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Title: The design and assembly of tailored oligosaccharides as polymer therapeutics for improved treatment of chronic respiratory disease
Author: Stokniene, Joana
ISNI:       0000 0004 7962 1984
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2019
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Healthcare-associated infections affect 4 million patients annually in the EU and result in an estimated 37,000 deaths per year. Of particular concern are the rapidly increasing resistance rates of Gram-negative bacterial pathogens to many, or even all, commonly used antibiotics, with a corresponding decrease in the design/development of new antibiotic compounds. Thus, there remains an urgent need for novel therapies for these 'hard to treat' infections. Fortunately, polypeptide antibiotics, such as colistin and polymyxin B, still retain potent antimicrobial activity against multidrug resistant (MDR) Gram-negative pathogens. In addition, polymer therapeutics are finding increasing utility as antimicrobial agents. The aim of this thesis was to generate and characterise an alginate oligosaccharide ("OligoG")-polymyxin conjugate library to optimise the antimicrobial functions of these last resort drugs. Reproducible conjugation of polymyxin to OligoG was achieved using amide or ester cross-linkers, producing conjugates with 6.1-12.9% (w/w) antibiotic loading and molecular weights of 14,500-27,000 g/mol (relative to pullulan MW standards). TNFα ELISA and MTT assays revealed that OligoG conjugation significantly decreased inflammatory cytokine production and cytotoxicity of colistin (2.2-9.3-fold) and polymyxin B (2.9-27.2-fold) from a human kidney cell line. Minimum inhibitory concentration (MIC) assays and bacterial growth curves demonstrated that antimicrobial activity of the OligoG-polymyxin conjugates was similar to that of the parent antibiotic, but with more sustained bacterial growth inhibition. Importantly, ester-linked conjugates showed full retention of the antibiotic's antimicrobial activity, while the MIC of the amide-linked conjugates increased by more than 2 log-fold. Confocal laser scanning microscopy revealed that both amide- and ester-linked colistin conjugates significantly disrupted the formation of P. aeruginosa biofilms and induced bacterial death. An in vitro 'time-to-kill' experiment using A. baumannii indicated that colistin and OligoG-ester-colistin conjugates reduced viable bacterial counts (~2 fold) after 4 h, with no significant activity observed with OligoG-amide-colistin conjugates. OligoG-induced disruption of the 3-dimensional architecture and clumping of P. aeruginosa and E. coli biofilms was demonstrated using a Transwell diffusion model and biofilm disruption assays, while fluorescent labelling of OligoG confirmed its rapid diffusion and distribution within the whole biofilm structure. These studies confirm that bi-functional polymer therapeutics such as OligoG-polymyxin conjugates have potential benefits in the treatment of MDR Gram-negative bacterial infections.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General)