Differential Penicillin-Binding Protein 5 (PBP5) Levels in the Enterococcus faecium Clades with Different Levels of Ampicillin Resistance

Abstract

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: Enterococcus faecium; ampicillin; penicillin-binding protein 5; protein levels; resistance.

FIG 1

Differential PBP5 levels in the E. faecium clades detected by Western blotting. Equivalent protein samples were separated by SDS-PAGE and transferred to a PVDF membrane, and PBP5 was detected with a polyclonal serum raised against rPBP5-S from strain Com15 or rPBP5-R from strain C68 (the antibodies used to detect PBP5-S were removed, and the membrane was reprobed with anti-rPBP5-R). The E. faecium strains and ampicillin (AMP) MICs are indicated above the blots (see Table 1 for detailed descriptions of the strains), while the relative quantification of the band intensity for PBP5 normalized to the RNA polymerase β subunit levels is shown below the blots; 10 ng each of recombinant PBP5-S and PBP5-R was loaded into the last two lanes as controls. See Fig. S1 for the results of an independent experiment with the rPBP5-R antiserum and a monoclonal antibody against the RNA polymerase subunit β protein (loading control).

FIG 2

pbp5 genetic environments found in the 16 E. faecium strains included in this study. (A) Com15_B, TX1330_B, 1.141.733_B, TX2050_B, E980_B, EnGen12_A2, and EnGen35_B; (B) EnGen21_A2 and TX16 (DO)_A1; (C) EnGen52_A2, EnGen24_A2, EnGen25_A2, and 1.231.502_A1; (D) TX82_A1 and 1.230.933_A1; (E) C68_A1.