STATE OF THE ART The Cystic Fibrosis Lung Microbiome Michael G. Surette1 1
Department of Medicine, Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada
Abstract The chronic colonization of the lower airways by bacterial pathogens is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). Pseudomonas aeruginosa is the most common CF pathogen, followed by Staphylococcus aureus. Improvements in airway clearance and more effective treatment of the conventional CF pathogens has led to the emergence of new airway pathogens such as Stenotrophomonas maltophilia, Mycobacterium abscessus, and Achromobacter. More recently, it has become appreciated that the lower airways in patients with CF are colonized by a more complex polymicrobial community composed primarily of bacteria found in
the upper respiratory tract. This includes obligate anaerobes, most commonly Prevotella. Expanded culturing methods and cultureindependent molecular methods are being used to characterize the composition and dynamics of these polymicrobial communities in patients with CF. The contribution of the CF microbiome to airway disease is actively being investigated and will present new opportunities for disease management in CF. However, there remain many challenges that must be overcome if microbiome profiling is going to inform clinical practice. Keywords: cystic fibrosis; microbiome; respiratory infections; Pseudomonas aeruginosa; polymicrobial infections
(Received in original form June 12, 2013; accepted in final form August 6, 2013 ) Supported by a Canada Research Chair in Interdisciplinary Microbiome Research and research grants from the Canadian Institutes of Health Research and Cystic Fibrosis Canada (M.G.S.). Correspondence and requests for reprints should be addressed to Michael G. Surette, Department of Medicine, Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, 1280 Main Street, HSC 3N 8F, Hamilton, ON, L8S 4K1 Canada. E-mail: [email protected] Ann Am Thorac Soc Vol 11, Supplement 1, pp 61–65, Jan 2014 Copyright © 2014 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201306-159MG Internet address: www.atsjournals.org
Cystic fibrosis (CF) is an autosomal recessive disease arising from mutation in the gene for an ATP-driven chloride ion channel (CF transmembrane conductance regulator, cftr) (1). It is the most common lethal genetic disease in the white population (2). The CFTR protein plays an important role on numerous mucosal surfaces, and CF is a multisystem disease. The life expectancy has increased from early childhood in the 1950s to a median life expectancy of 40 years or greater (in Canada, the median life expectancy is 47.5 yr) (3). The history of disease management in CF is overcoming one manifestation of the disease only to be confronted with another. Today greater than 90% of the morbidity and mortality of CF is due to lung failure associated with chronic airway infections (2, 4, 5). With improvements in management of airway
Surette: CF Microbiome
infections, patients with CF are living longer, and extrapulmonary complications are becoming an increasing challenge (6). These include CF-related diabetes, chronic kidney disease, osteoporosis, depression, and arthropathy (6). A role of the human microbiome has been implicated in most of these diseases (7–9), and the contribution of the gut microbiome in these complications of patients with CF is an area that will need to be addressed. The upper airways, in particular the oral cavity, represent the most diverse microbiome site in the body (10). In addition to resident microbiota, it is also an import site of interaction with the environment. In contrast, the lower airways of healthy individuals are believed to be effectively sterile, and although aerosolized microbes are constantly being delivered into the lungs, they are believed to be
quickly dealt with by host innate immune mechanisms (11). Whether there is a stable or transient lung microbiome in healthy individuals is unclear (11). Mutations in cftr result in an altered ion flux in the airways, reducing the volume of the airway surface fluid and compromising normal airway function (12). The altered airway milieu results in a thick mucus secretion that impairs normal innate immune defense, including impaired mucociliary clearance (2, 12). Consequently, the lower airways become colonized by bacteria. Traditionally, only a few organisms have been associated with chronic airway infections in CF (Table 1), and there is a progression of these from early childhood to adulthood. Haemophilus influenzae is an early colonizer of the CF airways, followed by Staphylococcus aureus. Pseudomonas aeruginosa is the most
S61
STATE OF THE ART Table 1. Conventional and emerging pathogens in cystic fibrosis lung infections Conventional CF Pathogens Pseudomonas aeruginosa Staphylococcus aureus Haemophilus influenzae Burkholderia cepacia complex
Emerging CF Pathogens Stenotrophomonas maltophilia Methicillin-resistant Staphylococcus aureus (MRSA) Mycobacterium abscessus Achromobacter spp Streptococcus milleri/anginosus group Aspergillus fumigatus*
Definition of abbreviation: CF = cystic fibrosis. *Fungal pathogen.
common CF airway pathogen, affecting up to three-fourths of adults, and is the primary CF pathogen (4, 5). As with other aspects of CF disease, improved management of airway infections and airway clearance has resulted in the emergence of new pathogens (Table 1). Many of these are intrinsically resistant to a wide spectrum of antibiotics, whereas others may have been present but overlooked by conventional clinical microscopy methods (4, 13). There is increasing prevalence in methicillinresistant Staphylococcus aureus (MRSA) in the CF population, mirroring its rise in the general population. Although S. aureus is not a new pathogen in CF, MRSA is more virulent and associated with a more rapid decline in lung function than methicillin-sensitive strains (14). Recently, Mycobacterium abscessus has been increasing in the CF population and is difficult to treat (15). It has also recently been shown that patients within one clinic may be colonized with the same strain, suggesting transmission between patients or from a common environmental source (16). It is now widely recognized that superimposed on the conventional view of CF microbiology, the lower airways of patients with CF are colonized by a more complex polymicrobial community (4, 17–20). Although undoubtedly shaped by the evolving landscape of clinical interventions and disease progression, the polymicrobial colonization of the lower airways is not a new development, but only recently has it been considered to be relevant to disease. The compromised lung function in individuals with CF results in an environment that can support bacterial growth. There are few data available on the lower airways of the very young, but it is likely that airway colonization coincides S62
with or maybe even precedes colonization by conventional pathogens. The microbial characterization of the lower airways in patients with CF has been performed primarily using cultureindependent molecular methods. The bacterial gene encoding the small ribosomal subunit 16S rRNA gene is widely used to define the composition of microbial communities. Earlier approaches relied on restriction endonuclease digestion of polymerase chain reaction–amplified 16S gene fragments. This generated terminal restriction length fragment polymorphisms (T-RFLP) that would differ between organisms. A limitation of T-RFLP is that many bacteria can have the same restriction pattern, and it is not by itself definitive for identification. Bruce and colleagues performed extensive characterization of the CF airway sputum by T-RFLP (18, 20), and their findings have largely been borne out by subsequent DNA sequencing–based studies. Alternatively, taxonomy can be inferred from direct sequencing of the amplified 16s rRNA gene fragment using clone libraries and now more commonly using massively paralleled DNA sequencing methods (17, 18). These approaches can provide hundreds to millions of sequences and provide in-depth analysis of community composition. The use of arraybased hybridization (Phylochip) has also been used to study the CF microbiome (18). One issue frequently raised in these studies is the contamination of the sputum during expectoration and the suggestion that the diverse community observed represents contamination rather than true colonization of the lower airways. Several studies have shown that the sputum or bronchoalveolar lavage microbiome is distinct in microbial composition and their relative abundance compared with the upper respiratory tract microbiome (21).
With respect to CF, sputum does seem to be a consistent sampling method for the lower airways. However, it should also be stressed that the lower airways are not uniform with respect to colonization, and a sputum sample is not representative of the whole airway. Significant spatial heterogeneity has been observed when explant and post mortem lungs have been examined. Studies have demonstrated spatial heterogeneity with respect to both bacteria (22) and viruses (22, 23). Spatially targeted sampling of regions of the lungs or methods for noninvasive monitoring of microbial populations would allow more accurate characterization of the spatial organization of the CF microbiome and also determine whether this spatial heterogeneity is stable. There are a growing number of studies of the CF microbiome. To date, most groups use different extraction protocols, regions of the 16S rRNA, sequencing technologies, and bioinformatics analysis. This limits our ability to make direct comparisons between the predicted communities in these various studies. There are two additional caveats of the culture-independent approaches that can bias what DNA is recovered to generate the inferred community. The CF airway contains a significant amount of DNA from dead cells, which can accumulate in the thick mucus. The DNA levels for P. aeruginosa do not always track accurately with viable bacteria as measured by cell counts (24). This limits the use of the current methodologies to follow short-term dynamics of the CF microbiome. Second, DNA extraction protocols vary widely, and some organisms can be underrepresented by some methods (25). This may account for some difference in relative abundances of some organisms between studies. Despite the differences in methodology between the various studies, several general conclusions have come from these numerous studies. There is consistent prediction about the main members of the CF microbiome. The 10 most common genera recovered are listed in Table 2. The abundance of obligate anaerobes in the lower airways, particularly Prevotella species, was an initial surprise, but this is consistent across all studies of the CF microbiome, and these same anaerobes (common in the upper airways) have been found as major constituents of the microbiome of lower airways in other
AnnalsATS Volume 11 Supplement 1 | January 2014
STATE OF THE ART Table 2. The top 10 most common genera recovered from the cystic fibrosis microbiome independent of the conventional pathogens Streptococcus Prevotella Veillonella Rothia Actinomyces Gemella Granulicatella Fusobacterium Neisseria Atopobium
disease states. Several studies have reported that a decrease in microbiome diversity is correlated with age and/or disease severity (26–28). In a small longitudinal study examining sputum samples over 8 years for six patients, it was shown that three patients with stable disease had more or less stable microbiomes as measured by Shannon diversity, but three patients with progressing disease had microbiomes that decreased in diversity over time (27). However, it should be stressed that the absolute diversity itself is not an indicator of disease progression, and these diversity
measurements would not accurately predict patient status in a cross-sectional study. Another observation is that there does not appear to be a significant decrease in bacterial load on antibiotic treatment (27, 29–31). This does contradict expectations, and it is possible that these observations are affected by DNA from nonviable cells and also skewed by the use of relative rather than absolute abundances. Organismspecific quantitative polymerase chain reaction does seem to capture the dynamics in populations expected from quantitative culture (32), suggesting this is a methodological problem. CF is a very heterogeneous disease influenced by the genetic defect, modifier genes, nutrition, treatment, and environmental exposures, as well as primary CF pathogens. It is not unexpected that the microbiomes will be unique to each individual. In a recent study by Fodor and colleagues (30), they demonstrated that samples from patients that had Pseudomonas, Burkholderia, or neither as the primary pathogen fell into three distinct groups. However, when the pathogen was removed from the analysis, there was no separation of samples. This suggests that the underlying community is
Figure 1. Multiple microbial pathways that may drive pulmonary exacerbations. In this simplified view, pulmonary exacerbations are characterized by a rapid drop in FEV1 and resolved by aggressive antibiotic therapy (red line). In the simplest case, the onset correlates with an increase in a pathogen (blue line). This could be a conventional cystic fibrosis (CF) pathogen such as Pseudomonas aeruginosa, an emerging pathogen (e.g., Stenotrophomonas maltophilia), or a pathogen overlooked by conventional clinical microbiology (e.g., Streptococcus constellatus). These would behave as typical single-organism infections. Other organisms may be present, but they may not contribute to acute disease. In the bottom panel, there is no change in the pathogen numerically, but it is stimulated by other organisms to increase its virulence. These would be examples of polymicrobial infections. For a more in depth discussion, see References 42 and 43.
Surette: CF Microbiome
somewhat independent of the primary pathogen. A recent study from Zemanick and colleagues (33) did an analysis of the relationship of inflammatory markers to the CF microbiome profiles for 21 patients. They observed that only Pseudomonas showed significant correlation with lower FEV1, serum C-reactive protein, and neutrophil elastase in sputum. This reinforces the primary role of P. aeruginosa in CF airways disease. The role of organisms other than the primary CF pathogens in disease progression has been suggested even in patients chronically colonized by P. aeruginosa. Using culture-based approaches, it was shown that members of the Streptococcus milleri/anginosus group (SMG) were the dominant microbe presenting at the onset of pulmonary exacerbations in up to 40% of adult patients in one clinic (34). This was not due to patient-to-patient spread within this community (35), and long-term management of the SMG in these patients controlled airway disease (36). It should be stressed that many of the patients in this study were colonized with SMG, but it was only associated with exacerbations when it was the numerically dominant organism in the community (34). That the complexity of CF microbiome is not apparent by conventional clinical microbiology has been equated with the suggestion that most of the CF microbiome is not readily cultured. However, it should be stressed that the culture conditions used are designed to be restrictive for the growth and identification of specific organisms. Several studies have demonstrated that most of the organisms in the lower airways are readily cultured, particularly the diverse anaerobes (37–40). A culture-enriched molecular profiling approach has demonstrated that all but the very low abundant organisms predicted from the direct molecular profiling of the same sputum samples could be cultured (38). Culturing of these organisms allows for assay of biological activities such as assessment of virulence and antibiotic susceptibilities. Reassembling communities in vitro or in animal models allows the measurement of polymicrobial interactions. This approach has been used to demonstrate that some seemingly benign commensal organisms can enhance the S63
STATE OF THE ART pathogenicity of P. aeruginosa (43). These polymicrobial interactions may explain exacerbations in patients where there is no change in the bacterial load of P. aeruginosa during exacerbations. Thus, there are likely multiple microbial drivers of exacerbations (Figure 1), and following the dynamics in individual patients is more likely to reveal the relevant players in specific patients than cross-sectional comparisons. It is still the early days in the study of the CF microbiome. More longitudinal studies as well as more studies addressing viral and fungal microbiomes are required. In addition to taxonomic profiling (“who is there?”), studies that start to address function and changing physiological states
of this community need to be performed. Metagenomic, metatranscriptomic, and metabolomic approaches will be required to start to address the biology of the CF airway microbiome. However, a more accurate understanding of the microbes that drive disease, particularly pulmonary exacerbation, should allow more effective use of existing therapies and can be translated to patients immediately. Identification of pathogens overlooked by conventional CF microbiology is an example of this (36). To make the CF microbiome relevant to clinical practice we need to have accurate quantitative measurements of the community that can be used to guide therapy and assess efficacy of treatment. This would be applicable to
References 1 Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245:1066–1073. 2 O’Sullivan BP, Freedman SD. Cystic fibrosis. Lancet 2009;373: 1891–1904. 3 The Canadian Cystic Fibrosis Registry 2012 annual report. 2012 [accessed June 3, 2013]. Available from: http://www.cysticfibrosis. ca/assets/files/pdf/CFC_AnnualReport2012_EN_FINAL_singles.pdf 4 Lipuma JJ. The changing microbial epidemiology in cystic fibrosis. Clin Microbiol Rev 2010;23:299–323. 5 Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 2002;15:194–222. 6 Quon BS, Aitken ML. Cystic fibrosis: what to expect now in the early adult years. Paediatr Respir Rev 2012;13:206–214. 7 Collins SM, Surette M, Bercik P. The interplay between the intestinal microbiota and the brain. Nat Rev Microbiol 2012;10:735–742. 8 Pflughoeft KJ, Versalovic J. Human microbiome in health and disease. Annu Rev Pathol 2012;7:99–122. 9 Shanahan F. The colonic microbiota in health and disease. Curr Opin Gastroenterol 2013;29:49–54. 10 Stearns JC, Lynch MD, Senadheera DB, Tenenbaum HC, Goldberg MB, Cvitkovitch DG, Croitoru K, Moreno-Hagelsieb G, Neufeld JD. Bacterial biogeography of the human digestive tract. Sci Rep 2011; 1:170. 11 Beck JM, Young VB, Huffnagle GB. The microbiome of the lung. Transl Res 2012;160:258–266. 12 Boucher RC. Airway surface dehydration in cystic fibrosis: pathogenesis and therapy. Annu Rev Med 2007;58:157–170. 13 Sibley CD, Parkins MD, Rabin HR, Duan K, Norgaard JC, Surette MG. A polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients. Proc Natl Acad Sci USA 2008;105:15070–15075. 14 Dasenbrook EC. Update on methicillin-resistant staphylococcus aureus in cystic fibrosis. Curr Opin Pulm Med 2011;17:437–441. 15 Hill UG, Floto RA, Haworth CS. Non-tuberculous mycobacteria in cystic fibrosis. J R Soc Med 2012;105:S14–S18. 16 Bryant JM, Grogono DM, Greaves D, Foweraker J, Roddick I, Inns T, Reacher M, Haworth CS, Curran MD, Harris SR, et al. Wholegenome sequencing to identify transmission of mycobacterium abscessus between patients with cystic fibrosis: A retrospective cohort study. Lancet 2013;381:1551–1560. 17 LiPuma J. The new microbiology of cystic fibrosis: it takes a community. Thorax 2012;67:851–852.
S64
all airway disease. With the advent of new therapies that rescue the CFTR function, such as ivacaftor (41), we should not become complacent about CF microbiology. These are transformative therapies, but for most patients who will receive these therapies, there will be some pre-existing airway damage and susceptibility to infections. Comprehensive monitoring of the microbiomes should become a routine part of standard care for patients with CF. n Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgment: The author thanks Dr. Harvey Rabin, Dr. Michael Parkins, and the Surette lab for helpful discussions.
18 Lynch SV, Bruce KD. The cystic fibrosis airway microbiome. Cold Spring Harb Perspect Med 2013;3:a009738. 19 Sibley CD, Rabin H, Surette MG. Cystic fibrosis: a polymicrobial infectious disease. Future Microbiol 2006;1:53–61. 20 Rogers GB, Stressmann FA, Walker AW, Carroll MP, Bruce KD. Lung infections in cystic fibrosis: deriving clinical insight from microbial complexity. Expert Rev Mol Diagn 2010;10:187–196. 21 Rogers GB, Skelton S, Serisier DJ, van der Gast CJ, Bruce KD. Determining cystic fibrosis-affected lung microbiology: comparison of spontaneous and serially induced sputum samples by use of terminal restriction fragment length polymorphism profiling. J Clin Microbiol 2010;48:78–86. 22 Willner D, Haynes MR, Furlan M, Schmieder R, Lim YW, Rainey PB, Rohwer F, Conrad D. Spatial distribution of microbial communities in the cystic fibrosis lung. ISME J 2012;6:471–474. 23 Willner D, Haynes MR, Furlan M, Hanson N, Kirby B, Lim YW, Rainey PB, Schmieder R, Youle M, Conrad D, et al. Case studies of the spatial heterogeneity of DNA viruses in the cystic fibrosis lung. Am J Respir Cell Mol Biol 2012;46:127–131. 24 Rogers GB, Marsh P, Stressmann AF, Allen CE, Daniels TV, Carroll MP, Bruce KD. The exclusion of dead bacterial cells is essential for accurate molecular analysis of clinical samples. Clin Microbiol Infect 2010;16:1656–1658. 25 Zhao J, Carmody LA, Kalikin LM, Li J, Petrosino JF, Schloss PD, Young VB, LiPuma JJ. Impact of enhanced staphylococcus DNA extraction on microbial community measures in cystic fibrosis sputum. PLoS ONE 2012;7:e33127. 26 Cox MJ, Allgaier M, Taylor B, Baek MS, Huang YJ, Daly RA, Karaoz U, Andersen GL, Brown R, Fujimura KE, et al. Airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients. PLoS ONE 2010;5:e11044. 27 Zhao J, Schloss PD, Kalikin LM, Carmody LA, Foster BK, Petrosino JF, Cavalcoli JD, VanDevanter DR, Murray S, Li JZ, et al. Decade-long bacterial community dynamics in cystic fibrosis airways. Proc Natl Acad Sci USA 2012;109:5809–5814. 28 Filkins LM, Hampton TH, Gifford AH, Gross MJ, Hogan DA, Sogin ML, Morrison HG, Paster BJ, O’Toole GA. Prevalence of streptococci and increased polymicrobial diversity associated with cystic fibrosis patient stability. J Bacteriol 2012;194:4709–4717. 29 Daniels TW, Rogers GB, Stressmann FA, van der Gast CJ, Bruce KD, Jones GR, Connett GJ, Legg JP, Carroll MP. Impact of antibiotic treatment for pulmonary exacerbations on bacterial diversity in cystic fibrosis. J Cyst Fibros 2013;12:22–28. 30 Fodor AA, Klem ER, Gilpin DF, Elborn JS, Boucher RC, Tunney MM, Wolfgang MC. The adult cystic fibrosis airway microbiota is stable over time and infection type, and highly
AnnalsATS Volume 11 Supplement 1 | January 2014
STATE OF THE ART
31
32
33
34
35
36
resilient to antibiotic treatment of exacerbations. PLoS ONE 2012;7: e45001. Stressmann FA, Rogers GB, van der Gast CJ, Marsh P, Vermeer LS, Carroll MP, Hoffman L, Daniels TW, Patel N, Forbes B, et al. Longterm cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience. Thorax 2012;67:867–873. Zemanick ET, Wagner BD, Sagel SD, Stevens MJ, Accurso FJ, Harris JK. Reliability of quantitative real-time PCR for bacterial detection in cystic fibrosis airway specimens. PLoS ONE 2010;5:e15101. Zemanick ET, Harris JK, Wagner BD, Robertson CE, Sagel SD, Stevens MJ, Accurso FJ, Laguna TA. Inflammation and airway microbiota during cystic fibrosis pulmonary exacerbations. PLoS ONE 2013;8:e62917. Sibley CD, Grinwis ME, Field TR, Parkins MD, Norgaard JC, Gregson DB, Rabin HR, Surette MG. McKay agar enables routine quantification of the ‘Streptococcus milleri’ group in cystic fibrosis patients. J Med Microbiol 2010;59:534–540. Sibley CD, Sibley KA, Leong TA, Grinwis ME, Parkins MD, Rabin HR, Surette MG. The Streptococcus milleri population of a cystic fibrosis clinic reveals patient specificity and intraspecies diversity. J Clin Microbiol 2010;48:2592–2594. Sibley CD, Parkins MD, Rabin HR, Surette MG. The relevance of the polymicrobial nature of airway infection in the acute and chronic management of patients with cystic fibrosis. Curr Opin Investig Drugs 2009;10:787–794.
Surette: CF Microbiome
37 Field TR, Sibley CD, Parkins MD, Rabin HR, Surette MG. The genus Prevotella in cystic fibrosis airways. Anaerobe 2010;16:337–344. 38 Sibley CD, Grinwis ME, Field TR, Eshaghurshan CS, Faria MM, Dowd SE, Parkins MD, Rabin HR, Surette MG. Culture enriched molecular profiling of the cystic fibrosis airway microbiome. PLoS ONE 2011;6: e22702. 39 Tunney MM, Field TR, Moriarty TF, Patrick S, Doering G, Muhlebach MS, Wolfgang MC, Boucher R, Gilpin DF, McDowell A, et al. Detection of anaerobic bacteria in high numbers in sputum from patients with cystic fibrosis. Am J Respir Crit Care Med 2008;177: 995–1001. 40 Tunney MM, Klem ER, Fodor AA, Gilpin DF, Moriarty TF, McGrath SJ, Muhlebach MS, Boucher RC, Cardwell C, Doering G, et al. Use of culture and molecular analysis to determine the effect of antibiotic treatment on microbial community diversity and abundance during exacerbation in patients with cystic fibrosis. Thorax 2011;66: 579–584. 41 Flume PA, Liou TG, Borowitz DS, Li H, Yen K, Ordonez CL, Geller DE. Ivacaftor in subjects with cystic fibrosis who are homozygous for the f508del-cftr mutation. Chest 2012;142:718–724. 42 Rabin HR, Surette MG. The cystic fibrosis airway microbiome. Curr Opin Pulm Med 2012;18:622–627. 43 Sibley CD, Surette MG. The polymicrobial nature of airway infections in cystic fibrosis: Cangene gold medal lecture. Can J Microbiol 2011; 57:69–77.
S65
Department of Medicine, Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada
Abstract The chronic colonization of the lower airways by bacterial pathogens is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). Pseudomonas aeruginosa is the most common CF pathogen, followed by Staphylococcus aureus. Improvements in airway clearance and more effective treatment of the conventional CF pathogens has led to the emergence of new airway pathogens such as Stenotrophomonas maltophilia, Mycobacterium abscessus, and Achromobacter. More recently, it has become appreciated that the lower airways in patients with CF are colonized by a more complex polymicrobial community composed primarily of bacteria found in
the upper respiratory tract. This includes obligate anaerobes, most commonly Prevotella. Expanded culturing methods and cultureindependent molecular methods are being used to characterize the composition and dynamics of these polymicrobial communities in patients with CF. The contribution of the CF microbiome to airway disease is actively being investigated and will present new opportunities for disease management in CF. However, there remain many challenges that must be overcome if microbiome profiling is going to inform clinical practice. Keywords: cystic fibrosis; microbiome; respiratory infections; Pseudomonas aeruginosa; polymicrobial infections
(Received in original form June 12, 2013; accepted in final form August 6, 2013 ) Supported by a Canada Research Chair in Interdisciplinary Microbiome Research and research grants from the Canadian Institutes of Health Research and Cystic Fibrosis Canada (M.G.S.). Correspondence and requests for reprints should be addressed to Michael G. Surette, Department of Medicine, Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, 1280 Main Street, HSC 3N 8F, Hamilton, ON, L8S 4K1 Canada. E-mail: [email protected] Ann Am Thorac Soc Vol 11, Supplement 1, pp 61–65, Jan 2014 Copyright © 2014 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201306-159MG Internet address: www.atsjournals.org
Cystic fibrosis (CF) is an autosomal recessive disease arising from mutation in the gene for an ATP-driven chloride ion channel (CF transmembrane conductance regulator, cftr) (1). It is the most common lethal genetic disease in the white population (2). The CFTR protein plays an important role on numerous mucosal surfaces, and CF is a multisystem disease. The life expectancy has increased from early childhood in the 1950s to a median life expectancy of 40 years or greater (in Canada, the median life expectancy is 47.5 yr) (3). The history of disease management in CF is overcoming one manifestation of the disease only to be confronted with another. Today greater than 90% of the morbidity and mortality of CF is due to lung failure associated with chronic airway infections (2, 4, 5). With improvements in management of airway
Surette: CF Microbiome
infections, patients with CF are living longer, and extrapulmonary complications are becoming an increasing challenge (6). These include CF-related diabetes, chronic kidney disease, osteoporosis, depression, and arthropathy (6). A role of the human microbiome has been implicated in most of these diseases (7–9), and the contribution of the gut microbiome in these complications of patients with CF is an area that will need to be addressed. The upper airways, in particular the oral cavity, represent the most diverse microbiome site in the body (10). In addition to resident microbiota, it is also an import site of interaction with the environment. In contrast, the lower airways of healthy individuals are believed to be effectively sterile, and although aerosolized microbes are constantly being delivered into the lungs, they are believed to be
quickly dealt with by host innate immune mechanisms (11). Whether there is a stable or transient lung microbiome in healthy individuals is unclear (11). Mutations in cftr result in an altered ion flux in the airways, reducing the volume of the airway surface fluid and compromising normal airway function (12). The altered airway milieu results in a thick mucus secretion that impairs normal innate immune defense, including impaired mucociliary clearance (2, 12). Consequently, the lower airways become colonized by bacteria. Traditionally, only a few organisms have been associated with chronic airway infections in CF (Table 1), and there is a progression of these from early childhood to adulthood. Haemophilus influenzae is an early colonizer of the CF airways, followed by Staphylococcus aureus. Pseudomonas aeruginosa is the most
S61
STATE OF THE ART Table 1. Conventional and emerging pathogens in cystic fibrosis lung infections Conventional CF Pathogens Pseudomonas aeruginosa Staphylococcus aureus Haemophilus influenzae Burkholderia cepacia complex
Emerging CF Pathogens Stenotrophomonas maltophilia Methicillin-resistant Staphylococcus aureus (MRSA) Mycobacterium abscessus Achromobacter spp Streptococcus milleri/anginosus group Aspergillus fumigatus*
Definition of abbreviation: CF = cystic fibrosis. *Fungal pathogen.
common CF airway pathogen, affecting up to three-fourths of adults, and is the primary CF pathogen (4, 5). As with other aspects of CF disease, improved management of airway infections and airway clearance has resulted in the emergence of new pathogens (Table 1). Many of these are intrinsically resistant to a wide spectrum of antibiotics, whereas others may have been present but overlooked by conventional clinical microscopy methods (4, 13). There is increasing prevalence in methicillinresistant Staphylococcus aureus (MRSA) in the CF population, mirroring its rise in the general population. Although S. aureus is not a new pathogen in CF, MRSA is more virulent and associated with a more rapid decline in lung function than methicillin-sensitive strains (14). Recently, Mycobacterium abscessus has been increasing in the CF population and is difficult to treat (15). It has also recently been shown that patients within one clinic may be colonized with the same strain, suggesting transmission between patients or from a common environmental source (16). It is now widely recognized that superimposed on the conventional view of CF microbiology, the lower airways of patients with CF are colonized by a more complex polymicrobial community (4, 17–20). Although undoubtedly shaped by the evolving landscape of clinical interventions and disease progression, the polymicrobial colonization of the lower airways is not a new development, but only recently has it been considered to be relevant to disease. The compromised lung function in individuals with CF results in an environment that can support bacterial growth. There are few data available on the lower airways of the very young, but it is likely that airway colonization coincides S62
with or maybe even precedes colonization by conventional pathogens. The microbial characterization of the lower airways in patients with CF has been performed primarily using cultureindependent molecular methods. The bacterial gene encoding the small ribosomal subunit 16S rRNA gene is widely used to define the composition of microbial communities. Earlier approaches relied on restriction endonuclease digestion of polymerase chain reaction–amplified 16S gene fragments. This generated terminal restriction length fragment polymorphisms (T-RFLP) that would differ between organisms. A limitation of T-RFLP is that many bacteria can have the same restriction pattern, and it is not by itself definitive for identification. Bruce and colleagues performed extensive characterization of the CF airway sputum by T-RFLP (18, 20), and their findings have largely been borne out by subsequent DNA sequencing–based studies. Alternatively, taxonomy can be inferred from direct sequencing of the amplified 16s rRNA gene fragment using clone libraries and now more commonly using massively paralleled DNA sequencing methods (17, 18). These approaches can provide hundreds to millions of sequences and provide in-depth analysis of community composition. The use of arraybased hybridization (Phylochip) has also been used to study the CF microbiome (18). One issue frequently raised in these studies is the contamination of the sputum during expectoration and the suggestion that the diverse community observed represents contamination rather than true colonization of the lower airways. Several studies have shown that the sputum or bronchoalveolar lavage microbiome is distinct in microbial composition and their relative abundance compared with the upper respiratory tract microbiome (21).
With respect to CF, sputum does seem to be a consistent sampling method for the lower airways. However, it should also be stressed that the lower airways are not uniform with respect to colonization, and a sputum sample is not representative of the whole airway. Significant spatial heterogeneity has been observed when explant and post mortem lungs have been examined. Studies have demonstrated spatial heterogeneity with respect to both bacteria (22) and viruses (22, 23). Spatially targeted sampling of regions of the lungs or methods for noninvasive monitoring of microbial populations would allow more accurate characterization of the spatial organization of the CF microbiome and also determine whether this spatial heterogeneity is stable. There are a growing number of studies of the CF microbiome. To date, most groups use different extraction protocols, regions of the 16S rRNA, sequencing technologies, and bioinformatics analysis. This limits our ability to make direct comparisons between the predicted communities in these various studies. There are two additional caveats of the culture-independent approaches that can bias what DNA is recovered to generate the inferred community. The CF airway contains a significant amount of DNA from dead cells, which can accumulate in the thick mucus. The DNA levels for P. aeruginosa do not always track accurately with viable bacteria as measured by cell counts (24). This limits the use of the current methodologies to follow short-term dynamics of the CF microbiome. Second, DNA extraction protocols vary widely, and some organisms can be underrepresented by some methods (25). This may account for some difference in relative abundances of some organisms between studies. Despite the differences in methodology between the various studies, several general conclusions have come from these numerous studies. There is consistent prediction about the main members of the CF microbiome. The 10 most common genera recovered are listed in Table 2. The abundance of obligate anaerobes in the lower airways, particularly Prevotella species, was an initial surprise, but this is consistent across all studies of the CF microbiome, and these same anaerobes (common in the upper airways) have been found as major constituents of the microbiome of lower airways in other
AnnalsATS Volume 11 Supplement 1 | January 2014
STATE OF THE ART Table 2. The top 10 most common genera recovered from the cystic fibrosis microbiome independent of the conventional pathogens Streptococcus Prevotella Veillonella Rothia Actinomyces Gemella Granulicatella Fusobacterium Neisseria Atopobium
disease states. Several studies have reported that a decrease in microbiome diversity is correlated with age and/or disease severity (26–28). In a small longitudinal study examining sputum samples over 8 years for six patients, it was shown that three patients with stable disease had more or less stable microbiomes as measured by Shannon diversity, but three patients with progressing disease had microbiomes that decreased in diversity over time (27). However, it should be stressed that the absolute diversity itself is not an indicator of disease progression, and these diversity
measurements would not accurately predict patient status in a cross-sectional study. Another observation is that there does not appear to be a significant decrease in bacterial load on antibiotic treatment (27, 29–31). This does contradict expectations, and it is possible that these observations are affected by DNA from nonviable cells and also skewed by the use of relative rather than absolute abundances. Organismspecific quantitative polymerase chain reaction does seem to capture the dynamics in populations expected from quantitative culture (32), suggesting this is a methodological problem. CF is a very heterogeneous disease influenced by the genetic defect, modifier genes, nutrition, treatment, and environmental exposures, as well as primary CF pathogens. It is not unexpected that the microbiomes will be unique to each individual. In a recent study by Fodor and colleagues (30), they demonstrated that samples from patients that had Pseudomonas, Burkholderia, or neither as the primary pathogen fell into three distinct groups. However, when the pathogen was removed from the analysis, there was no separation of samples. This suggests that the underlying community is
Figure 1. Multiple microbial pathways that may drive pulmonary exacerbations. In this simplified view, pulmonary exacerbations are characterized by a rapid drop in FEV1 and resolved by aggressive antibiotic therapy (red line). In the simplest case, the onset correlates with an increase in a pathogen (blue line). This could be a conventional cystic fibrosis (CF) pathogen such as Pseudomonas aeruginosa, an emerging pathogen (e.g., Stenotrophomonas maltophilia), or a pathogen overlooked by conventional clinical microbiology (e.g., Streptococcus constellatus). These would behave as typical single-organism infections. Other organisms may be present, but they may not contribute to acute disease. In the bottom panel, there is no change in the pathogen numerically, but it is stimulated by other organisms to increase its virulence. These would be examples of polymicrobial infections. For a more in depth discussion, see References 42 and 43.
Surette: CF Microbiome
somewhat independent of the primary pathogen. A recent study from Zemanick and colleagues (33) did an analysis of the relationship of inflammatory markers to the CF microbiome profiles for 21 patients. They observed that only Pseudomonas showed significant correlation with lower FEV1, serum C-reactive protein, and neutrophil elastase in sputum. This reinforces the primary role of P. aeruginosa in CF airways disease. The role of organisms other than the primary CF pathogens in disease progression has been suggested even in patients chronically colonized by P. aeruginosa. Using culture-based approaches, it was shown that members of the Streptococcus milleri/anginosus group (SMG) were the dominant microbe presenting at the onset of pulmonary exacerbations in up to 40% of adult patients in one clinic (34). This was not due to patient-to-patient spread within this community (35), and long-term management of the SMG in these patients controlled airway disease (36). It should be stressed that many of the patients in this study were colonized with SMG, but it was only associated with exacerbations when it was the numerically dominant organism in the community (34). That the complexity of CF microbiome is not apparent by conventional clinical microbiology has been equated with the suggestion that most of the CF microbiome is not readily cultured. However, it should be stressed that the culture conditions used are designed to be restrictive for the growth and identification of specific organisms. Several studies have demonstrated that most of the organisms in the lower airways are readily cultured, particularly the diverse anaerobes (37–40). A culture-enriched molecular profiling approach has demonstrated that all but the very low abundant organisms predicted from the direct molecular profiling of the same sputum samples could be cultured (38). Culturing of these organisms allows for assay of biological activities such as assessment of virulence and antibiotic susceptibilities. Reassembling communities in vitro or in animal models allows the measurement of polymicrobial interactions. This approach has been used to demonstrate that some seemingly benign commensal organisms can enhance the S63
STATE OF THE ART pathogenicity of P. aeruginosa (43). These polymicrobial interactions may explain exacerbations in patients where there is no change in the bacterial load of P. aeruginosa during exacerbations. Thus, there are likely multiple microbial drivers of exacerbations (Figure 1), and following the dynamics in individual patients is more likely to reveal the relevant players in specific patients than cross-sectional comparisons. It is still the early days in the study of the CF microbiome. More longitudinal studies as well as more studies addressing viral and fungal microbiomes are required. In addition to taxonomic profiling (“who is there?”), studies that start to address function and changing physiological states
of this community need to be performed. Metagenomic, metatranscriptomic, and metabolomic approaches will be required to start to address the biology of the CF airway microbiome. However, a more accurate understanding of the microbes that drive disease, particularly pulmonary exacerbation, should allow more effective use of existing therapies and can be translated to patients immediately. Identification of pathogens overlooked by conventional CF microbiology is an example of this (36). To make the CF microbiome relevant to clinical practice we need to have accurate quantitative measurements of the community that can be used to guide therapy and assess efficacy of treatment. This would be applicable to
References 1 Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245:1066–1073. 2 O’Sullivan BP, Freedman SD. Cystic fibrosis. Lancet 2009;373: 1891–1904. 3 The Canadian Cystic Fibrosis Registry 2012 annual report. 2012 [accessed June 3, 2013]. Available from: http://www.cysticfibrosis. ca/assets/files/pdf/CFC_AnnualReport2012_EN_FINAL_singles.pdf 4 Lipuma JJ. The changing microbial epidemiology in cystic fibrosis. Clin Microbiol Rev 2010;23:299–323. 5 Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 2002;15:194–222. 6 Quon BS, Aitken ML. Cystic fibrosis: what to expect now in the early adult years. Paediatr Respir Rev 2012;13:206–214. 7 Collins SM, Surette M, Bercik P. The interplay between the intestinal microbiota and the brain. Nat Rev Microbiol 2012;10:735–742. 8 Pflughoeft KJ, Versalovic J. Human microbiome in health and disease. Annu Rev Pathol 2012;7:99–122. 9 Shanahan F. The colonic microbiota in health and disease. Curr Opin Gastroenterol 2013;29:49–54. 10 Stearns JC, Lynch MD, Senadheera DB, Tenenbaum HC, Goldberg MB, Cvitkovitch DG, Croitoru K, Moreno-Hagelsieb G, Neufeld JD. Bacterial biogeography of the human digestive tract. Sci Rep 2011; 1:170. 11 Beck JM, Young VB, Huffnagle GB. The microbiome of the lung. Transl Res 2012;160:258–266. 12 Boucher RC. Airway surface dehydration in cystic fibrosis: pathogenesis and therapy. Annu Rev Med 2007;58:157–170. 13 Sibley CD, Parkins MD, Rabin HR, Duan K, Norgaard JC, Surette MG. A polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients. Proc Natl Acad Sci USA 2008;105:15070–15075. 14 Dasenbrook EC. Update on methicillin-resistant staphylococcus aureus in cystic fibrosis. Curr Opin Pulm Med 2011;17:437–441. 15 Hill UG, Floto RA, Haworth CS. Non-tuberculous mycobacteria in cystic fibrosis. J R Soc Med 2012;105:S14–S18. 16 Bryant JM, Grogono DM, Greaves D, Foweraker J, Roddick I, Inns T, Reacher M, Haworth CS, Curran MD, Harris SR, et al. Wholegenome sequencing to identify transmission of mycobacterium abscessus between patients with cystic fibrosis: A retrospective cohort study. Lancet 2013;381:1551–1560. 17 LiPuma J. The new microbiology of cystic fibrosis: it takes a community. Thorax 2012;67:851–852.
S64
all airway disease. With the advent of new therapies that rescue the CFTR function, such as ivacaftor (41), we should not become complacent about CF microbiology. These are transformative therapies, but for most patients who will receive these therapies, there will be some pre-existing airway damage and susceptibility to infections. Comprehensive monitoring of the microbiomes should become a routine part of standard care for patients with CF. n Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgment: The author thanks Dr. Harvey Rabin, Dr. Michael Parkins, and the Surette lab for helpful discussions.
18 Lynch SV, Bruce KD. The cystic fibrosis airway microbiome. Cold Spring Harb Perspect Med 2013;3:a009738. 19 Sibley CD, Rabin H, Surette MG. Cystic fibrosis: a polymicrobial infectious disease. Future Microbiol 2006;1:53–61. 20 Rogers GB, Stressmann FA, Walker AW, Carroll MP, Bruce KD. Lung infections in cystic fibrosis: deriving clinical insight from microbial complexity. Expert Rev Mol Diagn 2010;10:187–196. 21 Rogers GB, Skelton S, Serisier DJ, van der Gast CJ, Bruce KD. Determining cystic fibrosis-affected lung microbiology: comparison of spontaneous and serially induced sputum samples by use of terminal restriction fragment length polymorphism profiling. J Clin Microbiol 2010;48:78–86. 22 Willner D, Haynes MR, Furlan M, Schmieder R, Lim YW, Rainey PB, Rohwer F, Conrad D. Spatial distribution of microbial communities in the cystic fibrosis lung. ISME J 2012;6:471–474. 23 Willner D, Haynes MR, Furlan M, Hanson N, Kirby B, Lim YW, Rainey PB, Schmieder R, Youle M, Conrad D, et al. Case studies of the spatial heterogeneity of DNA viruses in the cystic fibrosis lung. Am J Respir Cell Mol Biol 2012;46:127–131. 24 Rogers GB, Marsh P, Stressmann AF, Allen CE, Daniels TV, Carroll MP, Bruce KD. The exclusion of dead bacterial cells is essential for accurate molecular analysis of clinical samples. Clin Microbiol Infect 2010;16:1656–1658. 25 Zhao J, Carmody LA, Kalikin LM, Li J, Petrosino JF, Schloss PD, Young VB, LiPuma JJ. Impact of enhanced staphylococcus DNA extraction on microbial community measures in cystic fibrosis sputum. PLoS ONE 2012;7:e33127. 26 Cox MJ, Allgaier M, Taylor B, Baek MS, Huang YJ, Daly RA, Karaoz U, Andersen GL, Brown R, Fujimura KE, et al. Airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients. PLoS ONE 2010;5:e11044. 27 Zhao J, Schloss PD, Kalikin LM, Carmody LA, Foster BK, Petrosino JF, Cavalcoli JD, VanDevanter DR, Murray S, Li JZ, et al. Decade-long bacterial community dynamics in cystic fibrosis airways. Proc Natl Acad Sci USA 2012;109:5809–5814. 28 Filkins LM, Hampton TH, Gifford AH, Gross MJ, Hogan DA, Sogin ML, Morrison HG, Paster BJ, O’Toole GA. Prevalence of streptococci and increased polymicrobial diversity associated with cystic fibrosis patient stability. J Bacteriol 2012;194:4709–4717. 29 Daniels TW, Rogers GB, Stressmann FA, van der Gast CJ, Bruce KD, Jones GR, Connett GJ, Legg JP, Carroll MP. Impact of antibiotic treatment for pulmonary exacerbations on bacterial diversity in cystic fibrosis. J Cyst Fibros 2013;12:22–28. 30 Fodor AA, Klem ER, Gilpin DF, Elborn JS, Boucher RC, Tunney MM, Wolfgang MC. The adult cystic fibrosis airway microbiota is stable over time and infection type, and highly
AnnalsATS Volume 11 Supplement 1 | January 2014
STATE OF THE ART
31
32
33
34
35
36
resilient to antibiotic treatment of exacerbations. PLoS ONE 2012;7: e45001. Stressmann FA, Rogers GB, van der Gast CJ, Marsh P, Vermeer LS, Carroll MP, Hoffman L, Daniels TW, Patel N, Forbes B, et al. Longterm cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience. Thorax 2012;67:867–873. Zemanick ET, Wagner BD, Sagel SD, Stevens MJ, Accurso FJ, Harris JK. Reliability of quantitative real-time PCR for bacterial detection in cystic fibrosis airway specimens. PLoS ONE 2010;5:e15101. Zemanick ET, Harris JK, Wagner BD, Robertson CE, Sagel SD, Stevens MJ, Accurso FJ, Laguna TA. Inflammation and airway microbiota during cystic fibrosis pulmonary exacerbations. PLoS ONE 2013;8:e62917. Sibley CD, Grinwis ME, Field TR, Parkins MD, Norgaard JC, Gregson DB, Rabin HR, Surette MG. McKay agar enables routine quantification of the ‘Streptococcus milleri’ group in cystic fibrosis patients. J Med Microbiol 2010;59:534–540. Sibley CD, Sibley KA, Leong TA, Grinwis ME, Parkins MD, Rabin HR, Surette MG. The Streptococcus milleri population of a cystic fibrosis clinic reveals patient specificity and intraspecies diversity. J Clin Microbiol 2010;48:2592–2594. Sibley CD, Parkins MD, Rabin HR, Surette MG. The relevance of the polymicrobial nature of airway infection in the acute and chronic management of patients with cystic fibrosis. Curr Opin Investig Drugs 2009;10:787–794.
Surette: CF Microbiome
37 Field TR, Sibley CD, Parkins MD, Rabin HR, Surette MG. The genus Prevotella in cystic fibrosis airways. Anaerobe 2010;16:337–344. 38 Sibley CD, Grinwis ME, Field TR, Eshaghurshan CS, Faria MM, Dowd SE, Parkins MD, Rabin HR, Surette MG. Culture enriched molecular profiling of the cystic fibrosis airway microbiome. PLoS ONE 2011;6: e22702. 39 Tunney MM, Field TR, Moriarty TF, Patrick S, Doering G, Muhlebach MS, Wolfgang MC, Boucher R, Gilpin DF, McDowell A, et al. Detection of anaerobic bacteria in high numbers in sputum from patients with cystic fibrosis. Am J Respir Crit Care Med 2008;177: 995–1001. 40 Tunney MM, Klem ER, Fodor AA, Gilpin DF, Moriarty TF, McGrath SJ, Muhlebach MS, Boucher RC, Cardwell C, Doering G, et al. Use of culture and molecular analysis to determine the effect of antibiotic treatment on microbial community diversity and abundance during exacerbation in patients with cystic fibrosis. Thorax 2011;66: 579–584. 41 Flume PA, Liou TG, Borowitz DS, Li H, Yen K, Ordonez CL, Geller DE. Ivacaftor in subjects with cystic fibrosis who are homozygous for the f508del-cftr mutation. Chest 2012;142:718–724. 42 Rabin HR, Surette MG. The cystic fibrosis airway microbiome. Curr Opin Pulm Med 2012;18:622–627. 43 Sibley CD, Surette MG. The polymicrobial nature of airway infections in cystic fibrosis: Cangene gold medal lecture. Can J Microbiol 2011; 57:69–77.
S65
