Biomarker identification and multiplex assay development for the detection of antimicrobial resistance

Abstract

Hospital admissions with antimicrobial resistant (AMR) infections and associated deaths are increasing at a rate that will see AMR infections as the biggest cause of death globally by 2050. Carbapenems are used as a last resort antimicrobial to treat infections with an extended-spectrum of resistance. Carbapenemases are enzymes that hydrolyse carbapenems as well as most beta-lactam antimicrobials making carbapenemase producing organisms (CPOs) difficult to treat. This has led to the Centers for Disease Control and Prevention (CDC) listing CPOs as two out of five of the most urgent threats. Five carbapenemases have been identified as the biggest threat to worldwide dissemination of antimicrobial resistance and are therefore classed as the ‘big five’. The ‘big five’ carbapenemases are as follows: Klebsiella pneumonia carbapenemase (KPC), Oxacillinase-48-like family (OXA-48), New-Delhi metallo-beta-lactamase (NDM), Verona integron-encoded metallo-beta-lactamase (VIM), and Imipenemase (IMP). All variants of the ‘big five’ carbapenemases were identified; 33, 14, 21, 55, and 70 variants of KPC, OXA-48, NDM, VIM, and IMP have been sequenced, respectively. Through sequence alignment conserved sequences were identified across 100 % of blaKPC, 100 % of blaOXA-48, 100 % of blaNDM, 98% of blaVIM, and 86 % of blaIMP variants which primers were designed against. Primer sets were characterised in recombinase polymerase amplification (RPA) reactions using agarose gel electrophoresis and densitometry. An optimal primer set for each target was identified and assessed in triplex RPA reactions followed by agarose gel electrophoresis which demonstrated that each primer set could successfully amplify the target genes. A bespoke bioinformatic script was developed to design six unique single stranded oligonucleotide tails with minimal complementarity towards the oligonucleotides within the proposed one-pot pentaplex assay. Using this script six single stranded oligonucleotide tails each fifteen nucleotides in length with four or less complementary base pairs towards the selected primers and cognate probes were designed. The optimal primer sets were modified with 5′ tails to mediate detection on a solid surface. Tailed primers were assessed in both RPA in solution and solid-phase RPA reactions followed by enzyme-linked oligonucleotide assays (ELONA) detection. The background signal observed when RPA reactions containing opposing primers in solution (RPA in solution) could not be eliminated and assays performed in this manner were too variable; conversely, anchoring the forward primer to a solid surface (solid-phase RPA) proved to be a more reproducible form of amplification when paired with ELONA readout. For the first time a pentaplex solid-phase RPA assay done in parallel was successfully performed producing calibration curves with the limits of detections of 157 fM, 2.88 pM, 702 fM, 557 fM, and 275 fM for blaKPC, blaOXA-48, blaNDM, blaVIM, and blaIMP targets, respectively. Resonance Raman spectroscopy was explored as an alternative form of readout to detect oxidised TMB produced from solid-phase RPA and ELONA reactions. A portable handheld Raman reader was used to detect oxidised TMB and produced calibration curves for blaOXA-48, blaVIM, and blaIMP with the LODs of 12.72 pM, 826 fM, and 5.11 pM, respectively. Progress has been made towards developing a point-of-care test (POCT) that targets the ‘big five’ carbapenemases. Assays using the primers designed within this project are sensitive, specific and provide a higher level of coverage than the most widely used commercial assays. Further steps to evaluate the primers in a one-pot multiplex reaction will need to be performed and alternative forms of read-out such as surface-enhanced Raman spectroscopy should be investigated alongside mechanising the assay onto either magnetic beads or lateral flow format.