Genetic and pharmacological inactivation of D-alanylation of teichoic acids sensitizes pathogenic enterococci to β-lactams

Background: Enterococci intrinsically resistant to cephalosporins represent a major cause of healthcare-associated infections, and the emergence of MDR makes therapeutic approaches particularly challenging. Objectives: Teichoic acids are cell wall glycopolymers present in Gram-positive bacteria. Teichoic acids can be modified by D-alanylation, which requires four proteins encoded by the dltABCD operon. Our objective was to evaluate the Dlt system as a druggable target to treat enterococcal infections. Methods: The susceptibility of a D-alanylation-deficient strain of Enterococcus faecalis to beta-lactam antibiotics individually and/or in combination was analysed. Moreover, a DltA inhibitor was synthesized to test pharmacological inhibition of D-alanylation in vivo and in host using the animal model Galleria mellonella with different clinical isolates of E. faecalis and Enterococcus faecium. Results: Most cephalosporins used as mono treatment had no impact on survival of the parental strain, but were slightly lethal for the dltA mutant of E. faecalis. Addition of a very low concentration of amoxicillin significantly increased killing of the dltA mutant under these conditions. The most spectacular effect was obtained with a combination of cefotaxime (1mg/L) and amoxicillin (0.03mg/L). In the presence of the inhibitor, the WT strain was as susceptible to this combination treatment as the dltA mutant. This molecule associated with the antibiotics was also effective in killing other E. faecalis clinical isolates and successfully prevented death of Galleria infected with either E. faecalis or E. faecium. Conclusions: The combined results support the potential usefulness of the Dlt system as a target to potentiate antibiotic combination therapies for the treatment of drug-resistant enterococci.