Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp.

Abstract

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.

Effects of exogenous lactic acid bacteria and maize meal on fermentation quality and microbial community of Orychophragmus violaceus silage

“…It was suggested that potential positive effects of ensiling fermentation on inhibition deleterious microorganisms might be due to a direct effect of rapid acidification, reduced availability of water, production of antimicrobial compounds, or a combination of factors ( Garde et al, 2014 ). Achromobacter , the main genus of family Alcaligenaceae, is non-fermentative and potential pathogen bacteria found ubiquitously in environmental reservoirs including rivers, ponds, residential water sources, soil, mud and some plants ( Price et al, 2020 ). Similar to Clostridium , the relative abundance of undesirable Achromobacter was also observed higher ( p < 0.05) in Y5 silage than other three groups.…”

Contamination of Hospital Surfaces with Bacterial Pathogens under the Current COVID-19 Outbreak

“…The identification and the quantification of DNA of methicillin-sensitive and methicillinresistant Staphylococcus aureus, as well as methicillin-resistant coagulase-negative Staphylococcus spp., were performed using the «AmpliSens ® MRSA-screen-titer-FL» reagent kit (FSB of the Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Russia) according to the manufacturer's instructions. The detection of Achromobacter spp., Burkholderia cepacia complex, Pseudomonas aeruginosa, and Klebsiella pneumonia was carried out as previously described (Table S1, Supplementary Materials) [30][31][32][33].…”