The Spread Of Antibiotic Resistance Mediated By Plasmid In Pathogens And Intervention Of Engineered Bacteriophages

Due to the threat of antibiotic resistance rising,new therapy strategies are required.Here,I focused on the spread of antibiotic resistance in pathogens and the intervention of engineered phages.Explore environmental and genetic determinants of the spread of plasmid-mediated drug resistance genes,to establish a theoretical basis for new strategies and provide targets for engineered phage intervention.Perform active bacteriophages prediction,to expand the phage resource required for new strategies.Create a phage engineering platform for new strategies.In terms of theoretical study,I performed genomic analysis including annotation and type classification on Pac Bio sequencing data of pathogenic Escherichia coli.And based on the association analysis of genotype and phenotype,the study found that plasmid incompatibility group strongly correlates with transfer efficiency.In terms of bacteriophage resource,based on the existing phage genomics data,I performed phage parts mining,de-duplicating and classification.Thus,I created the phage parts library covering eleven categories with about 100,000 parts.Besides,I screened four optimal parts INF（infection）,ASB（assembly）,HYP（hypothetical）,UNS（unsorted）,and harnessed amino acid sequences of above four parts to assist in the development of an active prophage prediction tool-Prophage Hunter.In terms of platform construction,I built a stepping-stone host assisted phage engineering platform（SHAPE）,which integrates theλ-Red recombination module,the CRISPR-Cas9 counter-selection module,and the rebooting module in stepping-stone host,realizing phage modification,screening and activation in one step.I succeeded to engineer four Klebsiella phage with gene knock-in or knock-out.Besides,I explored the codon usage bias and circular genome determinants of SHAPE rebooting efficiency.At present,Enterobacteriaceae,Pseudomonas aeruginosa and Acinetobacter baumannii phages have been rebooted on SHAPE.I designed BES-Phage carrying a novel genome editing module-BES system-to target toxin gene,resistance gene（bla SHV）and rep gene on plasmid.And I succeeded to engineer BES_tox-Phage and BES_bla-Phage.Understanding the environmental and genetic determinants of plasmid mobility in pathogenic Escherichia coli,predicting more active prophages by Prophage Hunter,and constructing a phage engineering platform are expected to effectively utilize to control the spread of drug resistance and the bacteriophage intervention.