The waste recycling workforce is growing across Europe. This study investigates the airborne exposure of workers recycling plastic, paper/cardboard, and electronic (e) waste, and examines whether serum levels of inflammatory markers correlate with exposure. Exposure was measured repeatedly and analysed for inhalable and respirable dust, inhalable endotoxin, fungi, and bacteria. Microorganisms were identified using MALDI-TOF MS on cultured microorganisms and bacteria by 16S rRNA marker-gene sequencing. Blood samples collected at the end of each workday were analysed for three markers of inflammation. Waste types/tasks had an impact on exposure levels for all exposures and temperature on exposure to anaerobic bacteria. Exposure levels to dust, endotoxin, and anaerobic bacteria differed between workers. Exposures were highest for those handling paper/cardboard and plastic waste. The alpha diversity indices for most exposures did not differ between types of waste handled, but eWaste was associated with a lower species richness. Beta diversity did not differ between indoor temperatures or waste types except for mesophilic bacteria and bacteria (NGS-data). The species Aspergillus niger, Penicillium brevicompactum, Bacillus cereus, and Staphylococcus equorum were frequently detected. Serum levels of inflammatory markers increased with increasing exposure to dust, fungi or Penicillium spp, and anaerobic bacteria, but did not correlate with bacterial biodiversity indices. The study suggests further investigations of the impact of daily inhalation of bacteria able to grow anaerobically and fungi. Based on the high exposure levels, and the association between exposure and biomarkers of inflammation, it is advisable to explore risk management strategies aimed at minimizing worker exposure.

Background:Culture-independent molecular techniques could potentially be used to measure microbiological efficacy in response to antibiotic treatment and improve understanding of the role of the airway microbiota in determining response in patients with chronic respiratory disease. Methods:Using molecular methods, we analysed changes in the sputum microbiota in samples from 107 participants with bronchiectasis recruited to the iBEST-1 study, and defined community endotypes based on response to tobramycin inhalation powder (TIP) treatment. The relationship between microbiota metrics in these endotypes and clinical and inflammatory biomarkers were also determined. Results:There was a significant reduction in Pseudomonas aeruginosa density, measured by quantitative polymerase chain reaction (qPCR), between Days 1 and 29 for participants in the TIP treatment (n=63; p<0.0001) but not placebo (n=20; p>0.05) group. Based on decrease in P. aeruginosa density (oprL copies·mL-1) over 28 days, two clusters of participants receiving TIP were observed and stratified as either responders (≥2Log10; n=26) or non-responders (<2Log10; n=37). In responders, a shift to a microbial community structure less dominated (p=0.018) by a pathogen was apparent and associated with a greater improvement in inflammatory and fewer participant exacerbations in the following 6 months (27% versus 49%; p=0.117) when compared to non-responders. Lung function was higher at Day 1 in responders (median=64.6% predicted) than non-responders (μ̃median=50.3% predicted) and independently predicted response to treatment with TIP (p=0.013). Conclusions:qPCR may be a useful, culture-independent microbiological efficacy end-point in clinical trials. Using qPCR, participants with bronchiectasis were stratified into endotpyes which predicted response to antimicrobial treatment, potentially allowing for a more personalised approach to therapy.

Background and Objective This study explored the relationship between total bacterial density, airway microbiota composition and clinical parameters in bronchiectasis. We determined changes with time during clinical stability and following antibiotic treatment of a pulmonary exacerbation. Methods We conducted a multicentre longitudinal cohort study of UK participants with CT confirmed bronchiectasis. Sputum samples and clinical parameters [FEV1% predicted, lung clearance index, C-reactive protein, white cell count and Quality of Life] were collected when participants were clinically stable and pre/post-antibiotic treatment of an exacerbation. Total bacterial density and microbiota community composition was measured by quantitative polymerase chain reaction and sequencing of the V4 region of bacterial 16S rRNA, respectively. Results Among 105 participants at baseline, 65 (62%) were female with a mean age of 65 years and FEV1 at 69% predicted. In participants who remained clinically stable (n=15), no significant changes were observed in bacterial density, microbiota diversity, richness, evenness, and dominance (p=0.30, 0.45, 0.54, 0.23 and 0.43; respectively) across four time points over a 1-year period. Similarly, for participants with paired pre/post-antibiotic treatment samples (n=19), no significant changes were observed (p=0.30, 0.46, 0.44, 0.71 and 0.58; respectively). However, considerable fluctuation in community composition between samples was apparent for most patients. Total bacterial density and microbiota composition did not correlate with clinical parameters at baseline (n=75). Conclusions Stability in bacterial density and microbiota diversity, richness, evenness and dominance was observed over time at a population level but considerable fluctuation was apparent in samples from individual patients.

Rationale: Replicate phase III trials of inhaled antibiotics in patients with bronchiectasis have produced inconsistent results. Objectives: This study investigated if different microbial and inflammatory endotypes are linked to antibiotic response in patients with chronic Pseudomonas aeruginosa infection. Methods: ORBIT-3 and ORBIT-4 were phase III trials of inhaled liposomal ciprofloxacin compared with placebo in patients with bronchiectasis with chronic P. aeruginosa infections. Baseline sputum from the trials were analyzed by 16S rRNA sequencing (LoopSeq) (n = 377), proteomics (n = 164), and Olink (n = 117). Relationships with clinical features and frequency of exacerbations during the trials were analyzed. Measurements and Main Results: Patients with P. aeruginosa infections demonstrated heterogeneous endotypes. Reduced microbiota diversity was associated with exacerbation frequency (P = 0.021) and quality of life (P = 0.012). Increased exacerbations were associated with increased Pseudomonas abundance and neutrophilic inflammation; decreases in the relative abundance of commensals, including Rothia; and B-cell responses. Geographical differences were observed, with increased microbiota diversity and decreased neutrophilic inflammation in Central Europe. Candidate biomarkers for treatment response were identified, including neutrophil elastase, LSP1, and Rothia relative abundance. Before adjustment, treatment estimates of the two trials varied (ORBIT-3 rate ratio [RR], 0.85 [0.65-1.12], ORBIT-4 RR, 0.63 [0.48-0.82]). After linear discriminant analysis to adjust for microbiota profile and geographical region, the treatment estimates of ORBIT-3 (RR, 0.81 [0.54-1.22]) and ORBIT-4 (RR, 0.82 [0.56-1.22]) were similar and consistent with previous meta-analyses of inhaled antibiotics in bronchiectasis. Conclusions: Patients with chronic P. aeruginosa have heterogeneous microbiota and inflammatory profiles, influencing antibiotic treatment responses in bronchiectasis. Future trials could be improved by patient stratification, including accounting for geographical differences and biomarkers to represent microbiota differences.

Bacterial biofilms present significant therapeutic challenges due to their resistance to conventional antimicrobial treatment. Mucins typically serve as a protective barrier against pathogens, yet certain bacteria, such as Pseudomonas aeruginosa (P. aeruginosa), can exploit these glycoproteins as attachment sites for biofilm formation. This study introduces boronic acid-functionalized polyethyleneimine (PEI-BA) as a promising antibiofilm agent that effectively blocks bacterial adhesion to mucin-rich surfaces. Through the multivalent presentation of boronic acid groups, PEI-BA reversibly forms boronate ester bonds with mucin glycans, creating a protective barrier. Our findings show that PEI-BA prevents bacterial attachment through a nonbactericidal mechanism, potentially reducing the risk of resistance development. Notably, PEI-BA synergizes with a conventional antibiotic, tobramycin, significantly enhancing biofilm inhibition compared to either treatment alone. Systematic evaluation of PEI-BA formulations identified optimal functionalization levels, balancing glycan-binding capability with solubility. From a biomaterials design perspective, we demonstrate how rational polymer modification can transform a potent but cytotoxic antimicrobial agent (i.e., PEI) into a safe and effective antibiofilm material, opening further possibilities for managing biofilm-associated infections in clinical settings. This work establishes boronic acid-based nanomaterials as promising candidates for biofilm prevention and antibiotic enhancement, particularly in conditions like cystic fibrosis, where mucin-bacterial interactions contribute to disease progression.

Rationale: Pseudomonas aeruginosa airway infection is associated with increased exacerbations and poor outcomes in bronchiectasis. Our prior study (GREAT-1) showed that gremubamab—a bivalent, bispecific mAb targeting Psl exopolysaccharide and PcrV T3SS component—enhanced neutrophil clearance of P. aeruginosa and reduced virulence ex-vivo. Here we report the efficacy and safety of gremubamab in a proof-of-concept trial in bronchiectasis patients with P. aeruginosa infection. Methods: In a multi-centre, multinational (UK and Spain), randomized, double-blind, placebo-controlled trial, people with CT-confirmed bronchiectasis and sputum positive for P. aeruginosa were randomised 1:1:1 to 1500mg or 500mg gremubamab intravenous infusion, or placebo, once every four weeks for 12 weeks. Serum and sputum samples were obtained on days 1, 7, 14, 28, 56, 84 (end-of-treatment;EoT), and 168 (12-week follow-up). The primary outcome was change from baseline in quantitative sputum cultures at day 84. Key secondary outcomes included change from baseline in the St. George's Respiratory Questionnaire (SGRQ) and the quality of life bronchiectasis questionnaire (QoL-B), time to first exacerbation, FEV1 and safety. As a proof-of-concept study, statistical significance was pre-specified at the 1-sided p<0.1 level. Results: 37 participants were randomised, n=12 in the gremubamab 1500mg group (age 65.7±13.8 [mean±SD], 66.7% female), n=13 in the 500mg group (age 60.3±16.7, 84.6% female), n=12 in the placebo group (age 65.9±14.4, 66.7% female). Gremubamab treatment resulted in a significant reduction in bacterial load at EoT (day 84) with the 500mg dose compared with placebo treatment ([estimate[80%CI]]; -1.25 log-CFU (-2.33 to -0.16); 1-sided p=0.071; ANCOVA), meeting the trial primary endpoint. A non-significant directional trend was observed for the 1500mg dose (-0.66(-1.71 to 0.39); p=0.2). The SGRQ was significantly improved versus placebo at EoT (500mg: -10.8(-4.93 to -16.7), p=0.02; 1500mg: -12.1(-6.5 to -17.7), p=0.008). The proportion of patients achieving a clinically significant SGRQ improvement was higher for both gremubamab 500mg (38.5%) and 1500mg (41.7%) versus placebo (8.3%). Clinically significant benefit in favour of gremubamab treatment at EoT was observed for multiple QoL-B domains. Time to first exacerbation was significantly prolonged at the 1500mg dose versus placebo (p=0.046; restricted mean survival time method). There was no effect on FEV1. Adverse events (AEs) were reported in 91.7%, 84.6% and 89.2% of the 1500mg, 500mg and placebo groups, respectively, and severe AEs in 0%, 15.4% and 16.7%. Conclusion: Gremubamab treatment significantly reduced P. aeruginosa airway bacterial load and improved patient-reported quality of life in patients with bronchiectasis, providing proof-of-concept for specific anti-microbial monoclonal antibody therapy.

BACKGROUND:This study explores the effectiveness and safety of microbiome-directed antimicrobial therapy versus usual antimicrobial therapy in adult cystic fibrosis pulmonary exacerbations. METHODS:A multicentre two-arm parallel randomised control trial conducted across Europe/North-America enrolled 223 participants (January 2015 to August 2017). All participants were chronically colonised with Pseudomonas aeruginosa and were randomised 1:1 into two study arms. The "usual therapy" group received 2 weeks of intravenous ceftazidime 3 g thrice daily (for allergies: aztreonam 2 g thrice daily) and tobramycin 5-10 mg·kg-1 once daily. The "microbiome-directed" group received the same usual therapy plus an additional antibiotic with greatest presumed activity against the second, third and fourth most abundant genera present in the sputum microbiome, selected by a consensus expert treatment panel. The primary outcome was change in percentage of predicted forced expiratory volume in 1 s (ppFEV1) at 14 days post initiation of antibiotics. Secondary outcomes examined ppFEV1 at 7 days, 28 days and 3 months; time to next exacerbation; symptom burden at 7 days; health-related quality of life (HRQoL) at 28 days; and number of exacerbations and i.v. antibiotic days at 12 months. RESULTS:149 participants had an eligible exacerbation (usual therapy n=83, microbiome-directed therapy n=66). There was no difference between the groups for ppFEV1 at day 14 (-1.1%, 95% CI -3.9-1.7%; p=0.46), or ppFEV1 measured at other time points, or for time to next exacerbation (microbiome-directed versus usual therapy hazard ratio 0.91, 95% CI 0.60-1.38; p=0.66). The microbiome-directed group trended to have more i.v. days (median 42 days versus 28 days; p=0.08) and more subsequent exacerbations (median three versus two; p=0.044) the following year. There were no appreciable differences in symptom burden; however, HRQoL subscores were consistently worse in the microbiome-directed group (-4.3 points versus usual therapy, 95% CI -8.3--0.3 points; p=0.033). CONCLUSION:The addition of a third antibiotic based on sputum microbiome sequencing analysis did not result in improved clinical outcomes.

A dry powder inhaled liposomal azithromycin formulation was developed for the treatment of chronic respiratory diseases such as cystic fibrosis and bronchiectasis. Key properties including liposome size, charge and encapsulation efficiency powder size, shape, glass transition temperature (Tg), water content and in vitro respiratory deposition were determined. Antimicrobial activity against cystic fibrosis (CF) respiratory pathogens was determined by MIC, MBC and biofilm assays. Cytotoxicity and cellular uptake studies were performed using A549 cells. The average liposome size was 105 nm, charge was 55 mV and encapsulation efficiency was 75%. The mean powder particle size d[v,50] of 4.54 µm and Mass Median Aerodynamic Diameter (MMAD) was 5.23 µm with a mean Tg of 76˚C and water content of 2.1%. These excellent physicochemical characteristics were maintained over one year. Liposomal loaded azithromycin demonstrated enhanced activity against P. aeruginosa clinical isolates grown in biofilm. The formulation was rapidly delivered into bacterial cells with >75% uptake in 1 hour. Rapid uptake into A549 cells via a cholesterol-dependent endocytosis pathway with no cytotoxic effects apparent. These data demonstrate that this formulation could offer benefits over current treatment regimens for people with chronic respiratory infection.

RATIONALE:Pulmonary exacerbations are clinically impactful events that accelerate cystic fibrosis (CF) lung disease progression. The pathophysiological mechanisms underlying an increased frequency of pulmonary exacerbations have not been explored. OBJECTIVES:To compare host immune response during intravenous antibiotic treatment of pulmonary exacerbations in people with CF who have a history of frequent versus infrequent exacerbations. METHODS:Adults with CF were recruited at onset of antibiotic treatment of a pulmonary exacerbation and were categorised as infrequent or frequent exacerbators based on their pulmonary exacerbation frequency in the previous 12 months. Clinical parameters, sputum bacterial load and sputum inflammatory markers were measured on day 0, day 5 and at the end of treatment. Shotgun proteomic analysis was performed on sputum using liquid chromatography-mass spectrometry. MEASUREMENTS AND MAIN RESULTS:Many sputum proteins were differentially enriched between infrequent and frequent exacerbators (day 0 n=23 and day 5 n=31). The majority of these proteins had a higher abundance in infrequent exacerbators and were secreted innate host defence proteins with antimicrobial, antiprotease and immunomodulatory functions. Several differentially enriched proteins were validated using ELISA and Western blot including secretory leukocyte protease inhibitor (SLPI), lipocalin-1 and cystatin SA. Sputum from frequent exacerbators demonstrated potent ability to cleave exogenous recombinant SLPI in a neutrophil elastase dependent manner. Frequent exacerbators had increased sputum inflammatory markers (interleukin (IL)-1β and IL-8) and total bacterial load compared to infrequent exacerbators. CONCLUSIONS:A diminished innate host protein defence may play a role in the pathophysiological mechanisms of frequent CF pulmonary exacerbations. Frequent exacerbators may benefit from therapies targeting this dysregulated host immune response.

It is increasingly recognized that interspecies interactions may modulate the pathogenicity of Pseudomonas aeruginosa during chronic lung infections. Nevertheless, while the interaction between P. aeruginosa and pathogenic microorganisms co-infecting the lungs has been widely investigated, little is known about the influence of other members of the lung microbiota on the infection process. In this study, we focused on investigating the impact of Prevotella species isolated from the sputum of people with cystic fibrosis (pwCF) on biofilm formation and virulence factor production by P. aeruginosa. Screening of a representative collection of Prevotella species recovered from clinical samples showed that several members of this genus (8 out 10 isolates) were able to significantly reduce biofilm formation of P. aeruginosa PAO1, without impact on growth. Among the tested isolates, the strongest biofilm-inhibitory activity was observed for Prevotella intermedia and Prevotella nigrescens, which caused a reduction of up to 90% in the total biofilm biomass of several P. aeruginosa isolates from pwCF. In addition, a strain-specific effect of P. nigrescens on the ability of P. aeruginosa to produce proteases and pyocyanin was observed, with significant alterations in the levels of these virulence factors detected in LasR mutant strains. Overall, these results suggest that non-pathogenic bacteria from the lung microbiota may regulate pathogenicity traits of P. aeruginosa, and possibly affect the outcome of chronic lung infections.

Objectives: Non-culture methods are increasingly used for the detection and quantification of pathogens [1Forbes et al.Front Microbiol. 2017; 8: 1069Crossref PubMed Scopus (182) Google Scholar]. qPCR allows absolute quantification of target genes, while 16S rRNA marker-gene MiSeq sequencing (NGS) provides taxonomic resolution to a genera level [2Janda JM Abbott SL J Clin Microbiol. 2007; 45: 2761-2764Crossref PubMed Scopus (1223) Google Scholar], with the relative abundance of each of these bacterial genera determined within a sample. The aim of this study was to compare qPCR and relative abundance of 16S rRNA amplicons for the detection and quantification of bacteria within sputum samples from PwCF. Methods: Sputum samples (n = 179) were collected as part of the Real-world Orkambi cohort Cork study (ROCK). Microbial community composition was determined by NGS and qPCR was carried out for selected bacteria. Correlation between methods was determined by calculation of Cohen's Kappa coefficient. TableDetection of target bacterial pathogens by qPCR and NGS and agreement between detection methodsGeneTarget bacterial pathogenNo. of samples positive by qPCRNo. of samples positive by NGSCohen's kappa coefficient16S rRNAAll bacteria1791791oprLPseudomonas aeruginosa1281560.51ecfX1340.49hpDHaemophilus influenzae17850.18smbB210.21lytAStreptococcus pneumoniae271760.04femAStaphylococcus aureus101970.84 Open table in a new tab Results: There was almost perfect agreement for detection of S. aureus between methods, with moderate agreement for P. aeruginosa, slight agreement for H. influenzae and no agreement for S. pneumoniae. There was a positive correlation between oprL (r = 0.93, p < 0.001) and ecfX (r = 0.94, p < 0.001) copy number and the relative abundance of Pseudomonas spp. determined by NGS and between femA (r = 0.93, p < 0.001) and Staphylococcus spp. relative abundance. There was a weaker positive correlation between hpD (r = 0.4, p < 0.001) and smpB (r = 0.42, p < 0.001) copy number and the relative abundance of Haemophilus spp.. Conclusion: Both qPCR and NGS can be used to detect pathogens in respiratory samples. Discordance between methods may be due to detection of species from the same genera by NGS which are not detected by species specific qPCR assays. Supported by EU/EFPIA IMI iABC grant n° 115721 We would also like to acknowledge funding from the European Commission for CFMATTERS, Grant agreement 603038

Previous research using agar well diffusion assays revealed that Pseudomonas aeruginosa (PA) isolates from chronic CF infection, grown aerobically and anaerobically, inhibited the growth of other clinically relevant species. This study aimed to determine if this observed inhibition was also exhibited in conditions more physiologically representative of the CF airways.