title: High-throughput single-nucleotide polymorphism-based typing of shared Pseudomonas aeruginosa strains in cystic fibrosis patients using the Sequenom iPLEX platform.
abstract: Shared strains of Pseudomonas aeruginosa are now well recognized in people with cystic fibrosis (CF), and suitable P. aeruginosa laboratory typing tools are pivotal to understanding their clinical significance and guiding infection control policies in CF clinics. We therefore compared a single-nucleotide polymorphism (SNP)-based typing method using Sequenom iPLEX matrix-assisted laser desorption ionization with time-of-flight mass spectrometry (MALDI-TOF MS) with typing methods used routinely by our laboratory. We analysed 617 P. aeruginosa isolates that included 561 isolates from CF patients collected between 2001 and 2009 in two Brisbane CF clinics and typed previously by enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as 56 isolates from non-CF patients analysed previously by multilocus sequence typing (MLST). The isolates were tested using a P. aeruginosa Sequenom iPLEX MALDI-TOF (PA iPLEX) method comprising two multiplex reactions, a 13-plex and an 8-plex, to characterize 20 SNPs from the P. aeruginosa housekeeping genes acsA, aroE, guaA, mutL, nuoD, ppsA and trpE. These 20 SNPs were employed previously in a real-time format involving 20 separate assays in our laboratory. The SNP analysis revealed 121 different SNP profiles for the 561 CF isolates. Overall, there was at least 96% agreement between the ERIC-PCR and SNP analyses for all predominant shared strains among patients attending our CF clinics: AUST-01, AUST-02 and AUST-06. For the less frequently encountered shared strain AUST-07, 6/25 (24%) ERIC-PCR profiles were misidentified initially as AUST-02 or as unique, illustrating the difficulty of gel-based analyses. SNP results for the 56 non-CF isolates were consistent with previous MLST data. Thus, the PA iPLEX format provides an attractive high-throughput alternative to ERIC-PCR for large-scale investigations of shared P. aeruginosa strains.
authors: Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland, Australia.|Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland, Australia.
authors: Melanie W
authors: MW
authors: Syrmis
authors: Sequenom Inc., Sequenom Asia Pacific, Herston, Queensland, Australia.
authors: Ralf J
authors: RJ
authors: Moser
authors: Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland, Australia.|Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland, Australia.
authors: Timothy J
authors: TJ
authors: Kidd
authors: Sequenom Inc., Sequenom Asia Pacific, Herston, Queensland, Australia.
authors: Priscilla
authors: P
authors: Hunt
authors: Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland, Australia.|Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland, Australia.
authors: Kay A
authors: KA
authors: Ramsay
authors: Department of Thoracic Medicine, The Prince Charles Hospital, Brisbane, Queensland, Australia.|Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland, Australia.
authors: Scott C
authors: SC
authors: Bell
authors: Queensland Children's Respiratory Centre, Royal Children's Hospital, Brisbane, Australia.|Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland, Australia.
authors: Claire E
authors: CE
authors: Wainwright
authors: Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland, Australia.|Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland, Australia.
authors: Keith
authors: K
authors: Grimwood
authors: Microbiology Division, Pathology Queensland Central Laboratory, Herston, Queensland, Australia.|Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland, Australia.|Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland, Australia.
authors: Michael D
authors: MD
authors: Nissen
authors: Microbiology Division, Pathology Queensland Central Laboratory, Herston, Queensland, Australia.|Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland, Australia.|Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland, Australia.
authors: Theo P
authors: TP
authors: Sloots
authors: Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, Queensland, Australia.|Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Brisbane, Queensland, Australia.
authors: David M
authors: DM
authors: Whiley
mesh_terms: D015373:Bacterial Typing Techniques; D003550:Cystic Fibrosis / Q000382:microbiology*; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D011552:Pseudomonas Infections / Q000175:diagnosis / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000235:genetics*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization