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Journal of Clinical Microbiology, May 2004, p. 2074-2079, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.2074-2079.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

PCR-Based Assay for Differentiation of Pseudomonas aeruginosa from Other Pseudomonas Species Recovered from Cystic Fibrosis Patients

Theodore Spilker,1 Tom Coenye,2 Peter Vandamme,2 and John J. LiPuma1*

Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan, 48109,1 Laboratorium voor Microbiologie, Universiteit Gent, Ghent, Belgium2

Received 1 December 2003/ Returned for modification 15 January 2004/ Accepted 20 January 2004

Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients. Much of CF patient management depends on accurate identification of P. aeruginosa from sputum culture. However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus- or species-level identification. Both assays yielded DNA fragments of the predicted size. We tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum. Based on these 85 strains, the specificity and sensitivity of both assays were 100%. To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates. The results indicated that preliminary phenotypic testing had misidentified several isolates. The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays. Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa. Both assays show 100% sensitivity and specificity.

* Corresponding author. Mailing address: University of Michigan Medical School, 8323 MSRB III, Box 0646, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0646. Phone: (734) 936-9767. Fax: (734) 764-4279. E-mail: jlipuma{at}umich.edu.

Journal of Clinical Microbiology, May 2004, p. 2074-2079, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.2074-2079.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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