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. aeruginosaspecific 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, realtime PCR and FISH allowed identification of morphologically nontypical isolates of P. aeruginosa within a few hours.Chronic bacterial colonization of the respiratory tract, leading to exacerbations of pulmonary infection, is the major cause of disease and death in Cystic Fibrosis (CF) patients. The most common pathogen in respiratory secretions of CF patients is Pseudomonas aeruginosa, and Staphylococcus aureus, Haemophilus influenzae, and members of the Burkholderia cepacia complex also play an important role in CF lung disease (13,14,17). Other gram-negative glucose nonfermenters such as Achromobacter xylosoxidans, Ralstonia pickettii, and Stenotrophomonas maltophilia are also occasionally recovered from CF respiratory samples, but their pathogenic significance remains to be fully clarified (4,14,17). Recent studies that applied molecular approaches for the identification of unusual pathogens in CF patients revealed the presence of various rarely or even newly described species belonging to the genera Bordetella, Comamonas, Inquilinus, Pandoraea, Ralstonia,16,22,23). Determination of the clinical relevance of gramnegative bacteria other than P. aeruginosa in CF patients is, however, hampered by the difficult identification of these pathogens by conventional laboratory techniques.Phenotypic identification of bacteria grown from CF patients carries a high risk of misidentification regarding both manual methods and...

We report a Cystic Fibrosis patient with chronic Burkholderia multivorans infection involving persistency of one strain and temporary, consecutive coinfection with two different strains. Comparison of the colony morphology with the genotype revealed no correlation. These data are important for interpretation of clinical outcome and transmission studies in CF patients.

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