No abstract

Sixty thousand of deaths among cultured Ezo abalone Haliotis discus hannai occurred within a few days at an abalone farm in Japan in the middle of August, 2002. Dead animals were characterized by a hemolymphatic edema around the major circulatory system. Vibrios showing swarming motility dominated in the edema. The pathogenic vibrios were identified as Vibrio harveyi based on a phylogenetic analysis and a phenotypic characterization. In both immersion and injection experiments, the swarming vibrios fulfilled Koch's postulates as a pathogen for Ezo abalone. Using a GFP-tagged V. harveyi S20, a clump of bacterium was detected on the gills of the abalone within 48 hours after contact with the bacterium. This is the first report of V. harveyi infection in Ezo abalone Haliotis discus hannai.

In fish sauce production, microorganisms are associated with the fermentation process; however, the sequential changes in the bacterial communities have never been examined throughout the period of fermentation. In this study, we determined the bacterial floras in a fish sauce mash over 8 months, using three different culture media and 16S rRNA gene clone library analysis. During the first 4 weeks, viable counts of non-halophilic and halophilic bacteria decreased and were dominated by Staphylococcus species. Between 4 and 6 weeks, halophilic and highly halophilic bacterial counts markedly increased from 10(7) to 10(8) cfu/g, and the predominant species changed to Tetragenococcus halophilus. The occurrence of T. halophilus was associated with an increase of lactic acid and a reduction of pH values. In contrast, non-halophilic bacterial counts decreased to 10(6) cfu/g by 6 weeks with Bacillus subtilis as the dominant isolate. Clone library analysis revealed that the dominant bacterial group also changed from Staphylococcus spp. to T. halophilus, and the changes were consistent with those of the floras of halophilic and highly halophilic isolates. This is the first report describing a combination approach of culture and clone library methods for the analysis of bacterial communities in fish sauce mash.

Aim:  Our goal was to develop a simple system for tagging wild‐type marine bacteria with gfp. Methods and Results:  Escherichia coli strain CC118λpir carrying the conjugative helper plasmid pEVS104 and the gfp‐containing plasmid pKV111 was used to transfer gfp to Vibrio recipients. Four different media were tested for their ability to support the growth of recipients, but not the E. coli donor, to allow powerful enrichment of gfp‐tagged wild‐type vibrios from mating mixes. Forty‐three vibrio strains, representing 39 different species, were successfully tagged with gfp using the conjugative transfer from E. coli followed by selective outgrowth at 15°C on ZoBell 2216E agar containing 0·5% sodium alginate. Using this outgrowth medium, colonies of GFP‐expressing vibrio clones were detectable within 4 days. The percentage of visibly fluorescent cells in three representative GFP‐tagged vibrios was higher at 15°C than at 20 or 25°C (c. 50% vs. 45% or 40%, respectively), and was also higher during the aerobic rather than the anaerobic culturing (c. 50% vs. 35%, respectively). Conclusions:  We found a simple selective outgrowth technique that enabled us to isolate a wide variety of GFP‐tagged marine vibrios following the conjugative transfer of gfp from E. coli. Significance and Impact of the Study:  Tagging cells with GFP and related fluorescent proteins is a powerful approach for investigating the bacteria in situ, particularly during the colonization of hosts. The simple and cost‐effective outgrowth condition described in this study could be applied to construct a wide variety gfp‐tagged marine bacteria.

No abstract