Role of Pseudomonas Aeruginosa Biofilms in Exacerbations in Patients With Bronchiectasis With and Without Chronic Obstructive Pulmonary Disease

Exacerbations, in particular during chronic Pseudomonas aeruginosa (PA) infection, are very important in the prognosis of patients with non-cystic fibrosis bronchiectasis (BE). In Cystic Fibrosis patients, PA biofilms are associated with chronic respiratory infections and are the primary cause of their increased morbidity and mortality. However, the presence and role in exacerbations of PA biofilms, microbiome dysbiosis and inflammatory biomarkers has not been studied in depth in BE patients. Our aim is to determine the association between PA chronic infection and its biofilms with the number of exacerbations in the next year (primary outcome), time until next exacerbation, quality of life, FEV1 and inflammatory biomarkers (secondary outcomes) in BE patients with or without chronic obstructive pulmonary disease (COPD). The investigators will include and follow up during 12 months post study inclusion, 48 patients with BE and 48 with BE-COPD, with a positive sputum culture of PA. During stability and follow up (and in each exacerbation) The investigators will collect 4 sputum, 4 serum samples, perform spirometry, and quality of life tests every three months. For the biomarkers subproject, 4 additional serum samples will be collected at: exacerbation, 3-5 days after treatment, at 30 days and three months post-exacerbation. Biomarkers will be measured by commercial kits and Luminex. The investigators will quantify PA colony forming units (CFU)/mL, their resistance pattern, their mutation frequency and isolate mucoid and non-mucoid colonies. In each sputum, the investigators will analyze by Confocal Laser Scanning Microscopy (CLSM) and Fluorescent in situ Hybridizatrion (FISH) PA biofilms, their size, bacterial density and their in situ growth rate. Specific serum antibodies against PA will be determined through Crossed Immunoelectrophoresis. In addition, the investigators will indentify potential respiratory microbiome and gene expression patterns predictive for exacerbations, or with a protective role against chronic PA infection, as well as their association with biofilms. Microbiome analysis will be performed through the Illumina Miseq platform. Finally, the investigators will explore the antimicrobial activity of novel combinations of antibiotics against PA, both in in vitro planktonic cultures and in a biofilm model, and will include testing of antibiotic-containing alginate nanoparticles.

This project will study the association between chronic PA infection in the airways and the formation of biofilms in the following patient populations: 1-bronchiectasis not associated with cystic fibrosis (BQ) and 2-bronchiectasis with chronic obstructive pulmonary disease (BQ-EPOC). The primary objective is to determine the association between chronic infection by PA, presence of biofilms of PA and the worst evolution of the disease measured mainly by the number of exacerbations per year (primary outcome) days until the next exacerbation, quality of Life and Forced Expiratory Volume (FEV1), and inflammatory biomarkers (secondary outcomes). For this, these parameters will be compared in patients with chronic infection by PA and with the presence of biofilms of PA in the sputum (identified by FISH-CLSM) vs those with PA without chronic infection and without biofilms (control negative) in each study population (BE and BE-COPD). The investigators will analyze, to the recruitment and sequentially (3 determinations), various aspects of the biofilms that are detailed in the methodology. In addition, by comparing the microbiome before, during and after each exacerbation, the investigators will try to identify those microbial patterns that entail a higher risk of exacerbation, those with a protective role against chronic PA infection and its relationship with biofilms.

The diagnosis of biofilms is not yet routinely implemented in clinical practice for Chronic Obstructive Pulmonary Disease (COPD) and NonCF Bronchiectasis (BE) infections. The first guidelines for the diagnosis and treatment of biofilm infections were published in 2015 and included many biofilm-associated infections, but not COPD or BE. There are in fact very few studies on the presence of PA biofilms in these populations and on their potential role in exacerbations. However, there is sound scientific evidence that PA biofilms are associated with chronic respiratory infections in Cystic Fibrosis (CF) patients, and are the primary cause of their increased morbidity and mortality. It has been reported that during chronic PA lung infection in CF, PA adapts to different niches in the lungs. Both mucoid (biofilms) and non-mucoid (planktonic) bacteria are present in sputum. Cells with higher mutation rate develop multi-resistance to many antibiotics and their in situ growth rate is lower, since bacteria decrease their metabolism conditioned by high polymorphonuclear oxygen consumption, As yet, however, none of this has been described in patients with BE; in fact, there are very few studies on the presence of PA biofilms in these populations, and their role in the exacerbations has not been demonstrated. In addition, there is little evidence of the association of the microbiome dysbiosis with chronic colonization by PA and during exacerbations.

Chronic colonization by PA is present in 12-27% of patients with BE and BE-COPD, and entails a worse prognosis (a 3-fold increase in mortality risk) and up to a 7-fold increase in the risk of hospital admission, with an average of one additional exacerbation per patient and per year. In these patients, eradication of PA is difficult, despite the fact that an adequate antimicrobial treatment reduces the bacterial load and the number of exacerbations and improves lung function and quality of life. One of the main challenges in patients with BE is the eradication of PA, especially during the early stages of colonization by this pathogen. In this situation, aggressive antibiotic treatment is recommended. However, this eradication fails in a notable percentage of cases. In addition to the increase in multiresistance in PA, even against quinolones, its ability to produce biofilms and survive in them is one of the reasons for the failed eradication. Although the presence of PA in sputum is a factor of poor prognosis in BE, biofilms have not yet been identified and characterized (i.e., their presence, size, amount of alginate and metabolic status) in sputum of these populations. Recently, the presence of biofilms in bronchoalveolar lavage (BAL) samples has been described by confocal laser scanning microscopy (CLSM) in pediatric patients with BE, even in those samples in which the culture was negative. This finding draws attention to the lack of studies on biofilm in BE and the importance of improving diagnostic tools for detection in respiratory samples, especially in patients with recurrent exacerbations despite antibiotic treatment.

Between 30 and 50% of patients with moderate or severe COPD have BE. Its prevalence increases with the severity of COPD, while 5-10% of patients with BE have COPD. Patients with COPD and BE form a clinical group with its own characteristics (increased sputum production and purulence, greater dyspnea and number of exacerbations), worse prognosis, possible therapeutic implications and higher mortality. Although the causes of exacerbations are not yet well understood, bacterial infections are thought to be involved in approximately one-half of cases PA is one of the most frequent pathogens isolated from these exacerbations and is associated with higher mortality. PA from sputum of chronically infected COPD patients tends to be less cytotoxic and motile and produces more biofilm than PA from blood samples. Therefore, some authors suggest that COPD, BE and CF present similar patterns of infection and evolution. Following this reasoning, our hypothesis is that PA biofilms could be one of the pathogenic mechanisms associated with both the persistence of infections and the frequency and severity of exacerbations in patients with BE (with or without COPD). However, further research using the recommendations of the current guidelines is needed to confirm this hypothesis.

Some authors who have studied microbial dysbiosis in COPD exacerbations have seen that, along with eosinophilic inflammation, it is associated with more severe exacerbations and a greater fall in lung function. Longitudinal studies of the microbiome in the sputum of patients with BE suggest that the management of chronic bronchial infection can be improved with a therapy that is specific for its microbiome, taking into account the burden of the pathogen, the stability of the community, and acute and chronic community responses to antibiotics. However, the fluctuations in the microbiome and its predictive role in the exacerbations of bronchiectasis have not yet been studied in depth. The study of variations in the microbiome before and during the exacerbation could reveal novel therapeutic targets that may have an impact on the management of patients. The results could help to determine the need to administer antimicrobial treatment in patients with negative microbiological cultures during the exacerbation. There is evidence that a balanced oral flora could have a protective role against the colonization of saliva by PA; however, a microbiome pattern with a protective effect on the upper respiratory tract against PA in BE patients and its relationship with PA biofilms has not yet been described.

Simultaneously, systemic biomarkers and inflammatory cytokines determine the potential importance of chronic PA infection and the productions of biofilms. Systemic CRP has been shown to be correlated with higher BE scores such as FACED and BSI. However, there are no longitudinal studies evaluating the intensity and duration of systemic inflammation caused by chronic PA infection.

The spread of antibiotic resistance and increasing prevalence of biofilm-associated infections is driving demand for new means to treat such difficult to eradicate bacterial infections. Recently, the WHO published a list of bacteria for with research on new antibiotics is urgently needed. Drug discovery and development is a tedious, long and costly endeavour that can take hundreds of millions EUR and 10-15 years from the bench discovery to the bedside. Chronic PA infections associated to BE are typically treated with combinations of up to three antimicrobials. Despite periodic courses of multidrug therapy, effective cure for these diseases is below optimal levels. It is clear then that new therapeutic options are needed to improve positive outcomes in these patients. Our approach focuses on the concept of synergy, i.e. identifying synergistic combinations of antibiotics, being together more effective that when used alone. The investigators aim to systematically evaluate the synergistic interactions of currently used antibiotics and explore the introduction of other clinically approved antibiotics. Since their pharmacology and toxicity has been already well documented, any potential synergistic antibiotic combination could progress to clinical trials at fraction of the standard development cost and in much shorter period of time. Along with this, the nanocarrier-mediated delivery strategy has been suggested as a promising approach in treatment of drug resistant infections. Nanotechnology may provide an innovative platform for addressing the challenge of PA chronic infections associated to bronchiectasis, with potential to manage difficult to treat infections involving multidrug-resistant (MDR) bacteria. In particular, polysaccharide (PS)-based nanocarriers are ideal vehicles for encapsulation of antimicrobials and alginate is an ideal carrier for pulmonary administration due to its mucoadhesive properties.

Eligibility Criteria:
Inclusion Criteria:

* BE criteria (with and without COPD)
* Isolation of PA in sputum in the stable phase. \* A prospective screening of BQ patients in outpatient consultations will be conducted to prospectively detect those with isolation of PA in the sputum.

Exclusion Criteria:

* CF
* Immunosuppression (primary and secondary, with the exception of cases of IgG deficiency in stable substitution treatment)
* Sarcoidosis
* Tuberculosis, active infection by nontuberculous mycobacteria
* Diffuse interstitial lung disease
* Altered state of consciousness or disability to understand the study and perform the tests provided by it, involving the patient in another intervention study (clinical trials).

  * Patients with CF are excluded because the role of PA biofilms in CF has been extensively studied, as argued in the background of the present proposal and the selected literature. It is also a totally different disease from the one we intend to study, with patients of much younger ages. Finally, this is a very vulnerable population in which it would be very difficult to obtain all the samples sequentially.