Assessment of phylogenetic affiliation using 16S rRNA gene sequence analysis for Pseudomonas aeruginosa in patients of lower respiratory tract infection

Sir,

Lower respiratory tract infections (LRTI) are among the most common infectious diseases of humans worldwide, and are the leading cause of morbidity and mortality in critically ill patients in developing countries123. Most common bacterial agents of LRTI are Pseudomonas, Acinetobacter, Klebsiella, Citrobacter, and Escherichia coli145. In India, P. aeruginosa is the most common organism of LRTI (30-50%) followed by Klebsiella spp6. P. aeruginosa infections of the lower respiratory tract can range in severity from colonization (without an immunological response) to a severe necrotizing bronchopneumonia7.

Genotype-based identification methods circumvent the problem of variable phenotype to provide more accurate species identification. However, the taxonomic complexity, uncertain phylogeny, and paucity of genomic sequence data of the dozens of species within the broad genus Pseudomonas present an obstacle to genotypic identification assays. The part of the DNA commonly used for taxonomic purposes for bacteria is the 16S rRNA gene8910111213, also designated 16S rDNA. We undertook this study to assess the phylogenetic affiliation of P. aeruginosa using 16S rRNA gene sequence analysis in patients with LRTI.

P. aeruginosa isolates (n=102) were obtained from 298 patients with a confirmed history of LRTI who attended the outpatient Department of Pediatrics and Pulmonary Medicine of Gandhi Memorial and Associated Hospital of K.G. Medical University, Lucknow, India, during December 2007 and December 2010. All Pseudomonas isolates were obtained from fresh sputum specimens cultured on Pseudomonas Isolation Agar (Hi Media, Mumbai, India). The identity of all isolates was confirmed by using biochemical testing such as oxidase, nitrate reduction, citrate utilization, oxidative fermentation, arginine, and growth at 42^°C14.

Bacterial genomic DNA was extracted from P. aeruginosa reference strain ATCC 27853 (Department of Microbiology, K.G. Medical University, Lucknow, India), as well as from 102 Pseudomonas isolates from the sputum of LRTI patients, using the Geni Preparation kit in accordance with the manufacturer's instructions (Bangalore, India). Extracted DNA was stored at -20^oC prior to PCR amplification. For each batch of extractions, negative and positive controls were run.

PCR was done for all 102 Pseudomonas isolates and reference strain ATCC 27853. Amplification reactions were set up as detailed by Millar et al15. The following 16S rRNA specific primer set was used (Sigma-aldrich, Bangalore, India): 16S forward primer: 5’-AGAGTRTGATCMTYGCTWAC-3’; 16S reverse primer: 5’-CGYTAMCTTWTTACGRCT-3’.

Following optimization, reaction mixes (100 μl) were set up as follows: 10 mM Tris/HCl, pH 8·3; 50 mM KCl; 2·5 mM MgCl₂; 200 μM (each) dATP, dCTP, dGTP and dTTP; 1·25U Taq DNA polymerase (Genei Bangalore, India); 0·1 μM (each) primer; and 4 μl DNA template. Reaction mixtures, following a ‘hot start’, were subjected to the following empirically optimized thermal cycling parameters: 94^°C for 5 min, followed by 35 cycles of 94^°C for 30 sec, 55^°C for 30 sec and 72^°C for 2 min, followed by a final extension at 72^°C for 5 min. Positive (P. aeruginosa ATCC 27853 DNA) and multiple negative (water) amplification controls were included in every set of PCRs.

To confirm PCR-based identification results, comparative 16S rDNA sequence analysis was performed. All P. aeruginosa PCR amplicons were purified and eluted in Tris/HCl (10 mM, pH 8·5) prior to sequencing to remove dNTPs, polymerases, salts and primers. Randomly 10 PCR positive samples with reference strain ATCC 27853 were sequenced in both directions on an automated sequencer (Chromous Biotech Pvt. Ltd., Bangalore) by using both forward and reverse primers. The resulting sequences were aligned and compared with those stored in GenBank (http://www.ncbi.hlm.nih.gov/genbank/) by using BLAST alignment software (NCBI) and phylogenetic tree was drawn.

PCR amplification of genomic DNA from extracted organisms generated an amplicon of the expected size (approx. 1.4 Kb) for all P. aeruginosa isolates. Phylogenetic analysis was done of 1,411 bp sequence product with Pseudomonas species as well as several other γ-Proteobacteria using BLAST program on NCBI. The comparison of the 16S rRNA gene sequences allows differentiation between organisms at the genus level across all major phyla of bacteria, in addition to classifying strains at multiple levels, including the species and subspecies levels. The occasional exceptions to the usefulness of 16S rRNA gene sequencing usually relate to more than one well-known species having the same or very similar sequences.

16S rDNA sequence has long been used as a taxonomic gold standard in determining the phylogenies of bacterial species16. Selective amplification of Pseudomonas 16S rDNA by PCR followed by restriction fragment length polymorphism analysis or denaturing gradient gel electrophoresis has been used to detect and differentiate Pseudomonas species from clinical and environmental sample17181920.

In this study, we took advantage of a reassessment of the phylogenetic affiliation of the pseudomonads to re-examine the rapidly expanding 16S rDNA sequence data available in public databases21. Based on an alignment of 136 16S rDNA sequences from 42 validly described Pseudomonas species as well as several other γ-Proteobacteria, we identified Pseudomonas genus-specific and P. aeruginosa-specific signature sequences.