Journal of Clinical Medicine Article Characterization of Microbiota in Bronchiectasis Patients with Different Disease Severities Sang Hoon Lee 1 , YeonJoo Lee 2, Jong Sun Park 2, Young-Jae Cho 2, Ho Il Yoon 2, Choon-Taek Lee 2 and Jae Ho Lee 2,* 1 Division of Pulmonology, Department of Internal Medicine, Severance Hospital, Institute of Chest Diseases, Yonsei University College of Medicine, Seoul 120-752, Korea; [email protected] 2 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine Seoul National University Bundang Hospital, 82 Gumi-ro, 173 Beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do 463-707, Korea; [email protected] (Y.L.); [email protected] (J.S.P.); [email protected] (Y.-J.C.); [email protected] (H.I.Y.); [email protected] (C.-T.L.) * Correspondence: [email protected]; Tel.: +82-317-877-054 Received: 10 October 2018; Accepted: 6 November 2018; Published: 9 November 2018 Abstract: The applications of the 16S rRNA gene pyrosequencing has expanded our knowledge of the respiratory tract microbiome originally obtained using conventional, culture-based methods. In this study, we employed DNA-based molecular techniques for examining the sputum microbiome in bronchiectasis patients, in relation to disease severity. Of the sixty-three study subjects, forty-two had mild and twenty-one had moderate or severe bronchiectasis, which was classified by calculating the FACED score, based on the FEV1 (forced expiratory volume in 1 s, %) (F, 0–2 points), age (A, 0–2 points), chronic colonization by Pseudomonas aeruginosa (C, 0–1 point), radiographic extension (E, 0–1 point), and dyspnoea (D, 0–1 point). Bronchiectasis was defined as mild, at 0–2 points, moderate at 3–4 points, and severe at 5–7 points. The mean age was 68.0 ± 9.3 years; thirty-three patients were women. Haemophilus (p = 0.005) and Rothia (p = 0.043) were significantly more abundant in the mild bronchiectasis group, whereas Pseudomonas (p = 0.031) was significantly more abundant in the moderate or severe group. However, in terms of the alpha and beta diversity, the sputum microbiota of the two groups did not significantly differ, i.e., the same dominant genera were found in all samples. Further large-scale studies are needed to investigate the sputum microbiome in bronchiectasis. Keywords: bronchiectasis; FACED score; microbiome 1. Introduction Bronchiectasis is a chronic, irreversible airway disease with abnormal dilatation of one or more bronchi, causing chronic cough and purulent sputum production. Impaired mucociliary clearance in bronchiectasis patients is associated with continuous or repeated respiratory infection, inducing a vicious cycle of blockage, inflammation, exacerbation, and damage in the affected bronchi [1]. Bronchiectasis is associated with extended hospitalizations and high mortality, causing a significant economic burden [2,3]. Prevention of exacerbation, reduction of respiratory symptoms, and stopping the progression of the disease are important for the management of bronchiectasis [4]. By improving the bronchial hygiene and decreasing bronchial inflammation, recurrent infection and frequent exacerbation can be prevented [5]. Therefore, the ability to precisely identify colonizing bacterial species, including potential pathogens, is important for clinicians who treat bronchiectasis patients. Conventional, culture-based microbiological analyses identified multiple bacterial pathogens in bronchiectasis patients, such as Pseudomonas aeruginosa, Haemophilus influenzae, Streptococcus J. Clin. Med. 2018, 7, 429; doi:10.3390/jcm7110429 www.mdpi.com/journal/jcm J. Clin. Med. 2018, 7, x FOR PEER REVIEW 2 of 10 Conventional, culture-based microbiological analyses identified multiple bacterial pathogens in bronchiectasisJ. Clin. Med. 2018 , 7patients,, 429 such as Pseudomonas aeruginosa, Haemophilus influenzae, Streptococcus2 of 10 pneumoniae, Staphylococcus aureus, and Moraxella catarrhalis. Importantly, previous studies showed thatpneumoniae the P., Staphylococcusaeruginosa colonization aureus, and Moraxellain bronchiectasis catarrhalis .was Importantly, linked to previous clinical, studies functional, showed and that radiographicthe P. aeruginosa deteriorationcolonization. Although in bronchiectasis standard culture-based was linked to diagnostic clinical, functional, methods are and widely radiographic used, chronicdeterioration. infections Although caused standard by anaerobes culture-based or certain diagnostic bacterial methods species that are widely barely used,grow chronicunder standard infections conditionscaused by anaerobesare difficult or certainto diagnose bacterial using species these that methods barely grow [6]. underThe application standard conditions of next generation are difficult sequencingto diagnose (NGS), using theseusing methods16S rRNA [6 ].gene The pyrose applicationquencing of nexthas expanded generation our sequencing understanding (NGS), of using the pathogenesis16S rRNA gene of pyrosequencingbronchiectasis and has is expanded helping ourphysicians understanding to select of appropriate the pathogenesis antibiotic of bronchiectasis treatments [7].and is helping physicians to select appropriate antibiotic treatments [7]. Martínez-GarcíaMartínez-García etet al.al. used used five five dichotomized dichotomized variables variables to develop to develop a scoring a systemscoring forsystem non-cystic for non-cysticfibrosis bronchiectasis, fibrosis bronchiectasis, known as theknown “FACED as the score”, “FACED which score”, considers which lung considers function, lung age, function, colonization age, colonizationby P. aeruginosa by P., radiographic aeruginosa, radiographic extension, and extension, dyspnoea and [8 dysp]. Thenoea authors [8]. The conducted authors aconducted multicenter, a multicenter,observational observational study, with study, eight hundredwith eight and hundre nineteend and bronchiectasis nineteen bronchiectasis patients who patients were who classified were classifiedaccording according to disease to severity, disease inseverity, relation in to relation the five-year to the all-causefive-year mortality.all-cause mortality. InIn thisthis study,study, we we employed employed culture-independent, culture-independent, DNA-based DNA-based molecular molecular techniques techniques for examining for examiningthe composition the composition of the bacterial of the microbiota bacterial inmicrobiota sputum samples, in sputum in relation samples, to diseasein relation severity, to disease which severity,we derived which using we thederived FACED using scoring the FACED system. scoring system. 2.2. Experimental Experimental Section Section 2.1.2.1. Study Study Population Population BronchiectasisBronchiectasis was was diagnosed diagnosed by by high-resolut high-resolutionion computed computed tomography tomography (HRCT). (HRCT). Patients Patients who who hadhad active active tuberculosistuberculosis oror trauma/tuberculosis-related trauma/tuberculosis-related destroyed destroyed lungs, lungs, were were excluded excluded from from the study. the study.Figure Figure1 shows 1 shows the patient the patient flow chart. flow Initially, chart. Initially, from 1 Aprilfrom 1 2017 April to 2017 31 August to 31 August 2017, a total2017, of a seventytotal of seventypatients patients with bronchiectasis with bronchiectasis agreed to agreed participate to pa inrticipate this prospective in this prospective study, but seven study, patients but seven were patientsexcluded were from excluded the study from because the study of incomplete because of data incomplete (n = 6), ordata a low (n = quantity 6), or a low of DNA quantity extracted of DNA for extractedthe analysis for (then = analysis 1). Therefore, (n = 1). atotal Therefore, of sixty-three a total patientsof sixty-three with bronchiectasispatients with bronchiectasis were investigated were in investigatedthis study. in this study. FigureFigure 1. 1. PatientPatient flow flow chart. chart. From From 1 1April April 2017 2017 to to 31 31 Au Augustgust 2017, 2017, a atotal total of of seventy seventy patients patients with with bronchiectasisbronchiectasis agreed agreed to to participate participate in in this this prospe prospectivective study, study, but but seven seven pati patientsents were were excluded excluded from from thisthis study study because because of of incomplete incomplete data data (n (n == 6) 6) or or an an insufficient insufficient quan quantitytity of of DNA DNA extracted extracted for for the the analysisanalysis (n (n == 1). 1). PFT, PFT, pulmonary pulmonary function function test. test. J. Clin. Med. 2018, 7, 429 3 of 10 The severity of bronchiectasis was classified using the FACED score as follows; percentage of predicted forced expiratory volume in 1 s (FEV1 in %) (F, cut-off 50%, 0–2 points); age (A, cut-off 70 years, 0–2 points); presence of chronic colonization by P. aeruginosa (C, dichotomic, 0–1 point); radiographic extension (E, number of lobes affected, cut-off two lobes, 0–1 point); and dyspnoea (D, cut-off grade II on the Medical Research Council scale, 0–1 point). Mild bronchiectasis was defined as 0–2 points, moderate was 3–4 points, and severe was 5–7 points [8]. Out of the sixty-three patients, forty-two had mild bronchiectasis, and twenty-one had moderate (n = 15) or severe (n = 6) bronchiectasis. Demographic data and clinical measurements were collected, including age, sex, body mass index (BMI), smoking status and amount, respiratory symptoms, pulmonary function test (PFT), chest CT findings, sputum culture study, and comorbidities.