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 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/PBP5 mRNA and 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 cover wide range of ampicillin MICs. Our results found that pbp5/PBP5 levels are increased in subclades A1 and A2 ampicillin-resistant strains as compared to 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 nucleotides 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 points to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.
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