Molecular identification and genotyping of Pseudomonas aeruginosa isolated from cystic fibrosis and non-cystic fibrosis patients with bronchiectasis.
There is no standard methodology for the molecular identification and genotyping of Pseudomonas aeruginosa which are frequently isolated in bronchiectasis patients. Hence, the main goal of this work was to propose a methodology capable to simultaneously identify and genotype, in less than 6 h, clinical P. aeruginosa collected from cystic fibrosis (CF) and non-CF patients with bronchiectasis. Molecular analyses were conducted in clinical isolates by testing the newly colony-PCR strategy and SNaPaer assay. A total of 207 isolates of P. aeruginosa were collected from clinical samples. To assess the assay specificity, other Gram-negative non-aeruginosa bacteria, namely Pseudomonas and Burkholderia, were tested. The complete group of 23 markers included in the SNaPaer panel was observed exclusively in P. aeruginosa; more than 18 markers failed in other bacteria. A total of 43 SnaP profiles were obtained for clinical P. aeruginosa, being the profiles highly patient-specific. Six CF patients were colonized with P. aeruginosa isolates with very distinct SnaP profiles, particularly following adjustments on antibiotic therapy, thus suggesting changes on the dynamics and dominance of these bacteria. SnaPaer proved to be a good and reliable tool for identification and genotyping of clinical P. aeruginosa in a single-tube multiplex PCR. Combined with the proposed colony-PCR strategy, SnaPaer assay facilitates the molecular analysis of P. aeruginosa.
IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal.
Nadia
N
Eusebio
Department of Pneumology, Faculty of Medicine of the University of Porto and Hospital S. João, Alameda Prof. Hernani Monteiro, 4200-319, Porto, Portugal.
Adelina A
AA
Amorim
Serviço de Pneumologia, Hospitais Universidade Coimbra-CHUC, EPE, Rua Fonseca Pinto, 3000-075 Coimbra, Portugal.
Fernanda
F
Gamboa
IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal ricjparaujo@yahoo.com.
Ricardo
R
Araujo
D001987:Bronchiectasis / Q000150:complications*; D003550:Cystic Fibrosis / Q000150:complications*; D014644:Genetic Variation; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D058889:Molecular Typing; D011552:Pseudomonas Infections / Q000175:diagnosis* / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
MLST; SNP; SNaPaer; bacterial identification; bronchiectasis; genetic diversity; multiplex primer extension assay