A comparison of two informative SNP-based strategies for typing Pseudomonas aeruginosa isolates from patients with cystic fibrosis

BMC Infectious Diseases

David M Whiley¹  Theo P Sloots⁶  Michael Nissen⁶  Keith Grimwood¹  Claire E Wainwright⁵  Scott C Bell²  Snehal Anuj⁴  Kristen M Gibson⁴  Kay A Ramsay¹  Ralf J Moser³  Timothy J Kidd¹  Melanie W Syrmis¹ 

[1] Queensland Paediatric Infectious Disease Laboratory, Block 28, Royal Children’s Hospital, Herston Road, Herston, Brisbane 4029, Queensland, Australia;Department of Thoracic Medicine, The Prince Charles Hospital, Brisbane, Queensland 4032, Australia;Sequenom Inc., Sequenom Asia Pacific, Brisbane, Queensland 4029, Australia;Queensland Children’s Medical Research Institute, The University of Queensland, Brisbane, Queensland 4029, Australia;Queensland Children’s Respiratory Centre, Royal Children’s Hospital, Brisbane, Queensland 4029, Australia;Microbiology Division, Pathology Queensland Central Laboratory, Brisbane, Queensland 4029, Australia

关键词: SNP;    MLST;    Cystic fibrosis;    Typing;    Pseudomonas aeruginosa;   

Others  :  1127651
DOI  :  10.1186/1471-2334-14-307

 received in 2013-10-28, accepted in 2014-05-28,  发布年份 2014

【 摘 要 】

Background

Molecular typing is integral for identifying Pseudomonas aeruginosa strains that may be shared between patients with cystic fibrosis (CF). We conducted a side-by-side comparison of two P. aeruginosa genotyping methods utilising informative-single nucleotide polymorphism (SNP) methods; one targeting 10 P. aeruginosa SNPs and using real-time polymerase chain reaction technology (HRM10SNP) and the other targeting 20 SNPs and based on the Sequenom MassARRAY platform (iPLEX20SNP).

Methods

An in-silico analysis of the 20 SNPs used for the iPLEX20SNP method was initially conducted using sequence type (ST) data on the P. aeruginosa PubMLST website. A total of 506 clinical isolates collected from patients attending 11 CF centres throughout Australia were then tested by both the HRM10SNP and iPLEX20SNP assays. Type-ability and discriminatory power of the methods, as well as their ability to identify commonly shared P. aeruginosa strains, were compared.

Results

The in-silico analyses showed that the 1401 STs available on the PubMLST website could be divided into 927 different 20-SNP profiles (D-value = 0.999), and that most STs of national or international importance in CF could be distinguished either individually or as belonging to closely related single- or double-locus variant groups. When applied to the 506 clinical isolates, the iPLEX20SNP provided better discrimination over the HRM10SNP method with 147 different 20-SNP and 92 different 10-SNP profiles observed, respectively. For detecting the three most commonly shared Australian P. aeruginosa strains AUST-01, AUST-02 and AUST-06, the two methods were in agreement for 80/81 (98.8%), 48/49 (97.8%) and 11/12 (91.7%) isolates, respectively.

Conclusions

The iPLEX20SNP is a superior new method for broader SNP-based MLST-style investigations of P. aeruginosa. However, because of convenience and availability, the HRM10SNP method remains better suited for clinical microbiology laboratories that only utilise real-time PCR technology and where the main interest is detection of the most highly-prevalent P. aeruginosa CF strains within Australian clinics.

【 授权许可】

   
2014 Syrmis et al.; licensee BioMed Central Ltd.

【 参考文献 】

• [1]O’Sullivan BP, Freedman SD: Cystic fibrosis. Lancet 2009, 373:1891-1904.
• [2]Hauser AR, Jain M, Bar-Meir M, McColley SA: Clinical significance of microbial infection and adaption in cystic fibrosis. Clin Microbiol Rev 2011, 24:29-70.
• [3]Fothergill JL, Walshaw MJ, Winstanley C: Transmissible strains of Pseudomonas aeruginosa in cystic fibrosis lung infections. Eur Respir J 2012, 40:227-238.
• [4]Ashish A, Shaw M, Winstanley C, Humphrey L, Walshaw MJ: Halting the spread of epidemic Pseudomonas aeruginosa in an adult cystic fibrosis centre: a prospective cohort study. J R Soc Med Short Rep 2013, 4:1.
• [5]Kidd T, Grimwood K, Ramsay K, Rainey P, Bell S: Comparison of three molecular techniques for typing Pseudomonas aeruginosa isolates in sputum samples from patients with cystic fibrosis. J Clin Microbiol 2011, 49:263-268.
• [6]Sullivan CB, Diggle MA, Clarke SC: Multilocus sequence typing: data analysis in clinical microbiology and public health. Mol Biotech 2005, 29:245-254.
• [7]Maiden M: Multilocus sequence typing of bacteria. Ann Rev Microbiol 2006, 60:561-588.
• [8]Pérez-Losada M, Cabezas P, Castro-Nallar E, Crandall KA: Pathogen typing in the genomics era: MLST and the future of molecular epidemiology. Infect Gen Evol 2013, 16:38-53.
• [9]Li W, Raoult D, Fournier PE: Bacterial strain typing in the genomic era. FEMS Microbiol Rev 2009, 33:892-916.
• [10]Anuj SN, Whiley DM, Kidd TJ, Ramsay KA, Bell SC, Syrmis MW, Grimwood K, Wainwright CE, Nissen MD, Sloots TP: Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis. Clin Microbiol Infect 2011, 17:1403-1408.
• [11]Huygens F, Inman-Bamber J, Nimmo GR, Munckhof W, Schooneveldt J, Harrison B, McMahon JA, Giffard PM: Staphylococcus aureus genotyping using novel real-time PCR formats. J Clin Microbiol 2006, 44:3712-3719.
• [12]Robertson GA, Thiruvenkataswamy V, Shilling H, Price EP, Huygens F, Henskens FA, Giffard PM: Identification and interrogation of highly informative single nucleotide polymorphism sets defined by bacterial multilocus sequence typing databases. J Med Microbiol 2004, 53:35-45.
• [13]Eusebio N, Pinheiro T, Amorim AA, Gamboa F, Saraiva L, Gusmão L, Amorim A, Araujo R: SNaPaer: a practical single nucleotide polymorphism multiplex assay for genotyping of Pseudomonas aeruginosa. PLoS One 2013, 8:e66083.
• [14]Syrmis MW, Moser RJ, Kidd TJ, Hunt P, Ramsay KA, Bell SC, Wainwright CE, Grimwood K, Nissen MD, Sloots TP, Whiley D: High-throughput single nucleotide polymorphism-based typing of shared Pseudomonas aeruginosa strains in cystic fibrosis patients using the Sequenom iPLEX platform. J Med Microbiol 2013, 62:734-740.
• [15]Kidd TJ, Ramsay KA, Hu H, Marks GB, Wainwright CE, Bye PT, Elkins MR, Robinson PJ, Rose BR, Wilson JW, Grimwood K, Bell SC: Shared Pseudomonas aeruginosa genotypes are common in Australian cystic fibrosis centres. Eur Respir J 2013, 41:1091-1100.
• [16]Logan C, Habington A, Lennon G, Grogan J, Byrne M, O'Leary J, O'Sullivan N: Genetic Relatedness of Pseudomonas aeruginosa isolates among a paediatric cystic fibrosis patient cohort in Ireland. J Med Microbiol 2012, 61:64-70.
• [17]Pseudomonas aeruginosa MLST Database Home Page [http://pubmlst.org/paeruginosa/ webcite]
• [18]Price EP, Inman-Bamber J, Thiruvenkataswamy V, Huygens F, Giffard PM: Computer-aided identification of polymorphism sets diagnostic for groups of bacterial and viral genetic variants. BMC Bioinform 2007, 8:278. BioMed Central Full Text
• [19]Winsor GL, Lam DK, Fleming L, Lo R, Whiteside MD, Yu NY, Hancock RE, Brinkman FS: Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res 2011, 39:D596-D600.
• [20]De Soyza A, Hall AJ, Mahenthiralingam E, Drevinek P, Kaca W, Drulis-Kawa Z, Stoitsova SR, Toth V, Coeyne T, Zlosnik JEA, Burns JL, Sá-Correia I, De Vos D, Pirnay JP, Kidd T, Reid D, Manos J, Klockgether J, Wiehlmann L, Tümmler B, McClean S, Winstanley C, on behalf of EU FP7 funded COST Action BM1003 “Cell surface virulence determinants of Cystic Fibrosis pathogens: Developing an international Pseudomonas aeruginosa reference panel. Microbiologyopen 2013, 2:1010-1023.