ramR Deletion in an Enterobacter hormaechei Isolate as a Consequence of Therapeutic Failure of Key Antibiotics in a Long-Term Hospitalized Patient

Abstract

Genome changes are central to the adaptation of bacteria, especially under antibiotic pressure. The aim of this study was to report phenotypic and genomic adaptations undergone by an Enterobacter hormaechei clinical strain that became highly resistant to key antimicrobials during a 4-month period in a patient hospitalized in an intensive care unit (ICU). All six clinical E. hormaechei strains isolated in one ICU-hospitalized patient have been studied. MICs regarding 17 antimicrobial molecules have been measured. Single nucleotide polymorphisms (SNPs) were determined on the sequenced genomes. The expression of genes involved in antibiotic resistance among Enterobacter cloacae complex strains were determined by reverse transcription-quantitative PCR (qRT-PCR). All the strains belonged to sequence type 66 and were distant by a maximum of nine SNPs. After 3 months of hospitalization, three strains presented a significant increase in MICs for ceftazidime, cefepime, temocillin, ertapenem, tigecycline, ciprofloxacin, and chloramphenicol. Those resistant strains did not acquire additional antibiotic resistance genes but harbored a 16-bp deletion in the ramR gene. This deletion led to upregulated expression of RamA, AcrA, AcrB, and TolC and downregulated expression of OmpF. The ΔramR mutant harbored the same phenotype as the resistant clinical strains regarding tigecycline, chloramphenicol, and ciprofloxacin. The increased expression of RamA due to partial deletion in the ramR gene led to a cross-resistance phenotype by an increase of antibiotic efflux through the AcrAB-TolC pump and a decrease of antibiotic permeability by porin OmpF. ramR appears to be an important adaptative trait for E. hormaechei strains.

Keywords: Enterobacter cloacae complex; OmpF; RND efflux pumps; chloramphenicol; fluoroquinolone; ramR; tigecycline; whole-genome sequencing.

FIG 1

Timeline of patient’s hospitalization. Infectious clinical history, bacterial strains isolated, and antibiotic treatments are presented. For each antibiotic treatment, the duration (in days) is indicated. The dotted line represents the apparition of the 16-bp deletion in ramR among the E. hormaechei clinical strains. SEPI, Staphylococcus epidermidis; FY, E. hormaechei clinical strains; TZP, piperacillin-tazobactam; IPM, imipenem; MEM, meropenem; CPT, ceftaroline; CZA, ceftazidime-avibactam; AMK, amikacin; LVX, levofloxacin; ERY, erythromycin; SXT, trimethoprim-sulfamethoxazole; LZD, linezolid; DAP, daptomycin; VAN, vancomycin. *, E. hormaechei clinical strains with a cross-resistance phenotype.

FIG 2

Differences of gene expression between strains FY06 to FY10 (group II) and FY01 to FY03 (group I) represented by a bar chart. Fold changes were calculated using the ΔΔC_T method using rpoB as a housekeeping gene. Error bars represent one standard deviation. Statistical analyses have been performed using the Student’s t test on GraphPad Prism version 8. Significant P value was considered lower than 0.05. This figure highlights that partial deletion of ramR led to the overexpression of seven genes, ramR, romA, ramA, micF, acrA, acrB, and tolC, whereas ompF was downregulated.