Amino acid substitution mutations and mRNA expression levels of the pbp5 gene in clinical Enterococcus faecium isolates conferring high level ampicillin resistance

In this study, clinical ampicillin-resistant Enterococcus faecium isolates with minimum inhibitory concentrations (MICs) for ampicillin in the ranges from 128 to >512 mu g/mL (n = 17) and two ampicillin-susceptible isolates (MIC 1 mu g/mL) were investigated. No beta-lactamase production was detected in these isolates. Alterations in the C-terminal part of pbp5 and levels of pbp5 mRNA expression were investigated by sequencing and quantitative real-time qRT-PCR, respectively. Sequencing analysis revealed five different pbp5 alleles (A to E) having differences in 18 amino acid positions spanning from residue 426 to 642. Allele A (V-462 -> A, H-470 -> Q, M-485 -> A, N-496 -> K, A-499 -> T, E-525 -> D, N-546 -> T, A-558 -> T, G-582 -> S, E-629 -> V, K-632 -> Q, and P-642 -> L) was the most frequent allele. The presence of just two susceptible isolates in allele E suggests a possible correlation between amino acid patterns and MIC, even if there is no discernible correlation with specific single amino acid differences. Also, these were the only isolates that showed much lower expression of class B penicillin-binding protein 5 (PBP5) compared to isolates with MIC of 128 or greater. Thus, ampicillin MICs were correlated with PBP5 expression.