Development and Validation of a PCR Assay To Detect the Prairie Epidemic Strain of Pseudomonas aeruginosa from Patients with Cystic Fibrosis

Abstract

The monitoring of epidemic Pseudomonas aeruginosa is important for cystic fibrosis (CF) infection control. The prairie epidemic strain (PES) is common in western Canadian CF clinics. Using whole-genome sequencing, we identified a novel genomic island and developed a PCR assay for PES. Against a collection of 186 P. aeruginosa isolates, the assay had 98% sensitivity and 100% specificity. Multiple epidemic strains of Pseudomonas aeruginosa have been described as infecting patients with cystic fibrosis (CF) (1-4). Many of these strains are associated with a poor prognosis, manifesting as increased rates of lung function decline, exacerbation frequency, and treatment burden; decreased quality of life; and hastened progression to end-stage lung diseases (5, 6). We recently described a novel epidemic P. aeruginosa strain, the prairie epidemic strain (PES) (multilocus sequence type 192) (7). PES was found in one-quarter of patients attending the Calgary Adult CF Clinic (CACFC) and from patients who were transferred from other Prairie-based clinics, suggesting broad endemicity in western Canada. Furthermore, its prevalence has been unchanged through 3 decades of patients who transitioned to the CACFC. PES is more likely to be antibiotic resistant than are unique strains of P. aeruginosa, and infection with PES has been associated with increased rates of lung function decline, pulmonary exacerbation frequency, and progression to death and/or transplantation (7,8).The importance of identifying epidemic P. aeruginosa strains in CF has long been recognized. Typing strategies that are commonly currently employed include pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and random amplified polymorphic DNA PCR (RAPD-PCR) (7, 9, 10). These modalities have, to varied degrees, the ability to distinguish bacterial strains from one another, identify known epidemic strains, and determine if novel strains are potentially shared. However, they are costly and laborious, precluding routine use in clinical laboratories. Accordingly, we sought to develop a simple PCR assay to distinguish PES from non-PES P. aeruginosa organisms for clinical and research purposes in areas where this strain is prevalent (western Canada). Similar assays have been developed for other epidemic P. aeruginosa strains, including Liverpool epidemic strain (LES), Midlands-1, Manchester, and Australia epidemic strain-1 (AUS-1), and they are used prospectively in infection control and research (1-4).We performed whole-genome sequencing on 8 nonsequential isolates of PFGE and MLST-confirmed PES from our prospectively maintained biobank. The isolates were sequenced on the Illumina HiSeq 2500 (150 bp, paired-end) and assembled using SPAdes 2.5.0 (11). These strains were sequenced as a part of a large sequencing study, and a more detailed description will be given in a forthcoming manuscript (M. G. Surette, unpublished data). We identified a novel 40-kb genomic island (GI) using IslandViewer (12) and GView (13) that was not present in other pub...

Epidemiology, Biology, and Impact of Clonal Pseudomonas aeruginosa Infections in Cystic Fibrosis

“…These assays are typically used for the real-time detection of new infections (given their ease and cost-effectiveness) in centers in which particular isolates exist at epidemic rates. Individual assays have been developed for LES (132,134,135), AUST-01 (133), the Prairie epidemic strain (PES) (136), MA (137), and Midlands-1 (138). The development of these assays requires a detailed understanding of the genetic background and specificity of genetic elements within each P. aeruginosa strain.…”

Long-term clinical outcomes of ‘Prairie Epidemic Strain’Pseudomonas aeruginosainfection in adults with cystic fibrosis

“…These infections occurred evenly through the study period, occurred within 2 years of entry into the CACFC, and most recently in 2013. In those experiencing strain displacement, we did not discover the PES strain on testing of any of the early isolates by PFGE or PCR 34. Of the two patients with PES infection with the absence of antecedent chronic P. aeruginosa infection, one underwent transplantation within 3 months of the event, and the second cleared the infection after eradication therapy.…”

“…Following this, patients with discordant PES isolates after the initial encounter were considered a priori to have a ‘superinfection’ wherein they underwent strain displacement. A random sample of isolates from patients with PES and unique P. aeruginosa strains identified by PFGE underwent confirmatory testing with either whole genome sequencing (WGS),32 33 MLST30 or a qualitative PES-specific PCR assay 34…”

Emergence of Antimicrobial Resistance among Pseudomonas aeruginosa: Implications for Therapy

“…155 Several distinct epidemic clones of MDR-PA have disseminated globally among CF patients and centers. [156][157][158][159] Epidemic and nonepidemic strains may differ in virulence, biofilm formation, and antimicrobial susceptibility. 160,161 The importance of PA in evolution of the CF lung lesion is fascinating but is beyond the scope of this article and will not be further discussed herein.…”

“…90,138,274 Several international clones disseminated from hospitals, 90,138 whereas other clones evolved from CF centers. 153,[156][157][158][159] In North America, rates of MDR are modest. 320,321 Data from the Eurofins' Surveillance Network evaluated > 205,000 PA isolates from patients with HAP or BSI from 217 hospitals in the United States from 2005 to 2011.…”