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Increasing rates of bacterial antimicrobial resistance (AMR), associated with almost 5 million deaths per annum as of 2019, mean that it has never been more crucial that all facets of this phenomenon are explored. An underappreciated and hence understudied aspect of the AMR crisis is the potential risk of cross-resistance. This is when bacteria develop resistance to another antimicrobial which may be related to one to which they have been exposed, or which they may never have encountered. Previous work by colleagues in the United Kingdom Health Security Agency Technology Development Group has established that cross-resistance to clinical antibiotics can occur in Gram-negative bacteria upon long-term bacterial exposure to biocides. Notably, upon exposure to the antiseptics octenidine and chlorhexidine, mutations are found not only in in genes controlling the expression of efflux pumps but also membrane remodelling genes and this causes wholesale changes in the bacterial cell envelope. However, it remains unclear whether exposure to biocides may lead to cross-resistance to human innate immune effectors, which may also act on the bacterial cell surface, and if so whether the same mechanisms might be involved. In humans, host defence peptides (HDPs) act to guide the early stages of infection by exerting both immunomodulatory and direct antimicrobial effects which they are thought to achieve, like antiseptics, by disrupting the bacterial cell envelope. LL-37 is a very well-studied human HDP and the sole human cathelicidin. In contrast, there are far fewer studies of its physiologically occurring N-terminally truncated fragments: RK-31, KS-30, and KR-20. These potentially play key roles in widening the antimicrobial activity of cathelicidins in humans, and due to their truncation their antibacterial properties and mechanism of action may be significantly distinct from that of the parent peptide. Here therefore we first present studies that explain the effect of LL-37 truncation on peptide biophysical properties & antimicrobial behaviour and then ask whether and how Gram-negative bacteria are susceptible to cathelicidin cross-resistance in the event of biocide exposure-as well as whether previously identified genes of interest may play a role in this phenomenon. Chapter 3 comprises a thorough characterisation of the conformational behaviour of the human cathelicidins, using circular dichroism spectroscopy to understand how the different HDPs respond to their environment. This includes temperature, pH, solvent, as well as bacterial metabolites which have a coordinating effect. Since these are differently produced by commensal vs pathogenic bacteria, they may modulate antibacterial activity. LL-37 and its fragments are variably sensitive to different bacterial metabolites, with the greatest induction of α-helix conformation occurring at low pH and in the presence of disodium succinate. To complement this approach, we employed molecular dynamics simulations to probe the conformational behaviour of the peptides in a model membrane environment. Chapter 4 describes changes in antibacterial activity caused by the truncation of LL-37. In vitro results showing the inability of any human cathelicidin peptides to kill Staphylococcus aureus are validated in vivo using Galleria mellonella infection models. In contrast, LL-37 and its fragments are active against Acinetobacter spp. and differences in in vitro pharmacodynamics properties such as cooperativity & maximum bacterial killing rate are revealed for the first time, demonstrating that the shortest fragment is the most cooperative across tested Acinetobacter species. N-terminal truncation of the parent peptide is shown to affect bactericidal rate, but not potency. We also demonstrate the consistently superior maximum bacterial killing rate and cooperativity of octenidine vs chlorhexidine against these organisms. In Chapter 5 we describe adaptation experiments, in which Gram-negative bacteria were serially passaged through increasing concentrations of antimicrobials to identify the genomic modifications arising from long-term exposure. In Pseudomonas aeruginosa, mainly pmrB mutations were identified upon adaptation to (non-cathelicidin) HDPs. Cross-resistance to HDPs was identified in biocide-adapted strains but not vice versa. The experiment was repeated with cathelicidins against a panel of four Acinetobacter strains, confirming that similar typologies of genes (involved in cell envelope remodelling and efflux) are mutated upon adaptation to these compounds. Finally, in the Appendix, we describe progress made in characterising the P. aeruginosa protein SmvR. The protein was cloned into a plasmid vector, expressed, purified, and studied by 2D nuclear magnetic resonance (NMR) spectroscopy. Preliminary spectra enabled detection of part of the expected resonances, opening a new avenue of research into these bacterial mediators.

Bacterial infections, which have been susceptible to standard antibiotic regimes for decades, are now becoming resistant to all major antimicrobial treatments, undermining the efficacy of available antibiotics. Bacteria can naturally develop resistance over time as they are exposed to antibiotics. However, due to poor economic incentives and challenging regulatory requirements, antibiotic resistance is accelerated mainly by the overuse and misuse of these medications and a lack of new drug development by the pharmaceutical industry. This highlights the need for novel therapeutic approaches to tackle resistant bacterial infections. Herein, different therapeutic strategies targeting antibiotic-resistant bacteria were investigated. The therapeutic approach based on the use of membrane-disrupting agents to enhance the effectiveness of existing antibiotics for the treatment of antibiotic-resistant bacterial infections is suggested. Antibiotics such as rifampicin are known to have poor activity against Gram-negative bacteria owing to the outer membrane, which prevents their efficient uptake. To overcome this challenge, two series of compounds were designed and synthesised, referred to as Lipoguanidines and photoswitchable guanidines. Lipoguanidine compounds consist of a guanidine moiety, which gives selectivity toward bacterial membrane with its positive charge, and a 'lipo' component which can be intercalated and interact with the hydrophobic cytoplasmic membrane of bacteria. Lipoguanidine compounds showed a broad spectrum of excellent membrane permeabilising activity as they reduced the Minimum Inhibitory Concentration of antibiotics with no toxicity observed in Galleria mellonella assays. The structure of photoswitchable guanidines is based on lipoguanidine but also contains photoswitchable moiety that can change conformation upon light irradiation, enhancing their membrane-disrupting activity. Its ability to modulate the abacterial membrane via light irradiation was also observed. The use of light-activated CO-releasing molecules (PhotoCORMs) against multi-drug-resistant bacteria was proposed. CORMs have recently attracted interest in microbiology as it has been shown that CO can exert rapid bactericidal activity when it is delivered through CORMs. Two series of photoactivable PhotoCORMs were designed and synthesised, phenanthroline and flavonols-based PhotoCORMs. Phenanthroline-based PhotoCORMs were effective at low concentrations against multidrug-resistant K. pneumoniae and A. baumannii when photoactivated with blue light. It has also been shown to inhibit the formation of biofilms. The antibacterial and biofilm inhibitory effects are plausibly due to the release of CO and the formation of phenanthroline photo by-products. Besides phenanthroline-based PhotoCORMs, organic molecule-based CORMs include flavonols and flavonoid metal complexes were also synthesised, and its biological activity were evaluated against bacteria. These novel therapeutic approaches demonstrate promising antibacterial activity to combat multidrug-resistant bacterial infection. These new approaches share the insight of sustainable use of antibiotics as they can extend the lifespan of current antibiotics by improving their efficient uptake via membrane-disrupting activity as well as the application of photopharmacology can avoid the emergence of antibiotic resistance via selective activation upon light irradiation.