Molecular identification of intestinal microflora in Takifugu niphobles

We investigated the intestinal microflora of coastal fish including Takifugu niphobles using both culture techniques and library cloning. As a result, the numbers of bacteria appeared on agar media were 1.0 × 104 to 1.4 × 109 CFU/g (colony forming units/gram), whereas those of total bacteria stained with 4′,6-diamidino-2-phenylindole were 4.7 × 1010 to 1.9 × 1011 cells/gram, irrespective of different fish species. In addition, the culture technique showed that the intestinal microflora in all specimens was mainly composed of the genus Vibrio. In contrast, the direct count method showed that spirochaetes with length of 2.5-4.5 μm were present in the intestinal contents of T. niphobles at high densities, whereas such bacteria could not be detected in those of other fish species. Library cloning yielded the sequences of 16S rRNA genes that were divided into seven taxonomic categories of bacteria including Actinobacteria, Bacilli, Clostridia, Gammaproteobacteria, Mollicutes, Spirochaetes and an unclassified bacterial group. These results demonstrate that the molecular diversity of the intestinal bacteria in T. niphobles based on the clone library method reflects the direct observation by fluorescence microscopy to some extent.