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Updated: Jul 20, 2025
Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance
Published on: May 2, 2018
In vitro development of imipenem/relebactam resistance in KPC-producing Klebsiella pneumoniae involves multiple
Eva Gato1, Paula Guijarro-Sánchez1, Isaac Alonso-García1
1Servicio de Microbiología and Instituto de Investigación Biomédica A Coruña (INIBIC), Complexo Hospitalario, Universitario A Coruña, A Coruña, Spain.
Objectives:
In order to inform and anticipate potential strategies aimed at combating KPC-producing Klebsiella pneumoniae infections, we analysed imipenem/relebactam and ceftazidime/avibactam single-step mutant frequencies, resistance development trajectories, differentially selected resistance mechanisms and their associated fitness cost using four representative high-risk K. pneumoniae clones.
Methods:
Mutant frequencies and mutant preventive concentrations were determined using agar plates containing incremental concentrations of β-lactam/β-lactamase inhibitor. Resistance dynamics were determined through incubation for 7 days in 10 mL MH tubes containing incremental concentrations of each antibiotic combination up to their 64 × baseline MIC. Two colonies per strain from each experiment were characterized by antimicrobial susceptibility testing, whole genome sequencing and competitive growth assays (to determine in vitro fitness). KPC variants associated with imipenem/relebactam resistance were characterized by cloning and biochemical experiments, atomic models and molecular dynamics simulation studies.
Results:
Imipenem/relebactam prevented the emergence of single-step resistance mutants at lower concentrations than ceftazidime/avibactam. In three of the four strains evaluated, imipenem/relebactam resistance development emerged more rapidly, and in the ST512/KPC-3 clone reached higher levels compared to baseline MICs than for ceftazidime/avibactam. Lineages evolved in the presence of ceftazidime/avibactam showed KPC substitutions associated with high-level ceftazidime/avibactam resistance, increased imipenem/relebactam susceptibility and low fitness costs. Lineages that evolved in the presence of imipenem/relebactam showed OmpK36 disruption, KPC modifications (S106L, N132S, L167R) and strain-specific substitutions associated with imipenem/relebactam resistance and high fitness costs. Imipenem/relebactam-selected KPC derivatives demonstrated enhanced relebactam resistance through important changes affecting relebactam recognition and positioning.
Conclusions:
Our findings anticipate potential resistance mechanisms affecting imipenem/relebactam during treatment of KPC-producing K. pneumoniae infections.
Insights
Imipenem/relebactam showed lower initial resistance emergence than ceftazidime/avibactam against KPC-producing Klebsiella pneumoniae. However, rapid resistance developed with imipenem/relebactam, involving specific KPC modifications and OmpK36 disruption.
Area of Science:
- Microbiology
- Infectious Diseases
- Antimicrobial Resistance
Background:
- Carbapenemase-producing Enterobacteriaceae (KPC) infections pose a significant global health threat.
- Klebsiella pneumoniae is a leading cause of KPC infections, necessitating novel therapeutic strategies.
- Imipenem/relebactam and ceftazidime/avibactam are crucial agents for treating KPC-producing K. pneumoniae.
Purpose of the Study:
- To analyze resistance development trajectories and mechanisms of imipenem/relebactam and ceftazidime/avibactam against high-risk K. pneumoniae clones.
- To identify differentially selected resistance mechanisms and their associated fitness costs.
- To inform strategies for combating KPC-producing K. pneumoniae infections.
Main Methods:
- Determined mutant frequencies and mutant preventive concentrations using agar-based methods.
- Assessed resistance dynamics through prolonged incubation with incremental antibiotic concentrations.
- Characterized evolved strains via antimicrobial susceptibility testing, whole genome sequencing, and competitive growth assays.
- Investigated KPC variants through cloning, biochemical experiments, and molecular modeling.
Main Results:
- Imipenem/relebactam exhibited lower single-step mutant emergence concentrations compared to ceftazidime/avibactam.
- Rapid resistance development and higher resistance levels were observed with imipenem/relebactam in three of four strains.
- Ceftazidime/avibactam-selected lineages showed KPC substitutions conferring high-level resistance and low fitness costs.
- Imipenem/relebactam-selected lineages displayed OmpK36 disruption, KPC modifications (S106L, N132S, L167R), and high fitness costs.
Conclusions:
- Findings anticipate potential resistance mechanisms against imipenem/relebactam in KPC-producing K. pneumoniae.
- Understanding these mechanisms is crucial for optimizing treatment strategies and preserving antibiotic efficacy.
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