Achromobacter spp. can establish occasional or chronic lung infections in patients with cystic fibrosis (CF). Chronic colonization has been associated with worse prognosis highlighting the need to identify markers of bacterial persistence. To this purpose, we analyzed phenotypic features of 95 Achromobacter spp. isolates from 38 patients presenting chronic or occasional infection. Virulence was tested in Galleria mellonella larvae, cytotoxicity was tested in human bronchial epithelial cells, biofilm production in static conditions was measured by crystal violet staining and susceptibility to selected antibiotics was tested by the disk diffusion method. The presence of genetic loci associated to the analyzed phenotypic features was evaluated by a genome-wide association study. Isolates from occasional infection induced significantly higher mortality of G. mellonella larvae and showed a trend for lower cytotoxicity than chronic infection isolates. No significant difference was observed in biofilm production among the two groups. Additionally, antibiotic susceptibility testing showed that isolates from chronically-infected patients were significantly more resistant to sulfonamides and meropenem than occasional isolates. Candidate genetic biomarkers associated with antibiotic resistance or sensitivity were identified. Achromobacter spp. strains isolated from people with chronic and occasional lung infection exhibit different virulence and antibiotic susceptibility features, which could be linked to persistence in CF lungs. This underlines the possibility of identifying predictive biomarkers of persistence that could be useful for clinical purposes.

Achromobacter spp. lung infection in cystic fibrosis has been associated with inflammation, increased frequency of exacerbations, and decline of respiratory function. We aimed to evaluate in vivo the inflammatory effects of clinical isolates exhibiting different pathogenic characteristics. Eight clinical isolates were selected based on different pathogenic characteristics previously assessed: virulence in Galleria mellonella larvae, cytotoxicity in human bronchial epithelial cells, and biofilm formation. Acute lung infection was established by intratracheal instillation with 10.5 × 10⁸ bacterial cells in wild-type and CFTR-knockout (KO) mice expressing a luciferase gene under control of interleukin-8 promoter. Lung inflammation was monitored by in vivo bioluminescence imaging up to 48 h after infection, and mortality was recorded up to 96 h. Lung bacterial load was evaluated by CFU count. Virulent isolates caused higher lung inflammation and mice mortality, especially in KO animals. Isolates both virulent and cytotoxic showed higher persistence in mice lungs, while biofilm formation was not associated with lung inflammation, mice mortality, or bacterial persistence. A positive correlation between virulence and lung inflammation was observed. These results indicate that Achromobacter spp. pathogenic characteristics such as virulence and cytotoxicity may be associated with clinically relevant effects and highlight the importance of elucidating their mechanisms.

The Achromobacter genus comprises 22 species and various genogroups. Some species with higher virulence or antibiotic resistance are more likely to cause chronic infections in people with cystic fibrosis (CF). Current identification methods often fail to accurately distinguish between the species or result in misidentifications due to biochemical similarities. This study aims to develop an accurate qPCR protocol for species-level identification that is applicable in clinical diagnostic laboratories. Whole-genome sequencing of clinical isolates from different Achromobacter species identified species-specific single-nucleotide polymorphisms (SNPs) in two 16S gene regions. Based on these SNPs, two sets of primers and qPCR probes were designed to generate unique identification profiles. Thermal profiles were optimized, and qPCR was performed on serial bacterial DNA dilutions to determine the detection limit (LOD). Four probes successfully identified three species: A. xylosoxidans, A. dolens, and A. insuavis. Two additional probes were designed for novel genotypes unrelated to publicly available sequences. The LOD ranged from 0.005 pg/µL to 1 pg/µL. Combined probes achieved 100% sensitivity, with specificity ranging from 97.95% to 100%. This qPCR protocol enables accurate species identification, overcoming the limitations of current methods, and represents a reliable tool for clinical diagnostics.

Achromobacter spp. are opportunistic pathogens in people with cystic fibrosis (PwCF), yet the role of the upper airways in their persistence and adaptation remains poorly understood. We investigated whether the sinonasal compartment may act as reservoir and evolutionary niche for Achromobacter spp. during airway infection. Twenty-two isolates obtained from paired nasal lavage and sputum samples of seven PwCF were analysed by whole-genome sequencing. Within each PwCF, identical clone types were detected in both airway compartments, supporting bacterial exchange between upper and lower airways. Despite clonal relatedness, substantial genomic diversification was observed between paired isolates. Genomic signatures indicative of elevated mutation rates were detected in a high number of isolates (73%) and in both airway compartments, highlighting widespread genomic diversification across the respiratory tract. Mobilome analysis revealed compartment-specific variations in insertion sequences, prophages, and integrative elements, suggesting genome plasticity. Additionally, mutation in an aspartate kinase gene was consistently associated with loss of biofilm formation in vitro, highlighting a potential link between this pathway and biofilm phenotype. Overall, our findings indicate that upper and lower airways represent interconnected but partially independent ecological niches where Achromobacter populations can diverge during colonization, supporting the view that both compartments contribute to their persistence and evolution in CF airways.