Cystic fibrosis (CF) is an autosomal recessive genetic disorder resulting from genetic defects in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR dysfunction in patients with CF leads to a number of pleiotropic manifestations with the prime pathology being mucus plugging in the airways and paranasal sinuses. Patients with CF are prone to polymicrobial infections and the airway microbiome in such patients changes continuously and evolves over time. The composition of the airway microbiome in CF patients is dependent on a number of factors including geographic variation, type of genetic mutation (e.g., ΔF508), antibiotic exposures, and chronic infection with certain pathogenic bacteria (e.g., Pseudomonas aeruginosa). Proteomic and genomic approaches to understanding the microbiome of patients with CF have provided new insights into the pathogenesis of this disease. High‐throughput pyrosequencing, Sanger sequencing, and phylogenetic microarray analysis have enabled the recognition of multiple lineages and clonal populations of a single bacterial species within the same patient. This provides a unique opportunity to explore novel therapeutic approaches to this disease (for instance, use of probiotics and environmental manipulation) and potentially translate them into bedside clinical interventions.
- cystic fibrosis
- Pseudomonas aeruginosa
- burkholderia cenocepacia
Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene . CF is most prevalent in the Caucasian population and is a common life‐limiting disease . CFTR is expressed on the apical surface of epithelial cells of the respiratory, gastrointestinal, pancreatic and reproductive tracts, and sweat glands . The prime function of CFTR ion channel is to transport chloride ions across epithelial surfaces in order to maintain the osmotic gradient. Chloride ions are actively pumped out into the luminal side of the gastrointestinal and respiratory tracts, which decrease water potential on the luminal side. Subsequently, water molecules move from a higher osmotic potential to a lower osmotic potential (down the osmotic gradient) and combine with mucin glycoproteins to keep them adequately hydrated. This in turn helps to maintain the thin consistency of the mucus layer, which is essential for optimal mucociliary function . Thick and viscid mucus caused by a defect in chloride‐conducting transmembrane channel results in stagnation of mucus. Moreover, CFTR channel also plays an important role in regulating the transepithelial transport of sodium and bicarbonate ions . Defective CFTR functioning leads to an increase in pH of the mucus layer, which compromises the innate immune system and promotes inflammation. Defects in innate immunity and chronic inflammation predispose patients to recurrent pulmonary infections, which result in permanent lung damage—the prime cause of morbidity and mortality . Pulmonary system is not the only organ‐system affected in CF; endocrine, gastrointestinal, and reproductive systems are also involved in this multisystem disorder .
The human microbiome project aims to identify and characterize microbial flora of healthy and diseased individuals . Understanding the role of infectious pathogens in the pathogenesis of CF in general and pulmonary exacerbations and lung damage in particular has enabled the scientific community to devise new treatment modalities for CF patients, which can potentially improve outcomes and survival in such patients. In patients with CF, different bacteria inhabit different parts of the lung at various stages of the disease and persistent inflammation in the lungs can change and modify the composition of the microbiome . For instance, methicillin‐sensitive
2. The microbiome
As mentioned previously, the human microbiome project aims to identify and characterize microbial flora of healthy and diseased individuals . There is a diversity of microbes in every single human being i.e., diversity being defined as the number and distribution of a particular type of organism in a body habitat. Every human has particular and distinct microbes; dysbiosis (alteration in composition and balance) of these microbes is now thought to underlie the pathogenesis of many diseases, such as inflammatory bowel disease,
Another example of a disease where microbiota plays a major role in pathogenesis is Crohn's disease. The exact cause of Crohn's disease is unknown; however, evidence suggests that microbiota contribute to the underlying pathology and disease development . No single bacterium has been convincingly shown to contribute to the overall pathogenesis of Crohn's disease. Instead, dysbiosis (bacterial imbalance) is more widely accepted as a leading factor in the disrupted host immune system cross‐talk that results in subsequent intestinal inflammation . Depletion of symbiont (beneficial) microbes (including Firmicutes, Bifidobacteriaceae, and Clostridia) in conjunction with an increase in pathobiont (harmful) microbes (such as Bacteroidetes and Enterobacteriaceae) is a striking feature observed in Crohn's disease. No single factor has been definitely identified as driving this dysbiosis; instead, a host of environmental factors—such as the diet, antibiotic exposures and possible early life infections—in the presence of underlying genetic susceptibilities may contribute to the overall pathogenesis of Crohn's disease .
In CF patients, composition of the microbiome of pulmonary and gastrointestinal tracts changes over time, presumably as a consequence of inflammation . Most research studies have demonstrated the influence of inflammation in negatively selecting against potential pathogens. Moreover, some bacterial species may also have the ability to exploit inflammatory byproducts for their benefit, which may promote their natural selection in inflamed habitats . Reactive nitrogen species produced during inflammatory responses can be exploited by pathogens for their growth. Moreover, inflammatory mediators can provide an environment for some bacteria to grow and use these inflammatory mediators for their survival . Examples of such bacteria include
3. Heterogeneity of the CF airway microbiome
Due to defects in innate immunity, CF patients are prone to polymicrobial infections and their airway microbiome changes continuously and evolves over time. The primary cause of death in CF patients is respiratory failure due to persistent and recurrent pulmonary infections with different pathogenic organisms . Over the past decade, the median survival for such patients stands at 37 years despite increases in life expectancy . MSSA and
Microbes of the lower airways in all humans exist in a dynamic state. Literature published on microbiome of CF patients has shown a complex and dynamic interaction between different organisms in the airways of such patients . Organisms within a single patient are genetically and phenotypically diverse and heterogeneity is detectable even in different parts of the same lung. Over a period of time, community diversity of bacteria declines in CF patients as pulmonary function declines and lung disease progressively worsens. Studies have shown that diversity of microbial communities correlates positively with pulmonary function and outcome . Such diversity was previously unrecognized as most studies relied solely on culture‐based methods of culturing bacteria. However, novel state‐of‐the‐art molecular techniques (such as Sanger sequencing of clone libraries, terminal restriction fragment length polymorphism [RFLP] analysis and microarray hybridization) have enabled the detection of subtle molecular diversity among seemingly similar bacterial species . This diversity may be influenced by a number of factors including the patient's age, sex, type of CFTR mutation, antibiotic exposures, environmental factors, and extent and severity of lung disease. In a study by Zhao et al., sputum samples were collected from six CF patients over a period of 10 years. Of a total of 126 sputum samples, 662 operational taxonomic units (OTU) were identified and each patient had 5–114 different OTUs . Similarly, in another observational study, sputum samples of patients with acute infective exacerbation of non‐CF related bronchiectasis were collected. Sputum cultures from each patient contained large quantities of multiple bacterial species with a single predominant pathogenic species . In one study, polymerase chain reaction (PCR)‐temporal temperature gel electrophoresis (PCR‐TTGE) was used to evaluate intraspecific and intragenomic 16S rDNA variability among commonly isolated respiratory pathogens from CF patients . Significant discordance in intraspecific and intragenomic variability was noted among different bacterial species with
4. Composition of the CF microbiome and its determinants
The composition of the airway microbiome in CF patients is dependent on a number of factors including geographic variation (more common in white population), type of genetic mutation (e.g., ΔF508), antibiotic exposures, and chronic infection with certain pathogenic bacteria (e.g.,
The microbiome in patients with CF evolves as patients grow older, and this is a consequence of the wide adaptability of pathogenic bacteria. Clustering of phylogenetically similar bacterial communities and loss of the architectural diversity of the airway microbiome is a key feature of late‐stage CF airway disease. Moreover, the type of bacterial species predominating at a particular age group is also of immense importance. In one study, phylogenetic diversity of CF airway microbiota in patients of different age groups was studied using microarray analysis .
In another study , CF patients with progressive lung disease were noted to have a decrease in bacterial diversity with increasing age, but the total bacterial density remained stable over time. Antibiotic exposures in conjunction with recurrent pulmonary exacerbations were proposed as a possible contributing factor toward this observation. In a study by Tunney et al., several anaerobic species (including a number of Veillonella and Prevotella species) constituted a significant portion of the CF airway microbiota . In a unique study, next generation sequencing was used to study the microorganisms of gastric juice among patients with CF and non‐CF controls . CF gastric juice was noted to have an abundance of Pseudomonas spp. and a relative paucity of normal gut bacteria (such as Bacteroides and Faecalibacterium), which was in contrast with normal gastric juice samples. These results suggest that CF patients possess a unique aerodigestive microbiome that is inter‐related. This explanation seems plausible as the factors that influence the airway microbiome (for instance, antibiotic exposures) are also likely to influence the microbiota of gut and other organ‐systems of the body .
In patients with CF, different bacterial colony morphotypes can be isolated from a single sputum sample. There is some evidence to suggest that these different morphotypes arise from a single bacterial strain . Microbes in the lungs of CF patients are capable of constantly adapting to selection pressures. Some of the mechanisms that enable the evolution of microbes include motility, type III secretion systems, lipopolysaccharide, plasmids (encoding for antibiotic resistance), biofilm formation, small colony variants, quorum sensing, and hypermutability. As a consequence of these mechanisms, different phenotypes arise from a single bacterial species and, over time, a single bacterial strain with dominating features may evolve . Given that different bacterial strains have differing capacities to evolve, multiple lineages of bacterial colonies evolve and coexist . Some studies have shown that complexity of bacterial communities inversely correlates with patient age, antibiotic exposures, and presence of
The interaction among different bacterial colonies has also become a subject of intense research and genomic and proteomic approaches are currently being used to understand their interrelationships. In an experimental study, production of 4‐hydroxy‐2‐heptylquinoline‐N‐oxide (HQNO) by a strain of
In the recent literature, an increasing number of unusual microbes have been reported as the cause of infective exacerbations of CF. Such bacteria include multidrug resistant pathogens like
With the widespread use of antistaphylococcal antibiotics, incidence of Gram‐negative infections among CF patients has increased and MSSA has become less common among adult patients. Overall, the most common cause of chronic lung infections in CF patients is
4.3. Hemophilus influenzae
Similar to the general population, colonization of the upper respiratory tract of CF patients with
4.4. Pseudomonas aeruginosa
The main reservoir of
The effects of
The CF airway provides a pathological milieu and a scaffold for chronic infection with resistant organisms, the most notable of them being
4.5. Burkholderia cepacia complex
More than 60 species belonging to the genus Burkholderia are not pathogenic to humans, but some of the remaining species are implicated in serious infections in CF patients. Using 16S rDNA and recA gene analysis, 17 species of this genus have been grouped together as the Burkholderia cepacia complex (BCC). BCC is a group of virulent pathogens that are frequently implicated in infective exacerbations in CF patients with end‐stage lung disease. Colonization with BCC in CF patients indicates a poor prognosis and has been shown to be associated with a requirement for lung transplantation. This worse prognosis is due to the inherent antibiotic resistance possessed by these organisms and their ability to rapidly spread from patient to patient. In some cases, infection with BCC can lead to the development of cepacia syndrome—a rapid fulminating pneumonia that often leads to bacteremia and sepsis. Given their virulent nature, strict infection control measures are essential to prevent outbreaks of BCC in CF clinics and centers . A report of rapid spread and outbreak of BCC infection was reported in a CF center in Toronto . This center reported the development of cepacia syndrome in many patients, being characterized by rapidly deteriorating pulmonary function, fever, leukocytosis, elevated markers of inflammation, and BCC bacteremia. Furthermore, in another report, cepacia syndrome occurred in approximately 20% of infected patients and had a case fatality rate of 62% .
Outside of the BCC group, a few other species of the Burkholderia genus are also implicated in infective exacerbations. These species include
Most infected CF patients harbor genotypically distinct strains of the BCC. Strains of Burkholderiaspp. that are shared by multiple CF patients are very uncommon. This suggests that most Burkholderia infections in CF patients result from acquisition of strains from the natural environment [92, 96]. In this regard,
In the CFF patient registry, prevalence of BCC was reported to have declined from 9% in 1985 to 4% in 2005. Incidence of BCC was also found to be reduced from 1.3% in 1995 to 0.8% in 2005 . This has not changed significantly over the past decade as shown by data published in 2016 . Ramette et al. analyzed 285 confirmed isolates of BCC using restriction analysis of recA and identified seven different BCC species in the environment . Healthcare‐associated outbreaks of BCC infections as a consequence of contaminated medical devices and products (such as mouthwashes, ultrasound gels, skin antiseptics, and medications) have been reported previously. While most of these outbreaks have generally involved non‐CF patients, the potential for developing such outbreaks among CF patients remains a hazard . Infection of the respiratory tract with BCC species in CF patients often results in a chronic persistent infection . In most such cases, a single strain of Burkholderia spp. colonizes the respiratory tract.
Infection with BCC species has been associated with a worse prognosis. In one study, CF patients who were infected with
4.6. Anaerobic bacteria
Anaerobic bacteria have been described in the airways of people with healthy lungs and are generally not considered to be pathogenic. In patients with CF, anaerobic bacteria are persistent members of the lower airway community as the anaerobic conditions (and steep oxygen gradients) in the lower airways provide an ideal environment for their growth [88, 103]. However, in the CF lung, anaerobic bacteria can produce virulence factors and damage the lung parenchyma (perhaps as a consequence of impaired innate immunity), which may worsen pulmonary function and exacerbate the inflammatory response. Short‐chain fatty acids produced by anaerobic bacteria can increase production of interleukin‐8 (IL‐8) by upregulating expression of the short‐chain fatty acid receptor GPR41 . Moreover, in the CF microbiome, anaerobic bacteria can interact with other established pathogens and lead to progressive pulmonary damage . Previously, anaerobic bacteria were thought to be an infrequent cause of CF exacerbation; however, with the advent of novel (culture‐independent) microbial detection methods [106–109], anaerobes have been isolated from more frequently. In one study, 23.8% of sputum specimens from CF patients grew more than 105 colony forming units (CFU) per milliliter of anaerobic bacteria . In another study, 15 genera of obligate anaerobes were identified in 91% of CF patients with counts (CFU/ml) being comparable to that of
4.7. Nontuberculous mycobacteria
Traditionally, the frequency of CF patients infected with NTM has been reportedly low. In the CFF patient registry, the prevalence of NTM infections among CF patients has been estimated to be 2.2%. Nevertheless, the prevalence of NTM has been increasing slowly over the past few decades. The prevalence of NTM infection in 1999 among CF patients was 0.85%, which increased to 2.18% in 2008 . More recent data published in 2016 shows that the prevalence of NTM may be as high as 11.9% . The most common NTM species have been reported to be Mycobacterium avium‐intracellulare (MAI) complex and
5. Implications for further research
Cystic fibrosis is a monogenetic multisystem disorder, but, pulmonary disease is the leading cause of morbidity and mortality. Recurrent pulmonary infections with pathogenic bacteria can lead to progressive pulmonary damage and eventually lead to death. Therefore, understanding the CF airway microbiome has immense importance for understanding the overall pathology of the disease. Disruption of the CF airway microbiome under the influence of environmental factors and antibiotic exposures is a crucial step in the development of end‐stage pulmonary disease in such patients . Colonization of the lower airways with pathogenic bacteria, such as
As the CF airway microbiome evolves under the influence of antibiotic exposures, microbes undergo a number of mutations and changes in their genome . While these genetic mutations are an evolutionary mechanism for microorganisms (for instance, to acquire resistance to antibiotics), they create potential vulnerabilities that may be exploited in unique therapeutic approaches. Traditionally, the approach to management of CF pulmonary exacerbations has been through employment of antibiotics. While antibiotics are useful in the short run, multidrug resistant microbes eventually evolve and become a challenge to tackle. In view of this, novel approaches to the management of CF pulmonary disease have been proposed, which involve manipulating patients’ microbial consortia . From a theoretical perspective, such an approach aims to maintain the architecture of the CF airway microbiome and avoids the use of antimicrobials, thereby circumventing the problem of destroying the community structure of a patient's microbiome. Such a novel treatment approach is based on the principles of personalized medicine and aims to tailor treatment according to each patient's individual microbiome . By manipulating and restoring the structure of a patient's airway microbiome, the complex metabolomic profile of the patient's sputum (and other body fluids) can be altered, which may have long‐lasting and pleiotropic consequences .
Novel treatment approaches for the treatment of CF patients hold theoretical promise, but their practical applicability and clinical efficacy remains to be established . A recent pilot study compared the use of a probiotic (
|ABPA||Allergic bronchopulmonary aspergillosis|
|ATS||American Thoracic Society|
|BCC||Burkholderia cepacia complex|
|CFF||Cystic Fibrosis Foundation|
|CFTR||Cystic fibrosis transmembrane conductance regulator|
|CFU||Colony forming units|
|FAFLP||Fluorescent amplified fragment length polymorphism|
|FEF25||Forced expiratory flow at 25% of vital capacity|
|FEV1||Forced expiratory volume in first second|
|FMT||Fecal microbiota transplantation|
|MRSA||Methicillin‐resistant Staphylococcus aureus|
|MSSA||Methicillin‐sensitive Staphylococcus aureus|
|OTU||Operational taxonomic units|
|PCR||Polymerase chain reaction|
|PBP‐2A||Penicillin binding protein‐2A|
|PFGE||Pulsed‐field gel electrophoresis|
|RAPD||Random amplified polymorphic DNA|
|RFLP||Restriction fragment length polymorphism|
|RNI||Reactive nitrogen intermediates|
|ROS||Reactive oxygen species|
|VISA||Vancomycin‐intermediate Staphylococcus aureus|