Genomic context as well as sequence of both psr and penicillin-binding protein 5 contributes to β-lactam resistance in Enterococcus faecium

Penicillin-binding protein 5 (PBP5) of Enterococcus faecium (Efm) is vital for ampicillin resistance (AMP-R). We previously designated three forms of PBP5, namely, PBP5-S in Efm clade B strains [ampicillin susceptible (AMP-S)], PBP5-S/R (AMP-S or R), and PBP5-R (AMP-R) in clade A strains. Here, pbp5 deletion resulted in a marked reduction in AMP minimum inhibitory concentrations (MICs) to 0.01-0.09 mu g/mL for clade B and 0.12-0.19 mu g/mL for clade A strains; in situ complementation restored parental AMP MICs. Using D344SRF (lacking ftsW/psr/pbp5), constructs with ftsW(A)/psr(A) (from a clade A1 strain) cloned upstream of pbp5-S and pbp5-S/R alleles resulted in modest increases in MICs to 3-8 mu g/mL, while high MICs (>64 mu g/mL) were seen using pbp5 from A1 strains. Next, using ftsW +/- psr from clade B and clade A/B and B/A hybrid constructs, the presence of psr(B), even alone or in trans, resulted in much lower AMP MICs (3-8 mu g/mL) than when psr(A) was present (MICs >64 mu g/mL). qRT PCR showed relatively greater pbp5 expression (P = 0.007) with pbp5 cloned downstream of clade A1 ftsW/psr (MIC >128 mu g/mL) vs when cloned downstream of clade B ftsW/psr (MIC 4-16 mu g/mL), consistent with results in western blots. In conclusion, we report the effect of clade A vs B psr on AMP MICs as well as the impact of pbp5 alleles from different clades. While previously, Psr was not thought to contribute to AMP MICs in Efm, our results showed that the presence of psr(B) resulted in a major decrease in Efm AMP MICs.