A genome wide analysis of Escherichia coli responses to fosfomycin using TraDIS-Xpress reveals novel roles for the phosphonate and phosphate transport systems

Abstract

Fosfomycin is an antibiotic which has seen a revival in use due to its unique mechanism of action and resulting efficacy against isolates resistant to many other antibiotics. Mechanisms of resistance have been elucidated and loss of function mutations within the genes encoding the sugar importers, GlpT and UhpT are commonly selected for by fosfomycin exposure in E. coli. There has however not been a genome wide analysis of the basis for fosfomycin sensitivity reported to date. Here we used TraDIS-Xpress a high-density transposon mutagenesis approach to assay the role of all genes in E. coli in fosfomycin sensitivity. The data confirmed known mechanisms of action and resistance as well as identifying a set of novel loci involved in fosfomycin sensitivity. The assay was able to identify sub domains within genes of importance and also revealed essential genes with roles in fosfomycin sensitivity based on expression changes. Novel genes identified included those involved in glucose metabolism, the phosphonate import and breakdown system, phnC-M and the phosphate importer, pstSACB. The impact of these genes in fosfomycin sensitivity was validated by measuring the susceptibility of defined inactivation mutants. This work reveals a wider set of genes contribute to fosfomycin sensitivity including core sugar metabolism genes and two transport systems previously unrecognised as having a role in fosfomycin sensitivity. The work also suggests new routes by which drugs with a phosphonate moiety may be transported across the inner membrane of Gram-negative bacteria. ImportanceThe emergence and spread of antibiotic resistant bacteria had resulted in increased use of alternative drugs which retain efficacy against isolates resistant to other classes of drugs. One example is fosfomycin; an old drug which has found greatly increased use in recent years. We studied the mechanisms of fosfomycin resistance by applying a genome wide screen based on comparing the fitness of a massive library of transposon mutants in the presence of fosfomycin. This approach identified the previously known mechanisms of resistance but also identified a number of new pathways which contribute to fosfomycin sensitivity including two importer systems. This information advances our knowledge about an increasingly important antibiotic and identifies new potential routes to resistance.