EDITORIALS Expanding Our Understanding of Respiratory Microbiota in Cystic Fibrosis John J. LiPuma Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan
Therapeutic advances in medicine are driven by clinical observation tied to scientific insight. This paradigm certainly applies to cystic fibrosis (CF), in which therapeutic advances, based on decades of study of disease mechanism fueled by astute descriptive analyses, have seen life expectancy approach 40 years. Strategies such as gene therapy and CFTR modifiers hold the promise of extending both well-being and life expectancy further. Nevertheless, respiratory failure resulting from chronic infection and inflammation of the airways still represents the cause of death for most individuals with CF, and as such, a better understanding of infectionrelated pathology of the CF airways is needed. The obvious starting point in this effort is to gain a better appreciation of the repertoire of microbes that inhabit CF airways, and to then convincingly link their presence to disease progression. Although the list of “typical CF respiratory pathogens” seems to have grown steadily during the last couple decades, it still represents a relatively restricted set of bacterial species cultured from respiratory secretions. In addition to the human pathogens Haemophilus influenzae and Staphylococcus aureus, opportunistic species including Pseudomonas aeruginosa, Burkholderia cepacia complex, Achromobacter xylosoxidans, and Stenotrophomonas maltophilia remain the focus of conventional diagnostic microbiology in CF (1). Work as early as 2002 using DNA sequence-based analyses showed that a host of “unusual” bacterial species could be identified in cultures of respiratory specimens from persons with CF (2).
Studies in the years since have employed strategies in which nucleic acid signatures are used to define the species present in biologic samples without the need for microbial culture. This work has shown that a complex set of microbial species, or microbiota, are typically present in respiratory samples from children and young adults with CF (3–7), particularly during the early and intermediate stages of lung disease. Together with ecological bioinformatic analyses to assist in data interpretation, several studies have shown intriguing associations between airway microbial community structure and activity and clinical outcomes (5–10). The accelerating pace of studies of the human respiratory microbiome allows us to address unresolved questions regarding the use of culture-independent analyses in expanding our understanding of CF airway microbiology. Goddard and colleagues (11) reported that the CF airway microbiota sampled directly from explanted lungs at the time of transplantation were much less diverse than those observed in studies using bronchoalveolar lavage or sputum samples. One interpretation of this observation was that contamination of respiratory samples during transit from the lower airways by bacterial species present in the oropharynx made a marked contribution to the microbiota signatures detected. However, other studies have suggested that the contribution of upper airway microbiota to the bacterial communities detected in bronchoalveolar lavage or sputum samples is marginal (12, 13), and further studies have demonstrated that as patient age and lung disease increase, the diversity of CF airway microbial communities markedly
decreases, resulting in end-stage communities as constrained as those observed by Goddard and colleagues (9, 11, 14). Nevertheless, questions regarding the types of microbes that truly exist within CF lower airways persist. Another unsettled issue pertains to the difficulty of untangling the causal relationships between microbial community structure and activity, airway disease progression, and antimicrobial therapy. Although studies have shown strong associations between CF airway microbiota and clinical parameters such as lung function and antibiotic use (8–10), these features represent a confounded set of variables that challenge definition of causal links. Yet, linking microbiota, either in part or whole, with specific pathophysiological effects is a critical step toward the mechanistic studies that will ultimately advance therapy. The two articles published by Hoffman and colleagues in this month’s issue of the Annals provide further insight into these issues. In the first article (15), the authors provide a case study of a 3-year-old child with CF who had onset of clinical symptoms and radiographic evidence of severe lung disease (persistent cough and opacification and bronchiectasis of the right middle and right lower lobe) from the age of 3 months. When this proved recalcitrant to standard therapy, the right lower lobe was resected; pathologic examination was consistent with severe, focal CF lung disease. The composition of the microbiota in several sections of the surgically excised tissue was assessed by DNA sequencing and found to differ between sections: In the most proximal section, H. influenzae was common, whereas in more distal sections, Propionibacterium acnes and Ralstonia pickettii were both
(Received in original form July 7, 2014; accepted in final form July 10, 2014 ) Correspondence and requests for reprints should be addressed to John J. LiPuma, M.D., 1150 W. Med. Cntr. Dr., 8323 MSRB III, SPC 5646, Ann Arbor, MI 48109. E-mail: [email protected] Ann Am Thorac Soc Vol 11, No 7, pp 1084–1085, Sep 2014 Copyright © 2014 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201407-303ED Internet address: www.atsjournals.org
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EDITORIALS abundant. As typically reported in sputumproductive adults with CF, anaerobic species (e.g., from the genus Veillonella) were also detected, forming part of a complex mix of species. None of these species was recovered in routine culture of tracheal aspirate or oropharyngeal swab, and none of the most abundant species identified in lung tissue were detected in oropharyngeal swab by sequencing. A key element of this work is that because the lung tissue was surgically removed, sectioned, and sampled under sterile conditions, the diverse microbiota detected therein could not have resulted from oropharyngeal contamination. The degree to which the detected species contributed to lung damage or were selected for by damaged lung tissue remains unclear, although it seems hard to argue that the presence of these species did not play a role in contributing to lung disease. Nevertheless, these results provide solid evidence that diverse microbiota can reside in diseased lower airways, that finding certain species (eg, anaerobic species) in BAL or sputum specimens is not merely a result of contamination from the oropharynx, that species recovered in culture or by sequencing from oropharyngeal swab may poorly reflect species in diseased lower
airways, and that severe CF lung disease is not necessarily associated with infection with “traditional CF pathogens.” Whether the species detected in lung by sequencing would also have been detected in deep sequencing of a sputum sample could not be assessed in this nonexpectorating patient. The second article by Hoffman and colleagues (16) compares the airway microbiota in sets of children and adults with CF and non-CF bronchiectasis, as well as in children with protracted bacterial bronchitis. This study again combined sequencing and ecological statistical processing to identify species that were common and abundant (“core”) in each of the study sets. The authors found that although the core microbiota of all three sets of pediatric samples, representing three distinct lung diseases, were highly similar, there was marked divergence in microbial community composition between these conditions by adulthood. Although limited by cross-sectional design, the results suggest that early chronic airway infection, regardless of underlying pathology, involves a suite of common bacterial species. As lung disease progresses, the pathophysiologic features that distinguish
References 1 LiPuma JJ. The changing microbial epidemiology in cystic fibrosis. Clin Microbiol Rev 2010;23:299–323. 2 Coenye T, Goris J, Spilker T, Vandamme P, LiPuma JJ. Characterization of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and description of Inquilinus limosus gen. nov., sp. nov. J Clin Microbiol 2002;40:2062–2069. 3 Rogers GB, Carroll MP, Serisier DJ, Hockey PM, Jones G, Bruce KD. Characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16s ribosomal DNA terminal restriction fragment length polymorphism profiling. J Clin Microbiol 2004;42:5176–5183. 4 Harris JK, De Groote MA, Sagel SD, Zemanick ET, Kapsner R, Penvari C, Kaess H, Deterding RR, Accurso FJ, Pace NR. Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis. Proc Natl Acad Sci USA 2007;104:20529–20533. 5 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. 6 Klepac-Ceraj V, Lemon KP, Martin TR, Allgaier M, Kembel SW, Knapp AA, Lory S, Brodie EL, Lynch SV, Bohannan BJ, et al. Relationship between cystic fibrosis respiratory tract bacterial communities and age, genotype, antibiotics and Pseudomonas aeruginosa. Environ Microbiol 2010;12:1293–1303. 7 Stressmann FA, Rogers GB, van der Gast CJ, Marsh P, Vermeer LS, Carroll MP, Hoffman L, Daniels TW, Patel N, Forbes B, et al. Long-term cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience. Thorax 2012;67:867–873. 8 van der Gast CJ, Walker AW, Stressmann FA, Rogers GB, Scott P, Daniels TW, Carroll MP, Parkhill J, Bruce KD. Partitioning core and
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each condition, as well as the antimicrobial therapy used as “standard” in each disease, appear to select for distinct sets of species, resulting in divergent communities in later stages of disease. Observations such as these, linking specific microbiota to distinct diseases and disease stages, move us closer to defining the causal relationships between microbe and lung disease. Both articles reflect the paradigm of clinical observation and scientific insight. Although there is no immediate resulting therapeutic advance, the information and hypotheses that are generated from both reports are important for this rapidly growing field. There are clear parallels for respiratory microbiota research to the years after the identification of the CFTR gene and subsequent work on CFTR function that have led, in turn, to developments in gene therapy and CFTR modifiers. Articles such as those by Hoffman and colleagues reinforce the importance of microbiota research toward achieving real translational benefits. n Author disclosures are available with the text of this article at www.atsjournals.org.
satellite taxa from within cystic fibrosis lung bacterial communities. ISME J 2011;5:780–791. 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. Carmody LA, Zhao J, Schloss PD, Petrosino JF, Murray S, Young VB, Li JZ, LiPuma JJ. Changes in cystic fibrosis airway microbiota at pulmonary exacerbation. Ann Am Thorac Soc 2013;10:179–187. Goddard AF, Staudinger BJ, Dowd SE, Joshi-Datar A, Wolcott RD, Aitken ML, Fligner CL, Singh PK. Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota. Proc Natl Acad Sci USA 2012;109:13769–13774. Rogers GB, Carroll MP, Serisier DJ, Hockey PM, Jones G, Kehagia V, Connett GJ, Bruce KD. Use of 16S rRNA gene profiling by terminal restriction fragment length polymorphism analysis to compare bacterial communities in sputum and mouthwash samples from patients with cystic fibrosis. J Clin Microbiol 2006;44:2601–2604. Whiteson KL, Bailey B, Bergkessel M, Conrad D, Delhaes L, Felts B, Harris JK, Hunter R, Lim YW, Maughan H, et al. The upper respiratory tract as a microbial source for pulmonary infections in cystic fibrosis: parallels from island biogeography. Am J Respir Crit Care Med 2014;189:1309–1315. VanDevanter DR, LiPuma JJ. Microbial diversity in the cystic fibrosis airways: where is thy sting? Future Microbiol 2012;7:801–803. Brown PS, Pope CE, Marsh RL, Qin X, McNamara S, Gibson R, Burns JL, Deutsch G, Hoffman LR. Directly sampling the lung of a young child with cystic fibrosis reveals diverse microbiota. Ann Am Thorac Soc 2014;11: 1049–1055. van der Gast CJ, Cuthbertson L, Rogers GB, Pope C, Marsh RL, Redding GJ, Bruce KD, Chang AB, Hoffman LR. Three clinically distinct pediatric airway infections share a common core microbiota. Ann Am Thorac Soc 2014;11:1309–1048.
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Therapeutic advances in medicine are driven by clinical observation tied to scientific insight. This paradigm certainly applies to cystic fibrosis (CF), in which therapeutic advances, based on decades of study of disease mechanism fueled by astute descriptive analyses, have seen life expectancy approach 40 years. Strategies such as gene therapy and CFTR modifiers hold the promise of extending both well-being and life expectancy further. Nevertheless, respiratory failure resulting from chronic infection and inflammation of the airways still represents the cause of death for most individuals with CF, and as such, a better understanding of infectionrelated pathology of the CF airways is needed. The obvious starting point in this effort is to gain a better appreciation of the repertoire of microbes that inhabit CF airways, and to then convincingly link their presence to disease progression. Although the list of “typical CF respiratory pathogens” seems to have grown steadily during the last couple decades, it still represents a relatively restricted set of bacterial species cultured from respiratory secretions. In addition to the human pathogens Haemophilus influenzae and Staphylococcus aureus, opportunistic species including Pseudomonas aeruginosa, Burkholderia cepacia complex, Achromobacter xylosoxidans, and Stenotrophomonas maltophilia remain the focus of conventional diagnostic microbiology in CF (1). Work as early as 2002 using DNA sequence-based analyses showed that a host of “unusual” bacterial species could be identified in cultures of respiratory specimens from persons with CF (2).
Studies in the years since have employed strategies in which nucleic acid signatures are used to define the species present in biologic samples without the need for microbial culture. This work has shown that a complex set of microbial species, or microbiota, are typically present in respiratory samples from children and young adults with CF (3–7), particularly during the early and intermediate stages of lung disease. Together with ecological bioinformatic analyses to assist in data interpretation, several studies have shown intriguing associations between airway microbial community structure and activity and clinical outcomes (5–10). The accelerating pace of studies of the human respiratory microbiome allows us to address unresolved questions regarding the use of culture-independent analyses in expanding our understanding of CF airway microbiology. Goddard and colleagues (11) reported that the CF airway microbiota sampled directly from explanted lungs at the time of transplantation were much less diverse than those observed in studies using bronchoalveolar lavage or sputum samples. One interpretation of this observation was that contamination of respiratory samples during transit from the lower airways by bacterial species present in the oropharynx made a marked contribution to the microbiota signatures detected. However, other studies have suggested that the contribution of upper airway microbiota to the bacterial communities detected in bronchoalveolar lavage or sputum samples is marginal (12, 13), and further studies have demonstrated that as patient age and lung disease increase, the diversity of CF airway microbial communities markedly
decreases, resulting in end-stage communities as constrained as those observed by Goddard and colleagues (9, 11, 14). Nevertheless, questions regarding the types of microbes that truly exist within CF lower airways persist. Another unsettled issue pertains to the difficulty of untangling the causal relationships between microbial community structure and activity, airway disease progression, and antimicrobial therapy. Although studies have shown strong associations between CF airway microbiota and clinical parameters such as lung function and antibiotic use (8–10), these features represent a confounded set of variables that challenge definition of causal links. Yet, linking microbiota, either in part or whole, with specific pathophysiological effects is a critical step toward the mechanistic studies that will ultimately advance therapy. The two articles published by Hoffman and colleagues in this month’s issue of the Annals provide further insight into these issues. In the first article (15), the authors provide a case study of a 3-year-old child with CF who had onset of clinical symptoms and radiographic evidence of severe lung disease (persistent cough and opacification and bronchiectasis of the right middle and right lower lobe) from the age of 3 months. When this proved recalcitrant to standard therapy, the right lower lobe was resected; pathologic examination was consistent with severe, focal CF lung disease. The composition of the microbiota in several sections of the surgically excised tissue was assessed by DNA sequencing and found to differ between sections: In the most proximal section, H. influenzae was common, whereas in more distal sections, Propionibacterium acnes and Ralstonia pickettii were both
(Received in original form July 7, 2014; accepted in final form July 10, 2014 ) Correspondence and requests for reprints should be addressed to John J. LiPuma, M.D., 1150 W. Med. Cntr. Dr., 8323 MSRB III, SPC 5646, Ann Arbor, MI 48109. E-mail: [email protected] Ann Am Thorac Soc Vol 11, No 7, pp 1084–1085, Sep 2014 Copyright © 2014 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201407-303ED Internet address: www.atsjournals.org
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AnnalsATS Volume 11 Number 7 | September 2014
EDITORIALS abundant. As typically reported in sputumproductive adults with CF, anaerobic species (e.g., from the genus Veillonella) were also detected, forming part of a complex mix of species. None of these species was recovered in routine culture of tracheal aspirate or oropharyngeal swab, and none of the most abundant species identified in lung tissue were detected in oropharyngeal swab by sequencing. A key element of this work is that because the lung tissue was surgically removed, sectioned, and sampled under sterile conditions, the diverse microbiota detected therein could not have resulted from oropharyngeal contamination. The degree to which the detected species contributed to lung damage or were selected for by damaged lung tissue remains unclear, although it seems hard to argue that the presence of these species did not play a role in contributing to lung disease. Nevertheless, these results provide solid evidence that diverse microbiota can reside in diseased lower airways, that finding certain species (eg, anaerobic species) in BAL or sputum specimens is not merely a result of contamination from the oropharynx, that species recovered in culture or by sequencing from oropharyngeal swab may poorly reflect species in diseased lower
airways, and that severe CF lung disease is not necessarily associated with infection with “traditional CF pathogens.” Whether the species detected in lung by sequencing would also have been detected in deep sequencing of a sputum sample could not be assessed in this nonexpectorating patient. The second article by Hoffman and colleagues (16) compares the airway microbiota in sets of children and adults with CF and non-CF bronchiectasis, as well as in children with protracted bacterial bronchitis. This study again combined sequencing and ecological statistical processing to identify species that were common and abundant (“core”) in each of the study sets. The authors found that although the core microbiota of all three sets of pediatric samples, representing three distinct lung diseases, were highly similar, there was marked divergence in microbial community composition between these conditions by adulthood. Although limited by cross-sectional design, the results suggest that early chronic airway infection, regardless of underlying pathology, involves a suite of common bacterial species. As lung disease progresses, the pathophysiologic features that distinguish
References 1 LiPuma JJ. The changing microbial epidemiology in cystic fibrosis. Clin Microbiol Rev 2010;23:299–323. 2 Coenye T, Goris J, Spilker T, Vandamme P, LiPuma JJ. Characterization of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and description of Inquilinus limosus gen. nov., sp. nov. J Clin Microbiol 2002;40:2062–2069. 3 Rogers GB, Carroll MP, Serisier DJ, Hockey PM, Jones G, Bruce KD. Characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16s ribosomal DNA terminal restriction fragment length polymorphism profiling. J Clin Microbiol 2004;42:5176–5183. 4 Harris JK, De Groote MA, Sagel SD, Zemanick ET, Kapsner R, Penvari C, Kaess H, Deterding RR, Accurso FJ, Pace NR. Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis. Proc Natl Acad Sci USA 2007;104:20529–20533. 5 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. 6 Klepac-Ceraj V, Lemon KP, Martin TR, Allgaier M, Kembel SW, Knapp AA, Lory S, Brodie EL, Lynch SV, Bohannan BJ, et al. Relationship between cystic fibrosis respiratory tract bacterial communities and age, genotype, antibiotics and Pseudomonas aeruginosa. Environ Microbiol 2010;12:1293–1303. 7 Stressmann FA, Rogers GB, van der Gast CJ, Marsh P, Vermeer LS, Carroll MP, Hoffman L, Daniels TW, Patel N, Forbes B, et al. Long-term cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience. Thorax 2012;67:867–873. 8 van der Gast CJ, Walker AW, Stressmann FA, Rogers GB, Scott P, Daniels TW, Carroll MP, Parkhill J, Bruce KD. Partitioning core and
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each condition, as well as the antimicrobial therapy used as “standard” in each disease, appear to select for distinct sets of species, resulting in divergent communities in later stages of disease. Observations such as these, linking specific microbiota to distinct diseases and disease stages, move us closer to defining the causal relationships between microbe and lung disease. Both articles reflect the paradigm of clinical observation and scientific insight. Although there is no immediate resulting therapeutic advance, the information and hypotheses that are generated from both reports are important for this rapidly growing field. There are clear parallels for respiratory microbiota research to the years after the identification of the CFTR gene and subsequent work on CFTR function that have led, in turn, to developments in gene therapy and CFTR modifiers. Articles such as those by Hoffman and colleagues reinforce the importance of microbiota research toward achieving real translational benefits. n Author disclosures are available with the text of this article at www.atsjournals.org.
satellite taxa from within cystic fibrosis lung bacterial communities. ISME J 2011;5:780–791. 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. Carmody LA, Zhao J, Schloss PD, Petrosino JF, Murray S, Young VB, Li JZ, LiPuma JJ. Changes in cystic fibrosis airway microbiota at pulmonary exacerbation. Ann Am Thorac Soc 2013;10:179–187. Goddard AF, Staudinger BJ, Dowd SE, Joshi-Datar A, Wolcott RD, Aitken ML, Fligner CL, Singh PK. Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota. Proc Natl Acad Sci USA 2012;109:13769–13774. Rogers GB, Carroll MP, Serisier DJ, Hockey PM, Jones G, Kehagia V, Connett GJ, Bruce KD. Use of 16S rRNA gene profiling by terminal restriction fragment length polymorphism analysis to compare bacterial communities in sputum and mouthwash samples from patients with cystic fibrosis. J Clin Microbiol 2006;44:2601–2604. Whiteson KL, Bailey B, Bergkessel M, Conrad D, Delhaes L, Felts B, Harris JK, Hunter R, Lim YW, Maughan H, et al. The upper respiratory tract as a microbial source for pulmonary infections in cystic fibrosis: parallels from island biogeography. Am J Respir Crit Care Med 2014;189:1309–1315. VanDevanter DR, LiPuma JJ. Microbial diversity in the cystic fibrosis airways: where is thy sting? Future Microbiol 2012;7:801–803. Brown PS, Pope CE, Marsh RL, Qin X, McNamara S, Gibson R, Burns JL, Deutsch G, Hoffman LR. Directly sampling the lung of a young child with cystic fibrosis reveals diverse microbiota. Ann Am Thorac Soc 2014;11: 1049–1055. van der Gast CJ, Cuthbertson L, Rogers GB, Pope C, Marsh RL, Redding GJ, Bruce KD, Chang AB, Hoffman LR. Three clinically distinct pediatric airway infections share a common core microbiota. Ann Am Thorac Soc 2014;11:1309–1048.
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