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Use of 16S rRNA gene profiling by terminal restriction fragment length polymorphism analysis to compare bacterial communities in sputum and mouthwash samples from patients with cystic fibrosis.

ABSTRACT: The bacterial communities present in the oral cavity and the lungs of 19 adult cystic fibrosis (CF) patients were compared by using terminal restriction fragment length polymorphism analysis of 16S rRNA gene PCR products amplified from nucleic acids extracted directly from bacteria in clinical samples. Sputum samples were not found to be subject to profound contamination by oral cavity bacteria. Evidence of colonization of the CF lung by certain oral bacterial species was found.

SUBMITTER: Rogers GB

PROVIDER: S-EPMC1489498 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Use of 16S rRNA gene profiling by terminal restriction fragment length polymorphism analysis to compare bacterial communities in sputum and mouthwash samples from patients with cystic fibrosis.

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

Journal of clinical microbiology 20060701 7

The bacterial communities present in the oral cavity and the lungs of 19 adult cystic fibrosis (CF) patients were compared by using terminal restriction fragment length polymorphism analysis of 16S rRNA gene PCR products amplified from nucleic acids extracted directly from bacteria in clinical samples. Sputum samples were not found to be subject to profound contamination by oral cavity bacteria. Evidence of colonization of the CF lung by certain oral bacterial species was found. ...[more]

PMID: 16825392

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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.

Project description:BackgroundBronchiectasis is accompanied by chronic bronchial infection that may drive disease progression. However, the evidence base for antibiotic therapy is limited. DNA based methods offer better identification and quantification of microbial constituents of sputum than standard clinical culture and may help inform patient management strategies. Our study objective was to determine the longitudinal variability of the non-cystic fibrosis (CF) bronchiectasis microbiome in sputum with respect to clinical variables. Eighty-five patients with non-CF bronchiectasis and daily sputum production were recruited from outpatient clinics and followed for six months. Monthly sputum samples and clinical measurements were taken, together with additional samples during exacerbations. 16S rRNA gene sequencing of the sputum microbiota was successful for 381 samples from 76 patients and analysed in conjunction with clinical data.ResultsMicrobial communities were highly individual in composition and stability, usually with limited diversity and often containing multiple pathogens. When compared to DNA sequencing, microbial culture had restricted sensitivity in identifying common pathogens such as Pseudomonas aeruginosa, Haemophilus influenzae, Moraxella catarrhalis. With some exceptions, community characteristics showed poor correlations with clinical features including underlying disease, antibiotic use and exacerbations, with the subject showing the strongest association with community structure. When present, the pathogens mucoid Pseudomonas aeruginosa and Haemophilus influenzae may also shape the structure of the rest of the microbial community.ConclusionsThe use of microbial community analysis of sputum added to information from microbial culture. A simple model of exacerbations driven by bacterial overgrowth was not supported, suggesting a need for revision of principles for antibiotic therapy. In individual patients, the management of chronic bronchial infection may be improved by therapy specific to their microbiome, taking into account pathogen load, community stability, and acute and chronic community responses to antibiotics.

Dataset Information

Use of 16S rRNA gene profiling by terminal restriction fragment length polymorphism analysis to compare bacterial communities in sputum and mouthwash samples from patients with cystic fibrosis.

Publications

Use of 16S rRNA gene profiling by terminal restriction fragment length polymorphism analysis to compare bacterial communities in sputum and mouthwash samples from patients with cystic fibrosis.

Similar Datasets

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