Penicillin-Binding Protein 5 Sequence Alterations in Clinical Isolates of Enterococcus faecium with Different Levels of  -Lactam Resistance

Abstract

The low-affinity penicillin-binding protein (PBP) 5 is the main beta-lactam target and is responsible for resistance to this class of antibiotics in Enterococcus faecium. The PBP 5 variants of 15 clinical isolates (including 8 resistant to vancomycin) with different levels of beta-lactam resistance were analyzed. Most of the highly beta-lactam-resistant isolates produced small quantities of PBP 5 of low affinity. This was associated with particular amino acid substitutions: an Ala or Ile for Thr-499, a Glu for Val-629, and a Pro for Ser-667. A change of Met-485 to Thr or Ala (adjacent to the conserved SDN box) was observed in isolates with MICs of ampicillin of 64 or 128 microg/mL, respectively. In the 2 most resistant isolates, with MICs of ampicillin of 256 microg/mL, an additional Ser was present just after Ser-466. Thus, particular point mutations in PBP 5 and combinations thereof may lead to high-level beta-lactam resistance in E. faecium.

Molecular Characterization of Ampicillin-Resistant Enterococcus faecium Isolates from Hospitalized Patients in Norway

“…Whether this reflects a causal association needs to be elucidated. Other studies have associated the presence of point mutations in the C-terminal region with certain levels of resistance (17,24). We also found statistically significant associations between point mutations in ampicillin-resistant and -susceptible isolates in positions that earlier have been related to ampicillin resistance, such as 485M3T, 496N3K, 499A3T, 525E3D, 586V3L, and 629E3V.…”

“…This is most likely not a clonal phenomenon, since some of these were clonally unrelated as determined by PFGE and AFLP, but an indication that the aspartic acid and serine insertion at this position may affect the affinity of beta-lactam antibiotics for PBP5. Insertions of aspartic acid and serine at this position in strains with an increased level of resistance to ampicillin have been described previously (24,39). Whether this reflects a causal association needs to be elucidated.…”

The 2.4-Å crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin

“…Insertion of a serine in position 466¢ (residue 466 is also a serine) in the most resistant strains emphasizes the importance of the 451 -465 loop on the left-hand side of the active site. This insertion is associated with a twofold-increased MIC for penicillin (strains AR9, 6885 and H80721) and seems to be independent of Met485 mutations [13]. As mentioned above, Val465 is close to the thiazolidine ring of the penicilloyl moiety and the structure rigidity of the left side of the active-site cleft rests upon the Arg464 -Asp481 salt bridge.…”

“…of some clinical isolates highly resistant to ampicillin, not by means of PBP5fm overproduction but by a reduction of the protein affinity for the antibiotic. Following an analysis by Rybkine et al [13] who studied a series of clinical isolates with various levels of resistance to ampicillin (well correlated with the resistance to benzylpenicillin), a first level of resistance is associated with PBP5fm overproduction but higher levels of resistance can be achieved by mutations of Met485 (fig. 4) or the insertion of an additional serine in position 466¢.…”

“…However, overproduction is not the only path to resistance within resistant strains of E. faecium. Point mutations in the PBP5fm further reduce the affinity of the protein for b-lactams, leading to very high levels of resistance [13,17]. PBP5fm is thought to be able to take over the role of the other HMMPBPs in peptidoglycan synthesis when these proteins are inhibited by an antibiotic [10,11,18,19].…”