In Vitro and In Vivo Characterization of the Pseudomonas aeruginosa Cyclic AMP (cAMP) Phosphodiesterase CpdA, Required for cAMP Homeostasis and Virulence Factor Regulation▿ †

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

The Adult Cystic Fibrosis Airway Microbiota Is Stable over Time and Infection Type, and Highly Resilient to Antibiotic Treatment of Exacerbations

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

Lung Microbiota and Bacterial Abundance in Patients with Bronchiectasis when Clinically Stable and during Exacerbation

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

Prevalent taxa are also abundant taxa.

<p>Plot showing the log transformed (log₁₀) average normalized sequence counts for each taxon compared to the number of samples in which the taxon is present. Averaged values only include samples in which the taxon is present. Only taxa present in two or more samples (155 OTUs) are plotted. Raw data used to generate used for this analysis are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045001#pone.0045001.s001" target="_blank">Table S7</a>. Red symbols indicate recognized dominant CF pathogens.</p

CF is a polymicrobial disease.

<p>Phylogenetic tree of the 169 OTUs identified in the CF sputum dataset. Tree construction was achieved by mapping consensus sequences from each OTU to the SILVA reference tree (see Methods). Each leaf of the tree represents a consensus OTU labeled with the most closely related genus assigned by the RDP classifier.</p

Antibiotic regimens used to treat acute pulmonary exacerbations in this study¹.

1<p>Table indicates antibiotic regimens used to treat each of the 26 exacerbations that occurred during the period of study. Of the 23 patients enrolled, three patients experienced two separate exacerbations. For these three patients, the first and second exacerbations were treated with different antibiotic combinations.</p

Total viable counts by culture show significant but non-linear agreement with relative sequence abundance.

<p>TVC for (a) <i>P. aeruginosa</i> and (b) <i>B. cepacia</i> complex species plotted against the fraction of sequences assigned to the corresponding genera in each sputum sample. Black lines represent linear regression by least squares fitting. Values for <i>Pseudomonas</i> (r² = 0.71, p<0.001) and <i>Burkholderia</i> (r² = 0.86, p<0.001) indicate a significant correlation. Red lines are intended to illustrate a potential non-linear relationship and are based on the two-parameter Michaelis-Menten function with arbitrarily selected parameters.</p

Mouthwash and sputum samples have a highly similar distribution of taxa.

<p>To determine whether oral flora contribute to CF airway microbial communities, we compared the normalized average sequence abundance of all OTUs found in 22 paired mouthwash and sputum samples from 9 patients. For each taxon, normalized average sequence abundance values are plotted as a logarithm to the base 10 (log₁₀). Red symbols indicate taxa that had significantly different distribution between sample types at a 10% false discovery rate from a parametric t-test in which the 22 samples were treated as independent measurements.</p

Low species richness in mouthwash samples is associated with decreased lung functions.

<p>Shown is a plot of the average FEV₁ compared to average microbial richness in mouthwash samples for nine patients. FEV₁ and microbial richness values were averaged across all timepoints for each patient. Numbers next to each symbol indicate patient ID. Results from linear regression analysis (red line) showed a modest correlation with lung function (r² = 0.42, p = 0.057).</p

There is broad agreement between qualitative bacterial culture results and 454 pyrosequencing for dominant CF pathogens.

<p>The fraction of sequence assigned to the genus (a) <i>Pseudomonas</i> or (b) <i>Burkholderia</i> are plotted for each patient and sample timepoint as a function of the patient' s reported culture status for the recognized CF pathogens <i>P. aeruginosa</i> and <i>B. cepacia</i> complex species. Patient samples are color-coded by timepoint (green, onset of exacerbation; red, end-of-treatment for exacerbation with intravenous antibiotics; blue, clinically stable interval). Patient 19 was culture negative for both <i>P. aeruginosa</i> and <i>B. cepacia</i>.</p