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Journal of Clinical Microbiology, August 2005, p. 4070-4075, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.4070-4075.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Superiority of Molecular Techniques for Identification of Gram-Negative, Oxidase-Positive Rods, Including Morphologically Nontypical Pseudomonas aeruginosa, from Patients with Cystic Fibrosis

Nele Wellinghausen,* Juliane Köthe, Beate Wirths, Anja Sigge, and Sven Poppert

Department of Medical Microbiology and Hygiene, University of Ulm, Ulm, Germany

Received 3 February 2005/ Returned for modification 16 March 2005/ Accepted 29 April 2005

Phenotypic identification of gram-negative bacteria from Cystic Fibrosis (CF) patients carries a high risk of misidentification. Therefore, we compared the results of biochemical identification by API 20NE with 16S rRNA gene sequencing in 88 gram-negative, oxidase-positive rods, other than morphologically and biochemically typical P. aeruginosa, from respiratory secretions of CF patients. The API 20NE allowed correct identification of the bacterial species in 15 out of 88 (17%) isolates investigated. Agreement between the API and the 16S rRNA gene sequencing results was high only in isolates with an API result classified as "excellent identification." Even API results classified as "very good identification" or "good identification" showed a high rate of misidentification (67% and 84%). Fifty-two isolates of morphological and biochemical nontypical Pseudomonas aeruginosa, representing 59% of all isolates investigated, were not identifiable or misidentified in the API 20NE. Therefore, rapid molecular diagnostic techniques like real-time PCR and fluorescence in situ hybridization (FISH) were evaluated in this particular group of bacteria for identification of the clinically most relevant pathogen, P. aeruginosa. The LightCycler PCR assay with a P. aeruginosa-specific probe showed a sensitivity and specificity of 98.1% and 100%, respectively. For FISH analysis, a newly designed P. aeruginosa-specific probe had a sensitivity and specificity of 100%. In conclusion, molecular methods are superior over biochemical tests for identification of gram-negative, oxidase-positive rods in CF patients. In addition, real-time PCR and FISH allowed identification of morphologically nontypical isolates of P. aeruginosa within a few hours.

* Corresponding author. Mailing address: Department of Medical Microbiology and Hygiene, University of Ulm, Robert-Koch-Str. 8, D-89081 Ulm, Germany. Phone: 49-(0)731-500 24602. Fax: 49-(0)731-500 23473. E-mail: nele.wellinghausen{at}medizin.uni-ulm.de.

Journal of Clinical Microbiology, August 2005, p. 4070-4075, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.4070-4075.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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