SACM - Australia
Permanent URI for this collectionhttps://drepo.sdl.edu.sa/handle/20.500.14154/9648
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Item Restricted Assessing isothermal technology for detection of antimicrobial pathogens(Saudi Digital Library, 2024) Abdulrahman Khalid, Ayfan; David, WhileyThis thesis aims to provide solutions for antibiotic resistance by developing rapid point-of-care tests for key bacteria and associated resistances that have been flagged by the World Health Organization (WHO) and Centre for Disease prevention and Control (CDC). The studies initially focused on detection of New Delhi Metallo (NDM), and Verona Integrated Metallo (VIM) Beta Lactamase genes among carbapenemase-producing organisms Gram negative bacteria. The project then expanded to detect Neisseria gonorrhoeae (by targeting the gonococcal porA pseudogene) and associated resistances to ciprofloxacin (by the detection of a single nucleotide polymorphism [SNP] within the gonococcal gyrA gene) and ceftriaxone (by targeting the gonococcal penA 60.001 gene). The technology used for detection was iso-thermal recombinase polymerase amplification (RPA) and the detection was coupled with a lateral flow detection (LFD) system. Recognising the complexity for SNP detection by RPA (e.g., its ability to accommodate several mismatches in assay targets), this thesis explored several approaches to tackle this, including adding additional artificial mismatches in oligonucleotides, as well as using blocker primer and probe approaches. Assays performances compared well to traditional methods of detection (e.g., polymerase chain reaction; PCR), and time needed to perform/read results was less than thirty minutes compared to ninety minutes using PCR. Unlike PCR, RPA can be performed at a relatively low temperature, and assays can be successfully conducted by holding the reaction tubes in the palm of a hand. In addition to the wet-lab experiments, extensive in-silico sequence-based analyses were conducted to help further accelerate the development of point of care (POC) assays; these included improving our understanding of gonococcal gyrA diversity to inform target selection (using publicly available databases; National Centre for Biotechnology Information (NCBI), Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR), Pathogen Watch) as well as to identify new potential N. gonorrhoeae diagnostic gene targets (noting that, for example, the gonococcal porA pseudogene can lead to false-negative results for certain N. gonorrhoeae strains). Overall, this thesis provides further potential POC solutions for antibiotic resistance; specifically, the ability to detect gram negative bacteria-harbouring blaNDM and blaVIM genes, and N. gonorrhoeae gyrA and penA 60.001 resistance targets. These advances will provide valuable information to help clinicians and healthcare teams to tailor antibiotic therapy in a timely manner, with limited skills and training needed to perform and read results.9 0Item Restricted Next Generation Antibiotic Susceptibility Testing (NGAST)(The University of Queensland, 2018) Abdulrahman Khalid, Ayfan; Zowawi, HosamAntimicrobial Resistance (AMR) is a worldwide issue that needs to be tackled. The lack of a rapid tool for the detection of antimicrobial resistance is needed. Recently Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-TOF MS) have revolutionized the routine clinical labs for the bacterial identification with providing rapid, accurate and reliable results within hours in comparison to the conventional methods of detections. Also, MALDI-TOF MS seems to have the potentiality to be used as an antibiotic susceptibility testing tool. In recent times, one of the developed testings to detect AMR by this MS-platform. MALDI is the MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). In this project, The MBT-ASTRA was used for testing bug-drug combination from preliminary studies to validate the robustness of this technique on a MALDI-TOF MS (AB SCIEX). 4 strains of Klebsiella pneumoniae and P. aeruginosa (including 2 sensitive strains and 2 resistant strains for each bug) were tested against 64μg/ml of meropenem with an incubation time of one and two hours respectively. Also, Escherichia coli was tested against Ciprofloxacin at a concentration of 32μg/ml with two hours of an incubation. Secondly, the project also involved in optimizing four new bug-drug combinations which included E. coli against colistin and gentamicin and P. aeruginosa against gentamicin and ciprofloxacin. The results from the testing showed a clear discrimination between the different strains. As well as a significant discrimination for E. coli against gentamicin within one hour of an incubation time at a concentration of 64μg/ml. This study demonstrates the ability of the MBT-ASTRA in the rapid detection of AMR.6 0