title: A comparison of two informative SNP-based strategies for typing Pseudomonas aeruginosa isolates from patients with cystic fibrosis.
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.
authors.forename: Melanie W
authors.initials: MW
authors.lastname: Syrmis
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kidd
authors.forename: Ralf J
authors.initials: RJ
authors.lastname: Moser
authors.forename: Kay A
authors.initials: KA
authors.lastname: Ramsay
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Gibson
authors.forename: Snehal
authors.initials: S
authors.lastname: Anuj
authors.forename: Scott C
authors.initials: SC
authors.lastname: Bell
authors.forename: Claire E
authors.initials: CE
authors.lastname: Wainwright
authors.forename: Keith
authors.initials: K
authors.lastname: Grimwood
authors.forename: Michael
authors.initials: M
authors.lastname: Nissen
authors.forename: Theo P
authors.initials: TP
authors.lastname: Sloots
authors.affiliation: Queensland Children's Medical Research Institute, The University of Queensland, Brisbane, Queensland 4029, Australia. d.whiley@uq.edu.au.
authors.forename: David M
authors.initials: DM
authors.lastname: Whiley
mesh_terms: D001315:Australia; D015373:Bacterial Typing Techniques / Q000379:methods*; D003550:Cystic Fibrosis / Q000150:complications* / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D058885:Multilocus Sequence Typing / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011552:Pseudomonas Infections / Q000175:diagnosis / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial