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Open Access Research article

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

Melanie W Syrmis12, Timothy J Kidd12, Ralf J Moser3, Kay A Ramsay12, Kristen M Gibson1, Snehal Anuj1, Scott C Bell14, Claire E Wainwright15, Keith Grimwood12, Michael Nissen126, Theo P Sloots126 and David M Whiley12*

Author Affiliations

1 Queensland Children’s Medical Research Institute, The University of Queensland, Brisbane, Queensland 4029, Australia

2 Queensland Paediatric Infectious Disease Laboratory, Block 28, Royal Children’s Hospital, Herston Road, Herston, Brisbane 4029, Queensland, Australia

3 Sequenom Inc., Sequenom Asia Pacific, Brisbane, Queensland 4029, Australia

4 Department of Thoracic Medicine, The Prince Charles Hospital, Brisbane, Queensland 4032, Australia

5 Queensland Children’s Respiratory Centre, Royal Children’s Hospital, Brisbane, Queensland 4029, Australia

6 Microbiology Division, Pathology Queensland Central Laboratory, Brisbane, Queensland 4029, Australia

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BMC Infectious Diseases 2014, 14:307  doi:10.1186/1471-2334-14-307

Published: 5 June 2014

Abstract

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.

Keywords:
Pseudomonas aeruginosa; Typing; Cystic fibrosis; MLST; SNP