Achromobacter species are increasingly isolated from the respiratory tract of cystic fibrosis patients and often a chronic infection is established. How Achromobacter sp. adapts to the human host remains uncharacterised. By comparing longitudinally collected isolates of Achromobacter sp. isolated from five CF patients, we have investigated the within-host evolution of clonal lineages. The majority of identified mutations were isolate-specific suggesting co-evolution of several subpopulations from the original infecting isolate. The largest proportion of mutated genes were involved in the general metabolism of the bacterium, but genes involved in virulence and antimicrobial resistance were also affected. A number of virulence genes required for initiation of acute infection were selected against, e.g. genes of the type I and type III secretion systems and genes related to pilus and flagellum formation or function. Six antimicrobial resistance genes or their regulatory genes were mutated, including large deletions affecting the repressor genes of an RND-family efflux pump and a beta-lactamase. Convergent evolution was observed for five genes that were all implicated in bacterial virulence. Characterisation of genes involved in adaptation of Achromobacter to the human host is required for understanding the pathogen-host interaction and facilitate design of future therapeutic interventions.

Identification and directed development of non-organic catalysts with apparent pan-enzymatic mimicry into nanozymes for efficient prodrug conversion

Achromobacter species have attracted attention as emerging pathogens in cystic fibrosis. The clinical significance of Achromobacter infection is not yet fully elucidated; however, their intrinsic resistance to antimicrobials and ability to form biofilms renders them capable of establishing long-term chronic infections. Still, many aspects of Achromobacter biofilm formation remain uncharacterized. In this study, we characterized biofilm formation in clinical isolates of Achromobacter and investigated the effect of challenging the biofilm with antimicrobials and/or enzymes targeting the extracellular matrix. In vitro biofilm growth and subsequent visualization by confocal microscopy revealed distinctly different biofilm morphotypes: a surface-attached biofilm morphotype of small aggregates and an unattached biofilm morphotype of large suspended aggregates. Aggregates consistent with our in vitro findings were visualized in sputum samples from cystic fibrosis patients using an Achromobacter specific peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) probe, confirming the presence of Achromobacter biofilms in the CF lung. High antibiotic tolerance was associated with the biofilm phenotype, and biocidal antibiotic concentrations were up to 1000 fold higher than for planktonic cultures. Treatment with DNase or subtilisin partially dispersed the biofilm and reduced the tolerance to specific antimicrobials, paving the way for further research into using dispersal mechanisms to improve treatment strategies.