Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)

Authors


  • Present addresses: Ingrid Huber, GeneScan Analytics GmbH, Engesser Str. 4, D-79108 Freiburg, Germany.

  • Bettina Spanggaard, Novozymes A/S, Hallas Allé, DK-4400 Kalundborg, Denmark.

  • Torben Nielsen, Siljeager 1, DK-7300 Jellinge, Denmark.

Lone Gram, Danish Institute for Fisheries Research, Department of Seafood Research, Søltofts Plads, c/o Technical University of Denmark, bldg. 221, DK-2800 Kgs. Lyngby, Denmark (e-mail: gram@dfu.min.dk).

Abstract

Aims:  To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine.

Methods and Results:  Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4,6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were ≤2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50–90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were ≤2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium.

Conclusions:  Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish.

Significance and Impact of the Study:  Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.

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