|Year : 2013 | Volume
| Issue : 4 | Page : 557-559
Assessment of phylogenetic affiliation using 16S rRNA gene sequence analysis for Pseudomonas aeruginosa in patients of lower respiratory tract infection
Piyush Tripathi1, Gopa Banerjee1, Mahendra Kumar Gupta1, Shivani Saxena1, PW Ramteke2
1 Department of Microbiology, K.G. Medical University, Lucknow 226 003, India
2 Sam Higginbottom Institute of Agriculture, Technology & Sciences, Allahabad, India
|Date of Web Publication||19-Nov-2013|
Department of Microbiology, K.G. Medical University, Lucknow 226 003
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Tripathi P, Banerjee G, Gupta MK, Saxena S, Ramteke P W. Assessment of phylogenetic affiliation using 16S rRNA gene sequence analysis for Pseudomonas aeruginosa in patients of lower respiratory tract infection. Indian J Med Res 2013;138:557-9
|How to cite this URL:|
Tripathi P, Banerjee G, Gupta MK, Saxena S, Ramteke P W. Assessment of phylogenetic affiliation using 16S rRNA gene sequence analysis for Pseudomonas aeruginosa in patients of lower respiratory tract infection. Indian J Med Res [serial online] 2013 [cited 2020 Oct 27];138:557-9. Available from: https://www.ijmr.org.in/text.asp?2013/138/4/557/121555
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 countries ,, . Most common bacterial agents of LRTI are Pseudomonas, Acinetobacter, Klebsiella, Citrobacter, and Escherichia coli,, . In India, P. aeruginosa is the most common organism of LRTI (30-50%) followed by Klebsiella spp  . P. aeruginosa infections of the lower respiratory tract can range in severity from colonization (without an immunological response) to a severe necrotizing bronchopneumonia  .
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 gene ,,,,, , 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 o C  .
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 o C 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 al . 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 ΅l) were set up as follows: 10 mM Tris/HCl, pH 8·3; 50 mM KCl; 2·5 mM MgCl 2 ; 200 ΅M (each) dATP, dCTP, dGTP and dTTP; 1·25U Taq DNA polymerase (Genei Bangalore, India); 0·1 ΅M (each) primer; and 4 ΅l DNA template. Reaction mixtures, following a 'hot start', were subjected to the following empirically optimized thermal cycling parameters: 94C for 5 min, followed by 35 cycles of 94C for 30 sec, 55C for 30 sec and 72C for 2 min, followed by a final extension at 72C 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 Pseudomonas0 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 species  . 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 sample ,,, .
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 databases  . 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.
| Acknowledgment|| |
Authors acknowledge Department of Science and Technology (DST), New Delhi, for the financial support.
| References|| |
|1.||Navaneeth BV, Belwadi MR. Antibiotic resistance among gram-negative bacteria of lower respiratory tract secretion in hospitalized patients. Indian J Chest Dis Allied Sci 2002; 44 : 173-6. |
|2.||Pittet D. Nosocomial pneumonia: Incidence, morbidity and mortality in the intubated-ventilated patients. Schweiz Med Woechensch 1994; 124 : 227-35. |
|3.||Kumari HBV, Nagarathna S, Chandramuki A. Antimicrobial resistance pattern among aerobic gram negative bacilli of lower respiratory tract specimens of intensive care unit patients in a neurocentre. Indian J Chest Dis Allied Sci 2007; 49 : 19-22. |
|4.||Gonlugur U, Bakici MZ, Akkurt I, Efeoglu T. Antibiotic susceptibility patterns among respiratory isolates of Gram negative bacilli in Turkish University Hospital. BMC Microbiol 2004; 4 : 32-4. |
|5.||Mukhopadhyay C, Bhargava A, Ayyagari A. Role of mechanical ventilation and development of multidrug resistant organisms in hospital acquired pneumonia. Indian J Med Res 2003; 118 : 229-35. |
|6.||Gagneja D, Goel N, Aggarwal R, Chaudhary U. Changing trend of antimicrobial resistance among Gram-negative bacilli isolated from lower respiratory tract of ICU patients: A 5-year study. Indian J Crit Care Med 2011; 15 : 164-7. |
|7.||Banerjee D, Stableforth D. The treatment of respiratory pseudomonas infection in cystic fibrosis: what drug and which way? Drugs 2000; 60 : 1053-64. |
|8.||Bottger EC. Rapid determination of bacterial ribosomal RNA sequences by direct sequencing of enzymatically amplified DNA. FEMS Microbiol Lett 1989; 65 : 171-6. |
|9.||Garrity GM, Holt JG. The road map to the manual. In: Garrity GM, editor. Bergey's manual of systematic bacteriology. New York: Springer-Verlag; 2001. p. 119-66. |
|10.||Harmsen D, Karch H. 16S rDNA for diagnosing pathogens: a living tree. ASM News 2001; 70 : 19-24. |
|11.||Kolbert CP, Persing DH. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Curr Opin Microbiol 1999; 2 : 299-305. |
|12.||Palys T, Nakamura LK, Cohan FM. Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data. Int J Syst Bacteriol 1997; 47 : 1145-56. |
|13.||Tortoli E. Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s. Clin Microbiol Rev 2003; 16 : 319-54. |
|14.||Oberhofer TR. Growth of nonfermentative bacteria at 42 degrees C. J Clin Microbiol 1979; 10 : 800-4. |
|15.||Millar BC, Xu J, Moore JE. Risk assessment models and contamination management: implications for broad-range ribosomal DNA PCR as a diagnostic tool in medical bacteriology. J Clin Microbiol 2002; 40 : 1575-80. |
|16.||Woese CR. Bacterial evolution. Microbiol Rev 1987; 51 : 221-71. |
|17.||Duineveld BM, Kowalchuk GA, Keijzer A, van Elsas JD, van Veen JA. Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA. Appl. Environ Microbiol 2001; 67 : 172-8. |
|18.||Porteous LA, Widmer F, Seidler RJ. Multiple enzyme restriction fragment length polymorphism analysis for high resolution distinction of Pseudomonas (sensu stricto) 16S rRNA genes. J Microbiol Methods 2002; 51 : 337-48. |
|19.||Schabereiter-Gurtner C, Maca S, Rolleke S, Nigl K, Lukas J, Hirschl A, et al. 16S rDNA-based identification of bacteria from conjunctival swabs by PCR and DGGE fingerprinting. Invest Ophthalmol Vis Sci 2001; 42 : 1164-71. |
|20.||Widmer F, Seidler RJ, Gillevet PM, Watrud LS, Di Giovanni GD. A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples. Appl Environ Microbiol 1998; 64 : 2545-53. |
|21.||Anzai Y, Kim H, Park JY, Wakabayashi H, Oyaizu H. Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Int J Syst Evol Microbiol 2000; 50 : 1563-89. |