Mutations in the gene are a clinically relevant mechanism for β-lactam resistance in meticillin-resistant lacking determinants.

In , resistance to β-lactamase stable β-lactam antibiotics is mediated by the penicillinbinding protein 2a, encoded by or by its homologues or . However, a substantial number of meticillin-resistant isolates lack known genes and, thus, are called meticillin resistant lacking (MRLM). This study aims to identify the genetic mechanisms underlying the MRLM phenotype. A total of 141 MRLM isolates and 142 meticillin-susceptible controls were included in this study. Oxacillin and cefoxitin minimum inhibitory concentrations were determined by broth microdilution and the presence of genes was excluded by PCR. Comparative genomics and a genome-wide association study (GWAS) approach were applied to identify 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. GWAS revealed that mutations in were significantly associated with the MRLM phenotype. GdpP is a phosphodiesterase enzyme involved in the degradation of the second messenger cyclic-di-AMP in . A total of 131 MRLM isolates carried truncations, insertions or deletions as well as amino acid substitutions, mainly located in the functional DHH-domain of GdpP. We experimentally verified the contribution of these mutations to the MRLM phenotype by heterologous complementation experiments. The mutations in had no effect on transcription levels of ; however, cell sizes of MRLM strains were reduced. The impact on biofilm formation was highly strain dependent. We report mutations in as a clinically relevant mechanism for β-lactam resistance in MRLM isolates. This observation is of particular clinical relevance, since MRLM are easily misclassified as MSSA (meticillin-susceptible ), which may lead to unnoticed spread of β-lactam-resistant isolates and subsequent treatment failure.