Culture-Based and Culture-Independent Bacteriologic Analysis of Cystic Fibrosis Respiratory Specimens

Abstract

Cystic fibrosis (CF) is characterized by chronic infection and inflammation of the airways. In vitro culture of select bacterial species from respiratory specimens has been used to guide antimicrobial therapy in CF for the past few decades. More recently, DNA sequence-based, culture-independent approaches have been used to assess CF airway microbiology, although the role that these methods will (or should) have in routine microbiologic analysis of CF respiratory specimens is unclear. We performed DNA sequence analyses to detect bacterial species in 945 CF sputum samples that had been previously analyzed by selective CF culture. We determined the concordance of results based on culture and sequence analysis, highlighting the comparison of the results for the most prevalent genera. Although overall prevalence rates were comparable between the two methods, results varied by genus. While sequence analysis was more likely to detect Achromobacter, Stenotrophomonas, and Burkholderia, it was less likely to detect Staphylococcus. Streptococcus spp. were rarely reported in culture results but were the most frequently detected species by sequence analysis. A variety of obligate and facultative anaerobic species, not reported by culture, was also detected with high prevalence by sequence analysis. Sequence analysis indicated that in a considerable proportion of samples, taxa not reported by selective culture constituted a relatively high proportion of the total bacterial load, suggesting that routine CF culture may underrepresent significant segments of the bacterial communities inhabiting CF airways.

FIG 1

Concordance of culture-based and sequencing-based detection of bacteria. Euler diagrams (31) showing concordance between culture-based and sequencing-based detection of bacteria in CF sputum samples. Proportions are based on the percentage of 945 samples in which the indicated genus was not detected (white), detected by culture (light gray), detected by sequence analysis (dark gray), or detected by culture and sequence analysis (black).

FIG 2

Proportion of false-negative cultures based on the relative abundance of the indicated genus in DNA sequence analysis.

FIG 3

Prevalences and relative abundances of genera. The prevalences (dark bars) and mean (plus standard deviation) relative abundances (light bars) of the top 10 most abundant and/or prevalent genera detected in 945 sputum samples by sequence analysis are shown. Mean relative abundance in samples in which the specified genus was detected.

FIG 4

Box plot of relative abundance and proportion of OTUs not reported by culture. Boxes signify the upper and lower quartiles, black lines denote median values, whiskers show 1.5 interquartile range, and open circles designate outliers.

FIG 5

Box plot showing the relative abundances (summed) of the genera not reported by culture and grouped by Shannon diversity quartiles. The black line corresponds to the median summed relative abundance for each diversity quartile (1, low diversity; 4, high diversity). Each black dot represents one sputum sample.