PCR identification of Pseudomonas aeruginosa and direct detection in clinical samples from cystic fibrosis patients

Filho, Luiz Vicente Ferreira da Silva; Levi, Joséa Eduardo; Bento, Christina N. O.; Ramos, Scnia Regina Testa Da Silva; Rozov, Tatiana

doi:10.1099/00222615-48-4-357

Cited by 55 publications

(35 citation statements)

References 12 publications

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“…Genotypic identification methods would be expected to circumvent this problem, and molecular assays based on specific genes have been described. These include PCR assays targeting the P. aeruginosa oprL (11), algD (10,25), and exotoxin A genes (17,32). The performance of these assays in differentiating P. aeruginosa from other closely related Pseudomonas species has not been examined, however, and the paucity of sequence data for these loci from non-P. aeruginosa strains limits predictions based on in silico analyses.…”

Section: Discussionmentioning

confidence: 99%

PCR-Based Assay for Differentiation of Pseudomonas aeruginosa from Other Pseudomonas Species Recovered from Cystic Fibrosis Patients

et al. 2004

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Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients. Much of CF patient management depends on accurate identification of P. aeruginosa from sputum culture. However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus-or species-level identification. Both assays yielded DNA fragments of the predicted size. We tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum. Based on these 85 strains, the specificity and sensitivity of both assays were 100%. To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates. The results indicated that preliminary phenotypic testing had misidentified several isolates. The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays. Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa. Both assays show 100% sensitivity and specificity.

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Section: Discussionmentioning

confidence: 99%

PCR-Based Assay for Differentiation of Pseudomonas aeruginosa from Other Pseudomonas Species Recovered from Cystic Fibrosis Patients

et al. 2004

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“…Organisms were coded prior to testing and tested in a blind fashion. Three published gene sequences that have been reported for use in the identification of P. aeruginosa were targeted for PCR amplification: oprI (OPR) (3,4,5), algD (VIC) (2,19), and the exotoxin A gene (ETA) (9,17). The designated primer pairs are listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Use of Real-Time PCR with Multiple Targets To Identify Pseudomonas aeruginosa and Other Nonfermenting Gram-Negative Bacilli from Patients with Cystic Fibrosis

Qin¹,

et al. 2003

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Pseudomonas aeruginosa and other gram-negative isolates from patients with cystic fibrosis (CF) may be difficult to identify because of their marked phenotypic diversity. We examined 200 gram-negative clinical isolates from CF respiratory tract specimens and compared identification by biochemical testing and real-time PCR with multiple different target sequences using a standardized combination of biochemical testing and molecular identification, including 16S rRNA partial sequencing and gyrB PCR and sequencing as a "gold standard." Of 50 isolates easily identified phenotypically as P. aeruginosa, all were positive with PCR primers for gyrB or oprI, 98% were positive with exotoxin A primers, and 90% were positive with algD primers. Of 50 P. aeruginosa isolates that could be identified by basic biochemical testing, 100% were positive by real-time PCR with gyrB or oprI primers, 96% were positive with exotoxin A primers, and 92% were positive with algD primers. For isolates requiring more-extensive biochemical evaluation, 13 isolates were identified as P. aeruginosa; all 13 were positive with gyrB primers, 12 of 13 were positive with oprI primers, 11 of 13 were positive with exotoxin A primers, and 10 of 13 were positive with algD primers. A single false-positive P. aeruginosa result was seen with oprI primers. The best-performing commercial biochemical testing was in exact agreement with molecular identification only 60% of the time for this most difficult group. Real-time PCR had costs similar to those of commercial biochemical testing but a much shorter turnaround time. Given the diversity of these CF isolates, real-time PCR with a combination of two target sequences appears to be the optimum choice for identification of atypical P. aeruginosa and for non-P. aeruginosa gram-negative isolates.Pseudomonas aeruginosa is the most significant pathogen affecting patients with cystic fibrosis (CF). It is often acquired in the first few years of life and causes chronic airway infections, which ultimately result in death. While P. aeruginosa is not thought of as an organism that is difficult to identify in the microbiology laboratory, CF isolates often have unique phenotypes, including the loss of pigment production, synthesis of rough lipopolysaccharide devoid of O side chains, and development of mucoidy (18). While the presence of mucoidy may be helpful in identifying CF isolates of P. aeruginosa, some of the other characteristics may make these isolates more difficult to identify. Both commercial methods of identification and standard biochemical testing may be lengthy and inaccurate (10, 11). In addition, CF patients often have other nonfermenting gram-negative bacilli isolated from their respiratory secretions, making rapid isolation and identification of P. aeruginosa even more difficult (1, 7). While sequencing 16S rRNA genes in CF isolates of P. aeruginosa and other gram-negative bacilli has utility (6), it may be expensive and time-consuming.The Cystic Fibrosis Foundation-funded Therapeutic Development Network (...

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“…Although valuable, these assays by definition, however, do not characterize the total bacterial community. As such, potentially clinically important pathogens can remain unidentified (11,23,27,41). Methodologies, however, have been developed that allow the total bacterial community in a given environment to be characterized (24).…”

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confidence: 99%

Bacterial Diversity in Cases of Lung Infection in Cystic Fibrosis Patients: 16S Ribosomal DNA (rDNA) Length Heterogeneity PCR and 16S rDNA Terminal Restriction Fragment Length Polymorphism Profiling

et al. 2003

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The leading cause of morbidity and mortality in cystic fibrosis (CF) patients stems from repeated bacterial respiratory infections. Many bacterial species have been cultured from CF specimens and so are associated with lung disease. Despite this, much remains to be determined. In the present study, we characterized without prior cultivation the total bacterial community present in specimens taken from adult CF patients, extracting DNA directly from 14 bronchoscopy or sputum samples. Bacterial 16S ribosomal DNA (rRNA) gene PCR products were amplified from extracted nucleic acids, with analyses by terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), and sequencing of individual cloned PCR products to characterize these communities. Using the same loading of PCR products, 12 distinct T-RFLP profiles were identified that had between 3 and 32 T-RFLP bands. Nine distinct LH-PCR profiles were identified containing between one and four bands. T-RFLP bands were detected in certain samples at positions that corresponded to pathogens cultured from CF samples, e.g., Burkholderia cepacia and Haemophilus influenzae. In every sample studied, one T-RFLP band was identified that corresponded to that produced by Pseudomonas aeruginosa. A total of 103 16S rRNA gene clones were examined from five patients. P. aeruginosa was the most commonly identified species (59% of clones). Stenotrophomonas species were also common, with eight other (typically anaerobic) bacterial species identified within the remaining 17 clones. In conclusion, T-RFLP analysis coupled with 16S rRNA gene sequencing is a powerful means of analyzing the composition and diversity of the bacterial community in specimens sampled from CF patients.

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PCR identification of Pseudomonas aeruginosa and direct detection in clinical samples from cystic fibrosis patients

Cited by 55 publications

References 12 publications

PCR-Based Assay for Differentiation of Pseudomonas aeruginosa from Other Pseudomonas Species Recovered from Cystic Fibrosis Patients

PCR-Based Assay for Differentiation of Pseudomonas aeruginosa from Other Pseudomonas Species Recovered from Cystic Fibrosis Patients

Use of Real-Time PCR with Multiple Targets To Identify Pseudomonas aeruginosa and Other Nonfermenting Gram-Negative Bacilli from Patients with Cystic Fibrosis

Bacterial Diversity in Cases of Lung Infection in Cystic Fibrosis Patients: 16S Ribosomal DNA (rDNA) Length Heterogeneity PCR and 16S rDNA Terminal Restriction Fragment Length Polymorphism Profiling

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