92.0%), followed by HNCs (72.0%), and the HNAQs (43.0%). Among the sul genes detected, sul1 was the most prevalent gene in all three environments (57.0,33.0 and 60.0% in HNPs, HNAQS, and HNCs, respectively) followed by sul2 (51.0, 19.0, and 20.0%, respectively) and sul3 (14.0, 6.0, and 8.0%, respectively). All combinations of paired different sul genes were detected, with the combination between sul1 and sul2 being the most frequent in all three environments (20.0, 8.0, and 8.0% in HNPs, HNAQs, and HNCs, respectively). The combination of three sul genes was detected at low frequencies (2-3%) in the HNPs and HNAQs, and was absent in the HNCs. The sul genes were more frequently located on the chromosome than on plasmids. The identification of SR isolates positive for the sul genes and plasmids showed that Acinetobacter was the most dominant. our study revealed that the sul genes were common in SR bacteria from the aquatic environments we examined from northern Vietnam. Wastewater from swine farms might be "hot spots" of the sul genes and plasmids and may be reservoirs for the exchange of the sul genes among bacteria. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scitotenv.2008.06.023

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

DOI： 10.1093/jac/dkn209

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The tetracycline (TC) resistance gene team) was monitored in bacteria isolated from Japanese coastal and off-shore marine sediments. The high rate of occurrence of TC resistant (TC(r)) bacteria (120 mu g mL(-1) TC) was observed at frequency ranges between 0.0-0.08% in Tokyo Bay, 1.67-1.82% in Sagami Bay and 0.0-4.35% in the open Pacific Ocean. The tet(M) gene was PCR amplified from the TCr isolates, showing 127 of 209 isolates (60.8%) as positive. The rate of occurrence of tet(M)) was between 32.0-96.0%, 21.1-28.0% and 0.0-83.3% in the isolates from Tokyo Bay, Sagami Bay and the open Pacific Ocean, respectively. The team) positive isolates belonged to 4 orders of bacteria. Bacillales was the most dominant order (121 strains) among team) possessing bacteria, followed by Actinomycetales (three strains), Flavobacteriales (one strain) and Pseudomonadales (one strain). This indicates that team) is present in various bacterial species and suggests that marine sediments are a natural reservoir of the team) gene. Nucleotide sequence of the team) revealed that two genotypes of team) were found in the bacteria. The two genotypes were placed in genetically distant branches of the phylogenetic tree, suggesting that the two tet(M)s have different origins.

DOI： 10.1021/es702986y

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

In aquatic environments extracellular enzymes are bound to microbial cells or exist in a free and adsorbed state. Various filters have been used to fractionate these enzymatic activities, but enzymes may be readily adsorbed onto some materials, and such adsorption can induce errors in the estimation of enzymatic activity. In this study we examined three filters to determine the most suitable filter for fractionation when estimating proteolytic enzyme activity in seawater. We found that the polycarbonate Nuclepore membrane, widely used for size fractionation because of its pore-size accuracy, was the most favorable for this purpose, even though it adsorbed slightly more enzymes than the low-protein-binding polyethersulfone membrane. We also found that trypsin- and chymotrypsin-type enzymes were more easily adsorbed than aminopeptidases.

DOI： 10.1007/s10872-008-0029-x

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

The coding regions for the major epitopes of structural protein VP2 (vp2e) and structural protein VP3 were amplified from marine birnavirus (MABV) cDNA and efficiently expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. Polyclonal antibodies against VP2e and VP3 were raised in rabbits and fish using the purified proteins of GST/VP2e and GST/VP3. The rabbit anti-serum against VP3 was more sensitive than the rabbit anti-VP2e serum in detecting virus in MABV-infected fish, while fish anti-VP2e serum showed a stronger neutralization response than fish anti-VP3 serum.

DOI： 10.1111/j.1365-2761.2008.00909.x

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INTER-RESEARCH  

We analyzed potential activities of different proteolytic enzymes in size-fractionated seawater, and estimated the contribution of each size fraction (&lt; 0.2, 0.2-0.8, 0.8-5, and &gt; 5 mu m) to the bulk hydrolytic activity of each enzyme in the seawater. The activity of leucine-aminopeptidase was highly attributed to cell-associated size fractions, while the contribution of the dissolved fraction (&lt; 0.2 mu m) to the bulk activity was only 10 to 30 %. In contrast, the contribution of the dissolved fraction to the activities of the trypsin- and chymotrypsin-type endopeptidases was as high as 40 to 80 % of their bulk activities measured in unfiltered seawater. These results indicated the potential importance of free proteolytic enzymes in seawater, especially for endopeptidases. Significant enzymatic activity in the dissolved fraction was also detected from experiments with isolated bacteria, suggesting that direct secretion of proteases from marine bacteria into surrounding water could be at least one of the sources of the dissolved proteolytic enzymes in seawater. Generally, the natural bacterial community of seawater was able to hydrolyze all of the 16 tested substrates, but at different rates. Selected members of the community (3 bacterial isolates and Synechococcus) hydrolyzed only one or a few of the applied substrates, and the substrate preference varied among the strains. These results suggest that natural bacterial communities are composed of a great variety of bacterial species with different (specific) enzymatic properties, including dissolved endopeptidases and cell-associated aminopeptidase. The combined activities of these enzymes are responsible for an effective degradation and re-use of high molecular weight organic matter at the community level.

DOI： 10.3354/ame01169

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.marenvres.2007.06.006

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE  

Spatial monitoring of tetracycline (TC)-resistant bacteria in sediments of the Mekong River watershed revealed that the main waterway showed a high occurrence rate of TC-resistant bacteria, whereas Tonle Sap Lake and the Sai Gon estuary did not. The Shannon index (H'), an indicator of ecological diversity, was calculated from denaturing gradient gel electrophoresis (DGGE) profiles, which indicated that the main waterway of the Mekong River had high microbial diversity (high H') compared to Tonle Sap Lake and the Sai Gon estuary; this diversity was positively correlated with the occurrence rate of TC-resistant bacteria. Analysis of ribosomal protection protein (RPP) genes tet(M), tet(S) and tet(W) in the same area also revealed that high diversity was positively correlated with the occurrence rate of RPP genes, suggesting that RPP genes are well conserved across various bacterial species. Further evidence of different genotypes of tet(M) suggests that the drug resistance genes likely have various origins, and are mixed in the sediment. Sediments in this area are therefore potential reservoirs of drug resistance genes.

DOI： 10.1264/jsme2.23.149

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

A serious disease of the ascidian Halocynthia roretzi has been spread extensively among Korean aquaculture sites. To reveal the cause of the disease and establish a monitoring system for it, we constructed a cDNA microarray spotted with 2,688 cDNAs derived from H. roretzi hemocyte cDNA libraries to detect genes differentially expressed in hemocytes between diseased and non-diseased ascidians. We detected 21 genes showing increased expression and 16 genes showing decreased expression in hemocytes from diseased ascidians compared with those from non-diseased ascidians. RT-PCR analyses confirmed that the expression levels of genes encoding astacin, lysozyme, ribosomal protein PO, and ubiquitin-ribosomal protein L40e fusion protein were increased in hemocytes from diseased ascidians, while those of genes encoding HSP40, HSP70, fibronectin, carboxypeptidase and lactate dehydrogenase were decreased. These genes were expressed not only in hemocytes but also in various other tissues in ascidians. Furthermore, the expression of glutathione-S transferase omega, which is known to be up-regulated in H. roretzi hemocytes during inflammatory responses, was strongly increased in hemocytes from diseased ascidians. These gene expression profiles suggest that immune and inflammatory reactions occur in the hemocytes of diseased ascidians. These genes will be good markers for detecting and monitoring this disease of ascidians in Korean aquaculture sites.

DOI： 10.2108/zsj.24.1231

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE  

We found increased numbers of oxytetracycline (OTC)-resistant bacteria in sediment and seawater around a marine aquaculture site after OTC therapy. Samples were collected at an aquaculture site along the coast of the Seto Inland Sea, Japan in 2004. In April, the percentage of bacteria resistant to 60 mu g mL(-1) OTC in the surface sediment was 6.8%-20.0%. The percentages increased during OTC therapy in the summer reaching 53.3%-60.7% in September. Ninety-two days after drug cessation, the percentages decreased to below 22.9%. Tet(M)-positive bacteria were detected in the sediment and seawater samples. Tet(M) was evident in both Gram-positive and Gram-negative bacteria from various genera, and was newly identified in Paenibacillus, Sporosarcina, Shewanella, and Pseudoalteromonas. The dominant tet(M)-positive isolates were strains of Vibrio suggesting that this genus is an important reservoir for tet(M) in the marine environment. Two different alleles were found, tet(M)-A and tet(M)-B, each in isolates from five genera. The data suggests drug therapy used in the aquaculture acted as a selective pressure promoting increased numbers of resistant bacteria.

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The ribosomal protection proteins (RPPs) mediate the resistance to tetracycline (TC) in Gram-positive and Gram-negative bacteria. The RPPs display sequence similarity to translation elongation factors, EF-G/EF-2 and EF-Tu/EF-1 alpha. To determine the evolutionary origin of the RPPs, we constructed a composite phylogenetic tree of the RPPs, EF-G/EF-2 and EF-Tu/EF-1 alpha. This tree includes two universal trees for the EF-G/EF-2 and EF-Tu/EF-1 alpha, which form clusters corresponding to the respective two groups of proteins from three superkingdoms. The cluster of RPPs was placed at a point between the EF-G/EF-2 and EF-Tu/EF-1 alpha clusters. The branch length (substitutions/site) between the node for the RPP cluster and the primary divergence of the RPPs was statistically shorter than that between the node for this cluster and the primary divergence in the EF-G/EF-2 cluster. This indicates that the RPPs derived through duplication and divergence of the ancient GTPase before the divergence of the three superkingdoms. Furthermore, this suggests the RPPs' extant function occurred before the streptomycetes that include the TC-producing strains. Therefore, the RPPs evolved independent of the presence of TCs and serve a function other than antibiotic resistance. The RPPs may provide ribosomal protection against other chemical substances in the environment.

DOI： 10.1007/s00239-007-9006-z

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Tributyltin (TBT) is organotin compound that is toxic to aquatic life ranging from bacteria to mammals. This study examined the concentration of TBT in sediment from and near the Mekong River and the distribution of TBT-resistant bacteria. TBT concentrations ranged from &lt;2.4 to 2.4 ng/g (dry wt) in river sediment and &lt;2.4-15 ng g(-1) (dry wt) in harbor sediment. Viable count of total bacteria ranged from 2.0 x 10(4) to 1.4 x 10(7) cfu/g, and counts of TBT-resistant bacteria ranged &lt;1.0 x 10(2) to 2.5 x 10(4) cfu/g. The estimated occurrence rate of TBT-resistant bacteria ranged from &lt;0.01 to 34% and was highest in upstream sites in Cambodia. The occurrences of TBT in the sediment and of TBT-resistant bacteria were unrelated, and chemicals other than TBT might induce TBT resistance. TBT-resistant bacteria were more abundant in the dry season than in the rainy season. Differences in the selection process of TBT-resistant bacteria between dry and rainy seasons were examined using an advection-diffusion model of a suspended solid (SS) that conveys chemicals. The estimated dilution-diffusion time over a distance of 120 km downstream from a release site was 20 days during dry season and 5 days during rainy season, suggesting that bacteria at the sediment surface could be exposed to SS for longer periods during dry season. (C) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2007.03.033

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Seasonal changes in the infection state of marine birnavirus (MABV) in Japanese flounder Paralichthys olivaceus and in rearing sea water are described. Sea water and 10-11 healthy fish were sampled monthly from April 2002 to February 2003. The MABV genome was detected throughout the year in &gt; 80% of the fish examined at each sampling. The virus was isolated from the liver, kidney, and spleen, but not from the brain. The detection rate in each organ increased from April to October, and then decreased. Detection of virus antigen by the indirect fluorescent antibody technique also showed that the virus was present from spring to autumn (June-September) in the liver, kidney, and spleen, but not the brain. Sequence analysis of the MABV genome at the VP2-NS region revealed two specific mutations compared to the standard yellowtail strain (Y-6). It is suggested that the infection state of MABV in Japanese flounder changes to a latent or persistent infection after autumn. MABV was detected in sea water between September and February, suggesting that virus particles in the environment are relatively higher during cool seasons.

DOI： 10.1111/j.1444-2906.2007.01374.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Recently, a serious disease spread extensively in aquaculture sites of the ascidian Halocynthia roretzi in Korea. To understand circumstances of ascidians in Korean aquaculture sites, residue levels of organotin compounds were analyzed, and detection of a marine birnavirus (MABV) in tissues of H. roretzi was attempted. Korean H. roretzi showed high concentrations of butyltins (mono, di, and tributyltins), especially in the gill, hepatopancreas, and digestive tract. However, there was no significant difference in the residues of butyltins in the hepatopancreas between diseased and non-diseased ascidians. The positive rate of MABV detection was high in the hepatopancreas, but also no significant difference was observed between diseased and non-diseased individuals. These observations suggest that an accumulation of tributyltin and a latency of MABV in H. roretzi tissues does not directly relate to the occurrence of the disease.

DOI： 10.1111/j.1444-2906.2007.01332.x

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

We investigated the distribution and diversity of tetracycline resistance genes encoding ribosomal protection proteins (RPPs) in river and channel sediments of the Mekong Delta in Vietnam. The sediment samples were taken from nine sites in the Hau River in southern Vietnam and from 1 site in a channel in Can Tho City in May 2004 using an Ekman-Birge sediment surface sampler. The RPP genes were amplified using PCR with DNA templates obtained directly from the sediments. The tet(M), tet(S), and tet(W) genes were detected by PCR in most sediment samples. Denaturing gradient gel electrophoresis analysis of these genes and sequencing of the resulting bands showed that tet(S) and tet(W) had only one genotype each, but that tet(M) had at least two, which were tentatively called type 1 and type 2. Type 1 tet(M) was identical to the gene encoded in various plasmids and transposons of gram-positive and gram-negative bacteria, and type 2tet(M) was similar to the gene encoded in Tn1545 of Enterococcus faecalis (99% identity, 170 bp/171 bp). This study showed that various RPP genes were widely distributed in the river and channel sediments of the Mekong Delta.

DOI： 10.1111/j.1574-6941.2006.00244.x

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOLOGY SOC KOREA  

In this study we examined the multi-drug resistance profiles of the tetracycline (TC) resistant genus Vibrio to determine its susceptibility to two beta-lactams, ampicillin (ABPC), and mecillinam (MPC), as well as to macrolide, erythromycin (EM). The results showed various patterns of resistance among strains that were isolated from very close geographical areas during the same year, suggesting diverse patterns of drug resistance in environmental bacteria from this area. In addition, the cross-resistance patterns suggested that the resistance determinants among Vibrio spp. are acquired differently within the sediment and seawater environments.

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The degradation of tributyltin (TBT) and changes of bacterial number and community structures were investigated in microcosms using the sediment collected from the Mekong River, Vietnam. Concentrations of TBT in sediments were less than 0.62 ng/g (dry wt), lower than those reported from other areas. TBT-resistant bacteria were found in the three sampling sites, and the occurrence rates were 11-16% out of the total viable count. In this Microcosm experiment, initial concentration of TBT [1.0-1.4 mu g/g (dry wt)] decreased to 0.6 mu g/g (dry wt) during 150 days, whereas that in the control microcosm with autoclaved sediment did not change, indicating that Mekong River sediment contains high TBT-degrading activity by microorganisms. The occurrence of TBT-resistant bacteria and the bacterial community structures monitored by denaturing gradient gel electrophoresis were almost the same between test and control groups, indicating that the addition of TBT had little influence on microbial community structure. Mekong River sediment seems to have a stable microbial community against TBT pollution.

DOI： 10.1007/s00248-006-9079-z

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOLOGY SOC KOREA  

The tributyltin (TBT)-resistant bacterium, Pseudomonas aeruginosa 25W, which was isolated in seawater from the Arabian Sea, was subjected to transcriptome analysis in the presence of high concentrations of TBT. Only slight effects were observed at TBT concentration of 50 mu M, but exposure to 500 mu M resulted in the upregulation of 6 genes and the downregulation of 75. Among the 75 downregulated genes, 53% (40 out of 75) were of hypothetical function, followed by 14 transcriptional regulation- and translation-associated genes. The results of this study indicated that although the 25W strain was highly resistant to TBT, high concentrations of TBT result in toxic effect on the transcriptional and translational levels. The target genes likely belong to a specific category of transcription- and translation-associated genes rather than to other gene categories.

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VP5 is a 15-kDa nonstructural protein encoded by a small open reading frame in 5'-terminal of segment A of the Marine Birnavirus ( MABV) ( strainY-6) genome. Comparisons of the amino acid sequence of the VP5 with other Bcl-2 family member proteins indicated that the VP5 protein contains Bcl-2 homology (BH) domains BH1, BH2, BH3, and BH4, but without the transmembrane region. The VP5 gene from MABV was fused to enhancing green fluorescence protein (eGFP) gene and inserted into the baculovirus genome under the control of polyhedrin gene promoter, and then was highly expressed in insect cells. The expressed VP5 was capable of enhancing insect cell viability, prevented membrane blebbing and delayed DNA internucleosomal cleavage when cells were infected with the recombinant virus. The results suggested that the VP5 of MABV is a novel anti-apoptosis gene, which could regulate the cell apoptosis-off system.

DOI： 10.1007/s11262-005-1794-x

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC LIMNOLOGY OCEANOGRAPHY  

We assayed proteolytic enzymes in coastal surface seawater using 16 types of fluorogenic substrates, including those for aminopeptidase, trypsin, elastase, and chymotrypsin. Hydrolysis rates were similar or higher for substrates of trypsin and chymotrypsin than for those of aminopeptidase. Substrates for elastase were hardly hydrolyzed. The results strongly suggest trypsin-type and chymotrypsin-type endopeptidases and aminopeptidases were present in the seawater. In most previous studies of proteolytic enzymes in aquatic environments, leucine-aminopeptidase activity measured using a fluorogenic substrate has been used as a model of proteolytic activity. From the results of this study using various peptide analog fluorogenic substrates, the significance of endopeptidases, which could play a key role in downsizing of dissolved proteins and polypeptides to oligopeptides prior to microbial respiration, was confirmed.

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

A phylogenetic tree of aquabirnaviruses, including marine birnaviruses (MABV) and infectious pancreatic necrosis virus (IPNV), was developed based on the nucleotide sequences and deduced amino acid sequences of the polyprotein and VP5 genes of genomic segment A. In the polyprotein of MABV strains, the amino acid sequences were very similar, with identities of 98.3-99.7%. Twenty-one unique amino acid residues were found in the deduced amino acid sequences of the polyprotein gene of MABV strains. The phylogenetic tree based on the nucleotide sequence of genomic segment A and polyprotein sequences showed that 31 aquabirnavirus strains were clustered into seven genogroups. All MABV strains isolated in Japan and Korea were clustered into one genogroup which was distinct from other aquabirnaviruses. The seventh genogroup containing all MABV strains showed amino acid sequence similarities of 80.7-90.6% with other genogroups. In VP5, four unique residues were found in MABV strains when compared with IPNV strains. The MABV strains exhibited amino acid sequence similarities of 63.9-86.4% with IPNV strains. The amino acid sequences of VP5 were conserved among MABV strains, but differed from those of IPNV strains. The MABV strains isolated from different host species and different geographical areas were very similar to each other, suggesting that the MABV are distinct from the other genogroups.

DOI： 10.1111/j.1365-2761.2004.00585.x

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TERRA SCIENTIFIC PUBL CO  

To examine the possibility that outer membrane proteins (OMP) of Synechococcus sp. remain in seawater, we investigated the stability of OMPs in vitro and in situ. Some fractions prepared from Synechococcus sp. CSIRO-94 were treated with trypsin and proteinase K. Four tightly bound OMPs were separated from Synechococcus. We designated the two major OMPs of 52 kDa and 48 kDa as Omp52Sy and Omp48Sy, respectively. Degradation of the OMP in natural seawater was monitored in microcosms to which intact Synechococcus cells and outer membrane (OM) were added. Omp52Sy and Omp48Sy were the most stable against trypsin and proteinase K among the OMPs when they were embedded in the OM. However, in the microcosm experiment using intact cells, Omp52Sy and Omp48Sy were detected in the particulate fraction only during the first 4 days, after which they could not longer be detected. Omp52Sy and Omp48Sy were the most stable proteins among the Synechococcus OMPs in vitro, but they might be degraded in situ. This indicates that stability of Synechococcus porin differs depending on complex formation with other membrane molecules, which might cause different preservation of microbial membrane proteins in the dissolved protein pool in the ocean. This study suggests that Gram negative bacterial OM with thin peptidoglycan forms a lipid bilayer that proptects OMP, but Synechococcus OM with thick peptidoglycan cannot form a lipid bilayer. The incomplete bilayer might not be able to protect from protease attack in the natural environment.

DOI： 10.1007/s10872-004-5775-9

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Occurrence of tetracycline resistance genes encoding ribosomal protection proteins was examined in 151 tetracycline-resistant bacterial isolates from fish and seawater at coastal aquaculture sites in Japan and Korea. The tet(M) gene was detected in 34 Japanese and Korean isolates, which included Vibrio sp., Lactococcus garvieae, Photobacterium damsela subsp. piscicida, and unidentified Gram-positive bacteria. The majority of these bacterial isolates displayed high-level resistance with a minimum inhibitory concentrations (MICs) equal to or greater than 250 mug/ml of oxytetracycline and only four isolates had MICs less than 31.3 mug/ml. 16S rDNA RFLP typing of tet(M)-positive Vibrio isolates suggests that these are clonal populations of the same phylotype specific to a particular location. One Vibrio clone (phylotype III), however, is widely disseminated, being detected during different sampling years, at different locations, and in different fish species in both Japan and Korea. The tet(S) gene was detected in L. garvieae from yellowtail in Japan and in Vibrio sp. from seawater in Korea. This is the first report of tet(S) occurrence in Gram-negative facultative anaerobes. These results suggest that tet(M) and tet(S) genes are present in fish intestinal and seawater bacteria at aquaculture sites and could be an important reservoir of tetracycline resistance genes in the marine environment. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.femsle.2004.06.026

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INTER-RESEARCH  

We examined the inactivation kinetics of marine birnavirus (MABV) in a coastal sea, in seawater samples collected from 50 cm depth. MABV was added to both natural and autoclaved seawater at a concentration of 6 x 10(6.43) TCID50 (50% tissue culture infectious close) ml(-1), put in dialysis tubes and incubated at the original depth. The inactivation of MABV by solar UV radiation was examined using light and dark tubes. The infectivity titer of MABV was measured by the TCID50 method using CHSE-214 cells. Virus infectivity in natural seawater decreased quickly and was below the detection limit by 270 min in both light and dark conditions; however, virus infectivity was maintained in the autoclaved seawater until 420 min. These results suggest that the loss of virus infectivity is not caused by sunlight UV radiation.

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To determine the distribution of marine birnavirus (MABV) in cultured populations of different marine fish species, 1291 pooled tissue samples from 2672 fish belonging to 22 species and one hybrid were collected from Kagawa Prefecture, Japan, during 1999-2001. Using cell-culture MABV was isolated from three species: yellowtail, Seriola quinqueradiata Temminck & Schlegel (positive number/sample number, 10/419), amberjack, S. dumerili (Risso) (4/72), and Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel) (41/481). Using PCR on MABV-negative samples, the MABV genome was detected in the same three species [yellowtail (9/409), amberjack (4/68) and Japanese flounder (93/440)] and two additional species, spotted halibut, Verasper variegatus (Temminck & Schlegel) (5/11), and goldstriped amberjack, S. lalandi Valenciennes (1/5). These MABV-positive species can be taxonomically divided into two groups: the genus Seriola and flatfish. In Japanese flounder, MABV was detected during all seasons, and the infection rate was correlated with water temperature. Aquaculture sites with MABV-positive fish were evenly distributed over the surveyed area, suggesting that MABV is widely distributed at aquaculture sites in Kagawa Prefecture. The nucleotide sequence at the variable region, the VP2/NS junction, revealed that the 39th base mutation occurs host-specifically for flatfish. Flatfish are suspected to be the main reservoir of MABV and might be responsible for establishing the infection cycle in aquaculture environments.

DOI： 10.1111/j.1365-2761.2003.00518.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

Pseudomonas aeruginosa is a pathogenic bacterium that has been thoroughly investigated since the 19th century and is generally regarded as a freshwater or terrestrial organism. In 1995, it was reported that the OprP porin, an outer membrane protein corresponding to that of this bacterium, was widely distributed as a dissolved component in seawater. This finding led us to investigate the presence of P. aeruginosa in marine environments. Both culture-independent and -dependent methods were applied to seawater samples obtained in Tokyo Bay during four cruises. The DVC-FA (direct viable count-fluorescent antibody) technique showed that cells reactive to an antibody against P. aeruginosa were widely present in the bay, i.e., 10(3) to 10(4) cells/mL in the inner bay, and 10(2) to 10(3) cells/mL at the mouth. Bacterial cells isolated by selective medium were identified by three methods: the presence of oprI and oprL, two outer membrane lipoprotein genes specific to P. aeruginosa; the API20 NE kit; and 16S rDNA sequence analysis. The results confirmed that the majority of isolates from the bay were P. aeruginosa. Immuno-chemical analyses of the seawater results indicate that P. aeruginosa is commonly present in coastal marine environments and sheds OprP.

DOI： 10.1007/s00248-003-1032-9

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We isolated 2 virus-like agents that suppressed growth of Gymnodinium mikimotoi from coastal waters of the Uwa Sea, Japan. The agents found in the flagellate cells, named GM6 and GM7, were filterable in a 0.22-mum-pore filter with approximately 100-nm shapes. Electron microscopic observation showed the presence of virus-like particles in severely damaged G. mikimotoi cells infected by GM6. The growth-suppression activity of the agents (GM6 or GM7) was lost by heating at 50degreesC, with treatments of DNase and protease, and filtration through a 0.05-mum filter. Our results suggest that the agents are DNA viruses infectious to and virulent for G. mikimotoi. This is the first report of a virus-like agent specific to G. mikimotoi.

DOI： 10.1007/s10126-002-0085-y

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC FISH PATHOL DEPT FISHERIES-FAC AGR  

Species-specific polymerase chain reaction (PCR) targeting the 16S rRNA gene of Nocardia seriolae was developed. The PCR targeted nucleotide #609 to 1038 (Escherichia coli numbering), which gave a 432 bp-length product. This method could detect N. seriolae type strain (JCM3360) and eight clinical isolates of this species from yellowtail Seriola quinqueradiata and Japanese flounder Paralichthys olivaceus, but not those of other bacterial species including 5 other Nocardia spp. and 4 yellowtail pathogens. The detection limit of the PCR was 10(2) CFU. Eight diseased yellowtail were employed for detection of the bacterium by the PCR. Positive results were obtained from all fish.

File： Miyoshi2003FishPath_Nocardia PCR.pdf

DOI： 10.3147/jsfp.38.93

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The cDNA nucleotide sequence of the genome segment B encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp), was determined for 5 marine birnavirus (MABV) strains from different host or geographic origins and 1 infectious pancreatic necrosis virus (IPNV) strain AM-98. Segment B of the IPNV AM-98 strain and 4 MABV strains, Y-6, YT-01A, H1 and NC1, contained a 2535 bp ORF, which encoded a protein of 845 amino acid residues with a predicted MW of 94.4 kDa. Only the MABV AY-98 RdRp had 1 amino acid shorter RdRp. Pairwise comparisons were made among our data and 4 other known IPNV sequences. The nucleotide sequences of the 5 MABV strains were very similar each other, with identities of 98.3-99.7%. The highest divergence of the nucleotide level was between MABV strains and IPNV SP strain (serotype A2), with 20.4-20.8% divergences in the coding region, which gave 10.1-11.3% divergence in the amino acid level. The aquabirnavirus RdRp was noticeably conserved in amino acid sequences. Though the identities of the nucleotide sequences of encoding region were 85.1-85.9% between MABV strains and IPNV serotype A1 strains, they shared as high as 95.1-95.9% identities in amino acid level. A phylogenetic tree was constructed based on the amino acid sequences of the RdRp gene from different birnaviruses including avibirnavirus and entomobirnavirus. Ten aquabirnavirus strains were clustered into 3 Genogroups. The Genogroup I consisted of four IPNV A1 serotype strains. All MABV strains were clustered into Genogroup II. Only IPNV SP strain was clustered into an independent Genogroup III.

DOI： 10.1007/s00705-002-0951-y

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Marine birnaviruses (MABVs) infect a wide range of fish and shellfish, yet their mode of transmission is still unclear. To determine whether marine plankton serve as a vector for MABVs, we examined plankton collected from the Uwa Sea, Japan. The phytoplankton and zooplankton were collected monthly, at depths of 0 and 40 m, from May to November 2001. Detection of the MABV genome was carried out using 2-step PCR and virus isolation. Viral genome was detected in zooplankton collected at 0 m depth in September and at 40 m depth in November. The virus could not be isolated in the PCR-positive samples. These results suggest that zooplankton may act as a vector of MABVs, although the infective and/or accumulated virus titer in zooplankton was low.

DOI： 10.3354/dao054069

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In the present study an efficient method for sampling the marine birnavirus (MABV) gene from seawater was developed. MABV gene was monitored by a specific polymerase chain reaction. When Millipore filters were used, MABV was efficiently collected on a filter with 0.05-mum pore size. When both millipore and glass fiber filters were used, MABV was recovered from both filters. Use of plain glass fiber filters resulted in poor recovering efficiency. However, coating the glass fiber filters with 1% bovine serum albumin trapped MABV efficiently. Combining concentration on glass fiber filters with polymerase chain reaction is quantitative, economic and fast, suggesting that this method can be used to detect genetically identified fish disease viruses, algal viruses, and phages.

File： 03.Concentration of .pdf

DOI： 10.1007/s10126-002-0057-2

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The distribution of tet(34) was examined among oxytetracycline (OTC) resistant Vibrio strains isolated from Japan, Korea and the Philippines. tet(34) was detected in 10 isolates (6 in fish, 4 in seawater) from Japan and Korea, suggesting that it is widely distributed among fish and seawater bacteria in these countries. Ninety-eight percent of the Vibrio strains in the Philippines were sensitive to OTC, but none of the 60 isolates from the Philippines. The minimum inhibitory concentration (MIC) of OTC-resistant isolates increased 2 to 8 fold in the presence of Mg2+, and MICs were higher in the Japanese and Korean isolates than the Philippine isolates. The MIC for furaltadone (FD) was low in Japanese (23.9%) isolates at over 3.1 μg/ml and high in Korean (50%) and Philippine (56.7%) isolates. This was probably due to the different frequency of use of FD in these countries. Sequences of 16S rDNA of tet(34)-positive isolates were 100% identical, suggesting that tet(34) is conveyed in a particular Vibrio species. © 2003, Japanese Society of Microbial Ecology &amp
The Japanese Society of Soil Microbiology. All rights reserved.

DOI： 10.1264/jsme2.18.74

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This study examines the seasonal changes of marine birnavirus (MABV) in seawater and the Japanese pearl oyster Pinctada fucata reared at different depths (2 and 15 in). Oysters and seawater were collected in 1998, and a 2-step PCR was carried out to detect MABV. Virus isolation was performed on the PCR-positive samples in the oyster. The detection rate of the MABV genome in the oyster was low during June, but increased after July at both 2 and 15 in depths. MABV was not isolated until after September, when isolation rates of 10 to 28.6 % were recorded. The results suggest that growth of MABV in the oyster is similar at 2 and 15 in depth. In contrast, the MABV genome in seawater was present through the year at 15 in depth, but was not detected in summer at 2 in. This suggests that the virus is destroyed by UV and/or other factors at 2 in in summer, but is stable in deeper waters.

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

new oxytetracycline (OTC) resistance (Otc(r)) determinant,, Tet 34, was cloned from chromosomal DNA of Vibrio sp. no. 6 isolated from intestinal contents of cultured yellowtail (Seriola quinqueradiata). The transformant, containing cloned Tet 34, could grow in broth containing 25 jig of drug per nil with 10 mM MgCl2.. Tet 34 encoded an open reading frame (ORF) 154 amino acids long. The amino acid sequence of the ORF was homologous to sequences of several bacterial xanthine-guanine phosphoribosyltransferases (XPRTs), which Mg2+ binding site residues and the active site were highly conserved act in purine nucleotide salvage synthesis. in XTRT and the ORF of Tet 34. The results suggest that Tet 34 encodes a new Mg2+-dependent Ote(r) mechanism.

DOI： 10.1128/AAC.46.5.1550-1552.2002

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The distribution of a newly cloned oxytetracycline (OTC) resistance determinant, Tet 34, was examined among bacterial strains isolated from diseased fish. We analyzed 33 OTC-resistant strains isolated from 1998 to 2000 in Kagawa Prefecture. Tet 34 was detected in 3 of the strains, which were grouped in the genus Vibrio. Minimum inhibitory concentrations (MICs) of the 3 strains were higher than 500 μg/ml in the presence of MgCl2, and 125 or 250 μg/ml in its absence. Tet 34 was found in chromosomal DNA in all positive strains. Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis of 16S rDNA revealed the 3 strains to have the same migration profiles. The sequences of their PCR products were also identical, suggesting that the species were the same. It was concluded that Tet 34 has been present since at least 1998, and the determinant occurs only in Vibrio species. © 2002, Japanese Society of Microbial Ecology &amp
The Japanese Society of Soil Microbiology. All rights reserved.

DOI： 10.1264/jsme2.2002.26

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL VERLAG GMBH  

The effect of temperature on Aeromonas hydrophila infection in goldfish, Carassius auratus, was studied using A. hydrophila strain A-3500. After comparison of four different infection methods. subcutaneous injection was selected. Different test temperatures were also tested and higher mortality was observed at 17 and 25degreesC during a 15-day period. SDS-PAGE analysis of outer membrane proteins prepared from A. hydrophila cultured at 10, 17, 25 and 32degreesC in formulated salt water showed different protein profiles. For example, a 40-kDa band was found only at 17 and 25degreesC. Phagocytic rates of A. hydrophila by goldfish macrophages at 10, 17. 25 and 32degreesC were 20.46 +/- 2.07. 16.15 +/- 1.39, 15.94 +/- 1.85 and 22.22 +/- 2.49%, respectively. The results indicated that temperature affects both the cell membrane structure of A. hydrophila and phagocytic activity of goldfish macrophages. resulting in varying fish mortality when infected at different temperatures.

DOI： 10.1046/j.1439-0426.2001.00291.x

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To determine the infectivity of marine birnavirus (MABV) in various marine fish species, experimental infection was performed in combination groups of 5 fish species with 7 strains of MABV and 1 strain of infectious pancreatic necrosis virus (IPNV). Mortality was observed in yellowtail Seriola quinqueradiata and amberjack S. dumerili infected with MABV strains Y-6, Y-10K and H-1, but not in other infected species. MABV was reisolated from most combination groups, but the virus isolation rate and virus infectivity titer were often significantly different among groups with the same fish species or with the same virus strain. All MABV strains replicated well in makogarei Limanda yokohamae, but only slightly in tiger puffer Takifugu rubripes. IPNV also replicated in all fish species without causing death. The isolation rate and infectivity titer of IPNV were similar to or higher than those of non-virulent strains of MABV. In conclusion, the infectivity of MABV for different fish species is considered to change, which is an important factor in the development of the infection cycle of this virus among marine organisms.

File： 01.Infectivity of .pdf

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Pathogenicity of a marine birnavirus (AY-98) isolated from diseased ayu Plecoglossus altivelis was investigated. Cumulative mortalities in the intraperitoneally injected groups of ayu in two weeks were 70% (dose 10(5.4) TCID50/fish)-71%(10(5.8) TCID50/fish) in duplicated experiments. Histologically, the naturally and experimentally infected fish showed brain congestion and pancreatic necrosis. From the symptoms of body deformity and congestion in the brain, it was thought that AY-98 is similar to viral deformity virus, the birnavirus isolated from yellow tail Seriala quinqueradiata with deformity.

DOI： 10.3147/jsfp.36.99

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Marine birnavirus (MABV) is a member of Aquabirnavirus and an opportunistic pathogenic virus in eukaryotic marine organisms. This virus has a broad host range in wild and cultured fish and shellfish. In this study, the distribution of MABV in seawater from different areas was examined by PCR of the VP2/Ns junction region of the MABV genome. We have detected the MABV genome in three marine water columns
off the coast of Japan, in the Pacific Ocean and in the Mediterranean Sea, suggesting that MABV is widely distributed. The MABV genome was also detected in samples of zooplankton from the Pacific Ocean. A high nucleotide sequence similarity (more than 98.5%) was observed among the PCR products from MABV genomes. Such a prevalent distribution of genetically similar MABV suggests that birnavirus is one of the components of the RNA pool in ocean environments. © 2001, Japanese Society of Microbial Ecology &amp
The Japanese Society of Soil Microbiology. All rights reserved.

DOI： 10.1264/jsme2.2001.191

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：URBAN & FISCHER VERLAG  

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 degreesC for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10(4) cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 degreesC for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection.
The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.

File： 01.Formation of viable.pdf

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This study aims to determine the seasonal occurrence of marine birnavirus (MABV) at a coastal site in the Uwa Sea, Japan, in 1997 and 1998. To detect MABV from seawater, a simple method was developed for concentrating MABV by dialysis and ethanol precipitation. The concentrated virus was used for polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, and virus isolation. Viral genome was detected through the experimental period. The amount of PCR product varied; it was small in summer, but increased from fall to winter. Viral protein was also detected, and the amount in the January sample was equivalent to approximately 10(2) TCID50 (50% tissue culture infectious dose) of the virus. However, infectious viruses were not isolated. This suggested that MABV was released from hosts to environmental seawater in winter and possibly degraded after release.

File： 00.Occurrence of marine birnavirus.pdf

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This study examines the seasonal occurrence and infective state of marine birnavirus (MABV) in cultured Japanese pearl oyster (Pinctada fucata). Planted oysters were sampled monthly in 1997 and 1998. To detect MABV in the oysters, PCR and virus isolation were carried out. Also, the indirect fluorescent antibody technique (IFAT) was performed to know the organs expressing viral antigens. The detection rate of the MABV genome by PCR was low during July to October, but increased after November. This virus was isolated only after October, with a 10-40% isolation rate. Results of the IFAT showed that the specific fluorescence was observed in hemocytes in September. Fluorescence in hemocytes decreased in January, but increased in liver parenchymal cells. These results suggest that MABV persistently infected hemocytes in summer with a small amount of genome and protein, and then the virus spread in winter into the parenchymal cells.

File： 00.Seasonal change.pdf

DOI： 10.1007/s007050070036

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Bacterial porin homologues have recently been found in seawater as a dissolved organic matter. However, the bacteria of origin have not been identified. This manuscript describes the isolation of the marine bacteria having similar antigenic proteins to porin Omp35La of Vibrio anguillarum. Colony forming bacteria from seawater on Marine Broth 2216E plate were screened to determine whether they have Omp35La-like proteins by colony western blotting with anti-Omp35La antibody. Among the 37 final positive isolates, 11 isolates were classified into theVibrio-group, whereas 26 isolates were assigned to be non-Vibrio-group based on the biochemical properties. When outer membrane protein was purified and western blotting was performed, 8 out of 11 Vibrio-group and 10 out of 26 non-Vibrio-group isolates had antibody reactive proteins. This suggests that not only Vibrio but also other class bacteria are possibly the origin of the dissolved protein. The family Vibrionaceae specific PCR worked in 7 isolates of the Vibrio-group. However, V. anguillarum itself was not identified by PCR-RFLP assay. © 2000, Japanese Society of Microbial Ecology &amp
The Japanese Society of Soil Microbiology. All rights reserved.

DOI： 10.1264/jsme2.2000.189

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The occurrence of oxytetracycline (OTC) resistance was examined among bacteria isolated from intestinal contents of yellowtail, Seriola quinqueradiata, and the seawater environment where these animals are cultured. A high prevalence of OTC resistant bacteria was observed in all samples obtained before and after administration of OTC. The Vibrio/Aeromonas group were the predominant bacteria detected in the intestine samples. However, other genera also contained OTC-resistant bacteria. © 2000, Japanese Society of Microbial Ecology &amp
The Japanese Society of Soil Microbiology. All rights reserved.

DOI： 10.1264/jsme2.2000.223

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The molecular distribution of dissolved proteins in seawater from coastal marine environments in Uranouchi Bay, Kochi Prefecture, is first reported in this article. Occurrence of bacteria-derived dissolved proteins and their source bacteria were examined using a probe of the antibody (anti-Omp35La) against a porin outer membrane protein (Omp35La) of the fish pathogenic bacterium Vibrio (Listonella) anguillarum. The electrophoretograms of dissolved proteins from coastal seawater showed a large number of discrete and individual proteins overlapped each other over a wide range of molecular masses indicating active processes in coastal environments in transferring proteins from organisms to the inanimate dissolved protein pool. Among the dissolved proteins, 37 kDa- and 18 kDa-proteins reacted with the Omp35La. In order to isolate the source bacteria of such dissolved proteins, bacteria from seawater and diseased fish were screened by colony Western blotting with anti-Omp35La. The reactive strains were further examined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting to verify the presence of Omp35La homologues among the outer membrane proteins of such strains. Outer membrane proteins reacting with anti-Omp35La were detected in only 4 strains of the 129 strains that were positive in the colony Western blotting. The level of possible source bacteria of 37 kDa- and 18 kDa-dissolved proteins was suggested to be 5-6 orders of magnitude lower than the total bacterial count. The present study leads us to hypothesize that a minor portion of the bacterial assemblage is responsible for the dissolved proteins in the coastal waters.

DOI： 10.1023/A:1011109329434

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A birnavirus was recently isolated from cultured ayu Plecoglossus altivelis on Shikoku island, Japan. The diseased fish displayed vertebral or vertical curvature and mild haemorrhage around the brain. Cytopathic effects (CPE) of the virus, including cell roundness, filamentous change and cell lysis, were observed in CHSE-214, RTG-2 and RSBK-2 cells. The virus isolated from ayu, designated the AY-98 strain, was found to be antigenically related to the marine birnavirus (MABV) Y-6 strain that originated from yellowtail Seriola quinqueradiata. AY-98 had a bi-segmented RNA genome and the same nucleotide sequence in the 310 bp VP2/NS junction as MABV Y-6. At the same time that the ayu epizootics occurred, another birnavirus (AM-98) was isolated from amago salmon Oncorhynchus rhodurus which were cultured 66 km away from the ayu farm. AM-98 showed a similar CPE and had the same host cell ranges as AY-98. However, AM-98 was serologically similar to the VR-299 strain of infectious pancreatic necrosis virus (IPNV) and their nucleotide sequences in the VP2/ NS junction region showed 98% homology without changes at the amino acid level. In this study, the ayu strain AY-98 was grouped into MABV, whereas the amago salmon strain AM-98 was grouped into IPNV. This indicates that the 2 birnaviruses originated from different sources in spite of the fact that the places where they were isolated are close to one another. The results in this paper show a new aspect of the traditional consensus that the same serogroup of birnavirus distribute in close geographic areas.

File： 99.Isolation of different .pdf

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Thirteen wild molluscan shellfish species collected from 8 Prefectures in Japan were surveyed for the presence of marine birnavirus (MABV) by the polymerase chain reaction (PCR) technique and culture method. Approximately 60% of bivalves and 35% of gastropods tested had detectable MABV genome, although the prevalence of positive specimens varied among species. The PCR-positive shellfish were submitted to virus isolation. The isolation rate was low, suggesting that the MABV was in a state of persistent infection in these shellfish. Seventy four % of the examined virus strains were different in a position of the nucleotide sequences of the PCR products from that of MABV strains obtained from fish.

DOI： 10.3147/jsfp.34.121

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This study was undertaken to determine whether marine birnavirus (MABV) can induce apoptosis in vitro in cell lines established from four different fish species. MABV Y-6 strain was inoculated onto chinook salmon embryo (CHSE-214), red sea bream kidney (RSBK-2), fathead minnow caudal peduncle (FHM) and epithelioma papulosum cyprini (EPC) cell lines at a multiplicity of infection (m.o.i.) of 0.1. At 0, 12, 24, 36, 48, 72, and 96 h post-infection, the cells were harvested and used far DNA fragmentation analysis, apoptotic cell counts and determination of virus titer by plaque assay. MABV infection appeared to induce the typical features of apoptosis such as nuclear and cytoplasmic condensation, DNA fragmentation and formation of apoptotic bodies in CHSE-214 and RSBK-2 cells, but not in FHM and EPC cells. The concentration of free virus increased immediately after infection, whereas the apoptotic cell ratio and cell-associated virus titer scarecely increased until 24 h. Apoptotsis increased after 36 h when monitored by counting of apoptotic cells and analysis of DNA. The results suggest that MABV replicating to high concentrations during an early stage of infection induces apoptosis in later stages.

DOI： 10.3147/jsfp.34.73

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Fish Pathology  

DOI： 10.3147/jsfp.34.228

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC FISH PATHOL DEPT FISHERIES-FAC AGR  

It was reported in our previous paper that the starved cells of Aeromonas hydrophila were more virulent compared to the cells cultured in a medium. In this study, differences in their protein profiles were compared between the starved and cultured cells of the bacterium. Total proteins, outer membrane proteins (OMP) and S-layer proteins prepared from starved and cultured cells were analysed by SDS-PAGE. A different pattern was shown in the OMPs between the cells. Major bands of OMPs, 39, 52 and 97 kDa, were detected in the starved cells, however they were not detected in the cultured cells. In S-layer fraction, major band estimated at 91 kDa was found only in the cultured cells. Experimental result on phagocytosis of crucian carp macrophages against those cells revealed that phagocytic rate of the starved cells was lower than that of the cultured cells. These results indicate that starvation induces shifts in OMP and S-layer proteins of A. hydrophila which may enhance the resistance of the bacterium against phagocytosis of macrophages.

DOI： 10.3147/jsfp.33.275

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In birnaviruses, the viral capsid proteins VP2 and VP3, and the protease NS are translated as a polyprotein. In this study, the complete polyprotein gene was sequenced and the deduced amino acid sequence was analyzed for the marine birnavirus (MABV) strain Y-6. Comparison of VP2 region with infectious pancreatic necrosis virus (IPNV) strains revealed that the central part of VP2 contained a variable region whereas the N- and C-terminal parts appeared conserved among the MABV and IPNV strains. In the VP3 gene, hydrophilic residues were concentrated around the C-terminal region, suggesting that this region was exposed to the outside of the molecule. The N-terminal of NS appeared variable, however, the C-terminal appeared conserved. This result was similar to previous reports of other IPNV strains. The VP5 open reading frame (ORF) was found in 5'-end of the plyprotein ORF. Considerable variation was found in this region compared to other proteins. The amino acid composition of VP5 indicated a high, rich concentration of arginine suggesting that VP5 is a basic protein that may function in RNA binding. This is the first report of the complete analysis of the MABV polyprotein gene.

DOI： 10.2331/fishsci.64.428

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File： 1998アコヤからビルナ分離.pdf

DOI： 10.2331/fishsci.64.342

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Outer membrane proteins (OMP) of the marine fish pathogen, Vibrio (Listonella) anguillarum, were examined in a wild type strain and an oxytetracycline (OTC) resistant strain. When the OTC resistant strain was cultured in the presence of OTC, the concentration of a major porin, Omp35La, decreased whereas the concentration of an unknown 26 kDa OMP increased. The latter OMP was designated Omp26La. The change in the expression of Omp35La and Omp26La was unaffected by treatment with tetracycline, chlorotetracycline, maltose, rafinose and glucose. Additionally, heat shock treatment did not affect the OMP profiles. Although whether the regulation of both OMPs correlates or not is unclear, it is suggested that OTC specifically affected the expression of both OMPs in the OTC resistant strain. The nucleotide and deduced amino acid sequences of Omp26La were similar to the known OMP (OmpV) of Vibrio cholarae. From the amino acid composition and hydropathy profile, Omp26La was found to have a similar structure to porins such as OmpC and OmpF of E. coli. © 1998, Japanese Society of Microbial Ecology – The Japanese Society of Soil Microbiology. All rights reserved.

DOI： 10.1264/jsme2.13.197

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC SCI FISHERIES TOKYO UNIV FISHERIES  

Two strains of birnavirus were isolated from Agemaki (jack knife clam) Sinonovacula constricta originating in the Ariake sea, Japan, and from imported shells from Korea. The genome sequence of the VP2/Ns junction regions of virus genome revealed that the 2 strains are very similar to each other and also similar to other birnaviruses isolated from several marine fishes. Routine survey of the virus genome from Agemaki by PCR was performed in 1993 and 1994. The birnavirus genome was detected in high ratios using samples recollected from several fishing grounds where Korean shells were transplanted. The imported shells from Korea also possessed the virus genome. These findings suggest that birnavirus is widely present in Agemaki in the Ariake sea and on the Korean coast.

DOI： 10.2331/fishsci.63.563

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When Campylobacter jejuni was exposed to iron compounds, alterations in enterotoxin production were monitored by enzyme linked immunosorbent assay (ELISA) and Western blotting. By ELISA, treatment with 10 mu M haemin increased the ganglioside binding activity of the toxin in the extracellular fraction but not in the intracellular fraction. By Western blotting, a 68 kD subunit of the enterotoxin was observed to increase in concentration. The results suggest that haemin may have a positive regulation effect on enterotoxin maturation when released from bacterial cells.

File： 97.Effect of haemin.pdf

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Flavobacterium sp. RS-5LY is a causative bacterium of the Suminori disease of nori Porphyra sp. A pathogenic substance was partially purified from this strain by sequential gel filtration column chromatography and characterized. The substance separated on the thin layer chromatography plate was visualized as a yellow spot by the ninhydrin reaction, and the molecular weight was estimated to be 150-200 D. By treatment of nori thalli with 2,700 mu g/ml of the substance for 10 h, the peptization and necrosis ratio of cells reached 100%.

File： 97.Partial purification.pdf

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A serine protease was purified to homogeniety from the culture supernatant of a marine bacterium, Vibrio anguillarum by a 4-step procedure, ammonium sulfate precipitation, G-100 gel filtration, and two rounds of phenyl-Sepharose chromatography. The molecular weight of the enzyme was estimated to be 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The enzyme activity was inhibited by diisopropyl fluorophosphate, phenylmethyl sulfonyl fluoride, Leupeptin, and Chymostatin. The optimum temperature was 37°C and the optimum pH of the enzyme was 9.0. Mg2+ and Ca2+ at 10 mM enhanced the enzyme activity by 161 and 124 %, and stabilized the activity when exposed between 15 to 42°C. The enzyme hydrolyzed strongly butyloxycarbonyl-leucyl-seryl-threonyl-arginyl-4-methylcoumaryl-7-amide among 17 peptydyl-4-methylcoumaryl-7-amides, indicating narrow substrate specificity. The alkaline serine enzyme shows similar substrate specificity to a known metalloprotease. However, this novel serine protease did not show anticoagulation activity as detected in the metalloprotease, suggesting a different function in this bacterium. © 1997, Japanese Society of Microbial Ecology &amp
The Japanese Society of Soil Microbiology. All rights reserved.

DOI： 10.1264/jsme2.12.125

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

To detect marine birnaviruses (MBV) from fish samples, a two-step PCR assay was developed. The first step is a reverse transcription (RT) PCR using a primer set design based on the infectious pancreatic necrosis virus (IPNV) gene, The second step is a nested PCR using a primer set design based on the VP2/NS junction region of MBV. This method was specific for aquatic birnaviruses: heightened sensitivity was indicated in that 1 fg of viral genome could be detected when nested PCR was used. The birnavirus gene was detected from clinical samples with a high ratio even though the samples gave negative results in virus isolation. This method could be useful to survey MBV not only in diseased fish but also in nonsymptomatic carriers or reservoirs.

File： 97.Detection of aquatic.pdf

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A novel thiol protease hydrolyzing benzoyl-arginyl-4-methylcoumaryl-7-amide was purified from the culture supernatant of a marine bacterium Alteromonas haloplanktis strain S5B. The monomeric molecular weight of the protease was estimated to be 74 kDa by SDS-PAGE, and the native form was 76 kDa by gel filtration. The activity was inhibited by leupeptin and PCMB but not by DFP. The optimum pH for the activity was 8.0-9.0. The optimal temperature for the activity was 20 degrees C, and the activity over 30 degrees C was less than 20%. From these characteristics. the protease was identified as alkaline thiol protease having activity at low temperatures.

File： 97.Low-temperature-active.pdf

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In order to expand upon the discovery that specific proteins survive in seawater as dissolved protein and that the origin of these proteins is bacterial porin, we surveyed marine environments and cultured bacteria for the presence of homologues of 2 kinds of bacterial porins. Antisera against the N-terminus of the OprP porin of Pseudomonas aeruginosa and against the whole molecule of the Omp35La porin of Listonella (Vibrio) anguillarum were prepared and used as probes in Western blot analysis. In all samples collected in the subarctic and subtropical Pacific Ocean and the Antarctic Ocean, proteins reactive to the antisera were detected. The molecular masses of OprP and Omp35La are 48 and 33 to 37 kDa respectively; detected proteins in seawater samples were generally also of similar molecular mass. However, dissolved proteins as well as outer membrane proteins from cultured bacteria with different molecular masses were detected using the antisera. This indicates that dissolved proteins and bacterial outer membrane proteins distinct from OprP and Omp35La contain similar antigenic structures to OprP and Omp35La. Fluorescent-antibody staining revealed that bacterial cells that were stainable with antisera were present in natural bacterial assemblages throughout the entire water column. Present observations strongly suggest that bacterial porins are a major source of dissolved proteins.

DOI： 10.3354/meps158001

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Listonella (Vibrio) anguillarum, an important fish pathogen, is divided into 10 serotypes according to O-antigens present on the outer membrane. However, the biochemical and immunological properties of porin proteins have not been reported. In this study, the antigenicity and N-terminal amino acid sequence of the 35 kDa porin-like-major outer membrane protein (Omp35La) were compared among different serotypes of L. anguillarum as well as other bacteria. In Western blotting analysis, antisera against Omp35La from strains of J-O-1, -2 and -3 serotypes could detect Omp35La, but not other proteins, in most L. anguillarum strains and isolates of the genera Vibrio and Photobacterium. This antigenicity of Omp35La is unrelated to the serotype and is conserved in related organisms. An N-terminal sequence showed identification with OmpF and OmpC of Escherichia coli and Salmonella typhimurium. However, this similarity was lower when compared to other human pathogens. Thus it was concluded that Omp35La does not: contribute to the serotypes of L. anguillarum, although the N-terminal structure is well conserved among different serotypes.

File： 96.Antigenecity and N-terminal.pdf

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Yellowtail ascites virus and related strains isolated from marine fish have been shown to be similar to infectious necrosis virus (IPNV) in terms of biological and serological characteristics, This paper explores the relationship of aquatic birnaviruses at the genetic level. The junction region on the genome segment A coding viral capsid protein VP2 and viral protease NS was amplified by PCR in six marine strains. Analysis of nucleotide and the deduced amino acid sequences revealed that the six marine strains have amino acid variations in the possible amino terminus of NS when compared to IPNV. The six marine strains form a new genogroup which is distinguished from three serotypes of IPNV.

File： 96.Genogrouping og birnaviruses.pdf

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC FISH PATHOL DEPT FISHERIES-FAC AGR  

A metalloprotease was purified from a culture supernatant of the fish pathogen, Listonella (Vibrio) anguillarum. The molecular weight of the enzyme was estimated to be 36kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was inhibited by EDTA and o-phenanthroline, Zn2+ and Mn2+ at 10 mu M reactivated the enzyme which had been inactivated with 0.1 mu M EDTA. Ca2+ at 1 mM enhanced the enzyme activity by 170% and increased stability of the enzyme activity at high temperatures. One noteworthy finding is that the enzyme hydrolyzed only butyloxycarbonyl-leucyl-seryl-threonyl-arginyl-4-methylcoumaryl-7-amide (Boc-Leu-Ser-Thr-Arg-MCA, a substrate for plasma activated protein C (APC), an anticoagulation factor) among the 17 peptydyl-4-methylcoumaryl-7-amide. The enzyme extended the clotting time of rainbow trout plasma. The enzyme enhanced the hydrolyzing activity of Boc-Leu-Ser-Thr-Arg-MCA in trout plasma. These results indicate that the enzyme possesses an activity similar to APC and activates protein C of rainbow trout. The enzyme inhibited activities of Factor Xa and thrombin. From these findings, it was concluded that the metalloprotease produced by L. anguillarum plays a role as an anticoagulation factor similar to APC.

DOI： 10.3147/jsfp.31.9

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DOI： 10.2331/fishsci.62.148

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To clarify the production kinetics of antigenicity of virion polypeptides of yellowtail ascites virus (YAV), time course of the production of capsid proteins, VP2 and VP3, in infected CHSE-214 cells was surveyed by western blotting using polyclonal antisera against purified virion, VP2 or VP3. Pre-VP2 and VP3 were detected at 3 h after infection, whereas matured VP2 appeared after 7 h. The virus titer in the culture supernatant increased at 10h after infection. These results suggest that the cleavage of polyprotein to pre-VP2 and VP3 occurs in early period of infection and the VP2 maturation occurs after eclipse stage. In serological analysis, anti-VP3 serum did not neutralized the virus. Reactivity of anti-VP3 serum in ELISA increased when virion was treated with SDS.

DOI： 10.3147/jsfp.30.209

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A tributyltin chloride (TBTCl)-resistant bacterium, Alteromonas sp. M-1, was isolated from coastal seawater. This bacterium grew in medium containing 125 mu M TBTCl. TBTCl added to the medium was taken up by this bacterium, however, the amount of TBTCl in the cellular fraction was low after the logarithmic phase, suggesting the existence of a TBTCl-efflux system. A genetic library was constructed using plasmid vector pUC 19. Three positive clones were obtained, by which E. coli was transformed to TBTCl resistance. Of the three clones, the shortest fragment from HindIII-library was analyzed. This fragment was 1.8 kb long and contained one complete open reading frame. The predicted amino acid sequence of this open reading frame had a homologous domain to transglycosylases of bacteriophage and E. coli. TBTCl-tolerant marine bacteria other than Alteromonas sp. M-1 were obtained from natural seawater to which TBTCl was added. DNA-DNA hybridization was performed between the three cloned fragments from Alteromonas sp. M-1 and chromosomal DNA of the TBTCl-tolerant bacteria. Some strains hybridized with the fragments and some did not, suggesting that several genes are responsible for TBTCl tolerance.

File： 95.Tributyltin-resistant marine bacteria.pdf

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Vibriosis in ayu Plecoglossus altivelis has occurred in Lake Biwa, Japan, along with the blooming of cyanobacterial picoplankton. To search the factors accelerating the occurrence of vibriosis in ayu, the effect of picoplankton on the mortality of ayu was investigated. Pretreatment of ayu with Synechococcus sp. clone P before infection accelerated the occurrence of vibriosis in experimental infections. Moreover, a higher number of Listonella anguillara adhered to the skin which was taken from fish reared with clone P. These findings suggest that the acceleration of the occurrence of vibriosis by clone P is caused by facilitating the attachment of L. anguillara to the skin.

DOI： 10.2331/fishsci.60.713

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To determine how many kinds of proteolytic enzymes (proteases) are produced by Listonella anguillora, production profiles were surveyed using synthetic substrates, peptidyl-4-methylcoumaryl-7-amides (MCA substrates). In the qualitative assay, 19 strains of L. anguillara produced protease(s) on 0.5% skim milk agar plate. Ten strains out of 19 were selected to test for the substrate specificites among 13 kinds of MCA substrates. The strains were divided into 3 groups based on their substrate choice as follows: the first group showed high leucine aminopeptidase activity, the second showed high arginine aminopeptidase activity and the third high trypsin like-protease activity hydrolyzing t-butyloxycalbonyl-Leu-Ser-Thr-Arg-MCA (Boc-L-S-T-R-MCA). This indicates that L. anguillara produces several types of proteases. In addition, the growth and protease production of the organisms with time were examined. It was found that leucine aminopeptidase is a post-growth phase enzyme in all the 3 strains tested, whereas the secretion kinetics of arginine aminopeptidase and trypsin like-protease were different in different strains.

DOI： 10.2331/fishsci.60.741

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File： 94.Evidence for relatedness.pdf

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DOI： 10.2331/fishsci.60.353

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Resting cells of Campylobacter jejuni were spherical whereas growing cells were mainly spiral. Content of cadaverine increased with the decrease in spherical forms prior to growth commencing but production of spermidine increased in early log phase. Cadaverine and spermidine are possibly involved in changes in cell morphology and growth, respectively.

DOI： 10.1007/BF00414880

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The immunological properties of Campylobacter jejuni enterotoxin (CJT) and cholera toxin (CT) were compared by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis with antiserum against each toxin. Antibody against CJT recognized the 68, 54 and 43 kDa polypeptides of CJT and the 11 kDa subunit of CT, whereas antibody against CT recognized the 68 and 54 kDa polypeptides of CJT and 11 kDa subunit of CT. The immunological reactions between the heterogenous combinations of toxins and the antibodies were weaker than those between the homogenous systems. Thus, different antigenicity was found in CJT and CT at the subunit level, although they possessed cross-reactive epitope(s). The binding of CJT and CT to gangliosides was also examined. CJT and CT bound to GM1 ganglioside preferentially than to other ganglioside species. However, CJT did not bind to GD1b in spite of the fact that CT preferred GD1b. This suggests that both toxins recognize different receptors on the surface of the target cell. This study is the first demonstration of the different properties between CJT and CT in immunological character and ganglioside recognition.

DOI： 10.1016/0928-8244(94)90051-5

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Substrate specificities of proteases produced by two putrefactive marine bacteria, Shewanella putrefaciens and Alteromonas haloplanktis, were surveyed by using peptidyl-7-amino-4-methylcoumarin (MCA-substrates). Shewanella putrefaciens produced trypsin-like enzyme(s) showing broad spectrum specificity and chymotrypsin-like enzyme specifically hydrolysing Glt-Gly-Gly-Phe-MCA. Alteromonas haloplanktis produced high activity of aminopeptidase and trypsin-like enzyme(s) preferring Z-Phe-Arg-MCA, Bz-Arg-MCA and Boc-Leu-Ser-Thr-Arg-MCA. The two organisms would be able to utilize different proteins for their growth.

DOI： 10.1111/j.1472-765X.1994.tb00799.x

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Major outer membrane protein (MOMP) was prepared from fish pathogen Listonella anguillara. Triton X-100 treatment could extract the MOMP from the bacterium but not from Escherichia coli, suggesting loose association of the MOMP of L. anguillara to the membrane. Properties of purified MOMP from L. anguillara were similar to Omp C porin of E. coli. Similar antigenicity of the porin like-MOMP was found among different serotypes of L. anguillara, although the molecular sizes of the MOMP were different among the strains.

File： 94.Characteristics of porin-like.pdf

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The 16S rRNA of the tributyltin resistant marine bacterium, strain M-1, was partly sequenced to confirm the taxonomic status. The results indicated that this bacterium should be classified under the genus Alteromonas, instead of a previous report in which this strain was identified as a Vibrio. The genome size of this strain was also measured by pulsed field gel electrophoresis (PFGE) using a contoureclamped homogeneous electric field. The strain was found to contain a genome size of 2,240 kilo base pairs, whereas Alteromonas nigrifaciens and Shewanella putrefaciens had 2,040 and 2,383 kilo base pairs, respectively. This is the first report of the genome sizing of the genus Alteromonas by PFGE.

File： 94.The 16S rRNA.pdf

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File： 94.Purification and characterization.pdf

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File： 94.Occurrence of tributyltin.pdf

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The cytotoxic activity against cultured cells of nineteen clinically isolated strains of Campylobacter jejuni was tested. These strains were found to have different profiles in cytotoxin and enterotoxin production, and the characteristics of cytotoxin were further investigated. The cytotoxin showed cell killing toxicity against CHO and HeLa cells. Vacuole formation was observed in the case of rat hepatocyte primary culture. Treatment with trypsin at 80 degrees C for 30 min inactivated the cytotoxin activity, suggesting that the toxin was protein. The toxin was produced in the culture supernatant with high specific activity per protein, followed by polymyxin and CHAPS treatment fractions in this order. This suggests that the cytotoxin was a cell-releasing toxin and that the active toxin was present as a membrane-associated form. The cytotoxin activity was separated from the enterotoxin activity by DEAE-cellulose column chromatography. The washed fraction contained enterotoxin and cytotoxin, whereas the KCI eluted fraction showed mainly cytotoxic activity.

File： 94.Characteristics of cytotoxin.pdf

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File： 93.Cloning of gene.pdf

DOI： 10.1006/bbrc.1993.1883

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DOI： 10.3147/jsfp.28.91

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File： 93.Characterization of extracellular.pdf

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File： 93.Different pathways.pdf

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Naturally occurring amino acids and diamines such as arginine, agmatine and putrescine, inhibited proteases produced by the marine putrefactive bacteria Shewanella putrefaciens S29 and Alteromonas haloplanktis S5B, although the compounds did not inhibit bacterial growth. Among the amino acids and diamines tested, agmatine showed the greatest inhibitory effect, that is, 1 mM of agmatine inhibited 60% of the activity of the S. putrefaciens enzyme and 80% of the A. haloplanktis protease. Kinetic data indicated apparent competitive inhibition with the substrate (Bz-Arg-MCA) in the protease assay.

File： 93.Inhibition by agmatine.pdf

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Tributyltin chloride (TBTCl)-tolerant bacteria accounted for 90% of the flora in natural seawater to which TBTCl was added. These tolerant bacteria were insensitive to 250 nmol of TBTCl per disc, and all were Vibrio species. Total counts of viable bacteria did not decrease upon storage of the TBTCl-treated seawater, indicating that enrichment of tolerant strains took place. Addition of CdSO4 to seawater resulted in the occurrence of TBTCl-tolerant bacteria as well as Cd-tolerant bacteria, suggesting some correlation of Cd tolerance and TBTCl tolerance.

File： 92.Occurrence of tributyltin.pdf

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Tributyltin chloride (TBTCl)-resistant marine bacteria were isolated from coastal sea water. One of these bacteria (Vibrio M-1) was highly resistant when grown in medium containing 125-mu-M of TBTCl. This strain was sensitive to other metals. Two polypeptides, 30 kDa and 12 kDa, increased when the strain was cultured in the medium supplemented with TBTCl. Initially TBTCl was taken up by the cell; however, the amount of TBTCl determined in the cellular fraction was low after the exponential growth phase of Vibrio M-1, suggesting the existence of a TBTCl-efflux system.

DOI： 10.1016/0378-1097(92)90493-8

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DNA polymerase activities were surveyed in tumour tissue and normal tissue of cherry salmon (Oncorhynchus masou). High activity of DNA polymerase-alpha was detected in the tumour tissue but not in the normal tissue. This indicates that the tumour cells replicate prosperously. Viral DNA polymerase activity was detected only in the tumour tissue, indicating that Oncorhynchus masou virus (OMV) DNA should replicate there. DNA polymerase-beta activity was of same level in both tissues. This is the first evidence that herpesvirus DNA polymerase was detected in tumour tissue in association with herpesvirus.

File： 92.Detection of viral.pdf

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

1. Acute toxicity of ozone exposure to Japanese charr (Salvelinus leucomaenis) was studied histopathologically and hematologically on gill tissue and red blood cells (RBC) under different ozone conentrations (0-0.7 ppm).
2. Exposure of ozone above 0.7 ppm led to characteristic symptoms and all died of choking in 30 min.
3. Many swollen RBC were seen under the scanning electron microscope.
4. RBC congestion was serious in the gill where degeneration of lamellar epithelium was observed. However, injury to chloride cells was not clear.

File： 92.Acute toxicity.pdf

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Trimethylamine-N-oxide (TMAO) inhibited the growth of Staphylococcus aureus but not of S. epidermidis, which is the main flora in ripening squid, the Japanese traditional sea food Ika-shiokara. This selective inhibition of S. aureus was based on the inhibition of the electron transport system. The cytochrome fraction isolated from S. aureus was converted from the reduced to the oxidized form by the addition of TMAO. It is suggested that the inhibition occurred between cytochrome B and cytochrome o.

File： 92.Inhibition of the electron.pdf

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We have adopted the in vitro hepatocyte culture system of the duck infected with duck hepatitis B virus (HDBV) to an antiviral assay system. Using this method, we found that 2',3'-dideoxy-3'-azidothymidine (N3dT) and 2', 3'-dideoxy-3'-O-methylthymidine (OMeT) had antiviral effects against DHBV replication in the concentrations of 20-50-mu-mol/l and 4-40-mu-mol/l, respectively. The N3dT inhibited the single strand DNA formation (negative strand), which is an intermediate of virus replication. However, the inhibition of single strand DNA synthesis by OMeT was relatively weak. These two compounds may have different mechanisms of DHBV DNA replication inhibition. Two other 3'-substituted pyrimidine analogues tested were very weak inhibitors. Antiviral agents that inhibit the reverse transcriptase activity of the hepadnavirus DNA polymerase could be potential candidates for the chemotherapy of these viruses.

File： 91.Inhibition of duck.pdf

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File： 91.Synergistic inhibitory.pdf

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The phospholipid and fatty acid composition of Alteromonas putrefaciens S29 (non-halophilic type) and A. haloplanktis S5B (halophilic type) was determined. Major phospholipids of both strains were the same when they were grown in media containing optimum salt concentrations. However, the fatty acid composition of phospholipids in strain S29 was remarkably different from that of strain S5B. Strain S29 contained iso-C15:0 and eicosapentaenoic acid (20:5) as constituent fatty acids of phospholipids and also contained sterol ester and wax as neutral lipids. In contrast, strain S5B did not contain branched and polyunsaturated fatty acids, and neither sterol ester nor wax were detected.

File： 91.Phospholipid and fatty.pdf

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Ornithine decarboxylase (ODC) activity in cell extracts of Shewanella putrefaciens was surveyed. The pH dependency of the ODC activity revealed that the bacterium has two different ODC having optimum pH at 8.25 and 6.50. They were considered to be biosynthetic and biodegradative enzymes, respectively. Their activity ratio varied when the bacterium was cultured at pH 7.0 and 6.0. Both ODC activities were inhibited by alpha-difluoromethylornithine but cell growth was not affected.

DOI： 10.1111/j.1472-765X.1991.tb00518.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIRKHAUSER VERLAG AG  

DOI： 10.1007/BF01940675

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File： 90.Partial purification.pdf

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File： 90.Simultaneous determination.pdf

DOI： 10.1016/S0021-9673(00)91259-7

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File： 89.Different properties.pdf

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

DOI： 10.1128/AAC.33.3.336

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DOI： 10.1016/0378-1097(89)90337-6

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File： 89.Distribution and .pdf

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

File： 88.Inhibition of duck .pdf

DOI： 10.1016/S0006-291X(88)80752-6

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PubMed

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)  

File： 88.Profiles of polyamine.pdf

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

File： 87.Inhibitory effects.pdf

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC VET SCI  

DOI： 10.1292/jvms1939.49.403

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILLIAMS & WILKINS  

File： 87.A Proposed mechanism.pdf

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

DOI： 10.1016/0166-3542(87)90023-4

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PubMed

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

File： 86.Characterization of DNA.pdf

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Authorship：Lead author   Publishing type：Research paper (scientific journal)  

File： 86.Delay of herpesvirus.pdf

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

DOI： 10.1128/AAC.28.2.326

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

DOI： 10.1016/0042-6822(84)90119-3

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

DOI： 10.1016/0166-3542(83)90031-1

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

DOI： 10.1016/0166-3542(83)90030-X

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Total pages：xxix, 546 p.   Language：English  

CiNii Books

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Total pages：xv, 261p   Language：Japanese  

CiNii Books

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Total pages：xiii, 432p, 図版 [8] p   Language：Japanese  

CiNii Books

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Total pages：x, 508p   Language：Japanese  

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Total pages：xii, 252p   Language：Japanese  

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Total pages：ix, 204p, 図版 [8] p   Language：Japanese  

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Total pages：冊   Language：Japanese  

CiNii Books

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Total pages：viii, 237p   Language：Japanese  

CiNii Books

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Total pages：xxvii, 470 p.   Language：English  

CiNii Books

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Total pages：xvi, 343 p. :ill.   Language：English  

CiNii Books

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Total pages：282p   Language：Japanese  

CiNii Books

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Total pages：xvi, 326 p.   Language：English  

CiNii Books

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Total pages：xix, 379 p.   Language：English  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

File： 19.Drug resistant bacteria Ocean Newslett Selected.pdf

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

File： 18.海にもいる薬剤耐性菌 Ocean Newslett.pdf

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

File： 臨床微生物（合）2018s.pdf

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Language：Japanese   Publishing type：Lecture material (seminar, tutorial, course, lecture, etc.)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

File： 17.水環境に拡散する 公衆衛生.pdf

DOI： 10.11477/mf.1401208759

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

File： 14.水圏環境は薬剤耐性＿動物抗菌薬.pdf

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

File： 12.水圏環境におけるバイオサイエンスとインダストリ.pdf

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

File： 12.魚介類養殖環境における.pdf

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Rapid communication, short report, research note, etc. (bulletin of university, research institution)  

File： 11.Effect of exposure.pdf

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Other Link： http://iyokan.lib.ehime-u.ac.jp/dspace/handle/iyokan/1365

Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 10.Aeromonas molluscorum.pdf

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 10.Tetracycline resistance.pdf

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 10.Abundance of sulfonamide.pdf

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Authorship：Last author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 10.Distribution of mercury.pdf

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Authorship：Last author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 10.The effect of zinc.pdf

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Authorship：Corresponding author   Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (bulletin of university, research institution)  

File： 09.フミン酸分解菌.pdf

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Language：Japanese  

J-GLOBAL

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 09.Changes in proteolytic.pdf

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 09.Quantitative analysis.pdf

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 08.Cell-to-cell.pdf

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 08.Occurrence rates of sulfa-.pdf

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

File： 08.水環境における薬剤耐性菌.pdf

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Language：English   Publisher：JAPANESE SOC FISHERIES SCIENCE  

File： 07.水圏環境における日水誌.pdf

DOI： 10.2331/suisan.73.317

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Scopus

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Rapid communication, short report, research note, etc. (bulletin of university, research institution)   Publisher：Ehime University  

In the south Sumatra region of Indonesia, tiger grouper, Epinephelus fuscoguttatus, and humpbuck grouper, Cromileptes altivelis, are commercially important fish species. Recently, however, mass mortalities of the two species have occurred in the region and the cause of these mortalities has not been clarified. We surveyed the distribution of fish viruses including viral nervous necrosis virus (VNNV), marine birnavirus (MABV) and red seabream iridovirus (RSIV) in the two grouper species and MABV in silver the lip oyster, Pinctada maxima, by PCR. VNNV was detected from both healthy and diseased groupers whereas MABV was only detected in diseased groupers, suggesting that the mass mortalities of the two grouper species were caused by co-infection of VNNV and MABV. PCR product of RSIV was not observed in any fish samples. In addition, MABV was not detected in silver lip oysters from both healthy and those exhibiting poor growth.

File： 07.Virus detection.pdf

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Other Link： http://iyokan.lib.ehime-u.ac.jp/dspace/handle/iyokan/1352

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

File： 07.ナノサイズのウィルス.pdf

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Authorship：Last author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 06.Microbial degradation.pdf

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 06.Transfer of tetracycline.pdf

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 06.Occurrence of tetracycline.pdf

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 06.Tributyltin(TBT)　resistance.pdf

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution)  

File： 05.逆浸透膜を.pdf

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 05.Tetracycline resistance.pdf

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

File： 04.Effect of Tributyltin.pdf

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (bulletin of university, research institution)  

File： 03.十勝沖コア試料中.pdf

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Authorship：Last author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

File： 03.海洋環境に.pdf

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Language：Japanese   Publisher：地球環境財団  

File： 02.海洋における有機スズ化合物.pdf

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Language：Japanese   Publisher：愛媛大学農学部  

1994年から1999年まで、西日本各地でアコヤガイの大量斃死が続いたが、原因究明の過程で著者らは斃死状態のアコヤガイからMABVを分離した。アコヤガイから分離されたMABV(JPO-96株)は実験感染ではアコヤガイに対する病原性は弱いとされている。しかし、台湾で分離されたビルナウイルスはハマグリにおいて重金属や温度差などのストレス下で強い病原性を示すことが報告されていることから、西日本で起こったアコヤガイ大量斃死の場合も、貝に何らかのストレスが加わりMABVの病原性が増大した可能性がある。また、MABVは病気の魚介類のみならず、様々な健康な魚介類からも分離されており、宿主域が広く環境中にも遍在していることが考えられる。これらの点から、MABVの宿主内および環境中における動態を知ることは魚介類の防疫の面から重要である。しかし、MABVの水圏環境における動態を体系的に検討された報告はない。本稿では、宇和海におけるMABVの生態に関して、得られた知見を紹介する。

File： 02.宇和海のアコヤガイと.pdf

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010660878

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Authorship：Last author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

File： 01.宇和海のアコヤガイ漁場.pdf

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

File： 00.海洋細菌のタンパク質.pdf

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (bulletin of university, research institution)  

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Language：Japanese   Publisher：文永堂出版  

File： 99.魚介類ビルナウイルス.pdf

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

File： 99.ポーリンはなぜ.pdf

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Internal/External technical report, pre-print, etc.  

File： 98.アコヤガイでの通年動態.pdf

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper, summary (international conference)  

File： 98.Molecular ecology.pdf

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

File： 98.宿主域の広い.pdf

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Language：English  

File： 98.Experimental infection.pdf

CiNii Books

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Authorship：Last author,　Corresponding author   Publishing type：Internal/External technical report, pre-print, etc.  

File： 97.海産魚のビルナウイルス.pdf

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)  

File： 97.特集Vibrio.pdf