Unique microbial communities persist in individual cystic fibrosis patients throughout a clinical exacerbation

Abstract

Background: Cystic fibrosis (CF) is caused by inherited mutations in the cystic fibrosis transmembrane conductance regulator gene and results in a lung environment that is highly conducive to polymicrobial infection. Over a lifetime, decreasing bacterial diversity and the presence of Pseudomonas aeruginosa in the lung are correlated with worsening lung disease. However, to date, no change in community diversity, overall microbial load or individual microbes has been shown to correlate with the onset of an acute exacerbation in CF patients. We followed 17 adult CF patients throughout the course of clinical exacerbation, treatment and recovery, using deep sequencing and quantitative PCR to characterize spontaneously expectorated sputum samples

Results: We identified approximately 170 bacterial genera, 12 of which accounted for over 90% of the total bacterial load across all patient samples. Genera abundant in any single patient sample tended to be detectable in most samples. We found that clinical stages could not be distinguished by absolute Pseudomonas aeruginosa load, absolute total bacterial load or the relative abundance of any individual genus detected, or community diversity. Instead, we found that the microbial structure of each patient's sputum microbiome was distinct and resilient to exacerbation and antibiotic treatment.

Conclusion: Consistent with previously reported sputum microbiome studies we found that total and relative abundance of genera at the population level were remarkably stable for individual patients regardless of clinical status. Patient-by-patient analysis of diversity and relative abundance of each individual genus revealed a complex microbial landscape and highlighted the difficulty of identifying a universal microbial signature of exacerbation. Overall, at the genus level, we find no evidence of a microbial signature of clinical stage.

Figures

Figure 1

Neither absolute bacterial load nor absolute abundance of Pseudomonas correlates with clinical stage. (A) Absolute bacterial load in each sputum sample calculated by qPCR with universal primers to 16s rRNA gene normalized to gram of sputum extracted for this analysis (16s molecules (log10)/gram sputum). (B) Absolute abundance of P. aeruginosa in each sputum sample calculated by qPCR with primers to rplU, a reference gene validated as specific to P. aeruginosa, normalized to gram of sputum extracted for this analysis (rplU molecules (log10)/gram sputum). B, baseline; E, exacerbation; R, recovery; T, treatment.

Figure 2

Relative abundance of top twelve genera. (A) Stacked bar charts of relative abundance (left y-axis) of the top 12 genera for each patient across a clinical exacerbation show that 12 genera explain 90% of the complexity for all patient samples. (B) Relative abundance of the top 12 genera are plotted against the prevalence of each genus in the nine complete (BETR, BER) patient samples and show that bacteria that are highly abundant in a single patient are also highly prevalent across patients. The colored dots indicate those genera that are both highly abundant and highly prevalent, and the colors correspond to the legend shown in panel A. Colors in panels A and B correspond to genera as indicated by the legend beneath panel A. (C) The fraction of P. aeruginosa determined by qPCR (rplU detection/16s rRNA detection) correlates to the fraction of deep-sequencing reads assigned to the Pseudomonas genus. qPCR samples were analyzed six times and the median fraction values for each sample are shown. There is one outlier in this dataset (sample 206R, shown in red). When this outlier is removed from the analysis, the linear regression slope is 1.1 and R2 = 0.90. BER, baseline, exacerbation and recovery; BETR, baseline, exacerbation, treatment and recovery; qPCR, quantitative PCR.

Figure 3

Microbial communities cluster by patient, not by clinical stage. (A) Hierarchical clustering of top 12 genera found in patient samples. Each clinical BETR stage is designated by color (baseline, green; exacerbation, red; treatment, orange; recovery, blue) along the top of the diagram; patient number is given across the bottom. The relative abundance for each genus is colored in shades of red (low relative abundance) to yellow or bright white (high relative abundance) as shown in the color key (upper left). The x-axis of the color key (row Z-score) indicates the number of standard deviations from the mean relative abundance for each genus. The count histogram indicates the mean counts for all data in sample set. (B) Principal coordinate analysis of all patients. Clinical stages are represented by colored dots (baseline, green; exacerbation, red; treatment, orange; recovery, blue) and black lines connect the trajectories of each patient’s microbiome throughout the study. BETR, baseline, exacerbation, treatment and recovery.

Figure 4

Diversity does not correlate with clinical stage. (A) Simpson diversity index for the six patients with samples from all four stages (BETR) are color-coded. A mixed-effect linear model with treatment status as a categorical variable was used to identify significant differences in diversity as a function of clinical status. Analysis of variance showed no significant association between diversity and status. Aggregated Simpson diversity index (SDI) of the six patients with all four BETR samples (B) or from all seventeen patients (C). There is no statistically significant difference between stages for patients with all four BETR samples (ANOVA, Tukey post-test, P values all >0.05). Paired t-test for all patients reveals no statistical difference between any stage (P > 0.05). B, baseline; BETR, baseline, exacerbation, treatment and recovery; E, exacerbation; R, recovery; SDI, Simpson diversity index; T, treatment.

Figure 5

Changes in individual bacterial abundance at each clinical transition. (A) Relative abundance of Pseudomonas at baseline and exacerbation. Each point represents a different patient labeled with patient ID. The dotted line has a slope of 1. Points to the left of the 1 line indicate an increase in Pseudomonas at exacerbation compared to baseline; points to the right of the 1 line indicate a decrease in Pseudomonas. Coefficient estimates of each genus for (B) baseline to exacerbation, (C) exacerbation to treatment and (D) treatment to recovery. The top 12 genera from Figure 2 and genera with coefficient estimate significantly different than 1 (P < 0.05) are shown. Bars indicate mean coefficient estimate; error bars indicate 95% confidence interval of the mean.