Achromobacter xylosoxidans is an environmental opportunistic pathogen, which infects an increasing number of immunocompromised patients. In this study we combined genomic analysis of a clinical isolated A. xylosoxidans strain with phenotypic investigations of its important pathogenic features. We present a complete assembly of the genome of A. xylosoxidans NH44784-1996, an isolate from a cystic fibrosis patient obtained in 1996. The genome of A. xylosoxidans NH44784-1996 contains approximately 7 million base pairs with 6390 potential protein-coding sequences. We identified several features that render it an opportunistic human pathogen, We found genes involved in anaerobic growth and the pgaABCD operon encoding the biofilm adhesin poly-β-1,6-N-acetyl-D-glucosamin. Furthermore, the genome contains a range of antibiotic resistance genes coding efflux pump systems and antibiotic modifying enzymes. In vitro studies of A. xylosoxidans NH44784-1996 confirmed the genomic evidence for its ability to form biofilms, anaerobic growth via denitrification, and resistance to a broad range of antibiotics. Our investigation enables further studies of the functionality of important identified genes contributing to the pathogenicity of A. xylosoxidans and thereby improves our understanding and ability to treat this emerging pathogen.

A. xylosoxidans has been transmitted between patients from our clinic, but the recent increase in incidence is not caused by cross infections.

A multilocus sequence analysis (MLSA) scheme was developed for characterization of strains and species from the genus Achromobacter, which are increasingly recovered from patients with cystic fibrosis (CF). Five conserved housekeeping genes were selected for the MLSA, which was applied to a diverse collection of 77 strains originating from Europe, Asia, and South America and including type strains of the seven recognized Achromobacter species, six environmental strains, eight non-CF clinical strains, and 56 CF clinical strains. The discriminatory power of MLSA, based on 2,098 nucleotides (nt), was much superior to a 16S rRNA gene comparison based on 1,309 nt. Congruence was observed between single-gene trees and a concatenated gene tree. MLSA differentiated all seven current Achromobacter species and also demonstrated the presence of at least four novel potential species within the genus. CF isolates were predominantly Achromobacter xylosoxidans (64%), an undescribed Achromobacter species (18%), and Achromobacter ruhlandii (7%). A clone of Achromobacter, which has spread among patients from Danish CF centers in Aarhus and Copenhagen, was identified as Achromobacter ruhlandii. MLSA facilitates the specific identification of isolates of Achromobacter necessary for describing their role in clinical infections.

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.

Early treatment with inhaled antibiotics may prevent or postpone chronic infection with Achromobacter in CF patients.