Enterococcus faecium has six penicillin‐binding proteins (PBP), where PBP5 seems to be the main target for β‐lactam antibiotics. The PBP profiles of three imipenem‐resistant, ampicillin‐sensitive E. faecium strains, isolated from the same
patient, were studied using biotinylated ampicillin and chemiluminescence detection. Imipenem resistance in these strains was found to be associated with hyperproduction of PBP5 compared to the ampicillin‐ and imipenem‐susceptible strain ATCC 19434. PBP5 in the imipenem‐resistant
strains (S1, B2) exhibited a selectively decreased affinity for imipenem. An 854 bp DNA fragment, corresponding to the penicillin‐binding domain of pbp5fm, was studied in the resistant strains and the reference strain. Four amino acid substitutions were observed in the resistant
strains compared to the susceptible one. The contribution of these substitutions to the increased production of PBP5 in these strains is unclear since the substitution was observed also in a strain without increased production of PBP5. Our results suggest that the moderate imipenem resistance
observed in these strains is associated with increased production of PBP5 with relatively decreased affinity for imipenem, and that evolution of imipenem resistance in E. faecium is dinstinct from that of the other β‐lactams such as ampicillin.