RESUMEN
RATIONALE: Characterization of bacterial populations in infectious respiratory diseases will provide improved understanding of the relationship between the lung microbiota, disease pathogenesis, and treatment outcomes. OBJECTIVES: To comprehensively define lung microbiota composition during stable disease and exacerbation in patients with bronchiectasis. METHODS: Sputum was collected from patients when clinically stable and before and after completion of antibiotic treatment of exacerbations. Bacterial abundance and community composition were analyzed using anaerobic culture and 16S rDNA pyrosequencing. MEASUREMENTS AND MAIN RESULTS: In clinically stable patients, aerobic and anaerobic bacteria were detected in 40 of 40 (100%) and 33 of 40 (83%) sputum samples, respectively. The dominant organisms cultured were Pseudomonas aeruginosa (n = 10 patients), Haemophilus influenzae (n = 12), Prevotella (n = 18), and Veillonella (n = 13). Pyrosequencing generated more than 150,000 sequences, representing 113 distinct microbial taxa; the majority of observed community richness resulted from taxa present in low abundance with similar patterns of phyla distribution in clinically stable patients and patients at the onset of exacerbation. After treatment of exacerbation, there was no change in total (P = 0.925), aerobic (P = 0.917), or anaerobic (P = 0.683) load and only a limited shift in community composition. Agreement for detection of bacteria by culture and pyrosequencing was good for aerobic bacteria such as P. aeruginosa (κ = 0.84) but poorer for other genera including anaerobes. Lack of agreement was largely due to bacteria being detected by pyrosequencing but not by culture. CONCLUSIONS: A complex microbiota is present in the lungs of patients with bronchiectasis and remains stable through treatment of exacerbations, suggesting that changes in microbiota composition do not account for exacerbations.
Asunto(s)
Antibacterianos/uso terapéutico , Bronquiectasia/tratamiento farmacológico , Bronquiectasia/microbiología , Pulmón/microbiología , Metagenoma , Anciano , Estudios de Cohortes , Estudios Transversales , Femenino , Humanos , Estudios Longitudinales , Masculino , Esputo/microbiologíaRESUMEN
Cystic fibrosis (CF) is characterized by defective mucociliary clearance and chronic airway infection by a complex microbiota. Infection, persistent inflammation and periodic episodes of acute pulmonary exacerbation contribute to an irreversible decline in CF lung function. While the factors leading to acute exacerbations are poorly understood, antibiotic treatment can temporarily resolve pulmonary symptoms and partially restore lung function. Previous studies indicated that exacerbations may be associated with changes in microbial densities and the acquisition of new microbial species. Given the complexity of the CF microbiota, we applied massively parallel pyrosequencing to identify changes in airway microbial community structure in 23 adult CF patients during acute pulmonary exacerbation, after antibiotic treatment and during periods of stable disease. Over 350,000 sequences were generated, representing nearly 170 distinct microbial taxa. Approximately 60% of sequences obtained were from the recognized CF pathogens Pseudomonas and Burkholderia, which were detected in largely non-overlapping patient subsets. In contrast, other taxa including Prevotella, Streptococcus, Rothia and Veillonella were abundant in nearly all patient samples. Although antibiotic treatment was associated with a small decrease in species richness, there was minimal change in overall microbial community structure. Furthermore, microbial community composition was highly similar in patients during an exacerbation and when clinically stable, suggesting that exacerbations may represent intrapulmonary spread of infection rather than a change in microbial community composition. Mouthwash samples, obtained from a subset of patients, showed a nearly identical distribution of taxa as expectorated sputum, indicating that aspiration may contribute to colonization of the lower airways. Finally, we observed a strong correlation between low species richness and poor lung function. Taken together, these results indicate that the adult CF lung microbiome is largely stable through periods of exacerbation and antibiotic treatment and that short-term compositional changes in the airway microbiota do not account for CF pulmonary exacerbations.
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Fibrosis Quística/microbiología , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Metagenoma/efectos de los fármacos , Sistema Respiratorio/microbiología , Adulto , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Enfermedad Crónica , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/patología , Progresión de la Enfermedad , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Grampositivas/genética , Bacterias Grampositivas/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Metagenoma/genética , Filogenia , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/patología , Esputo/microbiologíaRESUMEN
The aim of this study was to determine whether geographical differences impact the composition of bacterial communities present in the airways of cystic fibrosis (CF) patients attending CF centers in the United States or United Kingdom. Thirty-eight patients were matched on the basis of clinical parameters into 19 pairs comprised of one U.S. and one United Kingdom patient. Analysis was performed to determine what, if any, bacterial correlates could be identified. Two culture-independent strategies were used: terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA clone sequencing. Overall, 73 different terminal restriction fragment lengths were detected, ranging from 2 to 10 for U.S. and 2 to 15 for United Kingdom patients. The statistical analysis of T-RFLP data indicated that patient pairing was successful and revealed substantial transatlantic similarities in the bacterial communities. A small number of bands was present in the vast majority of patients in both locations, indicating that these are species common to the CF lung. Clone sequence analysis also revealed that a number of species not traditionally associated with the CF lung were present in both sample groups. The species number per sample was similar, but differences in species presence were observed between sample groups. Cluster analysis revealed geographical differences in bacterial presence and relative species abundance. Overall, the U.S. samples showed tighter clustering with each other compared to that of United Kingdom samples, which may reflect the lower diversity detected in the U.S. sample group. The impact of cross-infection and biogeography is considered, and the implications for treating CF lung infections also are discussed.
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Bacterias/clasificación , Bacterias/aislamiento & purificación , Biodiversidad , Fibrosis Quística/microbiología , Pulmón/microbiología , Adolescente , Adulto , Bacterias/genética , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Reino Unido , Estados Unidos , Adulto JovenRESUMEN
Cyclic AMP (cAMP) is an important second messenger signaling molecule that controls a wide variety of eukaryotic and prokaryotic responses to extracellular cues. For cAMP-dependent signaling pathways to be effective, the intracellular cAMP concentration is tightly controlled at the level of synthesis and degradation. In the opportunistic human pathogen Pseudomonas aeruginosa, cAMP is a key regulator of virulence gene expression. To better understand the role of cAMP homeostasis in this organism, we identified and characterized the enzyme CpdA, a putative cAMP phosphodiesterase. We demonstrate that CpdA possesses 3',5'-cAMP phosphodiesterase activity in vitro and that it utilizes an iron-dependent catalytic mechanism. Deletion of cpdA results in the accumulation of intracellular cAMP and altered regulation of P. aeruginosa virulence traits. Further, we demonstrate that the cAMP-dependent transcription factor Vfr directly regulates cpdA expression in response to intracellular cAMP accumulation, thus providing a feedback mechanism for controlling cAMP levels and fine-tuning virulence factor expression.