Yvonne J. Huang, MD, Craig E. Nelson, PhD, Eoin L. Brodie, PhD, Todd Z

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Airway microbiota and bronchial hyperresponsiveness in patients with suboptimally controlled asthma Yvonne J. Huang, MD, Craig E. Nelson, PhD, Eoin L. Brodie, PhD, Todd Z. DeSantis, MS, Marshall S. Baek, BS, Jane Liu, MS, Tanja Woyke, PhD, Martin Allgaier, PhD, Jim Bristow, MD, Jeanine P. Wiener-Kronish, MD, E. Rand Sutherland, MD, MPH, Tonya S. King, PhD, Nikolina Icitovic, MAS, Richard J. Martin, MD, William J. Calhoun, MD, Mario Castro, MD, Loren C. Denlinger, MD, PhD, Emily DiMango, MD, Monica Kraft, MD, Stephen P. Peters, MD, PhD, Stephen I. Wasserman, MD, Michael E. Wechsler, MD, Homer A. Boushey, MD, Susan V. Lynch, PhD Journal of Allergy and Clinical Immunology Volume 127, Issue 2, Pages 372-381.e3 (February 2011) DOI: 10.1016/j.jaci.2010.10.048 Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 16S rRNA bacterial burden in bronchial brushings from asthmatic patients and healthy subjects. Of subjects analyzed by using PhyloChip (total n = 47), asthmatic patients exhibited greater bacterial burden (measured concentration of 16S rRNA PCR product) than healthy subjects (P = .008, Welch t test with log-transformed data). Median and interquartile ranges are noted. Journal of Allergy and Clinical Immunology 2011 127, 372-381.e3DOI: (10.1016/j.jaci.2010.10.048) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Relationships between variability in airway microbiota composition and community diversity with bronchial hyperresponsiveness. Each circle represents the microbiota present in a single sample (n = 44). A and B, NMDS ordination, based on Sorensen dissimilarity matrices, demonstrates that variability in community composition is most strongly related to bronchial hyperresponsiveness and bacterial burden. The NMDS axes have no inherent units and indicate spatial relationships of samples based on their phylogenetic (dis)similarity. Three samples from asthmatic patients were excluded due to extreme mean relative Sorensen distances from the pooled mean. C, Increased diversity (higher Shannon indices) is correlated with greater bronchial hyperresponsiveness (Pearson correlations with log-transformed data). Journal of Allergy and Clinical Immunology 2011 127, 372-381.e3DOI: (10.1016/j.jaci.2010.10.048) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Phylogenetic tree based on 16S rRNA representative gene sequences (GenBank accession numbers) of the approximately 100 bacterial taxa highly correlated with greater bronchial hyperresponsiveness (P < .01, q < 0.015). Colors represent different bacterial families. Asterisks denote taxa with member species previously associated with clinical disease or possessing notable functional features (see also Table E2 in this article’s Online Repository). Journal of Allergy and Clinical Immunology 2011 127, 372-381.e3DOI: (10.1016/j.jaci.2010.10.048) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Baseline airway microbiota diversity and change in bronchial hyperresponsiveness with clarithromycin treatment. Sixteen asthmatic patients with pretreatment samples analyzed by means of microarray received clarithromycin therapy. Subjects with significant improvement in bronchial hyperresponsiveness after clarithromycin (n = 9) possessed higher pretreatment airway bacterial diversity than those with no significant response (n = 3). Four subjects with incomplete data because of study discontinuation were not included. Journal of Allergy and Clinical Immunology 2011 127, 372-381.e3DOI: (10.1016/j.jaci.2010.10.048) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Diagram showing the sample evaluation process for PhyloChip analysis. Journal of Allergy and Clinical Immunology 2011 127, 372-381.e3DOI: (10.1016/j.jaci.2010.10.048) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions