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

Abstract

In Staphylococcus aureus , resistance to β-lactamase stable β-lactam antibiotics is mediated by the penicillinbinding protein 2a, encoded by mecA or by its homologues mecB or mecC. However, a substantial number of meticillin-resistant isolates lack known mec genes and, thus, are called meticillin resistant lacking mec (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 mec 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 gdpP were significantly associated with the MRLM phenotype. GdpP is a phosphodiesterase enzyme involved in the degradation of the second messenger cyclic-di-AMP in S. aureus . 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 gdpP mutations to the MRLM phenotype by heterologous complementation experiments. The mutations in gdpP had no effect on transcription levels of pbp4; however, cell sizes of MRLM strains were reduced. The impact on biofilm formation was highly strain dependent. We report mutations in gdpP 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 S. aureus ), which may lead to unnoticed spread of β-lactam-resistant isolates and subsequent treatment failure.

Comparative Genomics of Borderline Oxacillin-Resistant Staphylococcus aureus Detected during a Pseudo-outbreak of Methicillin-Resistant S. aureus in a Neonatal Intensive Care Unit

“…Two of the most prevalent BORSA sequence types, ST97 ( n = 5) and ST398 ( n = 5), have historically been contextualized as livestock-associated MRSA ( 34 – 38 ), though recent reports from France and the United States have described ST398 isolates of our predominating spa type, t1451, as human bloodstream infection (BSI)- and hospital-associated MSSA isolates ( 39 – 41 ), reflecting the demographics of our cohort. Moreover, a German study also found ST398 to be the third-most-common sequence type among their BORSA isolates of clinical origin ( 42 ). Yet, to the best of our knowledge, only five ST97 isolates have previously been annotated as BORSA ( 20 , 43 ).…”

A non-classical mechanism of β-lactam resistance in Methicillin-Resistant Staphylococcus aureus (MRSA) and its effect on virulence

“…MRSA is one of the most prominent agents contributing to the significant global antimicrobial resistance burden today (3). However, in recent years, the presence of β-lactam resistance in S. aureus strains without mecA have been reported (10, 22, 23). In previous studies, we have demonstrated the ability of PBP4 to mediate high-level β-lactam resistance via protein overexpression due to promoter-associated mutations in non- mecA strains (7, 17).…”

In-vivo detection of cyclic-di-AMP in Staphylococcus aureus

“…Recent studies have highlighted that increased CDA concentrations promote tolerance to β-lactam antibiotics and allow cell wall restructuring (8,11). Furthermore, a growing number of contemporary clinical surveillance studies have identified mutations in gdpP among β-lactam resistant or non-susceptible natural isolates of S. aureus (12)(13)(14)(15). Since many of these mutations have been either shown (8) or are predicted to attenuate the function of GdpP, causing increased CDA concentrations in cells, these findings underscored the clinical importance of CDA and highlighted the importance of accurate determination of its concentrations for both basic and clinical research settings.…”