Duplex real-time PCR assay for the simultaneous detection ofAchromobacter xylosoxidansandAchromobacterspp

Abstract

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

Bacterial microbiota of the contact lens surface and associated care behaviours

“…The main pathogens that were found in the present population were represented by 13 strains: Serratia surfactantfaciens (10%), Staphylococcus epidermidis (5%), S. pasteuri (65%), Stenotrophomonas maltophilia (5%), Pseudomonas aeruginosa (10%), Delftia tsuruhatensis (5%), Acinetobacter calcoaceticus (5%), Granulicatella adiacens (5%), Raoultella planticola (10%), R. ornithinolytica (5%), A. agilis (35%), Pseudomonas stutzeri (5%), and Acinetobacter junii (5%). Sources of contamination are normally residential water, rivers, soil, mud, and some plants [ 19 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ], which might be related to the CL care routines and nonhygenic environments. For contamination by nonpathogenic species, 13 strains were found: Brevundimonas albigilva , E. mori, Herbasprillum aquaticum, Kluyvera intermedia , Pseudomonas geniculata , Acinetobacter lactucae , Bacillus wiedmanni , Curtobacterium plantarum , Paludibacterium paludis, Kosakonia oryziphila , Rothia kristinae , Acinetobacter soli, and Streptococcus periodonticum.…”

“…(2017) [ 35 ]; A. agilis 35 rivers, ponds, residential water sources, soil, mud, some plants Price et al. (2020) [ 36 ] Agbaji et al. (2020) [ 37 ] Vandamme et al.…”

“…established on ocular surfaces in healthy adults represents as much as 73% of the bacterial community, especially S. epidermidis [ 48 ]. Aside from the skin flora discussed above, the gram-negative bacteria A. agilis showed a high abundance in the bacterial microbiota constituents in this study, which indicated that the contamination might be from pollutants in the surroundings [ 36 , 47 ]. The source of A. agilis was previously study and the strain was obtained from soil as a rhizobacterial flora [ 37 , 38 ].…”

What is the role of Achromobacter species in patients with cystic fibrosis?

“…However, as the differentiation of different species requires specific molecular-based methods, the true frequency of the various species in CF patients remains poorly defined [13]. Generally, Ac species isolated with conventional methods are reported as A. xylosoxidans [14,15].…”