Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium. The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium. We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.KEYWORDS ampicillin, Enterococcus faecium, penicillin-binding protein 5, protein levels, resistance A mpicillin-resistant Enterococcus faecium has emerged to be one of the leading pathogens in the hospital setting (1, 2). In the United States, the incidence of infections caused by ampicillin-resistant E. faecium strains has increased since the 1980s, and this has been accompanied by a progressive increase in the MICs over time (3, 4); currently, the majority of E. faecium isolates recovered in hospitals around the United States are resistant to this -lactam antibiotic (1, 2). The decreased susceptibility of E. faecium to ampicillin and other -lactams has been mainly attributed to the high-molecular-weight penicillin-binding protein 5 (PBP5) (3-5). The proposed mechanism of PBP5-mediated action is a low affinity for -lactam antibiotics, allowing peptidoglycan synthesis (transpeptidation) and bacterial growth when the other PBPs are inhibited by the drug (6, 7).