Approach to determine the diversity of Legionella species by nested PCR-DGGE in aquatic environments

Abstract

In this study, we describe a nested PCR-DGGE strategy to detect Legionella communities from river water samples. The nearly full-length 16S rRNA gene was amplified using bacterial primer in the first step. After, the amplicons were employed as DNA templates in the second PCR using Legionella specific primer. The third round of gene amplification was conducted to gain PCR fragments apposite for DGGE analysis. Then the total numbers of amplified genes were observed in DGGE bands of products gained with primers specific for the diversity of Legionella species. The DGGE patterns are thus potential for a high-throughput preliminary determination of aquatic environmental Legionella species before sequencing. Comparative DNA sequence analysis of excised DGGE unique band patterns showed the identity of the Legionella community members, including a reference profile with two pathogenic species of Legionella strains. In addition, only members of Legionella pneumophila and uncultured Legionella sp. were detected. Development of three step nested PCR-DGGE tactic is seen as a useful method for studying the diversity of Legionella community. The method is rapid and provided sequence information for phylogenetic analysis.

Fig 1. Graphic overview of the various PCR-DGGE tactics used to study the diversity of Legionella in river water samples analyzed.

(1) single step directed PCR-DGGE tactic; (2) two step PCR-DGGE tactic; (3) three step nested PCR-DGGE tactic. Comparative DGGE pattern analysis of PCR products gained by tactics1/2/3 makes it likely to infer the diversity of Legionella.

Fig 2. DGGE band patterns of 16S rRNA fragments gained after PCR amplification using various primers and DNA from river water samples.

Lanes: A-E, pattern of river water samples; Lanes: F and G, pattern of positive control samples (Legionella pneumophila ATCC 33823 and Legionella dumoffii ATCC 33279); Lanes: A, F and G, pattern gained with PCR products amplified using single step direct PCR method (strategy 1); Lanes: B and C, pattern gained from the product amplified using the two step PCR method (strategy2); D and E, pattern when primers specific to the Legionella species were used in the three step nested PCR method (strategy 3). DGGE patterns that were excised for DNA sequence analysis are numbered.

Fig 3. A phylogenetic tree produced with the neighbor-joining method based on the 16S rRNA gene sequences of Legionella from river water samples.

The numbers of the sequences in this tree refer to the numbers in the DGGE. The scale bar represents 200% of nucleotide sequence divergence. The numbers at the selected nodes indicate the levels of bootstrap support (percentage) based on 1000 re-sampled data sets (only values greater than 70% shown).