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.

中文翻译：

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用于实时检测木糖氧化无色杆菌和无色杆菌属的双重实时PCR分析。

革兰氏阴性环境细菌属无色杆菌属的几个成员与严重感染有关，其中木糖氧化无色杆菌是最常见的。尽管它们具有潜在的致病性，但对这些内在抗药性细菌及其在疾病中的作用了解甚少，导致诊断和管理欠佳。在这里，我们对158种无色杆菌进行了比较基因组学研究。基因组，以有力地识别物种边界，重新分配几个错误指定的分类单元，并鉴定无色杆菌属和木糖氧化过氧化物酶的遗传序列 。接下来，我们开发了一种基于Black Hole Quencher探针的双工实时PCR检测方法Ac-Ax，用于快速，同时检测无色杆菌属。和来自纯化菌落和多菌种临床标本的木糖氧化木霉。Ac-Ax在119种被鉴定为无色杆菌属的菌株上进行了测试。使用表型或基因型方法。与这些常规诊断方法相比，双重分析显示出无色杆菌属物种的出色鉴定。和A.木糖氧化，有五个无色杆菌菌株没有用Ac-AX扩增证实根据16S rRNA基因测序是不同的属。Ac-Ax定量检测两种无色杆菌 spp。和木糖氧化过氧化物酶（A. xylosoxidans）低至〜110个基因组当量，分别检测到低至〜12和〜1个基因组当量。广泛的计算机分析以及对34种非无色杆菌分离物和38种成年囊性纤维化痰液的实验室测试，证实了双重测定的特异性和敏感性。我们证明，Ac-Ax双工测定法提供了同时检测所有无色杆菌属物种的可靠，灵敏和具有成本效益的方法。和A. xylosoxidans，将有助于快速而准确地诊断这一重要的病原体群。