PBP 2a Mutations Producing Very-High-Level Resistance to Beta-Lactams

Abstract

Resistance to the beta-lactam class of antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is mediated by PBP 2a, a synthetic bacterial cell wall penicillin-binding protein with a low affinity of binding to beta-lactams that is encoded by mecA. Beta-lactams that bind to PBP 2a with a high affinity and that are highly active against MRSA are under development. The potential for the emergence of resistance to such compounds was investigated by passage of homogeneous MRSA strain COL in L-695,256, an investigational carbapenem. A highly resistant mutant, COL52, expressed PBP 2a in which a two-amino-acid deletion mutation and three single-amino-acid substitution mutations were present. To examine the effects of these mutations on the resistance phenotype and PBP 2a production, plasmids carrying (i) PBP 2a with two or three of the four mutations, (ii) wild-type PBP 2a, or (iii) COL52 PBP 2a were introduced into methicillin-susceptible COL variants COLnex and COL52ex, from which the staphylococcus cassette chromosome mec (SCCmec) has been excised, as indicated by the "ex" suffix. Two amino acids substitutions, E3K 237 within the non-penicillinbinding domain and V3E 470 near the SDN 464 conserved penicillin-binding motif in the penicillin-binding domain in COL52, were important for high-level resistance. The highest level of resistance was observed when all four mutations were present. The emergence of PBP 2a-mediated resistance to beta-lactams that bind to PBP 2a with a high affinity is likely to require multiple mutations in mecA; chromosomal mutations appear to have a minor role.Staphylococcal resistance to the beta-lactam class of antibiotics, termed methicillin resistance, is mediated by PBP 2a, a low-affinity penicillin-binding protein (PBP). The beta-lactam antibiotics that are used clinically do not bind to PBP 2a at therapeutic concentrations and therefore lack efficacy against infections caused by methicillin-resistant staphylococci. With the solution of the crystal structure of a soluble derivative of PBP 2a, low-affinity binding can be attributed at least in part to an energetically unfavorable acylation reaction of the active site serine due to the slippage of the beta-lactam molecule within a long groove that serves as the initial, reversible binding site in the formation of the Michaelis-Menten complex (16).Several cephalosporin and carbapenem derivatives that bind to PBP 2a with 100-fold or higher affinities than those of other beta-lactams and which are active in vitro and in vivo against methicillin-resistant staphylococci have been synthesized (2,6,7,13,20). These compounds have in common a relatively extended side chain attached to the ␣ ring of the beta-lactam compound core (3, 16). This structural feature permits the molecule to be positioned within the groove in such a way that the acylation reaction proceeds at a rate more rapid than that in its lower-affinity relatives. Two of these compounds, BAL 9141 and RWJ 54428, have been tested in phase I trials with humans (7, 17). It is qui...

Affinity of Ceftaroline and Other β-Lactams for Penicillin-Binding Proteins from Staphylococcus aureus and Streptococcus pneumoniae

“…Hence, the higher PBP2a affinity resulted in lower MICs. We did not see a strictly proportional correlation between IC 50 and the MIC for the ␤-lactams used in the study, which is a known phenomenon and reflects many other factors involved in the resistance level (7,19,26,30,49). We observed such differences between PBP2a affinity and the MIC for strain 25 for all antibiotics tested.…”

“…In strain 510, the PBP2a affinities for ceftriaxone (1 g/ml) and cefotaxime (Ͼ128 g/ml) varied compared to the MICs (Ͼ64 g/ ml), and this observation cannot be explained without additional experiments. The above-mentioned facts (a sufficient supply of peptidoglycan precursors and differences in peptidoglycan composition caused by altered gene expression involved in cell wall synthesis) may partially explain the differences as well as the possible mutations in the mecA gene encoding the PBP2a protein (30). It is important that affinity studies of this nature have not, to our knowledge, been pub- lished for hVISA and VISA strains, so there is no current basis for comparison of the results.…”

Rapid Detection of Staphylococcus aureus and Methicillin-Resistant S. aureus (MRSA) in Wound Specimens and Blood Cultures: Multicenter Preclinical Evaluation of the Cepheid Xpert MRSA/SA Skin and Soft Tissue and Blood Culture Assays

“…The MRSA/SA assays, used on the GeneXpert system, automate sample purification, nucleic acid amplification, and target sequence detection. The primers and probes in the Xpert MRSA/SA assays detect sequences within the staphylococcal protein A (spa) gene, the gene for methicillin resistance (mecA), and the staphylococcal cassette chromosome (SCCmec) inserted into the S. aureus chromosomal attB insertion site (12,13). The assays are the first commercial methods to simultaneously detect the mecA gene along with the attB insertion site.…”

Effects of Alterations in Staphylococcus aureus Cell Membrane and Cell Wall in Antimicrobial Resistance

“…The mecA gene is present on the staphylococcal cassete chromosome (SCC mec), a genomic island in staphylococci and other Gram-positive bacteria that encode methicillin resistance. Its mobile nature allows it to become widespread among microorganisms [57]. Generally, in susceptible S. aureus cells, the vancomycin molecules bind on the C-terminal D-Ala-D-Ala, thereby inhibiting the catalytic reactions of the transglycosylation and transpeptidation, mediated by PBPs.…”