Molecular Analysis of Intestinal Microbiota of Rainbow Trout (Oncorhynchus mykiss)

Chile has become the world’s second main producers of farmed salmonids. Over the last 15 years, production has been soaring, however more recently production has been negatively impacted by the increase of certain diseases; this may be due to negative interaction between salmonids and pathogens. It has been shown that some components of gastrointestinal (GI) microbiota can produce inhibitors for potential pathogens of fish and also control the colonisation of them.

Molecular methods have proven more efficient when studying the GI bacterial community of fishes. These methods include temporal temperature gradient gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) of DNA fragments which are dominant in the majority of bacterial species in environmental samples. The gene encoding 16S rDNA is normally used for analysis; however there are some disadvantages to using this, which include the heterogeneity of the different 16S rDNA genes and the poor discrimination between closely related bacteria. Due to these problems, the use of other genes has been proposed, such as the spacer region between 16S and 23S rDNA genes (known as the internal transcribed space [ITS]) and the rpoB gene.

The main aim of this study was to evaluate TTGE and restriction fragment length polymorphism (RFLP) based on the markers 16S rDNA, ITS and the rpoB gene, in order to study the composition of juvenile rainbow trout (O. mykiss) intestinal microbiota.

Bacterial counts were taken from samples. PCR was then completed in order to obtain the fingerprints presents in the intestinal samples and to amplify the 16S rDNA, ITS and the rpoB genes. RFLP and TTGE then took place, followed by sequencing analysis.

The dominant bacteria isolated from the samples of juvenile trout using cultural methods and 16S rRNA and rpoB gene sequencing included Citrobacter gillenii, Obesumbacterium proteus, Kluyvera intermedia, Shewanella sp., and L. lactis. The bacterial counts obtained in this study were similar to those previously reported in the GI tract. The bands excised from 16S rRNA gene- and rpoB-TTGE sequencing showed that all dominant bacteria in the samples could be cultured on TSA plates; however culturing methods are slower than molecular methods. Intestinal bacterial composition using 16S rRNA gene PCR-RFLP revealed that bands could be obtained from a single bacterial species and several bands could comigrate in the gel, making interpretation difficult. In other studies 16S rRNA gene-TTGE and –DGGE, have successfully characterised bacterial populations associated with certain species of fish, although in this study there were multiple bands for single strains. It was found that individual bands in the ITS-TTGE profiles should not be taken as exact measures of bacterial diversity in communities. This is because the number of bands cannot be strictly correlated to the number of distinct bacterial types. With the rpoB-TTGE profiles, bacterial strains exhibited one band per species. Moreover, the main strains present in the bacterial community could be identified using the rpoB PCR-TTGE method.

Overall, the PCR-TTGE and PCR-DGGE methods used were not quantitative, although it has been reported that the band intensity in TTGE profiles may be related to the bacterial abundance in the ecosystem. The rpoB-TTGE gene analysis was the more successful method in this study and should be used in the future when investigating the composition of intestinal microbiota of juvenile rainbow trout. I believe is a significant study as it shows methods which can be used in the future.

Navarette et al., 2009. ‘Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss)’. FEMS Microbiology Ecology. Volume 71. Issue 1. Pages 148-156.