Background: The respiratory microbiome is central to health and disease, requiring accurate sequencing for characterization. We compared Illumina NextSeq and Oxford Nanopore Technologies (ONT) for 16S rRNA profiling. Illumina produces short, highly accurate reads (∼300 bp) suited for genus-level classification but limited at species resolution. ONT generates full-length reads (∼1,500 bp), enabling higher resolution but with historically lower accuracy. Methods: We sequenced 34 respiratory samples from ventilator-associated pneumonia (VAP) patients (n=20) and a swine model (n=14) using Illumina (V3–V4) and ONT (whole gene). Data were processed with nf-core/ampliseq or epi2me-labs/wf-16s using the Silva 138.1 database. Diversity metrics and taxonomic profiles were compared. Results: Alpha diversity showed greater species richness with Illumina, while evenness was comparable. Beta diversity differences were significant in pigs but not humans, suggesting stronger platform effects in complex microbiomes. Illumina detected a broader taxonomic range, while ONT resolved dominant species. ANCOM-BC2 revealed platform-specific biases: ONT overrepresented Enterococcus and Klebsiella but underrepresented Prevotella and Bacteroides. Conclusions: Illumina is suited for broad surveys, while ONT excels in species-level resolution and real-time use. Hybrid approaches may best leverage both technologies in clinical and preclinical studies.

Prone positioning improves survival in patients with acute respiratory distress syndrome (ARDS) by reducing ventilator-induced lung injury and enhancing ventilation–perfusion matching. Whether these physiological benefits translate to patients supported with veno-venous extracorporeal membrane oxygenation (VV-ECMO) remains uncertain. This study compared the effects of prone versus supine positioning on the wet-to-dry lung weight ratio, gas exchange, respiratory mechanics, electrical impedance tomography, histopathology, microbiology, hemodynamics, and extracorporeal circuit function in pigs with severe ARDS undergoing VV-ECMO. Pigs with severe ARDS were placed on VV-ECMO according to EOLIA criteria and then randomized to either prone or supine positioning for 48 h, while receiving an ultraprotective ventilation strategy. Futility analyses were performed at half of the planned sample size using conditional power calculations, with early termination criteria set at < 10

The diagnostic performance of the BioFire® FilmArray® Pneumonia Panel Plus (FAPP) compared to standard microbial culture (SMC) during bronchiectasis (BE) exacerbations is unknown. To compare the microbiological diagnostic performance between FAPP and SMC during BE exacerbations. A prospective observational study was conducted in adults with a BE exacerbation at the Hospital Clinic of Barcelona (Spain) June 2020 to April 2022. All sputum samples underwent processing using both the FAPP and SMC (n = 109) but we focused in good quality samples (n = 73). The FAPP detected pathogens in a higher percentage (n = 64, 88

The respiratory microbiome plays a crucial role in health and disease, necessitating accurate characterization through high-throughput sequencing technologies. This study provides a comparative analysis of Illumina NextSeq and Oxford Nanopore Technologies (ONT) sequencing platforms for 16 S rRNA profiling of respiratory microbial communities. Illumina sequencing, known for its high accuracy and short-read lengths (~ 300 bp), is widely used for genus-level microbial classification but struggles with species-level resolution due to its limited read length. In contrast, ONT generates full-length 16 S rRNA reads (~ 1,500 bp), enabling higher taxonomic resolution but historically exhibiting higher error rates (5-15%). Analysis of alpha and beta diversity indicated that Illumina captured greater species richness, while community evenness remained comparable between platforms. Beta diversity differences were significant in pig samples but not in human samples, suggesting that sequencing platform effects are more pronounced in complex microbiomes. Taxonomic profiling revealed that Illumina detected a broader range of taxa, while ONT exhibited improved resolution for dominant bacterial species. ANCOM-BC2 differential abundance analysis highlighted platform-specific biases, with ONT overrepresenting certain taxa (e.g., Enterococcus, Klebsiella) while underrepresenting others (e.g., Prevotella, Bacteroides). These findings emphasize that platform selection should align with study Objective: Illumina is ideal for broad microbial surveys, whereas ONT excels in species-level resolution and real-time applications. Future research should explore hybrid sequencing approaches to leverage the strengths of both technologies, thereby improving microbiome characterization in both clinical and preclinical settings.

BACKGROUND:In patients with non-cystic fibrosis bronchiectasis (BE) Pseudomonas aeruginosa (PA) has been recently associated with low rather than high number of exacerbations without distinguishing chronic versus intermittent infection. The aim of our study was to determine whether the intermittent or chronic stage of P. aeruginosa (PA) infection is associated with the rate of exacerbations, quality of life and respiratory microbiome biodiversity after a one-year follow-up. METHODS:We conducted a longitudinal study, with 1-year follow-up, in patients with BE intermittently or chronically infected by PA involving sequential (3-monthly) measurements of microbiological (cultures, PA load, phenotype and biofilms presence) immunological (Serum IgGs against P. aeruginosa were measured by ELISA immunoassay) and clinical variables (Quality-of-Life and the number exacerbations). Additionaly, 16S sequencing was performed on a MiSeq Platform and compared between chronically infected patients with the mucoid PA versus intermittently infected patients with the non-mucoid PA. RESULTS:We collected 235 sputa and 262 serum samples from 80 BE patients, 61 with chronic and 19 with intermittent PA infection. Chronically compared to intermittently. Presented reduced quality of life but less hospitalized exacerbations after 1-year follow-up. Chronically infected patients presented reduced sputum biodiversity and higher systemic IgGs against P. aeruginosa levels that were associated to decreased number of hospitalized exacerbations. CONCLUSIONS:The assessment of Chronic versus intermittent P. aeruginosa infection has clinical implications such as quality of life, rate of hospitalized exacerbations and lung microbiome biodiversity. The distinction of these two phenotypes is easy to perform in clinical practice. TRIAL REGISTRATION:NCT04803695.

COVID-19 vaccination strategies are already available almost worldwide. However, it is also crucial to develop new therapeutic approaches, especially for vulnerable populations that may not fully respond to vaccination, such as the immunocompromised. In this project, we predicted 25 B-cell epitopes in silico in the SARS-CoV-2 Spike (S) protein and screened these against serum and plasma samples from 509 COVID-19 convalescent patients. The aim was to identify those epitopes with the highest IgG reactivity to produce monoclonal antibodies against them for COVID-19 treatment. We implemented Brewpitopes, a computational pipeline based on B-cell epitope prediction tools, such as BepiPred v2.0 and Discotope v2.0, and a series of antibody-epitope accessibility filters. We mapped the SARS-CoV-2 S protein epitopes most likely to be recognised by human neutralizing antibodies. Linear and structural epitope predictions were included and were further refined considering accessibility factors influencing their binding to antibodies like glycosylation status, localization in the viral membrane and accessibility on the 3D-surface of S. Blood samples were collected from 509 COVID-19 patients prospectively recruited 35 days after symptoms initiation, positive RT-qPCR or hospital/ICU discharge. Presence of IgG against SARS-CoV-2 was confirmed by lateral flow immunoassays. Epitopes immunogenicity was tested through the analysis of IgG levels and seropositivity in the convalescent serum and plasma samples and 126 pre-pandemic negative controls by Luminex to identify those with the highest reactivity. The seropositivity cut-offs for each epitope were calculated using a set of 126 pre-pandemic samples as negative controls (NC). Twenty-five SARS-CoV-2 S epitopes were predicted in silico as potentially the most immunogenic. These were synthesized and tested in a multiplex immunoassay against sera/plasmas from convalescent COVID-19 patients (5.7% asymptomatic, 35.6% mild, 13.8% moderate, 23% severe and 22% unknown because of anonymous donation). Among the 25 epitopes tested, 3 exhibited significantly higher IgG reactivity compared to the rest. The proportion of seropositive patients towards these 3 epitopes, based on median fluorescence intensity (MFI or Log10 MFI) above that from NC, ranged between 11 and 48%. Two out of the three most immunogenic epitopes were scaled up, resulting in the generation of two monoclonal antibodies (mAbs). These two mAbs exhibited comparable levels of S protein affinity to commercialized mAbs. Our data shows that the candidate S epitopes predicted in silico are recognised by IgG present in convalescent serum and plasma. This evidence suggests that our computational and experimental pipeline is able to yield immunogenic epitopes against SARS-CoV-2 S. These epitopes are suitable for the development of novel antibodies for preventive or therapeutic approaches against COVID-19.

The study of key Pseudomonas aeruginosa (PA) virulence factors, the molecular basis of pathogenicity, as well as their correlation with the immune response during exacerbations in patients with non-cystic fibrosis bronchiectasis can help to identify novel targets and biomarkers for clinical management. The objective was to compare P. aeruginosa virulence and the patient’s immune response during stable phases and exacerbations of bronchiectasis. We used polymerase chain reaction (PCR) and real-time quantitative PCR (qRT-PCR) to perform molecular characterization of the genomic islands and virulence genes present in 42 P. aeruginosa strains obtained from the sputum of patients with bronchiectasis during stability and exacerbations. Immunoglobulin (Ig) and interleukin (IL) levels in 32 serum samples were analyze by ELISA and Luminex assay. A greater presence of the conjugative element pKLC102, specific virulence genes (exoS, exoY) and pyoverdine production characterize the P. aeruginosa strains obtained during exacerbations. The expression levels of type III secretion system (exoS, exoY) showed an important role in the humoral immune response during exacerbations. Exacerbations were associated with high levels of IL-6. The presence of specific genomic islands, virulence genes, and increased IL-6 levels provide an accurate characterization on bronchiectasis exacerbations. These targets could be useful in the prevention, management and treatment of these exacerbations.

Background. Ventilator-associated pneumonia (VAP) is the second most common hospital-acquired infection and is associated with high morbidity and mortality. The insertion of the endotracheal tube (ETT) compromises the host’s bronchial defenses and facilitates the formation of microbial biofilms and VAP. Although antibiotics remain the cornerstone treatment, their efficacy is often compromised by poor biofilm penetration and the prevalence of multidrug-resistant bacteria. This reveals the need to develop novel non-antibiotic therapeutic strategies. We engineered a non-pathogenic strain of the pulmonary bacteria Mycoplasma pneumoniae CV2 to express anti-biofilm and bactericidal enzymes (VAP platform). The therapeutic effect was validated in a murine model of Pseudomonas aeruginosa infection and ex vivo in ETTs of patients with P. aeruginosa -induced VAP. Methods. We evaluated the safety of an improved version of CV2 (CV8, Mycochassis) in a miniature pig model, and its efficacy with the VAP therapeutic platform (CV8\_VAP) in a piglet model of P. aeruginosa -induced VAP. CV8\_VAP was administered via intralobe instillation or nebulization. We assessed the platform’s ability to degrade biofilms formed on ETTs in vivo and its impact on the airway microbiota. We evaluated bacterial load in the lungs, inflammatory responses, histopathological and the clinical outcomes to provide a comprehensive characterization of therapeutic efficacy and safety. Findings. VAP platform reduced biofilm thickness and P. aeruginosa load in ETTs and lungs, with the nebulized route showing enhanced efficacy. CV8_VAP contributed on microbiome diversity restoration. Inflammatory markers in the lung were markedly decreased, suggesting a local immunomodulatory effect. Interpretation. These findings support that CV8_VAP as a promising, non-antibiotic therapeutic strategy for treating VAP caused by P. aeruginosa . Funding. The project that gave rise to these results has received funding from “La Caixa” Foundation (HR18-00058), and the European Research Council under the European Union’s Horizon 2020 research and innovation program (670216). ### Competing Interest Statement The authors have declared no competing interest. La Caixa Foundation, HR18-00058 European Research Council under the European Union’s Horizon 2020 research and innovation program, 670216

Abstract Background Streptococcus pneumoniae, a primary cause of community-acquired pneumonia (CAP), is typically treated with β-lactams and macrolides or quinolones. Corticosteroids are now recommended as adjunctive therapy in severe CAP to improve outcomes. In this prospective randomized animal study, we evaluated the bactericidal efficacy of various antibiotic regimens combined with corticosteroids using a porcine pneumococcal pneumonia model. Results In 30 White-Landrace female pigs, pneumonia was induced by intrabronchial inoculation of macrolide-resistant S. pneumoniae 19A isolate. Animals were randomized to receive saline, ceftriaxone (CRO) with levofloxacin (LVX), CRO with azithromycin (AZM), or combinations of these with methylprednisolone (MP). The primary outcome, S. pneumoniae concentrations in lung tissue after 48 h of treatment, showed that the CRO + LVX, CRO + AZM, CRO + LVX + MP, and CRO + AZM + MP groups were equally effective in reducing bacterial load. However, complete bacterial eradication from lung tissue was achieved only in the CRO + AZM + MP group. Secondary outcomes, including bacterial burden in tracheal aspirates and bronchoalveolar lavage (BAL) samples, showed similar bactericidal activity across all treatment groups. The CRO + AZM + MP group demonstrated the most controlled inflammatory response, achieving baseline levels of inflammation, while other groups exhibited elevated inflammatory markers. Conclusions Despite using a macrolide-resistant S. pneumoniae isolate, the combination of CRO, AZM, and MP achieves similar or even superior results compared to other antibiotic combinations. This regimen provides both bactericidal and immunomodulatory benefits, suggesting its effectiveness in treating macrolide-resistant S. pneumoniae pneumonia.

Objectives: To evaluate the expression dynamics of biofilm genes in methicillin-resistant Staphylococcus aureus (MRSA) retrieved from endotracheal tubes (ETT) and to determine how gene regulation is attenuated in vitro where host–environmental factors are no longer present. Methods: Biofilm was grown (24 h) in tryptic broth soy plus 0.25% glucose for a clinical MRSA isolate in planktonic state and after sessile growth named ETT-MRSA (S2, S3, S4, S5, S6, S7). Gene expression of five biofilm-related genes (icaC, clfB, ebps, fnbB, and RNA III) was assessed consecutively from day 1 to day 4 after ETT growth through real-time PCR. 16S rRNA was used as a control. Results: The MRSA isolates retrieved from ETT were capable of producing biofilms dependent on ica. The gene expression dynamics of ETT-MRSA changed progressively compared to planktonic MRSA gene expression under both ambient air (p < 0.001) and ambient air with 5% CO2 (p < 0.001). Dynamic assessment of icaC expression in both atmospheric conditions showed progressive downregulation in vitro compared to in vivo ETT biofilms. The expression patterns of clfB and ebps genes were similar to icaC. In contrast, the expression of the RNA III gene showed progressive upregulation from day 1 to day 4 (p < 0.001). Conclusions: MRSA loses its biofilm gene expression in vitro, by adaptive features across multiple generations, as evidenced by the progressive downregulation of icaC and upregulation of RNA III. These findings underscore the significance of host–environment dependence in regulating bacterial biofilm genes, highlighting its importance in diagnostics. Bacterial strains lose their host-specific characteristics as they are cultured in vitro.

Background: Sputum colour is a useful clinical tool in non-cystic fibrosis bronchiectasis (BE) (Murray et al. 2009), but its ability to capture the quality of life (QoL) has barely been explored. Thus, the aim of this study was to assess the relationship between sputum colour and QoL in BE patients colonized by Pseudomonas aeruginosa (PA). Methods: In this cross-sectional observational study, sputum colour was recorded following Murray's scale, and QoL was evaluated by the Quality of Life Questionnaire-Bronchiectasis v3.1 (QOL-B) (Quittner et al. 2015). Fisher9s exact test and Kruskal-Wallis were used in the univariate analysis, whilst ordinal logistic regression was applied in the multivariate. Results: 57 patients were included; 23 women, 67,53±16,96 years. Purulent sputum showed significantly worse QoL in 3 domains compared to those mucopurulent (Table 1, Fig.1). Further, sputum colour and culture were significantly associated (mixed flora vs. non-mucoid PA vs. mucoid PA vs. other pathogenic microorganisms, p=0,04). No significant differences were found based on gender, disease phase (acute vs. stable), BSI, years of PA colonization, and lung function. These results were kept when considering sputum colour and culture concurrently. Conclusions: Sputum colour is a validated and simple tool that could be useful in BE to quickly identify vulnerable patients with a worsening of QoL, regardless of the phase of the disease.

Introduction: We recently demonstrated the potential of engineered live bacteria against Pseudomonasaeruginosaex vivo biofilms grown in endotracheal tubes (ETT) from patients with ventilator-associated pneumonia (VAP)1,2 (Fig.1A). Aim: To test the in vivo efficacy of the genetically modified Mycoplasmapneumoniae strain (CV8_HA_P1) against P. aeruginosa ETT-biofilms in mechanically ventilated pigs with VAP. Methods: Fourteen pigs previously infected with P. aeruginosa VAP3 were treated with PBS (Control, n=3), CV8 (non-pathogenic strain, n=6), or CV8_HA_P1 (non-pathogenic strain with antibiofilm and antimicrobial activity, n=5). At extubation, ETTs were analyzed for P. aeruginosa load, biofilm thickness (μm) and microbiome diversity. Results: After 39.0 [24.0-39.0] hours of mechanical ventilation, no significant differences were found in P. aeruginosa load between Control, CV8 and CV8_HA_P1 groups (p=0.32) (Fig.1B). However, a significant reduction in the ETT biofilm thickness was detected in the CV8_HA_P1 group compared to others (p<0.001) (Fig.1C, 1D). No significant microbiome diversity disbalances among groups were observed (pObserved=0.38, pShannon=0.78, pSimpson=0.87). Conclusions: Although genetically modified M. pneumoniae CV8_HA_P1 showed a reduction in biofilm thickness, further investigation is needed to determine its effectiveness in reducing the P. aeruginosa load in ETT biofilms.

Abstract Background Pseudomonas aeruginosa pneumonia is commonly treated with systemic antibiotics to ensure adequate treatment of multidrug resistant (MDR) bacteria. However, intravenous (IV) antibiotics often achieve suboptimal pulmonary concentrations. We therefore aimed to evaluate the effect of inhaled amikacin (AMK) plus IV meropenem (MEM) on bactericidal efficacy in a swine model of monolateral MDR P. aeruginosa pneumonia. Methods We ventilated 18 pigs with monolateral MDR P. aeruginosa pneumonia for up to 102 h. At 24 h after the bacterial challenge, the animals were randomized to receive 72 h of treatment with either inhaled saline (control), IV MEM only, or IV-MEM plus inhaled AMK (MEM + AMK). We dosed IV MEM at 25 mg/kg every 8 h and inhaled AMK at 400 mg every 12 h. The primary outcomes were the P. aeruginosa burden and histopathological injury in lung tissue. Secondary outcomes included the P. aeruginosa burden in tracheal secretions and bronchoalveolar lavage fluid, the development of antibiotic resistance, the antibiotic distribution, and the levels of inflammatory markers. Results The median (25–75th percentile) P. aeruginosa lung burden for animals in the control, MEM only, and MEM + AMK groups was 2.91 (1.75–5.69), 0.72 (0.12–3.35), and 0.90 (0–4.55) log10 CFU/g (p = 0.009). Inhaled therapy had no effect on preventing dissemination compared to systemic monotherapy, but it did have significantly higher bactericidal efficacy in tracheal secretions only. Remarkably, the minimum inhibitory concentration of MEM increased to > 32 mg/L after 72-h exposure to monotherapy in 83% of animals, while the addition of AMK prevented this increase (p = 0.037). Adjunctive therapy also slightly affected interleukin-1β downregulation. Despite finding high AMK concentrations in pulmonary samples, we found no paired differences in the epithelial lining fluid concentration between infected and non-infected lungs. Finally, a non-significant trend was observed for higher amikacin penetration in low-affected lung areas. Conclusions In a swine model of monolateral MDR P. aeruginosa pneumonia, resistant to the inhaled AMK and susceptible to the IV antibiotic, the use of AMK as an adjuvant treatment offered no benefits for either the colonization of pulmonary tissue or the prevention of pathogen dissemination. However, inhaled AMK improved bacterial eradication in the proximal airways and hindered antibiotic resistance.

Background: The Bronchiectasis Severity Index (BSI) and FACED score assess the prognosis and severity of bronchiectasis (BE). Yet, its relationship with the lung microbiome is unknown. The aim of this study was to explore the relationship between lung microbiota and severity in BE patients colonized by Pseudomonas aeruginosa. Methods: In this cross-sectional observational study, sputum was collected, and severity (BSI, FACED) was recorded. Alpha and beta diversities and linear discriminant analysis effect size (LEfSe) were estimated by 16S rRNA to determine differences among the severity groups. Results: 27 patients were included (Table 1). No significant differences were found between alpha diversity and BE severity, but LEfSe showed differential abundance of Actinomyces hyovaginalis in severe patients grouped by the FACED score (Fig. 1). Conclusions: Severity indexes do not reflect the loss of respiratory biodiversity. This is the first-ever description of A. hyovaginalis in human airways; in pigs, it has been associated with disseminated lung lesions (Aalbaek B et al. 2003). Thus, it could be a marker of severity and lung injury in patients with BE.

Animal models of acute respiratory distress syndrome (ARDS) do not completely resemble human ARDS, struggling translational research. We aimed to characterize a porcine model of ARDS induced by pneumonia—the most common risk factor in humans—and analyze the additional effect of ventilator-induced lung injury (VILI). Bronchoscopy-guided instillation of a multidrug-resistant Pseudomonas aeruginosa strain was performed in ten healthy pigs. In six animals (pneumonia-with-VILI group), pulmonary damage was further increased by VILI applied 3 h before instillation and until ARDS was diagnosed by PaO2/FiO2 < 150 mmHg. Four animals (pneumonia-without-VILI group) were protectively ventilated 3 h before inoculum and thereafter. Gas exchange, respiratory mechanics, hemodynamics, microbiological studies and inflammatory markers were analyzed during the 96-h experiment. During necropsy, lobar samples were also analyzed. All animals from pneumonia-with-VILI group reached Berlin criteria for ARDS diagnosis until the end of experiment. The mean duration under ARDS diagnosis was 46.8 ± 7.7 h; the lowest PaO2/FiO2 was 83 ± 5.45 mmHg. The group of pigs that were not subjected to VILI did not meet ARDS criteria, even when presenting with bilateral pneumonia. Animals developing ARDS presented hemodynamic instability as well as severe hypercapnia despite high-minute ventilation. Unlike the pneumonia-without-VILI group, the ARDS animals presented lower static compliance (p = 0.011) and increased pulmonary permeability (p = 0.013). The highest burden of P. aeruginosa was found at pneumonia diagnosis in all animals, as well as a high inflammatory response shown by a release of interleukin (IL)-6 and IL-8. At histological examination, only animals comprising the pneumonia-with-VILI group presented signs consistent with diffuse alveolar damage. In conclusion, we established an accurate pulmonary sepsis-induced ARDS model.