Precision-targeting of pathogens using bacteriophage and antimicrobial peptides

Salmonellosis is a major food-borne pathogen worldwide, frequently associated with contaminated poultry and eggs. Salmonella Infantis (SI) is the 4th most common serotype in human infections in the EU, and the most frequent serotype in broiler chickens. Worryingly, multidrug-resistant isolates are increasingly common in Europe, particularly those which have acquired a pESI megaplasmid encoding resistance to multiple classes of antibiotics as well as virulence genes and enhanced tolerance of heavy metals and biocides. Alternative approaches to controlling antimicrobial resistance in Salmonella and other pathogens are urgently needed. Two such approaches are biological control using viruses which specifically target bacteria (bacteriophages) and antimicrobial peptides (AMPs). Both approaches have unique advantages over antibiotics. Bacteriophage are self-replicating and self-limiting - reproducing only when susceptible bacteria are present. Unlike broad-spectrum antibiotics, they target a specific genus, species or strain of bacterium, avoiding potentially harmful dysbiosis in the patient. AMPs form the basis of effective innate immune responses to bacteria and other micro-organisms in all classes of life. The most common mode of action of these to bacterial cells is thought to be via a destabilisation of the bacterial cell membrane resulting in cell lysis. AMPs can vary in size from less than 10aas to 60-70aas, their small size enabling rapid diffusion, and penetration/destabilisation of bacterial biofilms. In this project we aim to combine the strengths of both approaches to control SI, with the ultimate aim of using this combination therapy in poultry, and potentially as a model for use in human medicine. The PhD study is divided into two strands: 1. Isolation and characterisation of antimicrobial peptides (AMPs) specifically targeting SI. Next Generation Peptide Phage Display (NGPD) will be used to isolate or modify peptides specifically targeting the surface of SI. NGPD couples the vast diversity of phage-peptide libraries with the screening power of next generation sequencing and bioinformatics. SI-targeting peptides will be coupled via peptide linkers to known broad specificity AMPs. These fusions will be expressed and characterised according to their MIC and biofilm penetration/inhibition ability against clinical and laboratory strains of SI, and potential host cell toxicity. The optimal combination of AMPs will be determined by iterative machine learning using parameters we identify as important for the performance of AMPs individually (e.g. MIC, stability, efficacy against a range of pathogenic strains). 2. Isolation and characterisation of SI-specific bacteriophages(phages) Phages specific for SI will be isolated from environmental samples, then purified, and screened for their ability to lyse a broad range of clinical and laboratory SI strains in our collection. The genomes of promising, strictly-lytic phage will be sequenced and analysed for therapeutic suitability before undergoing high throughput phenotypic and bioinformatic screening to determine the most effective combinations and titres. The final part of the project will be to combine the AMPs and phages identified from (1) and (2) to determine their synergistic action against SI in biofilm and planktonic cultures, and subsequently ex-vivo models of SI colonisation of chickens. A particular focus will be efficacy against pESI-carrying strains of SI.