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

Abstract

bCystic 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.A hallmark of cystic fibrosis (CF) is chronic bacterial infection of the respiratory tract. Infection and the associated inflammation contribute to progressive lung damage, which ultimately results in respiratory failure, the leading cause of death in CF. For the past few decades, an important aspect of CF care has been the use of intensive antimicrobial therapy. Although therapy in younger patients is often provided in an attempt to eradicate initial infection, antibiotics in older patients are typically prescribed to suppress chronic infection or to treat the intermittent exacerbations of respiratory symptoms characteristic of CF. In vitro culture of CF respiratory specimens (most often expectorated sputum) has been used to guide the choice of antibiotics and has focused on the selective recovery of a relatively small suite of bacterial species believed to contribute to lung disease in CF. Historically, this set included Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa (1). Since the 1990s, the list of CF species of interest has expanded to include a small number of other opportunistic bacterial pathogens, including Achromobacter xylosoxidans, Stenotrophomonas maltophilia, and the Burkholderia cepacia complex, which also have been associated with disease progression in CF (2).During the past decade, the microbiology of CF respiratory tract infection has been further explored through the use of method...

Microbial Interactions in the Cystic Fibrosis Airway

“…Second, we were limited to studying the eight organisms for which sufficient culture data are present in the CFFPR. This excluded direct study of many CF airway organisms that are infrequently present, underreported, not collected during the study period (such as nontuberculous mycobacteria), or identifiable only by nonculture methods (58). The use of culture data is subject to variable rates, by organism, of false-positive or false-negative results; however, similar difficulties affect the recovery of organisms by nonculture methods (58).…”

“…This excluded direct study of many CF airway organisms that are infrequently present, underreported, not collected during the study period (such as nontuberculous mycobacteria), or identifiable only by nonculture methods (58). The use of culture data is subject to variable rates, by organism, of false-positive or false-negative results; however, similar difficulties affect the recovery of organisms by nonculture methods (58). Furthermore, results from conventional sputum cultures for aerobic organisms are the data that drive clinical decisions in treating patients with CF, are correlated with results from cultureindependent methods for identifying the common aerobic infectious agents in CF analyzed in our study, especially P. aeruginosa (26,58), and provide the basis of prior reports showing associations between organisms and survival outcomes (6,9).…”

The effects of inhaled aztreonam on the cystic fibrosis lung microbiome

“…Many of these organisms are specifically not cultured or ignored entirely through traditional culture-based approaches [11]. However, the role of the CF microbiome at this point in health and disease is relatively unknown.…”

Cystic fibrosis year in review 2016

“…Non‐culture‐based identification of CF pathogens remains an exciting area of research. In 945 CF sputum specimens, compared to sputum culture, DNA sequencing performed well for detection of gram negative bacteria, but less well for detection of S. aureus and Staphylococcus species . Many bacterial sequences not found in culture were noted.…”