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. 2015 Dec 3;10(12):e0144022.
doi: 10.1371/journal.pone.0144022. eCollection 2015.

Genotypic Diversity within a Single Pseudomonas aeruginosa Strain Commonly Shared by Australian Patients with Cystic Fibrosis

Anna Sze Tai  1   2   3 , Scott Cameron Bell  2   4 , Timothy James Kidd  1   5 , Ella Trembizki  1   3 , Cameron Buckley  1 , Kay Annette Ramsay  1   3 , Michael David  1   6 , Claire Elizabeth Wainwright  1   7 , Keith Grimwood  1   8 , David Mark Whiley  1   9
Affiliations

Genotypic Diversity within a Single Pseudomonas aeruginosa Strain Commonly Shared by Australian Patients with Cystic Fibrosis

Anna Sze Tai et al. PLoS One. .

Abstract

In cystic fibrosis (CF), Pseudomonas aeruginosa undergoes intra-strain genotypic and phenotypic diversification while establishing and maintaining chronic lung infections. As the clinical significance of these changes is uncertain, we investigated intra-strain diversity in commonly shared strains from CF patients to determine if specific gene mutations were associated with increased antibiotic resistance and worse clinical outcomes. Two-hundred-and-one P. aeruginosa isolates (163 represented a dominant Australian shared strain, AUST-02) from two Queensland CF centres over two distinct time-periods (2001-2002 and 2007-2009) underwent mexZ and lasR sequencing. Broth microdilution antibiotic susceptibility testing in a subset of isolates was also performed. We identified a novel AUST-02 subtype (M3L7) in adults attending a single Queensland CF centre. This M3L7 subtype was multi-drug resistant and had significantly higher antibiotic minimum inhibitory concentrations than other AUST-02 subtypes. Prospective molecular surveillance using polymerase chain reaction assays determined the prevalence of the 'M3L7' subtype at this centre during 2007-2009 (170 patients) and 2011 (173 patients). Three-year clinical outcomes of patients harbouring different strains and subtypes were compared. MexZ and LasR sequences from AUST-02 isolates were more likely in 2007-2009 than 2001-2002 to exhibit mutations (mexZ: odds ratio (OR) = 3.8; 95% confidence interval (CI): 1.1-13.5 and LasR: OR = 2.5; 95%CI: 1.3-5.0). Surveillance at the adult centre in 2007-2009 identified M3L7 in 28/509 (5.5%) P. aeruginosa isolates from 13/170 (7.6%) patients. A repeat survey in 2011 identified M3L7 in 21/519 (4.0%) P. aeruginosa isolates from 11/173 (6.4%) patients. The M3L7 subtype was associated with greater intravenous antibiotic and hospitalisation requirements, and a higher 3-year risk of death/lung transplantation, than other AUST-02 subtypes (adjusted hazard ratio [HR] = 9.4; 95%CI: 2.2-39.2) and non-AUST-02 strains (adjusted HR = 4.8; 95%CI: 1.4-16.2). This suggests ongoing microevolution of the shared CF strain, AUST-02, was associated with an emerging multi-drug resistant subtype and possibly poorer clinical outcomes.

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Conflict of interest statement

Competing Interests: CEW declares income on a per patient basis derived from Pharmaceutical Studies - Vertex Pharmaceuticals Inc., & Boehringer-Ingelheim; Epidemiological Research Support from GlaxoSmithKline - Analysis of Bronchoalveolar lavage (BAL) fluid from children with respiratory disorders (2010 – 2012); Research Grant from Novo Nordisk Pharmaceuticals P/L- CF-IDEA Study. 2012–2013; and other reimbursement, as follows: Vertex Pharmaceuticals Inc. - Consultant on the Vertex Physician Pediatric CF Advisory Board and the Vertex Innovation Awards (VIA) Grants Committee; Gilead Sciences, Inc. - AZLI Advisory Board Honorarium 2010; Medscape - Consultation interview regarding CF Studies 2012; Vertex Pharmaceuticals 2013 San Francisco return flight and accommodation as Investigator in Lumacaftor study (104); Vertex Pharmaceuticals 2014 return flight and accommodation + honorarium as invited speaker at European CF Conference, Gothenburg, Sweden; Vertex Pharmaceuticals 2015 honorarium as speaker at a Vertex sponsored educational meeting series; Vertex Pharmaceuticals 2015 Chicago return flight and accommodation as Investigator in Lumacaftor study (109); Novartis Pharmaceuticals 2013 return travel and accommodation to give a symposium at European CF Conference in Lisbon; Novartis Pharmaceuticals Australia P/L 2013 honorarium to present symposium at Australasian CF Conference in Auckland; Novartis Pharmaceuticals 2014 return flight and accommodation + honorarium as invited speaker at National Pediatric Congress in Lebanon; Current Board Positions - Thorax Editorial Board /Associate Editor Respirology/ International Advisory Board Vertex Pharmaceuticals P/L. These do not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Flow diagram demonstrating patient and isolate selection for initial assessment of mexZ and lasR sequence diversity and antibiotic susceptibility.
Fig 2
Fig 2. Flow diagram showing patients included in the two cross-sectional surveys for ‘M3L7’ AUST-02 subtype at TPCH.
Fig 3
Fig 3. Clinical outcome of 166 TPCH adult patients within 3-years of participating in the 2007–2009 survey.
Fig 4
Fig 4. Kaplan-Meier survival analysis comparing unadjusted time to death or lung transplantation for 13 patients with M3L7, 57 with non-M3L7 AUST-02 and 96 patients with non-AUST-02 Pseudomonas aeruginosa strains.
See this image and copyright information in PMC

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Grants and funding

This study was funded by the Children’s Hospital Foundation [Queensland Children’s Medical Research Institute (QCMRI) Grant 50024], a Project Grant from The Prince Charles Hospital Foundation [MS2013-02 to SCB, DMW and TJK] (http://www.tpchfoundation.org.au) and a National Health Medical Research Council Project Grant [455919 to SCB & CEW] (www.nhmrc.gov.au). DMW is a recipient of a National Health and Medical Research Early Career Fellowship [APP1054129] (www.nhmrc.gov.au) and QCMRI Early Career Fellowship [50024] (www.qcmri.org.au). SCB is a recipient of Queensland Health Office of Health and Medical Research Fellowship [QCOS013795] (www.health.qld.gov.au/ohmr/html/funding/funding.asp). TJK is the recipient of an ERS–EU RESPIRE2 Marie Skłodowska-Curie Postdoctoral Research Fellowship (MC RESPIRE2 first round, 4571-2013) (www.ersnet.org/ers-funding/fellowships/post-doc/eu-co-funded-respire-2), which was partly funded by the People Programme of the European Union’s Seventh Framework Programme (FP7/2007–2013), under REA grant agreement 600368. KAR is the recipient of an Australian Postgraduate Award Scholarship [2127932] (https://education.gov.au/australian-postgraduate-awards). AST is the recipient of a National Health and Medical Research Council Post-graduate Medical and Dental Scholarship [APP1017517] (www.nhmrc.gov.au) and Australian Cystic Fibrosis Foundation Postgraduate Scholarship (www.cysticfibrosis.org.au/cfwa-scholarship). All aforementioned funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.