Mutations in the gdpP gene are a clinically relevant mechanism for ß-lactam resistance in methicillin resistant Staphylococcus aureus lacking mec determinants

Background: In Staphylococcus aureus resistance to ß-lactamase stable ß-lactam antibiotics is commonly mediated by the mecA-encoded penicillin binding protein 2a (PBP2a) or rarely by its homologues mecB or mecC. However, a substantial number of methicillin-resistant isolates lack known mec genes (methicillin resistant lacking mec, MRLM). This study aims to identify the genetic mechanisms underlying the MRLM phenotype. Methods: 141 MRLM isolates and 142 methicillin-susceptible controls (MSSA) were included in this study. Oxacillin and cefoxitin minimal inhibitory concentrations were determined by broth microdilution. The presence of mec genes was tested via PCR; whole genome sequencing (WGS) was performed for all isolates and a genome wide association (GWA) approach was applied to confirm genetic polymorphisms associated with the MRLM phenotype. The potential impact of such mutations on the expression of PBP4 as well as on cell morphology and biofilm formation was investigated. Results: GWAS revealed mutations in gdpP to be significantly associated with the MRLM phenotype. GdpP is a phosphodiesterase enzyme that degrades intracellular cyclic-di-AMP in S. aureus. 131 MRLM isolates carried truncations, insertions or deletions as well as amino acid substitutions, mainly located in the functional DHH-domain of GdpP. Mutations in gdpP had no effect on transcription levels of pbp4; however, cell sizes of MRLM strains were on average 13% reduced. The impact on biofilm formation was highly strain-dependent. Conclusions: We report mutations in gdpP as a clinically relevant mechanism for ß-lactam resistance in MRLM isolates. This observation is of particular clinical relevance, since MRLM are easily misclassified as MSSA, which may lead to unnoticed spread of ß-lactam resistant isolates and subsequent treatment failure.