Structural Alteration of OmpR as a Source of Ertapenem Resistance in a CTX-M-15-Producing Escherichia coli O25b:H4 Sequence Type 131 Clinical Isolate

Abstract

In this study, an ertapenem-nonsusceptible Escherichia coli isolate was investigated to determine the genetic basis for its carbapenem resistance phenotype. This clinical strain was recovered from a patient that received, 1 year previously, ertapenem to treat a cholangitis due to a carbapenem-susceptible extended-spectrum-β-lactamase (ESBL)-producing E. coli isolate. Whole-genome sequencing of these strains was performed using Illumina and single-molecule real-time sequencing technologies. It revealed that they belonged to the ST131 clonal group, had the predicted O25b:H4 serotype, and produced the CTX-M-15 and TEM-1 β-lactamases. One nucleotide substitution was identified between these strains. It affected the ompR gene, which codes for a regulatory protein involved in the control of OmpC/OmpF porin expression, creating a Gly-63-Val substitution. The role of OmpR alteration was confirmed by a complementation experiment that fully restored the susceptibility to ertapenem of the clinical isolate. A modeling study showed that the Gly-63-Val change displaced the histidine-kinase phosphorylation site. SDS-PAGE analysis revealed that the ertapenem-nonsusceptible E. coli strain had a decreased expression of OmpC/OmpF porins. No significant defect in the growth rate or in the resistance to Dictyostelium discoideum amoeba phagocytosis was found in the ertapenem-nonsusceptible E. coli isolate compared to its susceptible parental strain. Our report demonstrates for the first time that ertapenem resistance may emerge clinically from ESBL-producing E. coli due to mutations that modulate the OmpR activity.

Keywords: ESBL; OmpR; ST131; avibactam; carbapenem; envZ; temocillin.

FIG 1

OMP profiles of E. coli ErtR compared to its parental strain, E. coli ErtS. OMPs were profiled by SDS-PAGE. Lane 1, E. coli ErtR; lane 2, E. coli HB4 ΔompC-ompF strain (control strain); lane 3, E. coli ErtS. The horizontal arrows on the left indicate the positions of OMPs OmpC-OmpF and OmpA. Note the same amount of OmpA protein in the three lanes.

FIG 2

Multiple alignment of the amino acid sequences of various homologues of the OmpR-PhoB subfamily. Proteins studied include OmpR of E. coli ErtS (this study), OmpR of M. morganii (GenBank accession no. KJY03066), OmpR of S. marcescens (GenBank accession no. KFL01930), OmpR of C. freundii (GenBank accession no. AKL57621), OmpR of P. mirabilis (GenBank accession no. KGA91828), OmpR of K. pneumoniae (GenBank accession no. CDO12110), OmpR of E. cloacae (GenBank accession no. AHE72147), OmpR of P. aeruginosa (GenBank accession no. BAP53865), CheY of E. coli (GenBank accession no. BAA15698), NtrC of E. coli (GenBank accession no. ACT31142), NarL of E. coli (GenBank accession no. CAA48935), PhoP of E. faecalis (GenBank accession no. KGJ35834), CheY of E. faecalis (GenBank accession no. KOA04247), PhoB of E. coli (GenBank accession no. ACJ50526), DrrB of T. marina (PDB entry 1P2F), and CpxR of E. coli (GenBank accession no. BAE77397). Numbering of the amino acid residues is according to that of OmpR of E. coli (12). Shaded amino acids at positions 55 and 63 represent highly conserved residues among response regulators.

FIG 3

Representation of the secondary structures surrounding the aspartate residue (Asp) at position 50/55. The protein variant was modeled from the crystallographic structure of the DrrB response regulator of Thermotoga maritima (PDB entry 1P2F) by the introduction of an amino acid substitution followed by energy minimization. The amino acids belong to DrrB, whereas those italicized in parentheses are their corresponding residues in the homolog E. coli OmpR protein. The secondary structure in red refers to the loop lying between the α-3 helix and the β-3 strand. (Left) Regulator response DrrB of Thermotoga maritima with glycine residue at position 58 (corresponding to the Gly-63 residue in the native OmpR). (Right) Variant of DrrB with a Gly-58-Val substitution, corresponding to the Gly-63-Val replacement in the OmpR-G63V variant.

FIG 4

Growth curves of E. coli strains in LB. Growth of E. coli ErtS (in dotted line) and E. coli ErtR (in solid line). The lines are surperimposed. The error bars indicate the standard deviations of the means for five experiments. Values of the optical density (O.D.) at 600 nm were collected every 5 min. The maximum growth rate (MGR) of E. coli ErtS was 1.45 ± 0.12 h⁻¹, whereas the MGR of E. coli ErtR was 1.46 ± 0.14 h⁻¹.

FIG 5

Examples of virulence phenotypes observed in the D. discoideum amoeba model. Bacteria (10⁸ CFU) were plated on petri dishes containing HL5 agar with D. discoideum at variable concentrations and examined at day 6. (A) E. coli 536 with 10³ amoebae. Amoebae did not form lysis plaque, indicating a grazing-resistant phenotype. (B) E. coli ErtS with 10³ amoebae. Amoebae did not form lysis plaque. (C) E. coli ErtR with 10³ amoebae. Amoebae did not form lysis plaque. (D) E. coli B REL 606 with 10² amoebae. Amoebae fed on the lawn of bacteria, forming large lysis plaques, in agreement with a grazing-sensitive phenotype.