23Several members of the Gram-negative environmental bacterial genus, Achromobacter, are 24 associated with serious infections in immunocompromised individuals, of which 25Achromobacter xylosoxidans is the most common. Despite their pathogenic potential, 26comparatively little is understood about these intrinsically drug-resistant bacteria and their role 27 in disease, leading to suboptimal diagnosis and management of Achromobacter infections. 28Here, we performed comparative genomics of 158 Achromobacter spp. genomes to robustly 29 identify species boundaries, to reassign several incorrectly speciated taxa, and to identify 30 genetic sequences specific for the Achromobacter genus and for A. xylosoxidans. Next, we 31 developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the 32 rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both 33 purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates 34 identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to 35 these routine diagnostic methods, the duplex assay showed superior identification of 36 Achromobacter spp. and A. xylosoxidans, with five Achromobacter isolates failing to amplify 37with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax 38 quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents, 39 and detected down to ~12 and ~1 genome equivalent/s, respectively. In silico analysis, and 40 laboratory testing of 34 non-Achromobacter isolates and 38 adult CF sputa, confirmed duplex 41 assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a 42 robust, sensitive, and cost-effective method for the simultaneous detection of all 43Achromobacter spp. and A. xylosoxidans, and will facilitate the rapid and accurate diagnosis 44 of this important group of pathogens. 45

Several members of the Gram-negative environmental bacterial genus Achromobacter are associated with serious infections, with Achromobacter xylosoxidans being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 Achromobacter spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus Achromobacter and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive in silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans and will facilitate the rapid and accurate diagnosis of this important group of pathogens.