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

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

P-values derived from PCoA for comparisons between sampling timepoints.

1<p>All P-values are the result of the paired t-tests.</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

Abundance taxa are highly stable during exacerbation and in response to antibiotic treatment.

<p>To determine whether the relative abundance of specific taxa changed during exacerbation or following antibiotic treatment, the normalized average sequence abundance for all detected OTUs was compared (a) between exacerbation (n = 22) and end-of-treatment timepoints (n = 22) and (b) between the exacerbation (n = 22) and stable timepoints (n = 13). For each taxon, normalized average sequence abundance values are plotted as a logarithm to the base 10 (log₁₀). Red circles indicate taxa that had significantly lower normalized average sequence abundance following antibiotic treatment at a 10% false discovery rate. (c) Comparison of overall microbial richness at all three sampling timepoints indicates a slight, but transient decrease following antibiotic treatment. By pairwise t-tests, comparisons of richness between exacerbation and end-of-treatment (p = 0.06, n = 21), exacerbation and stable (p = 0.87, n = 13) and stable and end-of-treatment (p = 0.076, n = 13) timepoints all fail to reach statistical significance at a p≤0.05 threshold.</p

Lung function is not correlated with total bacterial abundance.

<p>Measurements of total bacterial abundance in sputum by (a) TVC and qPCR are well correlated (r² = 0.63, p<0.0001; n = 23). FEV₁ is not correlated with (b) TVC (r² = 0.017, p = 0.55; n = 23) or (c) qPCR (r² = 0.003, p = 0.8; n = 23) and only modestly correlated with TVC from (d) <i>B. cepacia</i> complex species (r² = 0.29, p = 0.13; n = 8) and (e) <i>P. aeruginosa</i> (r² = 0.26, p = 0.05; n = 14). Measurements for bacterial abundance and FEV₁ were averaged across timepoints for each of the 23 patients. Labels in each panel indicate patient ID. Lines indicate regression fit by linear least squares. Only TVC values >0 were included for <i>B. cepacia</i> and <i>P. aeruginosa</i> comparisons. TVC values represent log₁₀ of total bacterial colony forming units (CFUs) recovered per gram of sputum. qPCR values represent log₁₀ copies of the bacterial 16S rRNA gene detected per gram of sputum.</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

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