Role of penicillin-binding protein 5 in expression of ampicillin resistance and peptidoglycan structure in Enterococcus faecium

The contribution of penicillin-binding protein 5 (PBP 5) to intrinsic and a cquired beta -lactam resistance was investigated by constructing isogenic s trains of Enterocaccus faecium producing different PBP 5. The pbp5 genes fr om three E. faecium clinical isolates (BM4107, D344, and H80721) were clone d into the shuttle vector pAT392 and introduced into E. faecium D344S, a sp ontaneous derivative of E. faccium D344 highly susceptible to ampicillin du e to deletion of pbp5 (MIC, 0.03 mug/ml). Immunodetection of PBP5 indicated that cloning of the pbp5 genes into pAT392 resulted in moderate overproduc tion of PBP 5 in comparison to wild-type strains. This difference may be at tributed to a difference in gene copy number. Expression of the pbp5 genes from BM4107 (MIC, 2 mug/ml), D344 (MIC, 24 mug/ml), and H80721 (MIC, 512 mu g/ml) in D344S conferred relatively low levels of resistance to ampicillin (MICs, 6, 12, and 20 mug/ml, respectively). A methionine-to-alanine substit ution was introduced at position 485 of the BM4107 PBP 5 by site-directed m utagenesis. In contrast to previous hypotheses based on comparison of nonis ogenic strains, this substitution resulted in only a 2.5-fold increase in t he ampicillin MIC. The reversed-phase high-performance liquid chromatograph y muropeptide profiles of D344 and D344S were similar, indicating that dele tion of pbp5 was not associated with a detectable defect in cell wall synth esis. These results indicate that pbp5 is a nonessential gene responsible f or intrinsic resistance to moderate levels of ampicillin and by itself cann ot confer high-level resistance.