There is increasing evidence indicating that exposure to air pollutants may be associated with the onset of several respiratory diseases such as allergic airway disease and chronic obstructive pulmonary disorder (COPD). Many lung diseases demonstrate an outgrowth of pathogenic bacteria belonging to the Proteobacteria phylum, and the incidence of occurrence of these diseases is higher in heavily polluted regions. Within the human body, the lungs are among the first to be exposed to the harmful effects of inhaled pollutants and microbes. Research in the past few decades have expounded on the air-pollution-induced local and systemic inflammatory responses, but the involvement of the lung microbial communities has not yet been well-characterized. Lungs were historically considered to be sterile, but recent advances have demonstrated that the lower respiratory tract is replete with a wide variety of microorganisms - both in health and disease. Recent studies show that these lung microbes may play a significant role in modulating the immune environment by inducing IgA and mucus production. Air pollutants have previously been shown to alter intestinal bacterial populations that increase susceptibility to inflammatory diseases; however, to date, the effects of traffic-generated air pollutants on the resident microbial communities on the lungs have not been explored. The microbiome is influenced by several factors, including diet and environmental exposures. A large percentage of the Western world population consumes a high-fat (HF) diet which has resulted in the epidemic of obesity. Consumption of an HF diet has been shown to alter the intestinal microflora and increase baseline inflammation. We aimed to understand whether diet might also contribute to the alteration of the commensal lung microbiome, either alone or related to exposure. Thus, we investigated the hypothesis that exposure to air pollutants can alter the commensal lung microbiota, thereby promoting alterations in the lung's immune and inflammatory responses; in addition to determining whether these outcomes are exacerbated by a high fat-diet. We performed two studies with exposures to different components of air pollutant mixtures on C57Bl/6 mice placed on either a control (LF) diet or a high-fat (HF) diet. Our first exposure study was performed on C57Bl/6 mice with a mixture of gasoline and diesel engine emissions (ME: 30 µg PM/m3 gasoline engine emissions + 70 µg PM/m3 diesel engine emissions) or filtered air (FA) for 6h/d, 7 d/wk for 30 days. The ME study investigated the alterations in immunoglobulin A (IgA), IgG and IgM, and lung microbiota abundance and diversity. Our results revealed ME exposures alongside the HF diet causes a decrease in IgA and IgG when compared to FA controls, thereby decreasing airway barrier protection. This was accompanied by the expansion of bacteria within the Proteobacteria phylum and a decrease in the overall bacterial diversity and richness in the exposed vs. control groups. In our second study, we exposed C57Bl/6 mice to only the diesel exhaust particle component (35µg DEP, suspended in 35µl 0.9% sterile saline) or sterile saline only (control) twice a week for 30 days. We investigated immunoglobulin profiles by ELISA that revealed a significant increase in IgA and IgG in response to DEP. We also observed an increase in inflammatory tumor necrosis factor (TNF) - α, Interleukin (IL) -10, Toll-like receptors (TLR) - 2,4, nuclear factor kappa B (NF-κB) histologically and by RT-qPCR. Mucus production and collagen deposition within the lungs were also significantly elevated with DEP exposures. Microbial abundance determined quantitatively from the bronchoalveolar lavage fluid (BALF) by qPCR revealed an expansion of bacteria belonging to the Proteobacteria phylum in the DEP exposed groups on the HF diet. We also observed an increase in reactive oxygen and nitrogen species (ROS-RNS) products (nitrates), within the groups that revealed an expansion of Proteobacteria. These observations are most likely due to the unique metabolic capabilities of Proteobacteria to proliferate in inflammatory environments with excess nitrates. We assessed if treatments with probiotics could attenuate the DEP-induced inflammation by supplementing a separate group of study animals on the HF diet with 0.3 g/day of Winclove Ecologic® Barrier probiotics in their drinking water throughout the study. With probiotic treatments, we observed a significant decrease in ROS-RNS that was accompanied by complete elimination of Proteobacteria suggesting that in the absence of nitrates, the expansion of Proteobacteria is curbed effectively. We also observed a decrease in proinflammatory TNF-α and collagen deposition with probiotic treatments, and an increase in IgA levels within the BALF, suggesting that probiotics aid in balancing proinflammatory responses and enhance beneficial immune responses to efficiently mediate the DEP-induced inflammation. Both studies showed that air pollutants alter the immune defenses and contribute to lung microbial alterations with an expansion of Proteobacteria. The immunoglobulin profiles discordant between the two studies can be explained by the route and/or duration and composition of air pollutant exposure. Collectively these studies suggest that exposure to air pollutants alter immune responses and/or increase the availability of inflammatory by-products within the lungs that can enable the selective outgrowth of pathogenic bacteria. The observed detrimental outcomes are further exacerbated when coupled with the consumption of an HF diet. Importantly, these results may shed light on the missing link between air pollution-induced inflammation and bacterial expansion and also point to therapeutic alternatives to curb bacterial outgrowth in lung disease exacerbations observed in patient populations living and/or working in heavily polluted regions.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder involving a mutation in the CF transmembrane conductance regulator protein (CFTR), which causes dysfunctional transport of chloride ions across cell membranes. CF affects multiple body systems and a few of its symptoms include chronic cough, difficulty breathing, obstructive airway disease, bacterial pulmonary infections, maldigestion, malabsorption, pancreatitis, and male infertility. Until recently, treatment options have been limited to alleviating symptoms, but a new classification of drugs, CFTR modulators, provide an opportunity to slow the progression of the disease and improve clinical outcomes. The effect of CFTR modulators may be attributed to the reduction of persistently colonizing bacteria in CF lungs. Though, the effects of modulators on microbial communities colonizing the CF lung remains unknown, specifically with common respiratory pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. Particularly, previous CF studies have been limited in scope due to focusing on only one type of modulator and by using low-yield sequencing techniques. To address this gap, we seek to study the changes in CF respiratory pathogens of patients initiating CFTR modulator therapy at Nemours Hospital using long-read metagenomic sequencing (Oxford Nanopore) of longitudinally collected respiratory samples. We have optimized a protocol for host DNA depletion and microbial metagenomic sequencing to characterize the respiratory microbiome. This study focuses on utilizing these sequencing data to compare the microbiome among two healthy controls to pre-CFTR-treatment microbial communities of two recruited pediatric CF patients.

Récemment, les microbiotes pulmonaires bactériens d’un nombre très limité de patients atteints de mucoviscidose et de pneumopathies acquises sous ventilation mécanique (PAVM) ont été étudiés en utilisant l'amplification du gène 16S rDNA bactérien suivie par la construction de librairies de clones et différentes approches de séquençage. Ces études ont montré que la population microbienne de patients atteints de maladies respiratoires était plus diversifiée que prévue. Dans l'étude actuelle, nous utilisons une approche comparable pour identifier exhaustivement les agents pathogènes (bactéries, virus, et champignons) composant le microbiote pulmonaire associé aux pneumopathies développées en unités de réanimation. L'étude a inclus des patients admis en réanimation et présentant des formes de pneumopathies acquises sous ventilation mécanique (n = 106), de pneumopathies communautaires (n = 32), de pneumopathies nosocomiales sans ventilation mécanique (n = 22) et de pneumopathies d’aspiration (n = 25). Une cohorte de 25 patients admis en réanimation et ne présentant pas de symptômes de pneumopathie a été étudiée comme contrôle. Cette première partie du travail amènera ainsi à réaliser un catalogue exhaustif des agents de pneumopathies nosocomiales ; à connaître la prévalence des agents identifiés et d’identifier les co-infections fréquemment observées, et surtout à vérifier si ces agents peuvent être identifiés ou pas dans les prélèvements respiratoires profonds de patients non symptomatiques. Pour réaliser cette partie du travail, des séries de prélèvements, incluant des prélèvements de lavage broncho-alvéolaire (LBA), des prélèvements de sang et d'urine ont été étudiés. Ces prélèvements ont été testés par des moyens d’identification moléculaire moderne basés sur l’amplification de gènes conservés (gènes16S rDNA des bactéries et gène 18S rDNA des champignons) suivie par clonage et séquençage à grande échelle. D’autres pathogènes atypiques sont ciblés par des tests de PCR avec utilisation d’amorces spécifiques. Nous avons également inclus la culture, la co-culture d’amibes, la détection sérologique d'anticorps dirigés contre des agents sélectionnés et des tests d'antigène urinaire, afin de comparer ces tests de routine aux approches moléculaires. Comme résultats, les tests moléculaires nous ont permis d’identifier un vaste répertoire de 160 espèces bactériennes dont 73 n'ont jamais été précédemment rapportées à l’étiologie des pneumopathies. En outre, nous avons trouvé 37 phylotypes bactériens potentiellement nouveaux. Nous avons également identifié 24 espèces de champignons dont 6 n'ont pas été précédemment rapportées à l’étiologie des pneumopathies, 7 virus et étonnamment 6 espèces de plantes. De plus, certains agents pathogènes considérés comme typiques aux pneumopathies nosocomiales tels que Pseudomonas aeruginosa et des Streptococci ont été détectés chez les contrôles comme chez les patients. Cet étonnant résultat souligne l'existence d'un noyau de microbiote pulmonaire.Dans un deuxième travail, faisant suite aux travaux effectués dans notre laboratoire et qui ont pu mettre en évidence que 19% des pneumopathies nosocomiales étaient déterminées par des microorganismes associés aux amibes (MAAs) de l’eau préalablement ignorés ou négligés, nous avons utilisé un test d'immunofluorescence multiplexe pour tester la prévalence des anticorps contre les MAAs dans le sang de patients admis en réanimation et atteints de pneumopathies et la comparer à la prévalence au moment de l'admission. Comme résultat, nous démontrons que certains MAAs peuvent être plus fréquemment détectés après des épisodes de pneumopathies nosocomiales que lors de l’admission. En outre, la réponse immunitaire aux MAAs semble augmenter lorsque le séjour en réanimation est prolongé. Enfin, nous avons mis au point une stratégie de metagénomique pour tester les prélévements pour lesquels aucune étiologie n’a été retrouvée. [...] / Recently, bacterial microbiota from a limited number of patients with cystic fibrosis and ventilator-associated pneumonia (VAP) was studied using 16S rDNA gene amplification followed by clone libraries construction and sequencing. These studies have showed that the microbial population of patients with respiratory infections was more diverse than expected. In the current study, we use a similar approach to identify exhaustively the pathogens (bacteria, viruses, and fungi) comprising the microbiota associated with episodes of pneumonia developed in the intensive care units (ICU). Our study included patients admitted to ICUswith with episodes of ventilator-associated pneumonia (n = 106), community-acquired pneumonia (n = 32), nosocomial pneumonia without mechanical ventilation (n = 22) and aspiration pneumonia (n = 25). A cohort of 25 patients admitted to ICUs without symptoms of pneumonia were studied as controls. This first part of the work enables to prepare an exhaustive repertoire of nosocomial pneumonia pathogenes; to know the prevalence of the pathogens identified and to identify co-infections frequently observed, and especially to ascertain whether these agents can be identified or not in the respiratory samples of patients without symptoms of pneumonia. To perform this part of work, series of samples, including bronchoalveolar lavage (BAL) samples, blood samples and urine samples were collected. These samples were tested by means of modern molecular tools based on the amplification of conserved genes (bacterial 16S rDNA and fungal 18S rDNA genes), followed by highthroutput cloning and sequencing. The atypical pathogens are targeted by PCR tests using specific primers and probes. We also included culture, amoeba co-culture, serological detection of antibodies against selected agents and urinary antigen testing, to compare these routine tests to molecular approaches. Based on molecular testing, we identified a wide repertoire of 160 bacterial species of which 73 were never previously reported in pneumonia samples. Moreover, we found 37 putative new bacterial phylotypes. We also identified 24 fungal species of which 6 have not been previously reported in pneumonia, 7 viruses and surprisingly 6 plant species. Some pathogens considered being typical for ICU pneumonia such as Pseudomonas aeruginosa and Streptococcus species may be detected as commonly in controls as in pneumonia patients which strikingly highlight the existence of a core of pulmonary microbiota.In a second work, following previous works performed in our laboratory which were able to show that 19% of nosocomial pneumonia were determined by micro-organisms associated to amoebae (AAMs) previously ignored or neglected, we used a recent test based on multiplex serology to test for the prevalence of antibodies against the AAMs in the blood of patients admitted to ICU and developed episodes of pneumonia and compare it to the prevalence at the time of admission (controls). As a result, we demonstrate that some AAMs may be more frequently detected after episodes of nosocomial pneumonia than at the admission. In addition, the immune response to AAMS appears to increase when the ICU stay is prolonged.Finally, in order to explore samples for which no microbial aetiology was found, we have developed a subtractive hybridization metagenomic strategy and tested it on different clinical samples. The sensitivity of this strategy was also evaluated. We have demonstrated that our method, based on the detection of DNA and RNA of microorganisms in a single test, allows sensitive detection of different types of microorganisms.

La mucoviscidose est une maladie génétique autosomique causée par une mutation dans le gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Mon travail s’est décomposé en deux parties principales : d’une part j’ai réalisé une revue de la littérature sur l’analyse des génomes bactériens isolés de patients mucoviscidosiques comparativement aux génomes des mêmes espèces isolées dans d’autrescontextes et d’autre part j’ai analysé les génomes de trois espèces bactériennes (Microbacterium yannicii, Chryseobacterium oranimense et Haemophilus parahaemolyticus). L’analyse exhaustive des génomes bactériens issus de patients atteints de mucoviscidose a révélé une extraordinaire évolution de ces génomes en fonction du temps et des traitements reçus par ces patients qui témoigne de la capacité qu’ont ces bactéries à s’adapter à leur écosystème notamment par l’acquisition de nouveaux gènes par transfert latéral de gènes. Ce travail montre l’extraordinaire plasticité des génomes bactériens dans un milieu donné et à ce titre le poumon de patients atteints de mucoviscidose représente un modèle unique pour comprendre l’évolution des génomes bactériens. De plus, notre travail a permis d’identifier leurs mécanismes moléculaires de résistance aux antibiotiques. Les travaux à venir sur l’étude des métagénomes de prélèvements chez ces patients pourrait permettre de répondre à ces questions dans le futur. La découverte de nouvelles espèces et / ou émergentes va nous permettre d’avoir une image plus complète de la mucoviscidose qui pourrait conduire à une meilleure connaissance de la maladie et donc à une meilleure prise en charge thérapeutique. / Cystic fibrosis is an autosomal genetic disorder caused by a mutation in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. Pulmonary infection is the major problem faced by patients with cystic fibrosis. My work is divided into two main parts: first I made a review of the literature on the analysis of bacterial genomes isolated from CF patients compared to the genomes of the same species isolated in autrescontextes and other part I analyzed the genomes of three species of bacteria (Microbacterium yannicii, Chryseobacterium oranimense and Haemophilus parahaemolyticus). The comprehensive analysis of bacterial genomes from cystic fibrosis patients revealed an extraordinary evolution of these genomes with time and treatment received by these patients reflects the ability of these bacteria to adapt to their particular ecosystem the acquisition of new genes by lateral gene transfer. This work shows the extraordinary plasticity of bacterial genomes in a given environment and as the lungs of patients with cystic fibrosis represents a unique model for understanding the evolution of bacterial genomes. In addition, our work has identified their molecular mechanisms of resistance to antibiotics. Future work on the study of metagenomes sampling in these patients could help to answer these questions in the future. The discovery of new species and / or emerging will allow us to have a more complete picture of cystic fibrosis which could lead to a better understanding of the disease and thus a better therapeutic management.

Cystic Fibrosis (CF) is a genetic disorder, found with higher prevalence in the Caucasian population, affecting > 30,000 individuals in the United States and > 70,000 worldwide. Due to the astoundingly high rate of mortality among CF patients being attributed to respiratory failure brought on by chronic bacterial infections and subsequent airway inflammation, there has been a lot of focus on systematically analyzing CF lung airway communities. While it is observed traditionally that Pseudomonas aeruginosa is the most threatening and persistent CF colonizer due to high antibiotic resistance, recent studies have elicited the roles of other pathogens and it has been widely accepted the CF lung airway consists of a complex codependent community of bacteria, viruses, and fungi. To elucidate the interplay among the members of this community, within the constraint of lung uptake regime, I developed a community metabolic network model comprising of >380 metabolites obtained after modeling 39 most abundant bacterial genera across 279 sputum specimens collected from 79 individuals over 10 years from a study by LiPuma et. al. by 16S rRNA gene sequencing, accounting for >89% of reads across samples. The community metabolic model was contrasted with the 16S relative abundance data through standard data mining techniques employed for the analysis of multidimensional data. I further attempted to quantitatively analyze and elucidate the correlations among patient lung function, disease progression, community diversity, microbial compositions, and metabolic capabilities by standard classical hypothesis testing methods. Comparison through linear dimensionality reduction (PCA) of the 16S data and the model data revealed slightly higher variance explained by the model, indicating presence of relatively smaller number of metabolite-based than the 16S-based polymicrobial communities. A deeper analysis elucidated both the phenomena, consolidation of compositionally different communities due to metabolic closeness, as well as splitting of other communities into metabolically distinct clusters due to minor changes in composition and increase in diversity. Clustering of 16S-based relative abundance data and the model data revealed that the rare Burkholderia infections are metabolically distinct from other CF communities, and are heavily dominated by this genus. It was also reiterated that Achromobacter infections are highly resilient to treatment. Linear regression analysis between lung function and microbiota diversity revealed no strong correlation across the population, however, diversity was found to first increase and then subsequently decrease drastically with disease severity.