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.
Highlights
Initial pulmonary infections with Pseudomonas aeruginosa in patients with cystic fibrosis (CF) are typically intermittent and caused by non-mucoid environmental strains that are susceptible to anti-pseudomonal antibiotics [1,2,3]
Of the initial 201 P. aeruginosa isolates, 188 provided reliable sequence data for both mexZ and LasR regions (S1 Table) from 150 AUST-02, 17 AUST-01, 5 AUST-06, 1 AUST-11 and 15 unique strains. These 188 isolates came from 105 patients (33 children attending the Royal Children’s Hospital (RCH), 83 adults at The Prince Charles Hospital (TPCH) and included 11 children who provided isolates from both centres as they transitioned from the RCH to the TPCH between the first and second survey periods; Fig 1)
Mutations in mexZ (M3 type) were more likely in AUST-02 isolates in 2007–2009 (17/95, 17.9%) than 2001–2002 (3/55; 5.5%; odds ratio (OR) = 3.8; 95%confidence interval (CI): 1.1–13.5) and were only identified in isolates from adults (20/115 (17.4%) submitted AUST-02 isolates) attending the TPCH, being notably absent in AUST-02 isolates collected from children at the RCH clinic
Summary
Initial pulmonary infections with Pseudomonas aeruginosa in patients with cystic fibrosis (CF) are typically intermittent and caused by non-mucoid environmental strains that are susceptible to anti-pseudomonal antibiotics [1,2,3]. A single strain becomes established and develops a mucoid phenotype, which with increasing antibiotic resistance makes eradication from the airways difficult[4]. Once this event occurs, patients experience an accelerated pulmonary decline with increased exacerbation frequency and mortality [5]. Regional differences existed and AUST-02 was observed in 35%-40% of infected patients attending Queensland CF clinics [13, 14] This strain had increased carbapenem and aminoglycoside antibiotic resistance and was associated with greater treatment requirements than unique strains [13, 15]. The actual long-term clinical impact of AUST-02 remains uncertain
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