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.

SNaPaer: a practical single nucleotide polymorphism multiplex assay for genotyping of Pseudomonas aeruginosa.

Multilocus sequence typing (MLST) represents the gold standard genotyping method in studies concerning microbial population structure, being particularly helpful in the detection of clonal relatedness. However, its applicability on large-scale genotyping is limited due to the high cost and time spent on the task. The selection of the most informative nucleotide positions simplifies genomic characterization of bacteria. A simple and informative multiplex, SNaPaer assay, was developed and genotyping of Pseudomonas aeruginosa was obtained after a single reaction of multiplex PCR amplification and mini-sequencing. This cost-effective technique allowed the analysis of a Portuguese set of isolates (n = 111) collected from three distinct hospitals and the genotyping data could be obtained in less than six hours. Point mutations were shown to be the most frequent event responsible for diversification of the Portuguese population sample. The Portuguese isolates corroborated the epidemic hypothesis for P. aeruginosa population. SNaPaer genotyping assay provided a discriminatory power of 0.9993 for P. aeruginosa, by testing in silico several hundreds of MLST profiles available online. The newly proposed assay targets less than 0.01% of the total MLST length and guarantees reproducibility, unambiguous analysis and the possibility of comparing and transferring data between different laboratories. The plasticity of the method still supports the addition of extra molecular markers targeting specific purposes/populations. SNaPaer can be of great value to clinical laboratories by facilitating routine genotyping of P. aeruginosa.