bioRxiv preprint doi: https://doi.org/10.1101/2020.02.11.944942; this version posted February 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Duplex real-time PCR assay for the simultaneous detection of Achromobacter 2 xylosoxidans and Achromobacter spp. 3 4 Erin P. Price1,2#, Valentina Soler Arango2,3, Timothy J. Kidd4,5, Tamieka A. Fraser1,2, Thuy- 5 Khanh Nguyen5, Scott C. Bell5,6, and Derek S. Sarovich1,2 6 1GeneCology Research Centre, University of the Sunshine Coast, Sippy Downs, Queensland, 7 Australia 8 2Sunshine Coast Health Institute, Birtinya, Queensland, Australia 9 3School of Science and Engineering, University of the Sunshine Coast, Sippy Downs, 10 Queensland, Australia 11 4School of Chemistry and Molecular Biosciences, Faculty of Science, The University of 12 Queensland, St Lucia, Queensland, Australia 13 5QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia 14 6Adult Cystic Fibrosis Centre, The Prince Charles Hospital, Chermside, Queensland, Australia 15 16 # Corresponding author 17 University of the Sunshine Coast, Locked Bag 4, Maroochydore DC, Qld, 4558, Australia 18 Email: [email protected] 19 Phone: +61 7 5456 5568 20 Key Words 21 Achromobacter, Achromobacter xylosoxidans, comparative genomics, cystic fibrosis, 22 respiratory infections, polymicrobial infections, real-time PCR, diagnostics, sputum 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.11.944942; this version posted February 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 23 Abstract 24 Several members of the Gram-negative environmental bacterial genus, Achromobacter, are 25 associated with serious infections in immunocompromised individuals, of which 26 Achromobacter xylosoxidans is the most common. Despite their pathogenic potential, 27 comparatively little is understood about these intrinsically drug-resistant bacteria and their role 28 in disease, leading to suboptimal diagnosis and management of Achromobacter infections. 29 Here, we performed comparative genomics of 158 Achromobacter spp. genomes to robustly 30 identify species boundaries, to reassign several incorrectly speciated taxa, and to identify 31 genetic sequences specific for the Achromobacter genus and for A. xylosoxidans. Next, we 32 developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the 33 rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both 34 purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates 35 identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to 36 these routine diagnostic methods, the duplex assay showed superior identification of 37 Achromobacter spp. and A. xylosoxidans, with five Achromobacter isolates failing to amplify 38 with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax 39 quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents, 40 and detected down to ~12 and ~1 genome equivalent/s, respectively. In silico analysis, and 41 laboratory testing of 34 non-Achromobacter isolates and 38 adult CF sputa, confirmed duplex 42 assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a 43 robust, sensitive, and cost-effective method for the simultaneous detection of all 44 Achromobacter spp. and A. xylosoxidans, and will facilitate the rapid and accurate diagnosis 45 of this important group of pathogens. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.11.944942; this version posted February 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 46 Introduction 47 The Achromobacter genus comprises 19 officially designated species (1), all of which are 48 highly motile, Gram-negative, non-fermentative bacteria found ubiquitously in environmental 49 reservoirs including rivers, ponds, residential water sources, soil, mud, and some plants (2, 50 3). Achromobacter spp. are important community-acquired pathogens, particularly in people 51 with cystic fibrosis (CF), cancer, immunoglobulin deficiencies, renal disease, endocarditis, 52 diabetes, and those undergoing invasive procedures (4, 5). These opportunistic pathogens 53 can infect several organs, although the respiratory and urinary tracts are the most common 54 sites of infection (5). Members of this genus have been isolated from several usually sterile 55 hospital products such as disinfectants, ultrasound gel, dialysis fluids, contact lens fluid, 56 eardrops, incubators, respirators, humidifiers, and deionised water, consistent with the 57 adaptability of Achromobacter spp. to survive in diverse environments (2, 4, 6). 58 Naturally multidrug-resistant bacteria, including Achromobacter spp., are increasingly being 59 retrieved from CF airways due to the intensified implementation of aggressive antibiotic 60 therapies (7, 8). Achromobacter spp. prevalence in CF centres globally range from 3 to 30%; 61 of these, between 10 and 52% progress to a chronic infection. In addition to their intrinsic 62 antibiotic resistance towards aminoglycosides, aztreonam, tetracyclines, and some penicillins 63 and cephalosporins, Achromobacter spp. possess a similar denitrification system to 64 Pseudomonas aeruginosa, which facilitates their survival and proliferation in hypoxic and 65 anoxic environments such as those found in CF airways (5). 66 Although several Achromobacter species can infect CF airways (9), Achromobacter 67 xylosoxidans is the most common, comprising ~42-65% of all Achromobacter spp. identified 68 in CF respiratory secretions (4, 10-12). Up until recently, the role of Achromobacter spp. in 69 disease pathogenesis has been unclear; however, recent studies have shown that CF patients 70 with an Achromobacter spp. infection are in fact at greater risk of experiencing a pulmonary 71 exacerbation (9), and patients with chronic infections exhibit severe airway obstruction and 72 more rapid lung function decline (13-15). Further, these pathogens can cause a range of 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.11.944942; this version posted February 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 73 serious diseases such as pneumonia, meningitis, osteomyelitis, urinary tract infections, and 74 ocular infection in non-CF patients (16). Therefore, early and correct identification of 75 Achromobacter spp. and A. xylosoxidans is important for improving the treatment and 76 prognosis of disease. 77 Current diagnostic methods for identifying Achromobacter spp. and A. xylosoxidans, including 78 the commonly used VITEK® matrix-assisted laser desorption-ionisation time-of-flight mass 79 spectrometry platform (VITEK® MS), provide a reasonably accurate method for identifying 80 these organisms (17). However, all Achromobacter spp. are allocated as A. xylosoxidans/A. 81 denitrificans on this platform (18), thus providing limited capacity for accurate species-level 82 identification. Further, VITEK® MS and other mass spectrometry-based platforms require a 83 purified isolate to obtain an accurate speciation result, which limits the utility of this platform 84 as it cannot be used directly on polymicrobial clinical specimens such as sputum, resulting in 85 longer turn-around times, potentially incorrect antimicrobial treatment, and higher costs (19, 86 20). In addition, mass spectrometry-based equipment has a large upfront cost and footprint, 87 rendering this method out-of-reach for smaller, less-resourced laboratories. To address this 88 shortcoming, an automated multiplex PCR has recently been developed to detect four non- 89 fermentative Gram negative bacterial species, including A. xylosoxidans, directly from 90 respiratory samples using the BD MAX™ System (18). This multiplex assay detected A. 91 xylosoxidans with 97% specificity, but only 78% sensitivity (18), indicating suboptimal 92 diagnosis of this organism using this method. Further, the BD MAX™ multiplex assay was not 93 designed to identify other Achromobacter spp., meaning that ~50% of CF infections caused 94 by Achromobacter spp. cannot be diagnosed with this method. Other genotyping methods 95 such as amplified ribosomal DNA restriction analysis (ARDRA) (21), multilocus sequence 96 typing (22), nrdA gene sequencing (12), and whole-genome sequencing (WGS) provide robust 97 identification and speciation methods for Achromobacter spp., but are laborious and cannot 98 be performed in a rapid or cost-effective manner. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.11.944942; this version posted February 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under