ORIGINAL RESEARCH published: 30 June 2020 doi: 10.3389/fmicb.2020.01463 The Respiratory Microbiome in Cystic Fibrosis: Compartment Patterns and Clinical Relationships in Early Stage Disease Marian Garcia-Nuñez 1,2* , Miguel Garcia-Gonzalez 3,4,5 , Xavier Pomares 1,2,3 , Concepción Montón 1,3 , Laura Millares 1,2,6 , Sara Quero 1,2,6 , Elena Prina 1 , Oscar Asensio 3,4 , Montserrat Bosque 3,4 , Silvia Capilla 7 , Oscar Cuevas 4† and Eduard Monsó 1,2,5* 1 Department of Respiratory Medicine, Institut d’Investigació i Innovació Parc Taulí (I3PT), Hospital Universitari Parc Taulí, Edited by: Sabadell, Spain, 2 Centro de Investigación en Red en Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, John W. A. Rossen, Madrid, Spain, 3 Cystic Fibrosis Unit, Hospital Universitari Parc Taulí, Sabadell, Spain, 4 Department of Pediatrics, Institut University Medical Center Groningen, d’Investigació i Innovació Parc Taulí (I3PT), Hospital Universitari Parc Taulí, Sabadell, Spain, 5 Universitat Autonoma Netherlands de Barcelona, Barcelona, Spain, 6 Infectious and Respiratory Disease Research Group, Fundació Institut d’Investigació Germans Trias i Pujol, Badalona, Spain, 7 Department of Microbiology, Hospital Universitari Parc Taulí, Sabadell, Spain Reviewed by: Deirdre Gilpin, Queen’s University Belfast, We compared the bacterial microbiomes lodged in the bronchial tree, oropharynx and United Kingdom Thomas H. Hampton, nose of patients with early stage cystic fibrosis (CF) not using chronic antibiotics, Geisel School of Medicine, determining their relationships with lung function and exacerbation frequency. CF United States patients were enrolled in a cohort study during stability and were checked regularly *Correspondence: over the following 9 months. Upper respiratory samples (sputum [S], oropharyngeal Marian Garcia-Nuñez mgarcianu@tauli.cat swab [OP] and nasal washing [N]) were collected at the first visit and every 3 months. Eduard Monsó 16S rRNA gene amplification and sequencing was performed and analyzed with QIIME. emonso@tauli.cat † Present Seventeen CF patients were enrolled (16.6 SD 9.6 years). Alpha-diversity of bacterial address: Oscar Cuevas, communities between samples was significantly higher in S than in OP (Shannon index Department of Microbiology, Hospital median 4.6 [IQR: 4.1–4.9] vs. 3.7 [IQR: 3-1-4.1], p = 0.003/Chao 1 richness estimator de Getafe, Madrid, Spain median 97.75 [IQR: 85.1–110.9] vs. 43.9 [IQR: 31.7–59.9], p = 0.003) and beta- Specialty section: diversity analysis also showed significant differences in the microbial composition of both This article was submitted to respiratory compartments (Adonis test of Bray Curtis dissimilarity matrix, p = 0.001). Infectious Diseases, a section of the journal Dominant taxa were found at baseline in five patients (29.4%), who showed lower forced Frontiers in Microbiology expiratory volume in the first second (FEV1%, mean 74.8 [SD 19] vs. 97.2 [SD 17.8], Received: 17 March 2020 p = 0.035, Student t test). The Staphylococcus genus had low RAs in most samples Accepted: 04 June 2020 (median 0.26% [IQR 0.01–0.69%]), but patients with RA > 0.26% of Staphylococcus Published: 30 June 2020 in bronchial secretions suffered more exacerbations during follow-up (median 2 [IQR 1– Citation: Garcia-Nuñez M, 2.25] vs. 0 [0–1], p = 0.026. Mann–Whitney U test), due to S. aureus in more than a half Garcia-Gonzalez M, Pomares X, of the cases, microorganism that often persists as bronchial colonized in these patients Montón C, Millares L, Quero S, Prina E, Asensio O, Bosque M, (9/10 [90%] vs. 2/7 [28.6%], p = 0.034, Fisher’s exact test). In conclusion, the bronchial Capilla S, Cuevas O and Monsó E microbiome had significantly higher diversity than the microbial flora lodged in the (2020) The Respiratory Microbiome oropharynx in early stage CF. Although the RA of the Staphylococcus genus was low in in Cystic Fibrosis: Compartment Patterns and Clinical Relationships bronchial secretions and did not reach a dominance pattern, slight overrepresentations in Early Stage Disease. of this genus was associated with higher exacerbation frequencies in these patients. Front. Microbiol. 11:1463. doi: 10.3389/fmicb.2020.01463 Keywords: cystic fibrosis, microbiome, exacerbation, lung function, Staphylococcus aureus Frontiers in Microbiology | www.frontiersin.org 1 June 2020 | Volume 11 | Article 1463 Garcia-Nuñez et al. Microbiome in CF-Early Stages INTRODUCTION stability visits till the 9th month of follow-up. A second collection of OP was also obtained 6 months after the first Cystic fibrosis (CF) is a chronic respiratory disease characterized visit. Absence of acute symptoms, functional impairment or by defective mucociliary clearance and recurrent infections by antibiotic treatments in the previous month were the stability various potentially pathogenic microorganisms (PPMs) such as criteria required before every sampling. Patients treated with Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus chronic inhaled or systemic antibiotics at the moment of influenzae, Stenotrophomonas maltophila, Achromobacter enrolment were not included in the study. None of the xylosoxidans and the Burkholderia cepacia complex, some of patients used modulators during the study period. Acute which evolve into chronic colonization (Guss et al., 2011; Sibley episodes of increased sputum production and/or dyspnea et al., 2011). Culture-independent approaches have recently been during follow-up were identified as exacerbations when they introduced in the analysis of the respiratory microbiology in required antibiotics, and additional S samples were obtained for CF and have demonstrated that sputum (S) and oropharyngeal usual care culture in these episodes. Participants with two or (OP) cultures only identify a fraction of the microbial flora more exacerbations recorded during the study were considered lodged in the bronchial tree and the oropharynx, missing a frequent exacerbators. wide range of bacteria, which include anaerobic genera as Prevotella, Veillonella, Fusobacterium, and Streptococcus, that Sociodemographic and Clinical required specific cultures (Guss et al., 2011; Sibley et al., 2011; Fodor et al., 2012; Carmody et al., 2018). The diversity of this Measurements respiratory microbiome has a clinical meaning in CF, because Sociodemographic and clinical data, diagnostic tests and its decline, mainly due to the progressive loss of anaerobic flora treatments were recorded at enrollment. All patients performed and its partial substitution by PPMs that increase their relative a forced spirometry with reversibility testing at this first abundance, has been related to the lung function impairment visit, in the morning, with a dry rolling-seal spirometer commonly found in the disease (Delhaes et al., 2012; Fodor et al., (Sibelmed, Sibelgroup, Barcelona, Spain), in accordance 2012; Flight et al., 2015; Carmody et al., 2018). with standard techniques (American Thoracic Society, OP samples are easily obtained in CF patients and have 1995). Postbronchodilator forced vital capacity (FVC) and been often considered representative of bronchial secretions, and forced expiratory volume in the first second (FEV1) were recent series have shown a close similarity of the microbiomes measured and compared to age and height-adjusted reference in bronchi and oropharynx (Fodor et al., 2012; Boutin et al., values. Patients with FEV1% ≥ 70 were considered at early 2015). These studies have enrolled a restricted number of cases stage, while values ≥ 40–70 and <40 were identified as and included a high proportion of patients at intermediate suffering from intermediate and advanced disease, respectively or advanced stages of the disease (FEV1% < 70%) and/or (Caverly et al., 2019). chronic P. aeruginosa colonization. The relationships between the microbial composition of these upper airway sites and Sample Acquisition and Processing their clinical meaningfulness in early stages of the disease Spontaneous S samples were collected in a sterile container are mostly unknown. and a part of it was processed as usual care and cultured The objectives of this cohort study were to compare on agar plates (MacConkey agar, chocolate agar Polyvitex, the bacterial microbiomes at different sites of the upper Columbia agar + 5% sheep blood, Columbia CAN agar + 5% respiratory airway of early stage CF patients (FEV1% ≥ 70), sheep blood and Saboraud Chloramphenicol agar). MacConkey to determine its regional and temporal variability, and their cultures were incubated at 36◦ C, Saboraud Chloramphenicol relationships with clinical patterns of the disease, focusing agar at 30◦ C and all the other agar plates at 36◦ C in CO2 on lung function and exacerbation frequency. Samples from enriched conditions. Recovered bacteria were identified through sputum (S), oropharyngeal swabs (OP) and nasal lavages mass spectrometry using MALDI-TOF Biotyper version 3.0 (N) were examined and compared both cross-sectionally and (Bruker Daltonics, Billerica, MA, United States), reporting only longitudinally to attain the aims of the study. predominant PPMs. Patients with at least one positive S culture for a PPM during the study, and who had three or more positive results for that PPM during this period and/or the MATERIALS AND METHODS previous year were considered chronically colonized (Döring et al., 2000; Lee et al., 2003; Junge et al., 2016). The remnant Design and Patients of the S sample after the culture was stored for microbiome Between March 2013 and November 2014 CF all patients analyses. OP samples were obtained with a sterile cotton swab who regularly attended a reference university hospital and (Copan Diagnostics Inc., Murrieta, CA, United States) from the were able to produce spontaneous sputum were enrolled in posterior oropharynx, and N samples through a sterile Foley the cohort when clinically stable, and regularly checked every catheter after flushing 10 mL of pyrogenic free saline 0.9% 3 months over the following 9 months outside exacerbations. (Fresenius Kabi, Denmark) and recovery through aspiration. Clinical data, spirometry results and respiratory samples Sterile control samples were also collected for each type of (S, OP and N) for microbiome analysis were collected at sample: saline on unused S containers and after instillation and baseline, and followed by the collection of S at scheduled aspiration through a nasal catheter, and unused swabs. These Frontiers in Microbiology | www.frontiersin.org 2 June 2020 | Volume 11 | Article 1463 Garcia-Nuñez et al. Microbiome in CF-Early Stages samples were handled aseptically and processed in parallel with Sequence Analysis the clinical specimens. All samples with their controls were stored The Quantitative Insights Into Microbial Ecology (QIIME) at −80◦ C until use. pipeline 1.9.0 (Caporaso et al., 2010), was used for sequence processing to obtain taxonomic information using Greengenes DNA Extraction 13_8 sequence database as reference and RDP classifier Samples were manipulated in biosafety level 2 hoods with 2.2. The open reference operational taxonomic unit (OTU) laminar flow. All samples were thawed on ice prior to picking method was used with UCLUST and PyNAST DNA extraction. S samples were first incubated with four version 1.2.2 as alignment method. Chimeric sequences volumes of Sputasol (Oxoid, Hampshire, United Kingdom), were detected in QIIME with ChimeraSlayer and were removed mixed with four volumes of phosphate buffer solution and from the OTU table and the phylogenetic tree to perform centrifuged at 16000 g for 10 min. The pellet obtained was downstream analysis. mixed with 0.6 mL of a lysis buffer containing lysozime (final concentration 83 µg/mL; Sigma-Aldrich Corp., St. Louis, Identification of Procedural MO, United States), lysostaphin (final concentration 20 U/mL; Contaminants Sigma-Aldrich) and mutanolysin (final concentration 250 U/mL; To assess the influence of the procedure and reagent Sigma-Aldrich). OP samples were directly suspended on the contamination in our samples, we sequenced 6 extraction same volume of the lysis buffer. The same procedure was controls and one PCR negative control. We obtained a mean of followed with the pellet obtained from N samples after 85 (SD 10) sequences in these controls, which were also processed centrifugation. All samples were incubated during 2 h at 37◦ C in QIIME. Eighty-two genera were identified in these negative (Yuan et al., 2012). controls, 75 of which were also present in the clinical samples. Samples were transferred to a DNA dry bead tube (MO The number of reads per sample was significantly lower in BIO Laboratories, Carlsbad, CA, United States), and shaken control samples compared to N, OP, S samples (Kruskal–Wallis at 5000 rpm for 30 s in a Precellys Minilys homogenizer test, p < 0.001). (Bertin Technologies, Rockville, Washington, DC, United States). Identified genera were removed from the samples if After digesting with Proteinase K for 1 h at 60◦ C, samples their relative abundances (RAs) were higher in controls were again shaken in the homogenizer and DNA was purified (Mann–Whitney U test) or showed a significantly higher according to the manufacturer’s instructions. Genomic DNAs prevalence in them (Fisher’s exact test) (Bittinger et al., were quantified using the Qubit Fluorometer (Life Technologies. 2014). In low bacterial biomass samples (N and OP), Carlsbad, CA, United States). bacterial genera detected with RAs inversely correlated with the total bacterial DNA quantified in the amplicons PCR Amplification and Sequencing of (Spearman’s rho test) were also considered as contaminants 16S rRNA Gene and discarded (Jervis-Bardy et al., 2015). Downstream The 16S rDNA V3-V4 region was amplified following the 16S analyses to determine alpha and beta-diversity were Metagenomic Sequencing Library Preparation Illumina protocol performed after removing contaminant OTUs from the (Part # 15044223 Rev. A; Illumina, CA, United States). Illumina final OTU tables. adapter overhang nucleotide sequences were added to gene- specific sequences, selecting primers following Klindworth Data Analysis and cols (Klindworth et al., 2013). Using the standard Statistical analyses were performed using SPSS statistical software International Union of Pure and Applied Chemistry nucleotide version 19 (IBM SPSS Statistics, Armonk, NY, United States) nomenclature, the full-length primer sequences used to follow and R packages. Descriptive analyses obtained from categorical the protocol targeting this region were: 16S Forward primer = 50 - variables are expressed as absolute and relative frequencies and tcgtcggcagcgtcagatgtgtataagagacagcctacgggnggcwgcag-30 and results for continuous variables as means and standard deviations reverse primer = 50 -gtctcgtgggctcggagatgtgtataagagacaggactachvg (SD) when the distribution was normal, or as medians and ggtatctaatcc-30 . After 16S amplification, the multiplexing interquartile range (IQR) otherwise. step was performed using Nextera XT Index Kit (FC- Alpha diversity in bronchi, oropharynx and nose was 131-1096, Illumina). Extraction controls were amplified in calculated through Shannon index (Shannon, 1997) and Chao 1 parallel with the samples. Libraries were sequenced using a richness estimator (Chao, 1984) after subsampling with QIIME 2 × 300 bp paired-end run (MiSeq Reagent kit v3 MS-102- to avoid sequencing effort bias. Beta diversity and differences 3001, Illumina), on a MiSeq Sequencer in accordance with between the three compartments was assessed through Principal manufacturer’s instructions (Illumina). Quality assessment Coordinates Analysis (PCoA) with Bray-Curtis dissimilarity was performed by the use of the PRINSEQ-lite program index (Bray and Curtis, 1957) and Adonis testing. Dissimilarity (Schmieder and Edwards, 2011), on the following parameters: distances (θYC distance) among paired samples of S and OP were min_length: 50; trim_qual_right: 20; trim_qual_type: mean; also assessed (Yue and Clayton, 2005). trim_qual_window: 20. R1 and R2 from Illumina sequencing Samples were normalized to the percentage of total were joined using the fastq-join program, part of the ea-tools reads for the comparisons of their RAs. The RA of suite (Aronesty, 2011). each taxon was calculated by dividing its number of Frontiers in Microbiology | www.frontiersin.org 3 June 2020 | Volume 11 | Article 1463 Garcia-Nuñez et al. Microbiome in CF-Early Stages sequences by the total number of sequences in the sample. PPMs in 6 (35.3%). In 5 cases (23.8%) no PPM was identified A dominant genus in a sample was defined as the most in the exacerbation. Neither the appearance nor the number of abundant genus with at least twice the abundance of the exacerbations were significantly related to positive cultures for second most abundant genus (Coburn et al., 2015), and S. aureus at baseline (p = 0.6, Fisher exact test and p = 0.531, the identification of dominance either at the first or its Mann–Whitney U test, respectively), or to chronic colonization immediately successive sample was considered as baseline by this PPM (p = 0.28, Fisher exact test and p = 0.095, Mann– dominance pattern. Whitney U test, respectively). First, genetic characteristics, baseline lung function, previous and subsequent exacerbations and culture-based microbiology Sequencing were assessed. Second, alpha- and beta-diversity of the bacterial One hundred and seven samples were suitable for sequencing, 48 flora in bronchi, oropharynx and nose were examined and of them obtained at baseline (16 S, 17 OP and 15 N) and 59 during compared in both baseline and longitudinal samples. Finally, follow-up (45 S and 14 OP). After removal of low-quality reads relationships between the RAs of the identified genera in S and and procedure contaminants, a median of 3500 (IQR: 495–13386) clinical patterns were assessed, considering the median of RAs at reads were recovered from the processed samples, which allowed the baseline and its successive value as the reference (cut-off) RA the identification of a median of 43 genera per sample (IQR: 33– for these analyses. 57). For differential abundance analyses between groups, taxa At phylum level, OTUs with higher median RA in S were with median RAs above 0.1% in at least one type of sample Firmicutes (36.2%), Bacteroidetes (19.5%), Actinobacteria were included. Student t test, chi-square and Fisher’s exact (11.4%), Fusobacteria (10.3%) and Proteobacteria (7.5%). test, together with non-parametric Mann-Whitney U, Wilcoxon Gemellaceae genus (10.7%), Rothia (8.7%), Veillonella (7.5%), signed rank, Friedman and Kruskal–Wallis tests were used Prevotella (6.9%) and Fusobacterium (5.9%) were the genera with for testing, as required. Correction for multiple hypothesis the highest figures in the sample (Supplementary Table S1). testing was done by computing false discovery rates (FDR). These genera were also dominant in one or more of the S Tests producing a P value equal to or less than 0.05 were samples, together with Haemophilus and Porphyromonas. considered as significant. Topographical Analyses A comparative analysis of the three sampled compartments RESULTS was performed in the 15 patients with good-quality sequencing results available from the S, N and OP samples obtained Patient Characteristics at baseline. The diversity of bacterial communities between Respiratory samples were collected prospectively from 17 samples was significantly higher in S than in OP, when patients with CF in stable phase (6 adults and 11 children). Sixty- measured either with the Shannon index (median 4.6 [IQR: eight S samples were collected, as well as 33 OP and 17 N samples 4.1–4.9] vs. 3.7 [IQR: 3-1-4.1], p = 0.003) or the Chao 1 obtained on the same day as an S sample. Patients had a median richness estimator (median 97.75 [IQR: 85.1–110.9] vs. 43.9 age of 13 (IQR: 11–20) years and most presented delta F508 gene [IQR: 31.7–59.9], p = 0.003). Similar results were obtained mutations (15 patients, 88.2%, 14/1 hetero/homozygous). Mean when comparing S with N, for the Chao 1 richness estimator sweat test chloride at diagnosis was 95.2 (SD 20) mEq/L. Twelve (median 97.7 [IQR: 85.1–110.9] vs. 57.9 [41.7–78.6], p = 0.005) patients had lung volumes within normal ranges at enrollment (70.6%). One patient had used nebulized amikacin for more than 4 weeks the year before inclusion, but not during the month TABLE 1 | Patient characteristics at baseline. previous to enrollment (Table 1). N 17 Bacterial cultures of S were positive of S. aureus in six patients at baseline (35.3%), and positive results for this PPM were found Age (years), median (IQR) 13 (11–20) in follow-up stability samples from 5 additional patients who had Gender (male), n (%) 9 (52.9) previous negative S cultures (29.4%). These 11 patients showed Sweat test (Cl- mEq/L), mean (SD) 95.2 (20) recurrently positive cultures and attained the criteria for chronic CFTR genotype, n (%) colonization by S. aureus (64.7%), but repeatedly positive cultures 1F508-homozygous 3 (17.6) were not found for other PPMs. 1F508-heterozygous 12 (70.6) Positive cultures for S. aureus at enrolment were not Other mutation 2 (11.7) significantly associated to FEV1 impairment (FEV1% mean 79.8 FEV1 % predicted, mean (SD) 90.6 (20.5) [SD 25.1] vs. 96.5 [15.8], p = 0.110, Student t test), and an equal FVC% predicted, mean (SD) 92.9 (19.1) result was found for chronic colonization (FEV1% mean 88.4 FEV1/FVC%, mean (SD) 91.9 (12.3) [21.4] vs., 98.3 [17.8], p = 0.267, Student t test). Use of nebulized antibiotic during the previous year 1 (5.9) Twelve patients reported one or more exacerbations during (>1 month), n (%) follow-up (70.6%), and 7 of them were frequent exacerbators Exacerbations in the previous year, median (IQR) 1 (0–1) (41.2%). From the 21 exacerbations recorded, S cultures positive IQR, interquartile range; SD, standard deviation; CFTR, Cystic fibrosis were positive for S. aureus in 10 episodes (47.6%), and for other transmembrane conductance regulator. Frontiers in Microbiology | www.frontiersin.org 4 June 2020 | Volume 11 | Article 1463 Garcia-Nuñez et al. Microbiome in CF-Early Stages FIGURE 1 | Alpha diversity metrics across sample types. (A) Shannon index, (B) Chao1 richness estimator. S, sputum; OP, oropharyngeal swab; N, nasal lavage. with a clear overrepresentation of specific anaerobe genera in bronchial secretions. Longitudinal Study To determine the variability over time in the microbiome of the bronchial tree and oropharynx, we used data from paired S samples from the 17 patients studied, and paired OP samples from 14 patients suitable for sequencing. Alpha-diversity variation in the bacterial flora in S and OP was not statistically significant, using either the Shannon index or the Chao1 richness estimator (Figure 3). Dissimilarity (θYC distance) of bacterial communities was lower in S than in OP samples obtained after a 6-month interval (p = 0.017, Wilcoxon test) (Figure 4). This finding confirms the relative stability of the bacterial flora in the bronchial tree compartment, which was paralleled by the higher temporal variability of oropharyngeal flora, in these patients with early stage CF. FIGURE 2 | Beta-diversity analysis of the bacterial communities of different respiratory compartments. Principal coordinate analysis (PCoA) of sputum (yellow), oropharyngeal swab (blue), and nasal lavage samples (red). Adonis Bronchial Microbiome and Clinical test of Bray Curtis dissimilarity matrix, p = 0.001. Patterns Staphylococcus aureus was the main PPM recovered from S cultures at baseline and during the study period, in spite that Staphylococcus genus did not reach a dominance pattern in any (Figure 1). Beta-diversity analysis also showed significant S sample and showed low RAs in most of them (median 0.26% differences in the microbial composition of the three bacterial [IQR 0.01–0.69%]). communities (Figure 2) (Adonis test of Bray Curtis dissimilarity A dominance pattern of the genera Prevotella, Veillonella, matrix, p = 0.001). Gemellaceae genus, Porphyromonas, Rothia or Haemophilus was The predominant phyla in S samples were Firmicutes (32.4%), observed in five patients at baseline and was significantly Bacteroidetes (22.9%), Actinobacteria (14.5%), Fusobacteria associated with FEV1 impairment (FEV1%, mean 74.8 [SD 19] (10.0%) and Proteobacteria (8.32%). The most abundant vs. 97.2 [SD 17.8], p = 0.035, Student t test). Patients with higher genera in the bronchial compartment were Prevotella (12.9%), Staphylococcus median RAs (>0.26%) at baseline showed lower Rothia (11.7%), Gemellaceae genus (11.3%), Veillonella (8.8%), FEV1, but this difference did not reach statistical significance Fusobacterium (6.8%) and Porphyromonas (5.85), which showed (FEV1% mean 86.3 [SD 23.5] vs. 96.9 [14.6], p = 0.311, Student higher RAs in S than in samples from other respiratory t test) (Figure 5). Testing the other taxa with median RAs above compartments. This difference reached statistical significance for 0.1% in S at baseline (n = 31) gave similar non-significant results most of these genera (Table 2). (data not shown). These results suggest that in early stage CF patients there Patients with Staphylococcus RAs over 0.26% at baseline is higher diversity in the microbiome of the bronchial tree suffered more exacerbations (median 2 [IQR 1–2.25] vs. 0 [0– than in the microbial flora of other respiratory compartments, 1]) (Figure 5), often due to S. aureus (10/17, 58.8%), and higher Frontiers in Microbiology | www.frontiersin.org 5 June 2020 | Volume 11 | Article 1463 Garcia-Nuñez et al. Microbiome in CF-Early Stages TABLE 2 | Genera detected in bronchial tree (S), oropharynx (OP) and nose (N) (median [IQR]). Friedman test Wilcoxon test Phylum Genus Sputum (S) Oropharyngeal swab (OP) Nasal lavage (N) p S vs. OP S vs. N Acidobacteria Ellin6075_g 0 (0–0) 0 (0–0) 0.21 (0–2.65) 0.002 0.32 0.03 Actinobacteria Actinomyces 1.31 (0.43–1.91) 2.23 (0.06–3.3) 4.17 (0.21–6.38) 0.263 0.12 0.09 Actinobacteria Rothia 11.71 (5.85–23.34) 2.11 (0.8–12.35) 0.53 (0–1.99) 0.001 0.31 0.03 Actinobacteria 0319-7L14_f_g 0 (0–0) 0 (0–0) 1.05 (0–2.46) 0.001 1 0.03 Actinobacteria Corynebacterium 0.04 (0.01–0.11) 0.12 (0–0.64) 0.58 (0–1.57) 0.382 0.25 0.06 Actinobacteria Nocardioidaceae_g 0 (0–0) 0 (0–0.2) 0.28 (0–1.32) 0.002 0.13 0.03 Actinobacteria Propionibacterium 0 (0–0) 0 (0–0.2) 0.93 (0.17–1.99) 0.002 0.09 0.03 Actinobacteria Atopobium 0.34 (0.05–0.61) 0.23 (0–0.64) 0.93 (0–4.26) 0.789 0.98 0.29 Bacteroidetes [Prevotella] 2.2 (1.72–4.29) 1.66 (0.92–3.51) 0 (0–0) 0.002 0.98 0.03 Bacteroidetes Porphyromonas 5.79 (1.19–7.77) 0.7 (0.06–1.78) 0 (0–2.26) 0.002 0.03 0.03 Bacteroidetes Prevotella 12.88 (5.96–16.13) 8.28 (2.49–14.76) 6.38 (0.26–14.35) 0.024 0.07 0.16 Bacteroidetes Capnocytophaga 1.52 (0.68–2.78) 0.15 (0–0.67) 0 (0–0.01) 0.004 0.03 0.04 Chloroflexi Gitt-GS-136_o_f_g 0 (0–0) 0 (0–0) 0.53 (0–3.13) 0.002 1 0.03 Firmicutes Gemellaceae_g 11.32 (6.77–14.34) 2.56 (0.5–3.84) 0.01 (0–1.39) 0.001 0.08 0.03 Firmicutes Granulicatella 4.14 (2.46–5.81) 0.81 (0.32–2.48) 0 (0–1.57) 0.001 0.08 0.03 Firmicutes Streptococcus 0.78 (0.47–1.38) 2.3 (1.37–4.43) 1.06 (0.02–3.83) 0.021 0.03 0.19 Firmicutes Clostridiales_f_g 0.14 (0.02–0.25) 1.88 (0–3.18) 0 (0–0.35) 0.02 0.03 0.88 Firmicutes Veillonella 8.81 (3.73–12.3) 5.72 (3.76–7.71) 6.53 (0.03–11.5) 0.296 0.09 0.36 Firmicutes Staphylococcus 0.11 (0–0.66) 0 (0–0.03) 0.21 (0–1.58) 0.046 0.04 0.65 Firmicutes [Mogibacteriaceae]_g 0.25 (0–0.58) 0.82 (0–1.99) 0 (0–1.06) 0.144 0.05 0.96 Firmicutes Parvimonas 0.15 (0–0.17) 0 (0–0.01) 0 (0–0) 0.002 0.03 0.03 Firmicutes Lachnospiraceae_g 0.68 (0.32–1.07) 0.7 (0.12–1.94) 0.01 (0–3.28) 0.391 0.67 1 Firmicutes Catonella 0.36 (0.03–0.64) 0.1 (0–0.23) 0 (0–0) 0.002 0.09 0.03 Firmicutes Moryella 0.26 (0.03–0.76) 0.2 (0–0.35) 0 (0–0) 0.034 0.32 0.38 Firmicutes Oribacterium 0.86 (0.25–2.54) 0.35 (0–0.75) 0 (0–2.24) 0.077 0.04 0.22 Firmicutes Peptostreptococcus 0.29 (0.1–0.55) 0 (0–0.35) 0 (0–0) 0.002 0.2 0.18 Firmicutes Megasphaera 0.25 (0–1) 0.44 (0–3.31) 0 (0–0) 0.015 0.09 0.12 Firmicutes Selenomonas 0.14 (0.05–0.3) 0.4 (0.1–1.33) 0 (0–0.85) 0.014 0.07 1 Firmicutes Bulleidia 0.22 (0.09–0.71) 0.49 (0.1–2.71) 0 (0–0.7) 0.118 0.2 0.41 Firmicutes Aerococcaceae_other 0.12 (0–0.43) 0 (0–0) 0 (0–0) 0.012 0.05 0.09 Fusobacteria Fusobacterium 6.81 (2.82–9.76) 2.35 (1.29–7.44) 0 (0–1.28) 0.001 0.25 0.03 Fusobacteria Leptotrichia 3.61 (2.05–8.6) 0.99 (0.15–1.99) 0 (0–1.05) 0.001 0.03 0.03 Planctomycetes WD2101_f_g 0 (0–0) 0 (0–0) 0.87 (0–1.99) 0.002 1 0.03 Proteobacteria Campylobacter 1.29 (0.35–2.12) 1.38 (0–3.97) 0 (0–0) 0.002 0.46 0.03 Proteobacteria Haemophilus 1.73 (0.58–4.19) 0 (0–0.26) 0 (0–0) 0.001 0.03 0.03 Proteobacteria Lautropia 0.18 (0–0.87) 0 (0–0.03) 0 (0–0) 0.004 0.03 0.04 Proteobacteria Pseudomonadaceae_g 0 (0–0) 0 (0–0) 0.18 (0–1.99) 0.002 1 0.03 Spirochaetes Treponema 0.11 (0–0.37) 0 (0–0.12) 0 (0–0) 0.015 0.03 0.62 TM7 TM7-1_o_f_g 0 (0–0.05) 0 (0–0.12) 1.05 (0.01–4.63) 0.024 0.42 0.03 TM7 TM7-3_o_f_g 0.59 (0.07–1.25) 4.49 (0.89–30.09) 0.11 (0–1.48) 0.016 0.03 0.86 Taxa with relative abundances median > 0.1% in at least one type of sample included. RAs were also significantly associated chronic colonization by patterns, are clinically meaningful, because they are associated S. aureus (9/10 [90%] vs. 2/7 [28.6%], p = 0.034, Fisher’s with the subsequent appearance S. aureus-related acute episodes exact test). This trend was not observed when the relationship and the persistence of this PPM as colonizer, together with a with exacerbation frequency was tested for the other taxa tendency to suffer exacerbations more frequently. (Supplementary Table S2). Dominance patterns at baseline were not associated with higher exacerbation frequencies during follow-up (median 1 [IQR 1–2] vs. 1 [0–2], p = 0.701, Mann– DISCUSSION Whitney U test). These observations suggest that Staphylococcus genus RAs In the present cohort study, the microbiome of the bronchial tree above 0.26%, in spite of being low and far from dominance and two additional upper respiratory compartments, the nose Frontiers in Microbiology | www.frontiersin.org 6 June 2020 | Volume 11 | Article 1463 Garcia-Nuñez et al. Microbiome in CF-Early Stages FIGURE 3 | Variability of alpha-diversity over time in (A,C) sputum and (B,D) oropharyngeal swab samples. that the RA of the Staphylococcus genus was low in bronchial secretions and did not reach 1% in most patients, participants with RAs above 0.26% suffered more exacerbations, in most cases due to S. aureus, that often persists as chronic colonizer in the bronchial tree, confirming the clinical meaningfulness of small overrepresentations of the Staphylococcus genus, even at low RAs. In the present study Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria were the predominant phylum in bronchial secretions, with Prevotella, Rothia and Gemellaceae genus as the most abundant genera. The genera Pseudomonas and Burkholderia were not identified in the studied respiratory samples, in spite that P. aeruginosa and Burkholderia species are commonly cultured from CF patients (Guss et al., 2011; Fodor et al., 2012). Common PPMs are typically acquired in temporal succession in CF, beginning early in life with S. aureus and then FIGURE 4 | Intrasubject similarity (θYC distance) between bacterial communities recovered at baseline and after 6 months from sputum (S) and progressing to recurrent infections dominated by P. aeruginosa from the oropharyngeal swab (OP). and B. cepacia complex species (Razvi et al., 2009; Huang and Lynch, 2011), which often persist as colonizers and reach a pattern of dominance with RAs over 50% (Fodor et al., 2012). The frequent recovery of S. aureus from bronchial secretions in and the oropharynx, was assessed in early stage stable CF patients the present study in the absence of P. aeruginosa is expected not using chronic antibiotic therapy. S. aureus was recovered considering that enrolled patients showed early stage disease and from S cultures during the study in more than a half of the did not use chronic antibiotic treatments. participants, but all samples were negative for P. aeruginosa and The bronchial microbiome had higher diversity than the other common CF colonizers. The bronchial microbiome showed microbial flora in oropharynx and nose in the present study. a significantly higher diversity than oropharynx and nose. In spite These results emphasize the need to obtain bronchial samples Frontiers in Microbiology | www.frontiersin.org 7 June 2020 | Volume 11 | Article 1463 Garcia-Nuñez et al. Microbiome in CF-Early Stages FIGURE 5 | Relationship between the overrepresentation of Staphylococcus in bronchial secretions (relative abundance above 0.26% in sputum) and clinical measures: (A) forced expiratory volume in the first second as a percentage of the reference (FEV1%); and (B) exacerbation frequency during the study period. for an accurate evaluation of the bronchial flora in early stage exacerbations during follow-up, due to S. aureus in more than patients, to avoid any misevaluation that may derive from a half of the cases, which often persist as chronic colonization. the single assessment of oropharyngeal microbiome. In CF This finding suggests that small overrepresentations of the the bacterial composition of the oropharynx has been often Staphylococcus genus may have clinical significance, even when considered representative of the bronchial flora as in healthy showing low RAs. Additional studies would be needed to subjects (Fodor et al., 2012; Boutin et al., 2015; Dickson et al., confirm these results. 2015). However, previous studies on CF patients have included The present study has included 17 patients, that may not be a high proportion of patients at intermediate or advanced stages representative of the CF population as a whole, as other studies of the disease (FEV1% < 70%) often colonized by P. aeruginosa of the respiratory microbiome of CF with a similar number (Fodor et al., 2012; Boutin et al., 2015), a pattern mainly observed of participants (Fodor et al., 2012; Boutin et al., 2015). It is in adult patients (Coburn et al., 2015). The results of the present important also to consider that the study is restricted to early study suggests that the microbial composition of bronchi and stage CF, and does not include CF patients with intermediate and oropharynx have significant differences when the disease is at an advanced disease, and the results obtained cannot be extrapolated early stage in younger patients, through an overrepresentation to this population. In most cases these patients require chronic of specific genera in bronchial secretions, in accordance with and/or recurrent antibiotherapy, a treatment whose impact on previous suggestions (Zemanick et al., 2015, 2017). the respiratory microbiome has been demonstrated (Carmody A dominant pattern was observed at baseline in near a et al., 2018). The acceptance of this limiting selection criterion third of the participants and was related to lung function has the advantage of allowing a detailed study of respiratory flora impairment. Dominance was mainly due to increased RAs at earlier stages of the disease and outside the use of regular of different anaerobic genera as are Prevotella, Veillonella, antibiotics, a period that has been only marginally studied. Gemella, Porphyromonas or Rothia, in the present study, and To conclude, the bronchial microbiome shows higher diversity less often related to the overrepresentation of aerobic genus than the microbial flora lodged in the oropharynx and nose as Haemophilus. These results confirm the clinical meaning in early stage CF, mainly through an overrepresentation of of dominance patterns, even when unrelated to Pseudomonas, anaerobic genera in bronchial secretions. Different taxa may Burkholderia, Stenotrophomonas, and Achromobacter, the genera begin to show dominance patterns in this clinical stage, and, most commonly showing high RAs, which when dominant in spite of their variability, were associated with identifiable are often associated with diversity declines, low lung volumes functional impairments. S. aureus was often cultured from these and chronic colonization (Delhaes et al., 2012; Coburn et al., early stage CF patients, but the Staphylococcus genus shows only 2015; Flight et al., 2015). Dominance patterns were not related low RAs in bronchial secretions. However, patients with slight to exacerbation frequency in the present study, in agreement overrepresentations of this genus, even at rates below 1%, suffered with previous studies that have mainly related the appearance more exacerbations, mostly due to S. aureus, and frequently of exacerbations to changes in the microbial composition of evolve to a chronic colonization pattern by this PPM, findings bronchial secretions severe enough to be considered as disbiosis, that confirm the importance of the appearance of that genus in while dominant taxa are mainly observed is stable condition bronchial secretions, regardless of its abundance. when the disease is advanced (Carmody et al., 2013, 2015). Staphylococcus aureus was recovered from one third of the S cultures during the study period, even though the Staphylococcus DATA AVAILABILITY STATEMENT genus had RAs mostly below 1% and did not attain a dominant pattern. Patients with RAs over 0.26% for this genus did The datasets generated for this study can be found in the Bacterial not shown a lung function impairment, but suffered more 16S rRNA, data sets from this study are accessible in the European Frontiers in Microbiology | www.frontiersin.org 8 June 2020 | Volume 11 | Article 1463 Garcia-Nuñez et al. Microbiome in CF-Early Stages Nucleotide Archive under the reference PRJEB31332 with the analysis tools. MG-N and EM did the statistical analysis, prepared sample numbers ERS3193233–ERS3193339 (http://www.ebi.ac. the figures and tables and wrote the first draft which was reviewed uk/ena/data/view/ERS3193233-ERS3193339). and approved by all the co-authors. OA, MB, and MG-G critically reviewed and revised the manuscript for important intellectual content. All authors contributed to the interpretation of the ETHICS STATEMENT results and approved the final version for publication. All clinical investigations were conducted in accordance with the principles expressed in the Declaration of Helsinki. The studies FUNDING involving human participants were reviewed and approved by The Parc Taulí University Hospital Ethics Board. Written This work has been funded by the Fundació Catalana informed consent to participate in this study was provided de Neumología (FUCAP), Fundació Parc Taulí (I3PT), FIS by participants or by the participants’ legal guardian/next of PI18/00934, and Centro de Investigación Biomédica en Red de kin. Furthermore, protocols have been adhered to standard Enfermedades Respiratorias (CIBERES). CIBERES is an initiative biosecurity and institutional safety procedures in this study. of the Instituto de Salud Carlos III. Financial support was also provided by the CERCA Program/Generalitat de Catalunya. AUTHOR CONTRIBUTIONS SUPPLEMENTARY MATERIAL MG-N, MB, XP, and EM conceived and designed the study. XP, CM, and MB contributed to the recruitment of the participants. The Supplementary Material for this article can be found XP, CM, MG-G, and EP acquisition of the clinical data. MG-N, online at: https://www.frontiersin.org/articles/10.3389/fmicb. LM, SQ, SC, and OC contributed to the reagents, materials, and 2020.01463/full#supplementary-material REFERENCES Chao, A. (1984). Nonparametric estimation of the number of classes in a population. Scand. J. Stat 11, 265–270. American Thoracic Society (1995). Standardization of spirometry, 1994 update. Coburn, B., Wang, P. W., Diaz Caballero, J., Clark, S. T., Brahma, V., Donaldson, American thoracic society. Am. J. Respir. Crit. Care Med. 152, 1107–1136. S., et al. (2015). Lung microbiota across age and disease stage in cystic fibrosis. doi: 10.1164/ajrccm.152.3.7663792 Sci. Rep. 5:10241. doi: 10.1038/srep10241 Aronesty, E. 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R., Eshaghurshan, C. S., Faria, M. M., Dowd, Quero, Prina, Asensio, Bosque, Capilla, Cuevas and Monsó. This is an open-access S. E., et al. (2011). Culture enriched molecular profiling of the cystic fibrosis article distributed under the terms of the Creative Commons Attribution License airway microbiome. PloS One 6:e22702. doi: 10.1371/journal.pone.0022702 (CC BY). The use, distribution or reproduction in other forums is permitted, provided Yuan, S., Cohen, D. B., Ravel, J., Abdo, Z., and Forney, L. J. (2012). Evaluation the original author(s) and the copyright owner(s) are credited and that the original of methods for the extraction and purification of DNA from the human publication in this journal is cited, in accordance with accepted academic practice. No microbiome. PloS One 7:e33865. doi: 10.1371/journal.pone.0033865 use, distribution or reproduction is permitted which does not comply with these terms. 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