<i>Achromobacter xylosoxidans</i> isolates exhibit genome diversity, variable virulence, high levels of antibiotic resistance, and potential intrahost evolution

<i>Achromobacter xylosoxidans</i> is an emerging pathogen characterized by high levels of antibiotic resistance (AR) and increasing infection rates worldwide. This motile, opportunistic pathogen is widely distributed in the environment and can cause various infections, including pneumonia, bacteremia, endocarditis, meningitis, and others. In this study, we analyzed the population structure, AR profiles, and virulence factors of over 200 publicly available genomes. Core genome analysis revealed that <i>A. xylosoxidans</i> is highly adaptable, possessing a relatively small core genome. Antibiotic susceptibility testing of isolates from the United States revealed high resistance to multiple antibiotics. Our data show that imipenem/relebactam is as effective against <i>A. xylosoxidans</i> as imipenem alone, indicating that relebactam does not inhibit β-lactamase activity in <i>Achromobacter</i>. The species features multiple secretion systems, including the Type III secretion system of the YscN family, which is similar to those found in <i>Bordetella pertussis</i> and <i>Pseudomonas aeruginosa</i>. Isolates collected from the same patients showed changes in cytotoxicity, flagella motility, biofilm, and AR, suggesting their dynamic adaptation to the host environment. Intra-host evolved isolates, NIH-010, NIH-016, and NIH-018, demonstrated the loss of flagellar motility and variable cytotoxicity while exhibiting increased AR and enhanced biofilm formation. Sequence analysis suggests that NIH-016-3 has a tyrosine to histidine mutation at position 330 near the FlhF guanosine triphosphate-binding domain that may affect flagellar assembly. Interestingly, virulence assays showed significant variation in the ability of different <i>A. xylosoxidans</i> isolates to induce cell death in <i>in vitro</i> models, suggesting their dynamic adaptation to the host environment.IMPORTANCEThis study provides a comprehensive examination of <i>Achromobacter xylosoxidans</i>, an emerging pathogen of global concern due to its high antibiotic resistance (AR) and increasing clinical relevance. By analyzing over 200 genomes, we offer critical insights into the population structure, resistance mechanisms, and virulence factors of this species. The identification of a small core genome underscores its potential for genomic plasticity. The existence of multiple secretion systems highlights the great capacity of <i>A. xylosoxidans</i> as a pathogen. Variations in virulence among <i>A. xylosoxidans</i> isolates indicate the complexity of this pathogen, underscoring the need for further studies on its virulence mechanisms. Evolution within the host includes the loss of motility-associated systems and enhanced AR and biofilm formation. This work showed that <i>A. xylosoxidans</i> is resistant to relebactam when combined with imipenem, a combination effective in other bacteria. These findings emphasize the urgent need for targeted therapeutic strategies to combat this opportunistic pathogen.