Talbot, Nathan (2019) A Search for Novel Antimicrobials and Novel β-Lactamase Inhibitors. Doctoral thesis, University of Huddersfield.

For almost a century, antibiotics have been used as treatment agents in the combat of infections caused by pathogenic microbes.For a short while, antibiotics were heralded as miracle drugs; their discovery and introduction into the clinic has no doubt saved countless animal and human lives. The use of antibiotics in surgical procedures has ushered in the modern medical era where surgeons are afforded a whole host of cutting edge treatment and ground-breaking surgical techniques thanks to the benefits and treatment options antibiotics offer. The dark times of the pre-antibiotic era where many lives were lost through trivial illnesses have long been forgotten. However, what was once a trickle of reports of antibiotic resistance has since turned into a flood of pandemic proportions; the once lethal actions of antibiotics are threatened by the seemingly unstoppable march of resistance. Pathogenic microbes, once susceptible to the killing mechanisms of antibiotics, are now equipped with several mechanisms capable of shrugging off the previously deadly antibiotics. The main weapon in our antibiotic armamentarium is β-lactam antibiotics, owing to their cheapness, efficacy, and limited side effects. It is perhaps unsurprising then that the main driver of antibiotic resistance is by the production of β-lactamases, enzymes capable of efficiently catalysing the hydrolysis of the β-lactam ring present in β-lactam antibiotics, rendering them useless. Unfortunately, soil which was once a rich source of novel antibiotic producing bacteria has long been described as an over-mined resource and attempts to match the success of natural and semi synthetic antibiotics with truly synthetic antibiotics has largely been a failure.

It is known that the majority of bacteria in situ are unculturable in laboratory conditions. The work in this thesis explores novel methods to increase the in vitro growth rate of bacteria in an attempt to revitalise traditional antimicrobial discovery methods, by use of a novel incubation tool known as the isolation chip or “iChip” for short. The iChip is designed in the hope to increase the in vitro growth rate of previously unculturable bacteria. The work in this thesis describes the characterisation and antimicrobial activity of a previously little described strain of Pseudomonas baetica, a bacterium of little interest, only noted for its pathogenicity towards certain species of fish.

Given the dearth of novel antibiotics, β-lactamase inhibitors represent an alternative mechanism for overcoming antimicrobial resistance as compared to novel antimicrobials. This thesis also explores the potential of novel β-lactamase inhibitors, in the hope of extending the shelf life of existing “resistance vulnerable” β-lactam antibiotics. Several ring-opened carbamate ester analogues of avibactam, a novel β-lactamase inhibitor, were synthesised and their effectiveness as serine-β-lactamase inhibitors were assessed. One of the inhibitors was found to be even more effective than avibactam itself. Also, the effect of pH upon inhibition of serine-β-lactamases by these inhibitors and the catalytic activity of these enzymes was measured showing that the catalytic machinery is the same for both hydrolysis and inhibition.

Finally, several other existing compounds were tested for their inhibitory effectiveness against β-lactamases. Ellagic acid was found to be a good inhibitor of serine-β-lactamases and showed an interesting inhibitory profile dependent upon pH demonstrating that both the mono-and di-anions are effective inhibitors.

Talbot THESIS.pdf - Accepted Version
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