Abstract
The lung in cystic fibrosis (CF) is home to numerous pathogens that shorten the lives of patients. The aim of the present study was to assess changes in the lung bacteriome following antibiotic therapy targeting Pseudomonas aeruginosa in children with CF. The study included nine children (9–18 years) with CF who were treated for their chronic or intermittent positivity for Pseudomonas aeruginosa. The bacteriomes were determined in 16 pairs of sputa collected at the beginning and at the end of a course of intravenous antibiotic therapy via deep sequencing of the variable region 4 of the 16S rRNA gene, and the total bacterial load and selected specific pathogens were assessed using quantitative real-time PCR. The effect of antipseudomonal antibiotics was observable as a profound decrease in the total 16S rDNA load (p = 0.001) as well as in a broad range of individual taxa including Staphylococcus aureus (p = 0.03) and several members of the Streptococcus mitis group (S. oralis, S. mitis, and S. infantis) (p = 0.003). Improvements in forced expiratory volume (FEV1) were associated with an increase in Granulicatella sp. (p = 0.004), whereas a negative association was noted between the total bacterial load and white blood cell count (p = 0.007). In conclusion, the data show how microbial communities differ in reaction to antipseudomonal treatment, suggesting that certain rare species may be associated with clinical parameters. Our work also demonstrates the utility of absolute quantification of bacterial load in addition to the 16S rDNA profiling.
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References
Bag S, Saha B, Mehta O, Anbumani D, Kumar N, Dayal M, Pant A, Kumar P, Saxena S, Allin KH, Hansen T, Arumugam M, Vestergaard H, Pedersen O, Pereira V et al (2016) An improved method for high quality metagenomics DNA extraction from human and environmental samples. Sci Rep 6:26775. https://doi.org/10.1038/srep26775
Bera A, Herbert S, Jakob A, Vollmer W, Gotz F (2005) Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus. Mol Microbiol 55:778–787. https://doi.org/10.1111/j.1365-2958.2004.04446.x
Cameron SJS, Lewis KE, Huws SA, Hegarty MJ, Lewis PD, Pachebat JA, Mur LAJ (2017) A pilot study using metagenomic sequencing of the sputum microbiome suggests potential bacterial biomarkers for lung cancer. PLoS One 12:e0177062. https://doi.org/10.1371/journal.pone.0177062
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336
Cargill JS, Scott KS, Gascoyne-Binzi D, Sandoe JA (2012) Granulicatella infection: diagnosis and management. J Med Microbiol 61:755–761. https://doi.org/10.1099/jmm.0.039693-0
Carmody LA, Zhao J, Kalikin LM, LeBar W, Simon RH, Venkataraman A, Schmidt TM, Abdo Z, Schloss PD, LiPuma JJ (2015) The daily dynamics of cystic fibrosis airway microbiota during clinical stability and at exacerbation. Microbiome 3:12. https://doi.org/10.1186/s40168-015-0074-9
Carmody LA, Zhao J, Schloss PD, Petrosino JF, Murray S, Young VB, Li JZ, LiPuma JJ (2013) Changes in cystic fibrosis airway microbiota at pulmonary exacerbation. Ann Am Thorac Soc 10:179–187. https://doi.org/10.1513/AnnalsATS.201211-107OC
Caverly LJ, Zhao J, LiPuma JJ (2015) Cystic fibrosis lung microbiome: opportunities to reconsider management of airway infection. Pediatr Pulmonol 50(Suppl 40):S31–S38. https://doi.org/10.1002/ppul.23243
Coburn B, Wang PW, Diaz Caballero J, Clark ST, Brahma V, Donaldson S, Zhang Y, Surendra A, Gong Y, Elizabeth Tullis D, Yau YC, Waters VJ, Hwang DM, Guttman DS (2015) Lung microbiota across age and disease stage in cystic fibrosis. Sci Rep 5:10241. https://doi.org/10.1038/srep10241
Cox MJ, Allgaier M, Taylor B, Baek MS, Huang YJ, Daly RA, Karaoz U, Andersen GL, Brown R, Fujimura KE, Wu B, Tran D, Koff J, Kleinhenz ME, Nielson D et al (2010) Airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients. PLoS One 5:e11044. https://doi.org/10.1371/journal.pone.0011044
Cuthbertson L, Rogers GB, Walker AW, Oliver A, Green LE, Daniels TW, Carroll MP, Parkhill J, Bruce KD, van der Gast CJ (2015) Respiratory microbiota resistance and resilience to pulmonary exacerbation and subsequent antimicrobial intervention. ISME J. https://doi.org/10.1038/ismej.2015.198
Daniels TW, Rogers GB, Stressmann FA, van der Gast CJ, Bruce KD, Jones GR, Connett GJ, Legg JP, Carroll MP (2013) Impact of antibiotic treatment for pulmonary exacerbations on bacterial diversity in cystic fibrosis. J Cyst Fibros 12:22–28. https://doi.org/10.1016/j.jcf.2012.05.008
Doring G, Flume P, Heijerman H, Elborn JS (2012) Treatment of lung infection in patients with cystic fibrosis: current and future strategies. J Cyst Fibros 11:461–479. https://doi.org/10.1016/j.jcf.2012.10.004
Doring G, Hoiby N (2004) Early intervention and prevention of lung disease in cystic fibrosis: a European consensus. J Cyst Fibros 3:67–91. https://doi.org/10.1016/j.jcf.2004.03.008
Filkins LM, Hampton TH, Gifford AH, Gross MJ, Hogan DA, Sogin ML, Morrison HG, Paster BJ, O’Toole GA (2012) Prevalence of streptococci and increased polymicrobial diversity associated with cystic fibrosis patient stability. J Bacteriol 194:4709–4717. https://doi.org/10.1128/JB.00566-12
Fodor AA, Klem ER, Gilpin DF, Elborn JS, Boucher RC, Tunney MM, Wolfgang MC (2012) The adult cystic fibrosis airway microbiota is stable over time and infection type, and highly resilient to antibiotic treatment of exacerbations. PLoS One 7:e45001
Goddard AF, Staudinger BJ, Dowd SE, Joshi-Datar A, Wolcott RD, Aitken ML, Fligner CL, Singh PK (2012) Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota. Proc Natl Acad Sci U S A 109:13769–13774. https://doi.org/10.1073/pnas.1107435109
Gonzalez JM, Portillo MC, Belda-Ferre P, Mira A (2012) Amplification by PCR artificially reduces the proportion of the rare biosphere in microbial communities. PLoS One 7:e29973. https://doi.org/10.1371/journal.pone.0029973
Hauser AR, Jain M, Bar-Meir M, McColley SA (2011) Clinical significance of microbial infection and adaptation in cystic fibrosis. Clin Microbiol Rev 24:29–70. https://doi.org/10.1128/CMR.00036-10
Chotirmall SH, Smith SG, Gunaratnam C, Cosgrove S, Dimitrov BD, O’Neill SJ, Harvey BJ, Greene CM, McElvaney NG (2012) Effect of estrogen on pseudomonas mucoidy and exacerbations in cystic fibrosis. N Engl J Med 366:1978–1986. https://doi.org/10.1056/NEJMoa1106126
Kircher M, Sawyer S, Meyer M (2012) Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Res 40:e3. https://doi.org/10.1093/nar/gkr771
Kramna L, Kolarova K, Oikarinen S, Pursiheimo JP, Ilonen J, Simell O, Knip M, Veijola R, Hyoty H, Cinek O (2015) Gut virome sequencing in children with early islet autoimmunity. Diabetes Care 38:930–933. https://doi.org/10.2337/dc14-2490
Lee TW, Brownlee KG, Conway SP, Denton M, Littlewood JM (2003) Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. J Cyst Fibros 2:29–34. https://doi.org/10.1016/S1569-1993(02)00141-8
Li Z, Kosorok MR, Farrell PM, Laxova A, West SE, Green CG, Collins J, Rock MJ, Splaingard ML (2005) Longitudinal development of mucoid Pseudomonas aeruginosa infection and lung disease progression in children with cystic fibrosis. JAMA 293:581–588. https://doi.org/10.1001/jama.293.5.581
McMurdie PJ, Holmes S (2013) phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 8:e61217. https://doi.org/10.1371/journal.pone.0061217
Otto M (2009) Staphylococcus epidermidis—the ‘accidental’ pathogen. Nat Rev Microbiol 7:555–567. https://doi.org/10.1038/nrmicro2182
Pillarisetti N, Williamson E, Linnane B, Skoric B, Robertson CF, Robinson P, Massie J, Hall GL, Sly P, Stick S, Ranganathan S (2011) Infection, inflammation, and lung function decline in infants with cystic fibrosis. Am J Respir Crit Care Med 184:75–81. https://doi.org/10.1164/rccm.201011-1892OC
Price KE, Hampton TH, Gifford AH, Dolben EL, Hogan DA, Morrison HG, Sogin ML, O’Toole GA (2013) Unique microbial communities persist in individual cystic fibrosis patients throughout a clinical exacerbation. Microbiome 1:27. https://doi.org/10.1186/2049-2618-1-27
R Core Team (2016) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. url: https://www.R-project.org/
Rogers GB, Marsh P, Stressmann AF, Allen CE, Daniels TV, Carroll MP, Bruce KD (2010) The exclusion of dead bacterial cells is essential for accurate molecular analysis of clinical samples. Clin Microbiol Infect 16:1656–1658. https://doi.org/10.1111/j.1469-0691.2010.03189.x
Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541. https://doi.org/10.1128/AEM.01541-09
Spangler R, Goddard NL, Thaler DS (2009) Optimizing Taq polymerase concentration for improved signal-to-noise in the broad range detection of low abundance bacteria. PLoS One 4:e7010. https://doi.org/10.1371/journal.pone.0007010
Ulrich M, Beer I, Braitmaier P, Dierkes M, Kummer F, Krismer B, Schumacher U, Grapler-Mainka U, Riethmuller J, Jensen PO, Bjarnsholt T, Hoiby N, Bellon G, Doring G (2010) Relative contribution of Prevotella intermedia and Pseudomonas aeruginosa to lung pathology in airways of patients with cystic fibrosis. Thorax 65:978–984. https://doi.org/10.1136/thx.2010.137745
van der Gast CJ, Cuthbertson L, Rogers GB, Pope C, Marsh RL, Redding GJ, Bruce KD, Chang AB, Hoffman LR (2014) Three clinically distinct chronic pediatric airway infections share a common core microbiota. Ann Am Thorac Soc 11:1039–1048. https://doi.org/10.1513/AnnalsATS.201312-456OC
Weinstein MP, Mirrett S, Van Pelt L, McKinnon M, Zimmer BL, Kloos W, Reller LB (1998) Clinical importance of identifying coagulase-negative staphylococci isolated from blood cultures: evaluation of MicroScan rapid and dried overnight gram-positive panels versus a conventional reference method. J Clin Microbiol 36:2089–2092
Winstanley C, O’Brien S, Brockhurst MA (2016) Pseudomonas aeruginosa evolutionary adaptation and diversification in cystic fibrosis chronic lung infections. Trends Microbiol 24:327–337. https://doi.org/10.1016/j.tim.2016.01.008
Yuan S, Cohen DB, Ravel J, Abdo Z, Forney LJ (2012) Evaluation of methods for the extraction and purification of DNA from the human microbiome. PLoS One 7:e33865. https://doi.org/10.1371/journal.pone.0033865
Zemanick ET, Harris JK, Wagner BD, Robertson CE, Sagel SD, Stevens MJ, Accurso FJ, Laguna TA (2013) Inflammation and airway microbiota during cystic fibrosis pulmonary exacerbations. PLoS One 8:e62917. https://doi.org/10.1371/journal.pone.0062917
Zemanick ET, Sagel SD, Harris JK (2011) The airway microbiome in cystic fibrosis and implications for treatment. Curr Opin Pediatr 23:319–324. https://doi.org/10.1097/MOP.0b013e32834604f2
Zemanick ET, Wagner BD, Harris JK, Wagener JS, Accurso FJ, Sagel SD (2010) Pulmonary exacerbations in cystic fibrosis with negative bacterial cultures. Pediatr Pulmonol 45:569–577. https://doi.org/10.1002/ppul.21221
Zhao J, Schloss PD, Kalikin LM, Carmody LA, Foster BK, Petrosino JF, Cavalcoli JD, VanDevanter DR, Murray S, Li JZ, Young VB, LiPuma JJ (2012) Decade-long bacterial community dynamics in cystic fibrosis airways. Proc Natl Acad Sci U S A 109:5809–5814. https://doi.org/10.1073/pnas.1120577109
Acknowledgements
The authors wish to thank Drs. Klára Vilimovská-Dědečková, Veronika Skalicka, Jana Bartosova, and Tereza Jenik. Further, we would like to thank M. Antuskova for technical assistance and A. Bilkova for help with clinical data collection.
Funding
The study was funded by the Grant Agency of Charles University in Prague, grant no. 400213.
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L.K. processed the samples, prepared the libraries, performed sequencing, analyzed the data, and wrote the manuscript; P.D. conceived the study and helped in manuscript writing; J.L. and M.K. performed the statistical analysis; and O.C. supervised the molecular analysis protocol, analyzed the data, and helped in manuscript writing.
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Kramná, L., Dřevínek, P., Lin, J. et al. Changes in the lung bacteriome in relation to antipseudomonal therapy in children with cystic fibrosis. Folia Microbiol 63, 237–248 (2018). https://doi.org/10.1007/s12223-017-0562-3
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DOI: https://doi.org/10.1007/s12223-017-0562-3