Comparison of Three Molecular Techniques for Typing Pseudomonas aeruginosa Isolates in Sputum Samples from Patients with Cystic Fibrosis

Abstract

Monitoring the emergence and transmission of Pseudomonas aeruginosa strains among cystic fibrosis (CF) patients is important for infection control in CF centers internationally. A recently developed multilocus sequence typing (MLST) scheme is used for epidemiologic analyses of P. aeruginosa outbreaks; however, little is known about its suitability for isolates from CF patients compared with that of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). As part of a prevalence study of P. aeruginosa strains in Australian CF clinics, we compared the discriminatory power and concordance of ERIC-PCR, PFGE, and MLST among 93 CF sputum and 11 control P. aeruginosa isolates. PFGE and MLST analyses were also performed on 30 paired isolates collected 85 to 354 days apart from 30 patients attending two CF centers separated by 3,600 kilometers in order to detect within-host evolution. Each of the three methods displayed high levels of concordance and discrimination; however, overall lower discrimination was seen with ERIC-PCR than with MLST and PFGE. Analysis of the 50 ERIC-PCR types yielded 54 PFGE types, which were related by <6 band differences, and 59 sequence types, which were classified into 7 BURST groups and 42 singletons. MLST also proved useful for detecting novel and known strains and for inferring relatedness among unique PFGE types. However, 47% of the paired isolates produced PFGE patterns that within 1 year differed by one to five bands, whereas with MLST all paired isolates remained identical. MLST thus represents a categorical analysis tool with resolving power similar to that of PFGE for typing P. aeruginosa. Its focus on highly conserved housekeeping genes is particularly suited for long-term clinical monitoring and detecting novel strains.

Twenty-Five-Year Outbreak of Pseudomonas aeruginosa Infecting Individuals with Cystic Fibrosis: Identification of the Prairie Epidemic Strain

“…Patients who had isolates with discrepant PFGE profiles between FE and RE samples underwent multilocus sequence typing (MLST) to determine if relatedness existed despite changes in PFGE profiles, using a method developed by Curran et al (22). The procedure followed methods from previously published works (23)(24)(25). Sequences were compared to those publically available at the P. aeruginosa MLST online database (http://pubmlst.org/paeruginosa/) to identify allele and sequence types.…”

“…This population included 107 patients with chronic P. aeruginosa infection and 6 patients with transient infection. Of those included in the cohort, 72 (63%) patients are currently active within the CACFC cohort, 12 (11%) patients were transferred to another CF clinic, and 29 (26%) patients experienced respiratory failure and either received a life-saving lung transplant (24) or succumbed to CF (5). Among the members of the cohort, 101 (89%) patients had received prior care at a pediatric CF care center, and 12 were adults diagnosed with CF late in life, among which 9 received care only at CACFC.…”

“…Whereas other studies have assessed techniques such as random amplified polymorphic DNA PCR (RAPD-PCR) (25), enterobacterial repetitive intergenic consensus PCR (ERIC PCR) (24), and variable-number tandem repeat (VNTR) analysis (53) in combination with MLST and PFGE, we used a complementary approach. Whereas others have reported PFGE to have markedly discrepant profiles for up to 47% of paired patient isolates collected less than 1 year apart, we observed a high degree of conservation in our clinic cohort, as 87% (57/66 patients) of our patients had concordant profiles for up to 25 years.…”

SharedPseudomonas aeruginosagenotypes are common in Australian cystic fibrosis centres

“…Following DNA extraction, isolates were confirmed as P. aeruginosa by a duplex real-time PCR assay before performing DNA fingerprinting using the enterobacterial repetitive intergenic consensus (ERIC)-PCR typing technique [19,20]. Cluster analysis and genotype allocation of ERIC-PCR fingerprints were performed using FPQuest TM software (version 4.5; Bio-Rad Laboratories Pty Ltd, Hercules, CA, USA) as described previously [20].…”

Genotyping Techniques for Determining the Diversity of Microorganisms

“…Several studies have shown Rep-PCR to have good correlation with PFGE results but, in general, with slightly less discriminatory power and less reproducible (Kidd et al, 2011).…”