Show simple item record

dc.contributor.advisorHook, Sarah
dc.contributor.advisorVernall, Andrea
dc.contributor.advisorUssher, James
dc.contributor.authorOoi, Jocelyn May Fen
dc.identifier.citationOoi, J. M. F. (2021). Real-time in situ testing of antibiotic resistance (Thesis, Doctor of Philosophy). University of Otago. Retrieved from
dc.description.abstractAntimicrobial resistance (AMR) is one of the top ten threats to public health according to the World Health Organization (WHO), with approximately 700, 000 deaths recorded per year due to infections with drug-resistant bacteria. Furthermore, antibacterial treatments are often initiated with the wrong antibacterials without proper identification of the causative agent and/or empirical antimicrobial therapies are prescribed that can result in increased mortality and healthcare costs. Conventional methods used to diagnose infection and determine antimicrobial susceptibility often have long turnaround times, are expensive and labour intensive. There is a need for methods and technologies to enable rapid antimicrobial susceptibility testing (AST) with potential for point-of-care testing (POCT) to combat AMR. The hypothesis for this research was that a reporter/detector-based assay could be developed that would enable real-time, in situ, testing of antibacterial resistance. The reporter would be modified/activated by an enzyme that was upregulated in bacteria cells in response to the stress induced by exposure to an antimicrobial agent. The proposed AST would involve injecting a test dose of an antimicrobial agent into the patient along with the inactive reporter, co-encapsulated into a particulate delivery system. A highly sensitive assay would then be utilised to detect the modified/activated reporter in a drop of blood at the patient’s bedside. Four different enzymes known to be upregulated in stressed human pathogens (Escherichia coli and Staphylococcus aureus) were investigated; caseinolytic protease proteolytic (ClpP) subunit, the endoribonuclease toxin (MazF), recombination protein A (RecA) and sortase A (SrtA). The expression of the genes encoding these enzymes was investigated in antimicrobial susceptible and resistant clinical isolates of E. coli and S. aureus grown in vitro, after treatment with antibacterials, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Following treatment susceptible, but not resistant, S. aureus were found to upregulate all four genes. This upregulation was not detected in E. coli, likely due to assay-related issues. These findings were further investigated in both antibacterial-treated and untreated bacteria by a proteomics approach to assess protein levels. Proteomics results supported the upregulation of RecA in both E. coli and S. aureus; and ClpP and SrtA in antibacterial-treated S. aureus. A RecA protein assay was able to confirm the functional activity of RecA in antibacterial-treated susceptible, but not resistant, E. coli. In order to achieve co-delivery of antibacterial and reporter, a liposomal delivery system was developed. Optimisation of both cationic and neutral non-PEGylated and PEGylated liposomes was performed to produce liposomes with the required size, polydispersity, drug loading and zeta potential. Optimised cationic non-PEGylated liposomal-cefotaxime showed comparable in vitro antibacterial activity compared to free cefotaxime (a broad-spectrum antibiotic) when tested against E. coli. To further investigate the mechanism of uptake of liposomal-antibacterial in bacteria, a novel assay for the determination of subcellular drug delivery in E. coli lacking the TolC outer membrane efflux pump (tolC) was utilised. The bacteria were further modified such that streptavidin which has high affinity for biotin was localised into the periplasm or cytoplasm thereby allowing compartment-specific localisation of drug to be examined. Bacteria were incubated with a cyclooctyne-biotin probe which would attach to the localised streptavidin before being incubated with azido-containing compounds (luciferin or cefoxitin) delivered as free drug or encapsulated into liposomes. Any azido-containing compound taken up would undergo a click reaction with the cyclooctyne-biotin, with any unreacted cyclooctyne -biotin probe then being measured. Improved uptake of both azido compounds into the periplasm of tolC E. coli and decreased cytoplasmic localisation was observed for cationic liposomal formulations as compared to neutral formulations or free drug. This study provided the potential formulation to be used to deliver both antibacterial and reporter. The next steps in development of the in situ AST were the design and development of reporters for ClpP and RecA. High performance liquid chromatography (HPLC)-based techniques were developed and optimised to measure activation of the proposed reporters after exposure to the recombinant enzymes. A ClpP reporter consisting of a modified synthetic peptide was cleaved at Met-Ala bonds in the presence of acyldepsipeptide (ADEP)1 and ClpP. For RecA, a modified and truncated version of LexA protein was synthesised and purified to use as the reporter. Recombinant RecA was able to cleave the reporter in the presence of ssDNA and ATP-γ-S at Ala-Gly bonds. The detection of RecA-mediated cleavage of the LexA reporter was then further developed using aptamer-based technologies and mass spectroscopy. Analysis of antibacterial-treated and untreated E. coli samples by MS could detect the presence of the cleaved LexA reporter product, however the product was present in all samples. The background level of cleavage could be attributed to RecA expression in cultures due to the stress of in vitro culture. The aim of this study was to investigate if a real-time in situ AST could be developed. The thesis provides important data on the feasibility of this approach. A number of potential bacterial targets upregulated by antibacterial-treated susceptible bacteria were identified along with potential reporters. A formulation suitable for the co-delivery of antibacterial and the reporter was identified and the mechanism of uptake of the formulation was investigated. The need for a sensitive and quantitative assay for detection of the reporter was confirmed. Based on the work in this thesis, the findings confirmed the potential of this approach for the development of a rapid, point-of-care testing for AST in future, using the LexA reporter to detect in vitro and in vivo resistance of bacteria to killing with antimicrobial agents.
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectantimicrobial testing
dc.subjectantibiotic resistance
dc.subjectpoint-of-care testing
dc.subjectsusceptibility testing
dc.titleReal-time in situ testing of antibiotic resistance
dc.language.rfc3066en of Philosophy of Otago
otago.openaccessAbstract Only
 Find in your library

Files in this item


There are no files associated with this item.

This item is not available in full-text via OUR Archive.

If you are the author of this item, please contact us if you wish to discuss making the full text publicly available.

This item appears in the following Collection(s)

Show simple item record