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Title: Development of antisense/antigene agents based on synthetic oligonucleotides to inhibit extended spectrum β-lactamases and restore antibiotic sensitivity in resistant bacterial pathogens
Author: Readman, John Benedict
ISNI:       0000 0004 8499 040X
Awarding Body: Royal Holloway, University of London
Current Institution: Royal Holloway, University of London
Date of Award: 2016
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Increasingly prevalent are strains of bacteria that have a reduced susceptibility to multiple antibiotics, often leaving few therapeutic treatment options for bacterial infections. Extended spectrum β-lactamases confer resistance to β-lactam antibiotics and have been observed in a wide range of bacterial species. Synthetic antisense oligonucleotide analogues have been shown to specifically inhibit the translation of targeted bacterial genes and enable the possibility of the restoration of antibiotic sensitivity of resistant bacteria by the targeted inhibition of proteins responsible for reduced susceptibility, such as β-lactamases, thereby restoring antimicrobial efficacy. The current study designed and developed antisense oligomers to target CTX-M-group 1 extended-spectrum β-lactamase mRNA and inhibit enzyme expression via ribosomal hindrance. Expression plasmids harbouring blaCTX-M-15 cloned from field isolates were constructed for gene expression inhibition studies. Two assays for the quantification of β-lactamase activity were developed. Antisense oligomers significantly inhibited CTX-M-15 β-lactamase activity by 92 - 100% (P < 0.001) in a cell-free translation-transcription coupled system. An atypically permeable cell-wall mutant E. coli strain was transformed with expression plasmids harbouring blaCTX-M-15 to yield a resistant phenotype, and in cell growth inhibition assays sensitivity to cefotaxime was significantly increased. Antisense oligomers covalently attached to a cell-penetrating peptide significantly (P < 0.05) increased sensitivity to cefotaxime in clinical isolates in a dose dependent manner. A novel synthetic antisense oligomer delivery vehicle based on a 3D tetrahedral DNA structure was also developed and evaluated - a significant increase (P < 0.001) in the sensitivity of field and clinical isolates to cefotaxime was observed in a dose dependent manner. The current study has demonstrated the efficacy of targeted synthetic antisense oligomers in partially restoring cefotaxime sensitivity in previously resistant clinical E. coli isolates, and demonstrated the efficacy of a novel vector able to deliver antisense oligomers into bacteria.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available