Epidemiological insights into Haemophilus influenzae and Pseudomonas aeruginosa persistent colonization in non-cystic fibrosis bronchiectasis patients: a longitudinal and multicenter study

BACKGROUND: Bronchiectasis is a chronic respiratory disease characterized by recurrent exacerbations and persistent inflammation, often associated with bacterial pathogens such as Haemophilus influenzae and Pseudomonas aeruginosa. Phenotypic adaptations (e.g., antimicrobial resistance) complicate treatment and worsen a patient's quality of life. METHODS: Between 2019 and 2020, we isolated 52 H. influenzae and 48 P. aeruginosa strains from 62 non-CF bronchiectasis patients across three scheduled visits and during exacerbation episodes. Antimicrobial susceptibility (assessed by microdilution) and phenotyping assays (motility and hypermutability) were performed. Whole genome sequencing was applied for analyses of resistance determinants, virulence factors, and genetic diversity. RESULTS: Of the 62 patients, 31 were colonized by H. influenzae, 28 by P. aeruginosa, and 3 were co-colonized. Severe disease was predominantly linked to P. aeruginosa (70.6%), while exacerbations were more common with H. influenzae (81.8%). Multilocus sequence typing (MLST) revealed high genetic diversity, with ST1025 and ST253 most common in H. influenzae and P. aeruginosa, respectively. Antimicrobial resistance was low, but H. influenzae showed the highest resistance to cotrimoxazole (40.4%), while P. aeruginosa showed high resistance to aminoglycosides (27.1%) and fluoroquinolones (25%). Virulence profiling of P. aeruginosa identified 22.9% of strains as hypermutable, 27.1% as mucoid, 31.3% harboring the exoU gene, and 41.7% with impaired twitching motility. Persistent colonization occurred in 16 patients (25.8%), with antimicrobial resistance emerging following previous antimicrobial treatment in one case. CONCLUSIONS: In this cohort, H. influenzae and P. aeruginosa showed similar prevalence, high genetic diversity, and rare co-colonization. P. aeruginosa was associated with more severe disease, higher antimicrobial resistance, and hypermutability, whereas H. influenzae was associated with acute exacerbations.