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Characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16s ribosomal DNA terminal restriction fragment length polymorphism profiling.

ABSTRACT: Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients. A greater understanding of these bacterial infections is needed to improve lung disease management. As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data to characterize, without prior cultivation, the bacterial community in 71 sputa from 34 adult CF patients. Nineteen species from 15 genera were identified in 53 16S rRNA clones from three patients. Of these, 15 species have not previously been reported in CF lung infections and many were species requiring strict anaerobic conditions for growth. The species richness and evenness were determined from the T-RF length and volume for the 71 profiles. Species richness was on average 13.3 +/- 7.9 per sample and 13.4 +/- 6.7 per patient. On average, the T-RF bands of the lowest and highest volumes represented 0.6 and 59.2% of the total volume in each profile, respectively. The second through fifth most dominant T-RF bands represented 15.3, 7.5, 4.7, and 2.8% of the total profile volume, respectively. On average, the remaining T-RF bands represented 10.2% of the total profile volume. The T-RF band corresponding to Pseudomonas aeruginosa had the highest volume in 61.1% of the samples. However, 18 other T-RF band lengths were dominant in at least one sample. In conclusion, this reveals the enormous complexity of bacteria within the CF lung. Although their significance is yet to be determined, these findings alter our perception of CF lung infections.

SUBMITTER: Rogers GB

PROVIDER: S-EPMC525137 | biostudies-literature | 2004 Nov

REPOSITORIES: biostudies-literature

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characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16s ribosomal DNA terminal restriction fragment length polymorphism profiling.

Rogers G B GB Carroll M P MP Serisier D J DJ Hockey P M PM Jones G G Bruce K D KD

Journal of clinical microbiology 20041101 11

Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients. A greater understanding of these bacterial infections is needed to improve lung disease management. As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data to characterize, without prior cultivation, the bacterial comm ...[more]

PMID: 15528712

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Project description:ObjectiveBacteremia due to lactobacilli is uncommon, yet it is increasing in frequency, especially among immunosuppressed patients. In the clinical laboratory, lactobacilli must be subcultured from positive blood cultures before identification by traditional biochemical methods. Delays in diagnosis are significant because the organisms are inherently resistant to vancomycin, a drug frequently prescribed for empiric therapy for gram-positive bacteremia. Recently, we developed a rapid terminal-restriction fragment length polymorphism (T-RFLP) diagnostic assay based on species-specific variations in the bacterial 16S rRNA gene. We sought to apply this technique to the identification of Lactobacillus spp. from three cases of bacteremia.DesignThe results of the T-RFLP analysis are compared with two standard biochemical identification methods.MethodsLactobacillus strains were isolated from positive clinical blood cultures. Initial suspect cultures were subcultured and characterized using an automated substrate hydrolysis system and Lactobacillus carbohydrate fermentation profiles. Further biochemical and molecular analyses were performed from isolates propagated in Lactobacillus MRS broth. DNA was extracted and the 16S rRNA gene sequenced. Two sets of fluorescent labeled primers targeting the 16S rRNA gene were used for polymerase chain reaction (PCR) with chromosomal preparations from reference strains and blood isolates. The PCR products were digested with restriction enzymes and terminal-restriction fragment profile analysis performed.ResultsT-RFLP analysis correctly identified the Lactobacillus species in each case. T-RFLP analysis could be completed within 8 hours of obtaining a positive blood culture as compared to more than the 24 to 48 hours required for traditional culturing and biochemical characterizations.ConclusionT-RFLP analysis allows for rapid identification of Lactobacillus directly from positive blood cultures and circumvents the requirement for subculture. Reduced diagnostic time has implications for duration of infection, the cost of patient care, length of hospitalization, development of broad-spectrum antibiotic resistance, and mortality due to bacteremia. T-RFLP profiling represents a highly reproducible and predictive source for identification of many organisms associated with bacteremia.

Dataset Information

Characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16s ribosomal DNA terminal restriction fragment length polymorphism profiling.

Publications

characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16s ribosomal DNA terminal restriction fragment length polymorphism profiling.

Similar Datasets

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