Infection by Ralstonia species in cystic fibrosis patients: identification of R. pickettii and R. mannitolilytica by polymerase chain reaction

Abstract

The frequency of respiratory tract infections caused by Ralstonia species in persons with cystic fibrosis (CF) and the role of these species in CF pulmonary disease are not well documented. In part, this lack of documentation may be attributed to the difficulty in accurately identifying Ralstonia species; R. mannitolilytica and R. pickettii in particular may be misidentified as other closely related species, particularly those of the Burkholderia cepacia complex. We used polyphasic analyses to identify 42 Ralstonia isolates from sputum cultures from 38 CF patients. Several isolates that could not be identified to the species level may belong to novel Ralstonia species. We demonstrated chronic colonization by using genotyping of serial isolates recovered from the same patient. To facilitate identification of R. mannitolilytica and R. pickettii, we developed 16S ribosomal DNA-based polymerase chain reaction assays that allow sensitive and specific identification of these species.

Figure 1

Randomly amplified polymorphic DNA analysis of serial isolates from three patients. M: molecular weight marker; lanes 1, 2, and 3: serial Ralstonia mannitolilytica isolates from patient A in chronological order; lanes 4 and 5: serial R. mannitolilytica isolates from patient B in chronological order; and lanes 6 and 7: serial R. pickettii isolates from patient C in chronological order.

Figure 2

Polymerase chain reaction analysis of Ralstonia strains with primer pairs Rm-F1/Rm-R1 (lanes 1–8) and Rp-F1/Rp-R1 (lanes 9–16). M: 100-bp DNA ladder; lanes 1, 2, 3, 9, 10, and 11: R. mannitolilytica; lanes 4, 5, 6, 7, 12, 13, 14, and 15: R. pickettii; and lanes 8 and 16: R. gilardii.