Introduction

Tilmicosin (TIL) is a macrolide antibiotic that is licensed in pig farming for treating infections caused by pathogens other than Escherichia coli, which is intrinsically resistant to macrolides due to outer membrane impermeability. We previously demonstrated that peptidomimetics can be used to re-sensitize E. coli to TIL, suggesting potential for repurposing macrolides to address the shortage of effective therapeutic options for managing pig enteritis caused by E. coli.

Aims

To identify genes required for expressing intrinsic TIL resistance in E. coli, referred to collectively as the intrinsic TIL resistome, and use these genes as targets for TIL potentiation.

Methods

A clinical enterotoxigenic E. coli strain (ETEC5621) and a laboratory strain (K-12 MG1655) were used to construct transposon mutant libraries. Transposon Directed Insertion-site Sequencing (TraDIS) was used to explore non-essential genes involved in intrinsic TIL resistance. Lambda red recombineering was used to construct gene deletion mutants to test the effects of gene deletion on the minimal inhibitory concentration (MIC) of TIL.

Results

TraDIS analysis identified intrinsic TIL resistome genes in ETEC5621 and MG1655 after exposure to 1/8 MIC (n=15 and 16, respectively) and 1/4 MIC (n=38 and 32, respectively) of TIL. Genes common to both strains (n=23) were enriched in functions related to lipopolysaccharide biosynthesis, outer membrane assembly, the Tol-Pal system, efflux pump and peptidoglycan metabolism, suggesting that the integrity of cell envelope is crucial to intrinsic TIL resistance in E. coli. Deletion of seven of these genes led to a 64- to 2-fold reduction of TIL MIC in both strains. Notably, deletion of surA or waaG conferred the highest MIC reduction in the clinical strain, decreasing it to 16 µg/ml, a concentration that is achievable in the pig intestinal tract after oral administration.

Conclusion

Our findings contribute to a genome-wide understanding of the intrinsic TIL resistome in pig clinical E. coli and provide the proof of concept to re-sensitize this important veterinary pathogen to TIL by interfering with genes involved in cell envelope integrity.