Mechanistic Model of Rothia Mucilaginosa Adaptation Toward Persistence in the CF Lung, Based on a Genome Reconstructed from Metagenomic Data

Mechanistic Model of Rothia mucilaginosa Adaptation toward Persistence in the CF Lung, Based on a Genome Reconstructed from Metagenomic Data Yan Wei Lim1*, Robert Schmieder2, Matthew Haynes1¤, Mike Furlan1, T. David Matthews1, Katrine Whiteson1, Stephen J. Poole3, Christopher S. Hayes3,4, David A. Low3,4, Heather Maughan5, Robert Edwards2,6, Douglas Conrad7, Forest Rohwer1 1 Department of Biology, San Diego State University, San Diego, California, United States of America, 2 Computational Science Research Center, San Diego State University, San Diego, California, United States of America, 3 Department of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America, 4 Biomolecular Science and Engineering Program, University of California Santa Barbara, Santa Barbara, California, United States of America, 5 Ronin Institute, Montclair, New Jersey, United States of America, 6 Mathematics and Computer Science Division, Argonne National Laboratory, Argonne, Illinois, United States of America, 7 Department of Medicine, University of California San Diego, La Jolla, California, United States of America Abstract The impaired mucociliary clearance in individuals with Cystic Fibrosis (CF) enables opportunistic pathogens to colonize CF lungs. Here we show that Rothia mucilaginosa is a common CF opportunist that was present in 83% of our patient cohort, almost as prevalent as Pseudomonas aeruginosa (89%). Sequencing of lung microbial metagenomes identified unique R. mucilaginosa strains in each patient, presumably due to evolution within the lung. The de novo assembly of a near-complete R. mucilaginosa (CF1E) genome illuminated a number of potential physiological adaptations to the CF lung, including antibiotic resistance, utilization of extracellular lactate, and modification of the type I restriction-modification system. Metabolic characteristics predicted from the metagenomes suggested R. mucilaginosa have adapted to live within the microaerophilic surface of the mucus layer in CF lungs. The results also highlight the remarkable evolutionary and ecological similarities of many CF pathogens; further examination of these similarities has the potential to guide patient care and treatment. Citation: Lim YW, Schmieder R, Haynes M, Furlan M, Matthews TD, et al. (2013) Mechanistic Model of Rothia mucilaginosa Adaptation toward Persistence in the CF Lung, Based on a Genome Reconstructed from Metagenomic Data. PLoS ONE 8(5): e64285. doi:10.1371/journal.pone.0064285 Editor: James L. Kreindler, Abramson Research Center, United States of America Received November 30, 2012; Accepted April 13, 2013; Published May 30, 2013 Copyright: ß 2013 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health and Cystic Foundation Research Inc. through grants (1 R01 GM095384-01 and CFRI #09- 002) awarded to Forest Rohwer and grant U54 AI065359 awarded to Christopher Hayes and David Low. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ¤ Current address: DOE Joint Genome Institute, Walnut Creek, California, United States of America Introduction Previous sequencing of CF metagenomes revealed the presence of Rothia mucilaginosa at relatively high abundances in most patients Cystic fibrosis (CF) is a genetic disease caused by mutation of the [12]. R. mucilaginosa was first isolated from milk in 1900 as cystic fibrosis transmembrane conductance regulator (CFTR) gene Micrococcus mucilaginosus [15]. It was later re-isolated and further [1]. In CF lungs, the defective CFTR protein affects trans- studied by Bergan et al. in 1970 [16], and renamed Stomatococcus epithelial ion transport and consequently leads to the accumula- mucilaginosus in 1982 based on its 16S rDNA and biochemical tion of thick and static mucus. The resultant hypoxic microenvi- characteristics [17]. A recent study comparing S. mucilaginosus to ronment encourages the colonization of opportunistic microbes, Rothia dentocariosa and another unknown species (later known as viruses, and fungi (reviewed in [2]), causing acute and chronic Rothia nasimurium) led to the reclassification of S. mucilaginosus as R. infection. A few of the most commonly isolated pathogens are mucilaginosa [18]. Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, R. mucilaginosa is an encapsulated, Gram-positive non-motile and Burkholderia cepacia. However, an increasing number of coccus (arranged in clusters) belonging to the phylum Actinobac- microbial species have been detected in the CF airway using teria. It has variable catalase activity, reduces nitrate, and culture-independent methods such as metagenomic sequencing hydrolyses aesculin [17–19]. It is a facultative anaerobe commonly [3–7]. Metagenomics is a powerful approach that has been used to found in the human oral cavity and upper respiratory tract successfully characterize the microbial and viral communities in [16,20,21], and occasionally the gastrointestinal tract [22], small CF individuals [8–12]. These types of studies have illuminated the intestinal epithelial lining [23], tongue [24,25], teeth [26,27], complexity of microbial and viral communities, captured the vast colostrum [28], breast milk [29], and dental plaques [30,31]. diversity of functions encoded by these organisms, and have been Although R. mucilaginosa is commonly regarded as normal flora of used to trace the evolution of whole genomes [13,14]. the oral cavity and upper respiratory tract, its association with a PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e64285 Rothia mucilaginosa as an Opportunistic Pathogen wide range of diseases (Table S1) highlights its potential as an tion on which microbes and viruses are present, and their opportunistic pathogen, especially in immuno-compromised pa- metabolic capabilities, while metatranscriptomic data provide tients [32]. information on which organisms are metabolically active [12]. At the genus level, Rothia has been reported by Tunney et al. Such metabolism data will enable predictions of local lung [33] as an aerobic species that can be isolated from CF sputum chemistry that may impose patient-specific selective pressures on and pediatric bronchoalveolar lavage (BAL) samples. It has also the microbes. Data from eighteen microbiomes existing in six CF been detected under anaerobic culturing conditions and via 16S patients with different health statuses were analyzed here rRNA gene surveys [33]. Typically R. dentocariosa was the main (Supporting Information S1; Table S2). species identified in these studies [34]. In addition, Bittar et al. [5] The metagenomic data showed that 15/18 (83%) sputa and Guss et al. [7] have characterized R. mucilaginosa as a ‘‘newly’’ contained R. mucilaginosa and 16/18 (89%) sputa contained the emerging CF pathogen. Even so, R. mucilaginosa is usually treated common CF pathogen P. aeruginosa. Although P. aeruginosa was as part of the normal oral microbiota in the clinical lab. As a result, present in a greater number of samples, its abundance was lower the presence of R. mucilaginosa in CF lungs may be under-reported than that of R. mucilaginosa in 11 of the 14 samples where these and the significance of infection is underestimated. species co-existed. Both species abundances ranged from 1% to Here we confirm that R. mucilaginosa is present and metabolically 62% (Figure 1). The relative percentages of these two opportu- active in the lungs of CF patients. Comparisons with a non-CF nistic pathogens varied between patients and within the same reference genome revealed the presence of unique R. mucilaginosa patient as their health status changed. The results show no obvious strains in each patient. A near-complete genome was reconstruct- pattern of synergy or competition between the two pathogens. ed from the metagenomic reads of one patient; comparison of A health status of ‘Ex’ (for exacerbation) indicates a stark decline these sequence data with a non-CF reference genome enabled the in lung function that is typically treated with intravenous identification of unique genomic features that may have facilitated antibiotics. Thus, between a health status of ‘Ex’ and ‘Tr’, adaptation to the lung environment. patients will have been given antibiotics in addition to those that are often prescribed as continued therapy. The abundance data in Results and Discussion Figure 1 indicate that these exacerbation-associated antibiotic treatments did little to permanently exclude R. mucilaginosa from Mutations in the CFTR locus affect proper ion transport in lung CF lung communities. Patients CF1, CF4, CF6, CF7, and CF8 all epithelial cells, impairing the clearance of mucus in the airways had appreciable abundances of R. mucilaginosa by the last sampling and encouraging microbial colonization and persistence. Until time point. Only the R. mucilaginosa population in CF5 did not recently, most laboratory and clinical microbiology only focused recover from antibiotic treatment by the last sampling time point; on a few pathogens, particularly P. aeruginosa. This ‘‘one mutation, however, as this

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