(460) Achromobacter xylosoxidans as a Cystic Fibrosis Related Pathogen

Background: Achromobacter xylosoxidans (Ax) is a Gram-negative, opportunistic pathogen that is considered to be an emerging pathogen in cystic fibrosis (CF). Despite increasing prevalence in CF patients worldwide, little is known about the pathogenesis of Ax and its impact on CF disease progression, as this organism is not well studied in the context of pulmonary infection. We have obtained several Ax clinical isolates from CF patients and will investigate colonization and persistence in a mouse model, as well as important virulence determinants, such as adherence to immortalized CF bronchial epithelial cells (CFBEs), motility, and cytotoxicity. In addition, we have performed preliminary comparative genomics experiments to examine genetic relatedness of strains.

Methods: In order investigate colonization and persistence dynamics in a mouse model, BALB/cJ mice were intratracheally infected with Ax clinical isolates and euthanized at several time points (24, 48, and 72 hours post infection). Bacterial load of the lungs was assessed by viable colony counting. In addition, lungs were fixed and H&E stained in order to observe markers of infection severity, such as neutrophil infiltration. We also infected immortalized CFBE cells with these clinical strains of Ax to observe the percentage of bacterial inoculum that adhered. Cellular supernatant from infected epithelial cells was saved to perform cytotoxicity assays. These isolates were plated on low percentage agar plates in order to observe swimming and swarming motility. Ax was also grown in synthetic cystic fibrosis sputum media (SCFM2) to examine growth and gene expression. Strains were sequenced and the genomes were assembled and annotated to perform core genome analysis and to create a phylogenetic tree showing relatedness between strains.

Results: Clinical isolates of Ax persist in the lungs of infected mice for up to 72 hours post infection. Infection with clinical strains of Ax led to neutrophil infiltration and airway thickening in the lungs of infected mice, as seen in microscopy of stained lung sections. Ax clinical isolates also adhered to CFBE cells and exhibited cytotoxicity, with the highest percent cytotoxicity being around 20%. Swimming motility assays showed that one of the three strains tested was motile. Swarming motility assays showed that none of the examined isolates exhibited this type of motility. Growth curves in SCFM2 showed that Ax exhibits increased growth when compared to growth curves in a rich lab medium, such as LB broth.

Conclusions: Based on these results, we conclude that Ax colonizes and persists in the lungs and shows characteristics consistent with an opportunistic pathogen. Current work focuses on understanding which genes or gene sets contribute to these phenotypes (motility, cytotoxicity, adherence), and how these genes contribute to the pathogenesis and virulence of Ax. In addition, we plan to examine interactions that occur between Ax and Pseudomonas aeruginosa, since these two organisms are frequently isolated together.



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