Prevalence of streptococci and increased polymicrobial diversity associated with cystic fibrosis patient stability

Abstract

Diverse microbial communities chronically colonize the lungs of cystic fibrosis patients. Pyrosequencing of amplicons for hypervariable regions in the 16S rRNA gene generated taxonomic profiles of bacterial communities for sputum genomic DNA samples from 22 patients during a state of clinical stability (outpatients) and 13 patients during acute exacerbation (inpatients). We employed quantitative PCR (qPCR) to confirm the detection of Pseudomonas aeruginosa and Streptococcus by the pyrosequencing data and human oral microbe identification microarray (HOMIM) analysis to determine the species of the streptococci identified by pyrosequencing. We show that outpatient sputum samples have significantly higher bacterial diversity than inpatients, but maintenance treatment with tobramycin did not impact overall diversity. Contrary to the current dogma in the field that Pseudomonas aeruginosa is the dominant organism in the majority of cystic fibrosis patients, Pseudomonas constituted the predominant genera in only half the patient samples analyzed and reported here. The increased fractional representation of Streptococcus in the outpatient cohort relative to the inpatient cohort was the strongest predictor of clinically stable lung disease. The most prevalent streptococci included species typically associated with the oral cavity (Streptococcus salivarius and Streptococcus parasanguis) and the Streptococcus milleri group species. These species of Streptococcus may play an important role in increasing the diversity of the cystic fibrosis lung environment and promoting patient stability.

Fig 1

Characterization of the polymicrobial communities of sputum samples from cystic fibrosis inpatients and outpatients. (A) Fraction of 454 pyrosequencing reads assigned to each of the top-10 genera detected in the patient sample set as a whole is shown for sputum samples analyzed from inpatients (INPT; n = 13) and outpatients (OUTPT; n = 22) from a cross-sectional study. The legend indicates the color assigned to each indicated genus. (B) Heat map of patient samples based on Pearson hierarchical clustering of relative bacterial abundance (by deep sequencing) for the most prevalent four genera, which account for ∼86% of the total pyrosequencing reads. Patients can be described as one of four profiles: (i) high Pseudomonas, low Streptococcus; (ii) medium Pseudomonas, medium Streptococcus; (iii) low Pseudomonas, high Streptococcus; (iv) low Pseudomonas, low Streptococcus, but high other (primarily Fusobacterium or Prevotella). The group number is indicated in the bar at the bottom of the panel, and the inset key indicates the color associated with the fraction of reads for each patient and genera assigned.

Fig 2

Increased diversity correlates with outpatient status and is not impacted by tobramycin treatment. Box-and-whisker plots of the Simpson diversity index based on the complete deep-sequencing profile of each sputum sample (each read assigned to a single genus). Box parameters, the bold line represents median diversity, while the upper and lower ranges of the box represent the 75% and 25% quartiles, respectively; whisker parameters, 1.5× the interquartile range; open circles, data points that fall outside 1.5× the interquartile range. P values were calculated using the nonparametric Mann-Whitney U test. (A) Inpatient (INPT; n = 13) samples compared to outpatient (OUTPT; n = 22) samples. The asterisk indicates P values <0.05. (B) Comparison of sputum samples collected from patients off tobramycin antibiotic treatment (Off Tobi; n = 24) compared to on tobramycin (On Tobi; n = 11), irrespective of patient status. No significant difference in diversity was detected.

Fig 3

Increased Streptococcus fraction correlates with clinical patient stability. Box-and-whisker plots comparing the fraction of 454 pyrosequencing reads assigned to a single genus for inpatient and outpatient samples. Median fraction Streptococcus or Pseudomonas is represented by the bold middle line of the box. The extremities of the box represent the 75% and 25% quartiles. The whiskers of the plot indicate 1.5× the interquartile range. P values were calculated using the nonparametric Mann-Whitney U test. (A) Comparison of Pseudomonas fraction; (B) comparison of Streptococcus fraction. The asterisk indicates P values <0.05. (C) Transition plot comparing the fractional representation of taxonomic read assignments in inpatient samples to outpatient samples, based on 454 pyrosequencing results. Plotted circles correspond to the log₂-fold difference in fractional representation of an individual taxon between the inpatient cohort and the outpatient cohort. t test comparisons of relative abundance in inpatient versus outpatient samples for all genera detected in the sample set were performed, and the reported P values are Benjamini-Hochberg corrected for multiple hypothesis testing. Key: open circles, no significant difference in fractional representation, P > 0.05; gray circles, significantly higher fractional representation in outpatient samples, P ≤ 0.05. No detected genera were significantly enriched in inpatient samples compared to in outpatient samples after Benjamini-Hochberg correction. Genera are labeled in the order they are located on the transition plot (most significantly different at the bottom). Relative fractional representation in the total sample set is specified for each labeled genus.

Fig 4

qPCR analysis independently verifies 454 pyrosequencing detection of the most prevalent bacteria in cystic fibrosis patient sputum samples. (A) The fraction of Pseudomonas aeruginosa determined by qPCR (rplU detection/universal detection) correlates to the fraction of deep-sequencing reads assigned to the Pseudomonas genus. Pearson correlation = 0.968. (B) The fraction of Streptococcus determined by qPCR (tuf detection/universal detection) correlates to the fraction of deep-sequencing reads assigned to the Streptococcus genus. Pearson correlation = 0.941. All samples were analyzed in replicates of six for each primer set, with the exception of OUTPT 11 (n = 3). Median fraction values for each sample are plotted for qPCR detection and were used for the correlation analysis. Pearson correlation lines were calculated, excluding the outlier points. Legend: open circle, outpatient (OUTPT); black closed circle, inpatient (INPT); gray closed circle, outlier.

Fig 5

Oral streptococci and streptococcal species are prevalent in CF sputum samples. Shown is the assignment of relative abundance (score of 0 to 5) for each sample based on intensity of hybridization to each Streptococcus-specific 16S rRNA gene probe. Probes are species specific, group specific (hybridize 2 to 3 oral taxon), or cluster specific (hybridize multiple streptococcal oral taxon). Pearson hierarchical clustering was performed based on hybridization intensity in two dimensions: (i) probe and (ii) patient sample.