Overproduction of a low-affinity penicillin-binding protein and high-level ampicillin resistance in Enterococcus faecium

Abstract

Five ampicillin-resistant clinical isolates of Enterococcus faecium were analyzed for a correlation between overproduction of the low-affinity penicillin-binding protein (PBP 5) and the level of ampicillin resistance.Comparison was made with one susceptible clinical isolate and its ampicillin-resistant derivative obtained in the laboratory by selection with increasing concentrations of penicillin. Overproduction of the low-affinity PBP relative to the susceptible isolate was noted in moderately resistant strains (MIC, 32 ,ug/ml) but not in highly resistant strains (MIC, 128 ,ug/ml). Polyclonal antibodies specifically reacting with the low-affinity PBP of Enterococcus hirae, Enterococcusfaecalis, and Enterococcusfaecium (M. Ligozzi, M. Aldegheri, S. C. Predari, and R. Fontana, FEMS Microbiol. Lett. 83:335-340, 1991) were used to determine the amount of this PBP in the E. faecium isolates. In all strains, the antibody preparation reacted with a membrane protein of the same molecular mass as PBP 5. The amount of this protein was very small in the susceptible strain but large in all of the resistant strains. These results suggest that the highly resistant strains also overproduced the low-affinity PBP, which, compared with PBP 5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability.Despite several reports of enterococcal strains producing a P-lactamase (16,17,19), the main pathway of penicillin resistance development in these microorganisms still remains modification of penicillin-binding proteins (PBPs), in particular, overproduction of a low-affinity PBP which is a normal component of the PBP pattern of these bacteria (9, 10). The mechanism by which overproduction of PBP 5 confers resistance has been ascribed to the ability of this PBP to substitute the functions of the other PBPs (3,4,7,8,10). This proposal stems from the finding that ATCC 9790 mutants overproducing the low-affinity PBP grow normally in the presence of penicillin concentrations which saturate all PBPs but not PBP 5, which, in these conditions, is apparently the only active PBP. In addition, it has been shown that, in ATCC 9790 derivatives which carry a temperature-sensitive defect in the essential PBPs 1, 2, and 3 associated with a temperature-* Corresponding author. sensitive cell division, a second mutation causing overproduction of PBP 5 allows normal growth at the nonpermissive temperature (4).The recent cloning of the E. hirae PBP 5 gene (pbp5) (5) has provided insights into the genetic mechanism of penicillin resistance development (14). It has been shown that, in resistant mutants, PBP 5 overproduction is associated with a deletion in a genetic element, located 1 kb upstream of pbp5, which negatively controls PBP 5 synthesis (14). Hypersusceptibility to penicillin is associated with a point mutation inpbp5, which causes premature termination of translation. E. hirae pbpS does not hybridize with DNA of other enterococcal species, suggesting that the genes which code for low-affinity PBPs are not str...

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

“…Enterococcus faecium is the most penicillin resistant enterococcal species, with MICs ranging from 0.5 to more than 64 mg/ml for clinical isolates. MICs as high as 256 and 512 mg/ml have been found in very resistant strains [11,12]. Some highly blactam resistant E. faecium strains are also vancomycin resistant [12 -14], although vancomycin and b-lactam antibiotics can act in a synergistic way against vancomycinresistant enterococci [15].…”

“…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]. In E. faecalis, the peptidoglycan synthesized by PBP5 in the presence of benzylpenicillin is slightly different from that produced by the usual PBPs in the absence of benzylpenicillin.…”

FemABX Peptidyl Transferases: a Link between Branched-Chain Cell Wall Peptide Formation and β-Lactam Resistance in Gram-Positive Cocci

“…Their intrinsic ␤-lactam resistance is due to endogenous low-affinity PBP5, which is not essential (91). Acquired resistance to higher antibiotic concentrations is associated with the overproduction of PBP5 and/or a decreased affinity of PBP5 for the ␤-lactam (34). However, the role of PBP5 in high-level resistance is likely to depend on additional factors.…”

High-Level, Multiresistant Enterococci Associated with Systemic Disease: A Report of Two Cases

“…Resistance to antimicrobials in the enterococci is mediated in a number of ways. These include modification of penicillin-binding protein, 3 β-lactamase production, 4 the development of aminoglycoside-modifying enzymes, 3 and vancomycin resistance. [6][7][8] Vancomycin was introduced in 1956 and for nearly 30 years enjoyed an enviable status, exemplified by absence of development of resistance to the drug.…”

“…Their intrinsic resistance or relative resistance to β-lactams, clindamycin and aminoglycosides, and ability to acquire resistance to ciprofloxacin, erythromycin and tetracycline is well known. [3][4][5][6] The most recent and most disturbing trait to emerge is resistance to vancomycin. 7 Three years ago we had reported the first case of wound infection caused by vancomycin-resistant Enterococcus.…”