Date Published: December 31, 2015
Publisher: Public Library of Science
Author(s): Laura M. Filkins, George A. O’Toole, Donald C Sheppard.
Historically, H. influenzae and S. aureus were considered to be the primary organisms infecting the airways of infants and children with CF, followed by P. aeruginosa or Burkholderia cepacia complex during adulthood. These bacteria continue to play important roles in CF respiratory infections and clinical outcome; however, with the advent of improved culture methods and culture-independent approaches, including deep sequencing technology, it is clear that the airways of patients with CF are chronically colonized with complex, polymicrobial infections. In 2003, Rogers and colleagues revolutionized our understanding of CF lung infections through their identification of complex bacterial communities in sputum and bronchoscopy samples using a culture-independent, molecular-based approach, terminal restriction fragment length polymorphism profiling. This study was the first to recognize the role of highly prevalent and diverse bacterial species previously unrecognized in CF lung infections, including obligate anaerobes . Since then, several additional studies utilizing culture-independent approaches have confirmed that the airways of patients with CF are chronically colonized with diverse bacterial, fungal, and viral taxa [2–5]. These polymicrobial communities are highly individualized to each patient and promote intricate inter-microbial and host—pathogen interactions, which alter the lung environment, impact response to treatment, and direct the course of disease (summarized in Fig 1).
Contrary to common intuition, the lungs are not entirely aerobic—especially the airways of patients with CF. In CF, mutations in the cystic fibrosis transmembrane conductance regulator gene lead to decreased chloride ion secretion and dehydration of the airway surface liquid layer, which leads to deficient mucociliary clearance and development of a thick mucus layer. The combination of thickened mucus and decreased clearance further facilitates the formation of mucus plugs that can obstruct the airways and form a protected niche for microbes . Within this thick mucus, and particularly within the plugs, a steep oxygen gradient forms with hypoxic (low oxygen) or anoxic (no oxygen) regions (see Fig 1). Additionally, mucus hypoxia or anoxia may be further enhanced by oxygen consumption and growth of colonizing organisms, such as P. aeruginosa . The airways of patients with CF are heterogeneous in regard to the tissue environments (e.g., localized regions of high versus low oxygen and regional variation in inflammation) and microbial communities (e.g., the abundance of microbes and composition of communities in different regions of the airway) . This heterogeneity impacts localized host and microbial interactions and, ultimately, disease progression. Microbes equipped to survive under diverse host conditions, in particular low or varied oxygen concentrations, may have increased potential to chronically colonize the airways and impact patient outcome. Species traditionally thought of as aerobic microbes are often also equipped to survive and grow in low- or no-oxygen environments. P. aeruginosa, for example, can grow by respiring nitrate and nitrite in anoxic environments and has multiple terminal oxidases that support aerobic respiration in low oxygen conditions . P. aeruginosa also has the capability to ferment arginine and pyruvate to provide maintenance energy . Furthermore, the viscous mucous and low-oxygen environments may be advantageous for some species, such as P. aeruginosa, that experience enhanced antibiotic tolerance under such conditions .
In the absence of predominating P. aeruginosa, oral-associated microbes can be the most prevalent and abundant species found in the CF lung. Indeed, microbiome studies indicate that P. aeruginosa may only be predominant in approximately 50% of adult patients . Importantly, the oral cavity plays a significant role in seeding the lower respiratory tract with diverse microbes including Streptococcus spp., Prevotella spp., Veillonella spp., Fusobacterium spp., Rothia spp., and others (see Fig 1) [5,7,13]. Despite similar origins, the community structure of lung samples and corresponding mouth wash samples are distinct, indicating that the lung environment is unique from the upper respiratory tract and selects for a separate community .
Throughout their lives, patients with CF receive antibiotic treatment both intermittently, for chronic infection management, and aggressively, during hospitalization for pulmonary exacerbation. Despite this long-term exposure to a range of antimicrobial agents, microbiota of the CF lung are not cleared, as would be expected with other common bacterial infections. Viable bacterial cell counts generally only fall approximately 10-fold in sputum after antibiotic treatment for pulmonary exacerbation , and the impact of antibiotics on total airway bacterial populations is unknown. Molecular analyses of viable bacterial populations reveal decreased microbial diversity within 72 hours of initiating treatment ; however, the impact of antibiotic treatment is transient, and baseline communities generally recover within 30 days .
Throughout life, patients experience periodic pulmonary exacerbations characterized as flares of decreased lung function, increased cough and inflammation, chest pain, and often weight loss. Historically, it was speculated that pathogen blooms or increased total bacterial load are responsible for triggering these acute episodes. However, several recent independent studies have demonstrated that overall bacterial abundance, community composition, and diversity are largely unchanged when comparing paired samples from patients during clinical stability and onset of exacerbation (before antibiotic treatment;  and others). Furthermore, neither total viable cell counts nor P. aeruginosa viable counts are consistently altered during an exacerbation .
With the explosion of 16S rRNA gene deep sequencing studies performed over the past few years analyzing the microbial populations in the airways of CF patients, we face a new challenge: what does it mean? To begin to understand the impact of these polymicrobial infections on disease progression, we must study not only their composition but also their dynamics, the effects of inter-microbial and host—microbe interactions, the role of diverse host factors (e.g., genetics, immune response, and environment), and the impact of clinical intervention. Researchers are just beginning to tackle these issues. Most importantly, upon elucidation of complex, multifactorial disease mechanisms impacting CF lung infections, the ultimate challenge will be to use this information to develop new therapeutics, personalize care, and optimize treatment strategies.