dltA overexpression: A strain-independent keystone of daptomycin resistance in methicillin-resistant Staphylococcus aureus

The mechanisms leading to reduced susceptibility to daptomycin (DAP) are multifactorial and have not been fully elucidated. We analysed, by sequencing and expression studies, the role of the major molecular targets (cell-envelope charge genes, dltA, mprF, cls2; cell-wall turnover and autolysis genes, sceD, atl) involved in the emergence of DAP resistance in three series of isogenic clinical methicillin-resistant Staphylococcus aureus (MRSA) in which DAP resistance emerged after a heterogeneous glycopeptide-intermediate S. aureus (hGISA) step under teicoplanin and DAP therapy. All of the isolates had different genotypes and were δ-haemolysin negative, reflecting a strain proclivity to acquire DAP/glycopeptide non-susceptibility under antibiotic pressure. DAP exposure led to the emergence of DAP resistance after an hGISA step probably in parallel with the timing of the two antimicrobial administrations and, in two of three cases, in conditions of DAP underdosage. Real-time qPCR data revealed that all DAP-resistant (DAP-R) isolates had dltA overexpression, whereas mprF upregulation was found only in DAP-R strains with the S295L and T345I amino acid substitutions. Strains that were heteroresistant to DAP did not possess DAP-R-like characteristics. DAP-R strains presented high cls2 expression and no known cls2 mutations, and moreover exhibited sceD and atl upregulation. In conclusion, these findings highlight that dltA overexpression is the common pathway of resistance among genotypically different series of isolates and may represent the keystone of DAP resistance in MRSA, leading to electrostatic repulsion and, indirectly, to a reduction of autolysin activity. mprF mutations related to increased transcription may play a role in this complex phenomenon.