Investigating the potential of pre-clinical cell-wall synthesis inhibiting antibiotics
Creators: name: Lloyd, Daniel
Publisher: name: University of Lincoln
Year: 2023
Subjects: C500 - Microbiology

description: Antibiotic resistance in bacteria is reaching a tipping point, with rising numbers of infections untreatable with clinically available antibiotics. Reducing the human cost caused by increasing antibiotic resistance requires a new approach; combining the introduction of new antibiotic compounds with changing behaviours towards these miracle drugs. This work focuses on inhibitors of bacterial cell-wall synthesis, with a focus on two potential future antibiotics of clinical importance, teixobactin and moenomycin A. Despite differential mechanisms of action, both compounds are viewed to be recalcitrant to the development of resistance.Initially, the structure/activity relationship of teixobactin was investigated, biologically testing the activity of synthetic teixobactin analogues. Highlighting the importance of D-oriented amino acids in the peptide chain and identifying multiple analogues that retained activity, despite substitution of the rare L-allo-enduracididine. Following this, the role of individual residues in the peptide chain was assessed through ‘alanine scanning’, identifying residues amenable to modification and facilitating improved delivery to Gram-negative bacteria. With the establishment of a library of potent synthetic teixobactin analogues, their in-vivo activity was assessed using G. mellonella, identifying analogues with efficacy comparable to vancomycin.Access to biologically active synthetic teixobactin analogues facilitated other avenues of research. To assess overlapping antibiotic resistance pathways that develop from ‘target-adjacent’ mechanisms of bacterial inhibition, de-novo resistance was induced to both arg10-teixobactin and moenomycin A through multi-step serial passage. Over 45 days (~300 generations) the average tolerance of Staphylococcus aureus increased &gt;3-fold to arg10-teixobactin but increased ~1000-fold towards moenomycin A over the same period.Demonstrating that Staphylococcus aureus can develop significant resistance to arg10-teixobactin and moenomycin A within clinically meaningful timescales. The costs, consequences and genetic basis of resistance was assessed from the evolved strains, allowing interpretation of the mechanisms underpinning the acquired resistance.The first identification of acquired resistance to a teixobactin analogue led to an investigation into the pre-existing resistome of arg10-teixobactin. This utilised next-generation sequencing technology to identify changes in the soil microbiome following antibiotic treatment, focusing on the impact of wastewater-effluent on the associated soil resistome. This identified a core bacterial population that survived regardless of sampling location, though the mechanism of this ‘resistance’ or ‘persistence’ has not yet been determined.This work contributes to future efforts in counteracting antibiotic resistance. This has been achieved through the development and optimisation of synthetic teixobactin analogues, identifying pathways to resistance against clinically promising compounds (moenomycin A and arg10-teixobactin) and assessing the extent of pre-existing environmental resistance to arg10-teixobactin.Aims1. To develop and test novel teixobactin analogues, identifying structure/activity relationships to inform future analogue design2. To assess the rate and mechanisms of de novo resistance against arg10-teixobactin and moenomycin A3. To investigate the breadth of pre-existing resistance amongst culturable and non-culturable environmental bacteria to arg10-teixobactin