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

Abstract

Darehkordi H, Saffari F, Mollaei HR, Ahmadrajabi R. Amino acid substitution mutations and mRNA expression levels of the pbp5 gene in clinical Enterococcus faecium isolates conferring high level ampicillin resistance. APMIS 2019; 127: 115-122.In this study, clinical ampicillin-resistant Enterococcus faecium isolates with minimum inhibitory concentrations (MICs) for ampicillin in the ranges from 128 to ˃512 lg/mL (n = 17) and two ampicillin-susceptible isolates (MIC 1 lg/mL) were investigated. No b-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.

High-Resolution Genotyping Unveils Identical Ampicillin-Resistant Enterococcus faecium Strains in Different Sources and Countries: A One Health Approach

“…All 24 genomes analyzed had several mutations in the pbp5 gene ( Table S3 ) that have been associated with pbp5 clade A1R, which mostly comprises ampicillin-resistant isolates [ 21 ]. Even though most data seem to indicate that there are no consistent amino acid changes correlating with specific increases in the MICs of ampicillin [ 33 ], a recent study by Darehkordi et al established a link between high ampicillin MICs, high PBP5 expression levels, and specific pbp5 mutations [ 34 ]. A comparison between pbp5 -containing platforms (associated with AmpR) of our sequenced genomes with a large transferable pbp5 -containing platform that we have previously described in a clinical isolate [ 21 ] allowed the partial identification of highly similar genetic platforms carrying different metabolic and adaptive features, including virulence genes (e.g., sgrA or fms2 important in biofilm formation) in AmpR- Efm of different origins ( Figure 2 ).…”

Genomic Characterization and Phylogenetic Analysis of Linezolid-Resistant Enterococcus from the Nostrils of Healthy Hosts Identifies Zoonotic Transmission

“…Enterococcus faecium showing ampicillin resistance is an additional problem in the clinical chemotherapy of enterococci [ 36 ]. Chromosomal point mutations putatively conferring resistance to linezolid (in 23S rRNA and ribosomal proteins L3/L4/L22) were not detected in our isolates.…”

Enterococcus Virulence and Resistant Traits Associated with Its Permanence in the Hospital Environment

“…A higher resistance to these antibiotics is usually associated with a higher expression of PBP5 proteins [ 113 , 114 ] or to mutations in the amino acid gene sequence that lead to alterations of this protein’s molecular structure [ 114 , 115 , 116 ]. When evaluating the relation between pbp5 sequences of E. faecium and penicillin’s minimal inhibition concentration (MIC) presented by these isolates, a study by Galloway-Peña et al [ 115 ], corroborated later by another study by Pietta et al [ 116 ], concluded that isolates that presented a MIC ≤ 2 µg/mL and isolates that presented a MIC ≥ 16 µg/mL had a 5% difference in the pbp5 sequence, which remounted to the presence of two distinct allelic forms: a more susceptible (PBP5-S) and a more resistant (PBP5-R) form.…”

“…However, both studies concluded that the determination of which amino acid changes were responsible for this decrease in susceptibility to β-lactams was difficult. The divergence seen in results described in other studies also reinforces this conclusion, although some changes such as an additional serine after amino acid position 466 (Ser-466) or amino acid substitutions (for instance the substitution of a methionine, Met-485, for an alanine or threonine (Met-485 → Ala/Thr), the substitution of a glutamic acid, Glu-629, for a valine, and also the substitution of an aspartic acid, Asp-496, for a lysine) have been more frequently associated to a high-level of penicillin resistance than others [ 114 , 115 , 117 , 118 , 119 ]. Although it has been proven that this PBP is responsible for some degree of resistance to penicillins, high-level resistance cannot be solely justified by the increased expression or mutations of the pbp5 gene, which means that other factors should also play some kind of role in this adaptation [ 106 , 114 ].…”