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Patients with chronic obstructive pulmonary disease (COPD) benefit from the immunomodulatory effect of azithromycin, but long-term administration may alter colonizing bacteria. Our goal was to identify changes in and during azithromycin treatment. Fifteen patients were followed while receiving prolonged azithromycin treatment (Hospital Universitari de Bellvitge, Spain). Four patients (P02, P08, P11, and P13) were persistently colonized by for at least 3 months and two (P04 and P11) by H. parainfluenzae. Isolates from these patients (53 and 18 H. parainfluenzae) were included to identify, by whole-genome sequencing, antimicrobial resistance changes and genetic variation accumulated during persistent colonization. All persistent lineages isolated before treatment were azithromycin-susceptible but developed resistance within the first months, apart from those belonging to P02, who discontinued the treatment. isolates from P08-ST107 acquired mutations in 23S rRNA, and those from P11-ST2480 and P13-ST165 had changes in L4 and L22. In H. parainfluenzae, P04 persistent isolates acquired changes in rlmC, and P11 carried genes encoding MefE/MsrD efflux pumps in an integrative conjugative element, which was also identified in P11-ST147. Other genetic variation occurred in genes associated with cell wall and inorganic ion metabolism. Persistent strains all showed changes in licA and hgpB genes. Other genes (lex1, lic3A, hgpC, and fadL) had variation in multiple lineages. Furthermore, persistent strains showed loss, acquisition, or genetic changes in prophage-associated regions. Long-term azithromycin therapy results in macrolide resistance, as well as genetic changes that likely favor bacterial adaptation during persistent respiratory colonization. IMPORTANCE The immunomodulatory properties of azithromycin reduce the frequency of exacerbations and improve the quality of life of COPD patients. However, long-term administration may alter the respiratory microbiota, such as , an opportunistic respiratory colonizing bacteria that play an important role in exacerbations. This study contributes to a better understanding of COPD progression by characterizing the clinical evolution of in a cohort of patients with prolonged azithromycin treatment. The emergence of macrolide resistance during the first months, combined with the role of as a reservoir and source of resistance dissemination, is a cause for concern that may lead to therapeutic failure. Furthermore, genetic variations in cell wall and inorganic ion metabolism coding genes likely favor bacterial adaptation to host selective pressures. Therefore, the bacterial pathoadaptive evolution in these severe COPD patients raise our awareness of the possible spread of macrolide resistance and selection of host-adapted clones.

BackgroundAnalysis of microbiome data involves identifying co-occurring groups of taxa associated with sample features of interest (e.g., disease state). But elucidating key associations is often difficult since microbiome data are compositional, high dimensional, and sparse. Also, the configuration of co-occurring taxa may represent overlapping subcommunities that contribute to, for example, host status. Preserving the configuration of co-occurring microbes rather than detecting specific indicator species is more likely to facilitate biologically meaningful interpretations. In addition, analyses that utilize both taxonomic and predicted functional abundances typically independently characterize the taxonomic and functional profiles before linking them to sample information. This prevents investigators from identifying the specific functional components associate with which subsets of co-occurring taxa.ResultsWe provide an approach to explore co-occurring taxa using “topics” generated via a topic model and then link these topics to specific sample classes (e.g., diseased versus healthy). Rather than inferring predicted functional content independently from taxonomic abundances, we instead focus on inference of functional content within topics, which we parse by estimating pathway-topic interactions through a multilevel, fully Bayesian regression model. We apply our methods to two large publically available 16S amplicon sequencing datasets: an inflammatory bowel disease (IBD) dataset from Gevers et al. and data from the American Gut (AG) project. When applied to the Gevers et al. IBD study, we demonstrate that a topic highly associated with Crohn’s disease (CD) diagnosis is (1) dominated by a cluster of bacteria known to be linked with CD and (2) uniquely enriched for a subset of lipopolysaccharide (LPS) synthesis genes. In the AG data, our approach found that individuals with plant-based diets were enriched with Lachnospiraceae, Roseburia, Blautia, and Ruminococcaceae, as well as fluorobenzoate degradation pathways, whereas pathways involved in LPS biosynthesis were depleted.ConclusionsWe introduce an approach for uncovering latent thematic structure in the context of sample features for 16S rRNA surveys. Using our topic-model approach, investigators can (1) capture groups of co-occurring taxa termed topics, (2) uncover within-topic functional potential, and (3) identify gene sets that may guide future inquiry. These methods have been implemented in a freely available R package https://github.com/EESI/themetagenomics.

Trabajo presentado en el 7th European Conference on Prokaryotic and Fungal Genomics (ProkaGENOMICS), celebrado en Gottingen (Alemania), del 19 al 22 de septiembre de 2017), Introduction. The respiratory pathogen nontypeable Haemophilus influenzae (NTHi) persistently infects a significant proportion of Chronic Obstructive Pulmonary Disease (COPD) patients’ lower airways, and is usually associated with acute exacerbations of this chronic disease. Objectives. Based on the hypothesis that the COPD may be a unique niche favouring NTHi adaptation, this work aimed to unravel NTHi evolutionary dynamics in the COPD lungs over time, with the goal of identifying genomic patho-adaptive features.

Póster presentado en la XI Reunión del Grupo de Microbiología Molecular de la Sociedad Española de Microbiología (SEM), celebrada en Sevilla del 6 al 8 de septiembre de 2016., Haemophilus influenzae (Hi) es un patógeno oportunista causante de infección respiratoria crónica y de exacerbación periódica en pacientes con patologías respiratorias crónicas asociadas al tabaquismo como la Enfermedad Pulmonar Obstructiva Crónica (EPOC). El pulmón EPOC es un nicho ecológico complejo, con niveles elevados de inflamación, mucosidad, enfisema y fibrosis, expuesto a humo de tabaco y a fármacos. Todo ello genera una presión selectiva que modula la evolución, adaptación y persistencia pulmonar de Hi. Para entender la dinámica de la evolución pato-adaptativa de Hi en el pulmón EPOC, se dispuso de una colección prospectiva longitudinal de 86 aislados de Hi procedentes de muestras de esputo de 12 pacientes EPOC en exacerbación, generada entre los años 2005 y 2014. Para identificar rasgos fenotípicos y genotípicos de pato-adaptación, se analizaron crecimiento bacteriano, auto-agregación y resistencia a la muerte por péptidos antimicrobianos, y se realizó secuenciación genómica de la colección de aislados. El análisis bioinformático del material secuenciado estableció la existencia 20 series de aislados clonales. El análisis de polimorfismos en ORFs en las 20 series clonales identificó 231 SNPs no sinónimos en 180 genes, y reveló la existencia de 15 genes que presentan rasgos de evolución paralela (SNPs en varias series clonales aisladas de distintos pacientes). Entre ellos, 6 series clonales mostraron SNPs en el gen fadL (transiciones e inserciones), generando 12 variantes alélicas con cambios en loops extracelulares y 9 variantes alélicas truncadas. FadL es una proteína con función dual, (i) transportador de ácidos grasos exógenos de cadena larga (LCFA), y (ii) ligando del receptor eucariota Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 (hCEACAM1) que facilita la invasión epitelial por Hi. El significado biológico de la variabilidad alélica de FadL en ambas categorías funcionales se analizó mediante el diseño y utilización de (i) un medio mínimo con ácido oleico como fuente de carbono, y (ii) un ensayo de interacción con CEACAM1 mediante línea celular HeLa transfectada con hCEACAM1. En conjunto, este trabajo constituye el primer estudio evolutivo de un patógeno bacteriano adaptado al pulmón EPOC, e identifica rasgos de pato-adaptación y potenciales dianas que serán explotadas en el desarrollo de nuevos antimicrobianos.

The PLOS Pathogens Staff. Notice of Republication.This article was republished on May 13, 2016, to correct the title which was incorrectly published as “Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza”. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference., This work was supported by National Institutes of Health Ruth Kirschstein postdoctoral fellowship F32AI084427 (to JCM); a Canadian Institute of Health Research operating grant (to RJR); Genome British Columbia grant SOF122 (to RJR and JCM); the Faculty of Pharmaceutical Sciences, Canadian Foundation for Innovation (to CN); National Institutes of Heath R01 grant DC002873 (to JWSG); and (to JG) grants from Ministerio Economía y Competitividad-MINECO SAF2012-31166 and SAF2015-66520-R, Dpto. Salud Gobierno Navarra 359/2012 and Ministerio de Educación PRX12/00191.

Póster presentado en la International Pasteurellaceae Conference, celebrada en Prato (Italia) del 13 al 16 de mayo de 2014, Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

Trabajo presentado en el XXIV Congreso de la Sociedad Española de Microbiología, celebrado en L’Hospitalet de Llobregat (Barcelona) del 10 al 13 de julio de 2013., Se ha empleado la transformación natural como estrategia de cribado de genes/agrupamientos genéticos implicados en la invasión del epitelio respiratorio humano por el patógeno respiratorio H. Influenzae. Este trabajo constituye la primera búsqueda sistemática de invasinas de H. Influenzae mediante un cribado positivo de ganancia de función., MINECO SAF2012-31168; Departamento de Salud Gobierno Foral de Navarra; CIBERES, (España); Universidad British Columbia-UBC, (Canadá).