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 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).

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

“…On the other hand, since the complete pbp5 gene of these isolates has not been amplified and sequenced, other substitutions present in the N-terminal, non-penicillin binding domains may also contribute to the high level of resistance observed in these isolates. Regarding PBP5 expression, our results are in agreement with the results of the study conducted by Montealegre et al (21) on the expression of different PBP5 variants in the context of the background clade. Their results showed that there was a clear difference in the expression of PBP5 between clades, that is, comparing the PBP5 expression by clade A strains with its expression by clade B strains.…”

“…In our study, two susceptible isolates (allele E) were the only isolates that showed much lower expression of PBP5 compared to isolates with MIC ≥ 128. Thus, ampicillin MICs were correlated with PBP5 expression, consistent with those of Montealegre et al (21). According to our results, it is possible that pbp5 variants in various isolates confer different PBP5 affinity and as a result, posttranslational modification of PBP5 or different binding to ampicillin may occur (24).…”

“…Their results showed that there was a clear difference in the expression of PBP5 between clades, that is, comparing the PBP5 expression by clade A strains with its expression by clade B strains. However, within a clade, there was no clear correlation between MIC and expression of PBP5 (21). The pbp5 sequences shown in our study suggest that we dealt entirely with strains in clade A (perhaps both branches, A2 and A1).…”

“…amino acid changes which is paralleled in a general sense by increases in MIC and also with PBP5 expression reported by Montealegre et al (21). However, as these authors and Galloway-Pena et al (20) pointed out, nothing can be concluded about the impact of individual amino acid changes on ampicillin MIC within ampicillin-resistant strains.…”

Artificial Gene Amplification in Escherichia coli Reveals Numerous Determinants for Resistance to Metal Toxicity

“…This frequently involves the duplication of a gene and the concomitant overexpression of the encoded protein (Jensen 1976; Ohno 2013; Patrick et al 2007; Näsvall et al 2012; Kacar and Gaucher 2012; Herron and Doebeli 2013; Levin et al 2014; Hughes and Andersson 2015; Montealegre et al 2017; Notebaart et al 2018). …”