Aptamer beacon-based single-step fluorescence method for rapid detection of bacteria and antimicrobial resistance
Infectious disease and antimicrobial resistance have emerged as a major threat to public health and economy globally. Even though the development of resistance in bacteria is a natural process, the overuse or misuse use of antimicrobials have elevated the rise of antimicrobial resistance (AMR). The unnecessary antimicrobials prescriptions can be regulated by rapid near-patient diagnostic tool to differentiate viral and bacterial infections and select the most appropriate antimicrobials. The traditional microbiological culture based diagnostic test usually takes 24 to 72 hours to obtain the results because it requires culturing procedure to identify the species and antimicrobial susceptibility testing. Several rapid culture-independent methods, such as polymerase chain reaction (PCR), Next-generation sequencing, and mass spectrophotometry methods are available. However, those methods are expensive, involve multiple sample processing steps, and requires sophisticated equipment and skilled staff to perform the test. The global response to AMR requires rapid and affordable diagnostics that can be used at the point-of-care (PoC) settings which can rapidly determine the infection and antimicrobial resistance. Such test should quickly determine the bacterial infection and antimicrobial resistance and guide the clinicians to prescribe right antimicrobial therapy. The work presented in this thesis address the limitation of current diagnostic methods through the development of single-step detection of infection and antimicrobial resistance directly from complex samples. Aptamer beacons are short single-stranded oligonucleotide probe comprising dye and quencher at the terminals, that undergo structural switching while binding to the target. Such a binding-induced conformational change can be transduced by the changes of fluorescence signal. To date, aptamer beacons have been only explored for the detection of small molecules, peptides, and proteins. However, aptamer beacons for large molecules or whole bacteria were not explored due to the complexities in the beacon design for whole bacterial cell. In this thesis, an aptamer beacon was iv designed for the whole bacterial detection employing surface epitope as target. Usually, aptamer beacons were designed by inserting a short complimentary region to existing aptamer and destabilizing the native binding to form beacon. In this thesis, a stem-loop aptamer beacon was designed through a sequential approach involving six steps, (1) target identification, (2) aptamers selection against the target, (3) experimental assessment of aptamer against the target, (4) in-silico docking analysis of target and aptamer, (5) aptamer truncation and transformation to beacon design, (6) validation of aptamer beacon. The gram-negative lipopolysaccharide (LPS) was identified as target and aptamers against LPS was evaluated. The aptamer shows highest affinity against LPS was studied using bioinformatics tools to identify the LPS interacting residues. The identified LPS interacting residues was truncated to enhance the affinity and convert into stem-loop structured aptamer beacon. The designed aptamer beacon was validated and optimized for the detection LPS. The feasibility of the aptamer beacon based single-step method was evaluated using LPS of E. Coli O111:B4 as target due to its clinical importance in the management of urinary tract infection (UTI). In this thesis, the aptamer beacon was designed to target LPS of E. Coli O111:B4 and validated with a greater sensitivity by detecting 3.9ng/mL LPS directly in urine within 60 minutes. Following the successful validation for LPS detection, the aptamer beacon was extended to the detection of E. Coli O111:B4 in synthetic urine (SU) medium. Like synthetic urine, the aptamer beacon validation steps were explored in wound buffer medium for the detection of Pseudomonas aeruginosa in wound buffer (WB). The aptamer beacon sensitivity for the bacterial detection was determined by detecting lowest concentration of 103 cfu/mL directly in SU and WB. The kinetics response of aptamer beacons revealed the feasibility of phenotypic detection of bacteria within 5 minutes directly from SU and WB. Further, in this thesis, demonstrated the first feasibility of aptamer beacon based phenotypic antimicrobial sensitivity test within one hour directly from in SU and WB. Throughout the several stages of the thesis, the aptamers cross-reactivity towards the different strains of bacteria was studied. The detailed understanding of the LPS structure has allowed to identify a conserved epitope for the application of broad-gram negative bacteria detection. A similar aptamer beacon design pathway was followed and designed aptamer beacon for the conserved epitope on the LPS. The aptamer beacon targeting conserved epitope on the LPS was tested against v various gram-negative strains and observed consistency in the response for all the gram-negative strains. The knowledge gained through this thesis has allowed to transform and validate the aptamer beacons for various applications like bacteria detection from jet fuels and biofilms. Through this thesis, aptamer beacon-based singlet-step phenotypic detection of bacterial and antimicrobial susceptibility determination was successfully demonstrated. This single-step test can potentially be adopted in clinical practice as the first rapid point-of-care test for bacterial detection with phenotypic antibiotic resistance. This test should allow rapid definitive infectious disease diagnosis at the point of care, guiding precision antibiotic treatment timely, and contributing to a paradigm change the delivery of quality and affordable healthcare worldwide. The generic nature of the aptamer beacon design uniquely allows the flexibility to apply this method, in principle, to any pathogen with known surface epitopes, such as proteins or polysaccharides. The speed and simplicity of the assay using a handheld fluorescence reader offer this method the potential to be applied widely in low-skilled point-of-care settings.
- Aeronautical, Automotive, Chemical and Materials Engineering
- Chemical Engineering
Rights holder© Praveenkumar Kaveri
NotesA Doctoral Thesis. Submitted in partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy at Loughborough University.
Supervisor(s)Sourav Ghosh ; Guido Bolognesi
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