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Corynebacterium jeikeium is an emerging nosocomial pathogen responsible for vascular catheters infections, prosthetic endocarditis and septicemia. The treatment of C. jeikeium infections is complicated by the multiresistance of clinical isolates to antibiotics, in particular to beta-lactams, the most broadly used class of antibiotics. To gain insight into the mechanism of beta-lactam resistance, we have determined the structure of the peptidoglycan and shown that C. jeikeium has the dual capacity to catalyse formation of cross-links generated by transpeptidases of the d,d and l,d specificities. Two ampicillin-insensitive cross-linking enzymes were identified, Ldt(Cjk1), a member of the active site cysteine l,d-transpeptidase family, and Pbp2c, a low-affinity class B penicillin-binding protein (PBP). In the absence of beta-lactam, the PBPs and the l,d-transpeptidase contributed to the formation of 62% and 38% of the cross-links respectively. Although Ldt(Cjk1) and Pbp2C were not inhibited by ampicillin, the participation of the l,d-transpeptidase to peptidoglycan cross-linking decreased in the presence of the drug. The specificity of Ldt(Cjk1) for acyl donors containing a tetrapeptide stem accounts for this effect of ampicillin since the essential substrate of Ldt(Cjk1) was produced by an ampicillin-sensitive d,d-carboxypeptidase (Pbp4(Cjk)). Acquisition and mutational alterations of pbp2C accounted for high-level beta-lactam resistance in C. jeikeium.

Bypass of the penicillin-binding proteins by an L,D-transpeptidase (Ldt(fm)) confers cross-resistance to beta-lactam and glycopeptide antibiotics in mutants of Enterococcus faecium selected in vitro. Ldt(fm) is produced by the parental strain D344S although it insignificantly contributes to peptidoglycan cross-linking as pentapeptide stems cannot be used as acyl donors by this enzyme. Here we show that production of the tetrapeptide substrate of Ldt(fm) is controlled by a two-component regulatory system (DdcRS) and a metallo-D,D-carboxypeptidase (DdcY). The locus was silent in D344S and its activation was due to amino acid substitutions in DdcS or DdcR that led to production of DdcY and hydrolysis of the C-terminal D-Ala residue of the cytoplasmic peptidoglycan precursor UDP-MurNAc-pentapeptide. The T(161)A and T(161)M substitutions affected a position of DdcS known to be essential for the phosphatase activity of related sensor kinases. Complete elimination of UDP-MurNAc-pentapeptide, which was required specifically for resistance to glycopeptides, involved substitutions in DdcY that increased the catalytic efficiency of the enzyme (E(127)K) and affected its interaction with the cell envelope (I(14)N). The ddc locus displays striking similarities with portions of the van vancomycin resistance gene clusters, suggesting possible routes of emergence of cross-resistance to glycopeptides and beta-lactams in natural conditions.

Corynebacterium jeikeium is an emerging nosocomial pathogen responsible for vascular catheters infections, prosthetic endocarditis and septicemia. The treatment of C. jeikeium infections is complicated by the multiresistance of clinical isolates to antibiotics, in particular to beta-lactams, the most broadly used class of antibiotics. To gain insight into the mechanism of beta-lactam resistance, we have determined the structure of the peptidoglycan and shown that C. jeikeium has the dual capacity to catalyse formation of cross-links generated by transpeptidases of the d,d and l,d specificities. Two ampicillin-insensitive cross-linking enzymes were identified, Ldt(Cjk1), a member of the active site cysteine l,d-transpeptidase family, and Pbp2c, a low-affinity class B penicillin-binding protein (PBP). In the absence of beta-lactam, the PBPs and the l,d-transpeptidase contributed to the formation of 62% and 38% of the cross-links respectively. Although Ldt(Cjk1) and Pbp2C were not inhibited by ampicillin, the participation of the l,d-transpeptidase to peptidoglycan cross-linking decreased in the presence of the drug. The specificity of Ldt(Cjk1) for acyl donors containing a tetrapeptide stem accounts for this effect of ampicillin since the essential substrate of Ldt(Cjk1) was produced by an ampicillin-sensitive d,d-carboxypeptidase (Pbp4(Cjk)). Acquisition and mutational alterations of pbp2C accounted for high-level beta-lactam resistance in C. jeikeium.