Objectives: To establish an experimental platform in Staphylococcus aureus for identifying genetic loci that determine intrinsic antibiotic susceptibility and/or that have the potential to contribute to acquired antibiotic resistance. A near-saturation S. aureus transposon (Tn) library was screened for mutants exhibiting altered susceptibility to the antistaphylococcal agents daptomycin, vancomycin and nisin.
Methods: S. aureus SH1000 was mutagenized with Tn InsTet(G+)2(Cm) by electroporation of transposomes. Approximately 20500 transposants were screened for increased or reduced susceptibility to the three antistaphylococcal agents and Tn insertion sites were mapped by DNA sequencing in mutants of interest.
Results: Transposants exhibiting hypersusceptibility or reduced susceptibility were identified for all three antibacterial agents; mapping of Tn insertion sites in these mutants identified genetic determinants of intrinsic susceptibility and potential contributors to acquired resistance, respectively. Tn insertions in the dlt operon caused cross-hypersusceptibility to vancomycin, daptomycin and nisin. Daptomycin hypersusceptibility was also associated with disruption of genes directing lipoteichoic acid and riboflavin biosynthesis, apparent inactivation of a putative membrane protein encoded by SAOUHSC_00957 and truncation of the cell-division gene ezrA. Tn-mediated disruption of the vraDE- and SAOUHSC_02953/4-encoded ABC transporters conferred hypersusceptibility to nisin. Reduced susceptibility to both daptomycin and vancomycin was associated with Tn insertions in rpsU and upstream of yycFG. Several loci were associated with reduced susceptibility to nisin, including two genes encoding putative glycosyltransferases.
Conclusions: Tn library screening identified both known and novel modulators of antibacterial susceptibility in S. aureus and therefore represents a useful approach towards delineating the staphylococcal resistome.