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Updated: Apr 17, 2026
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Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance
Published on: May 2, 2018
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Development of a stable low-copy mini F plasmid derivative for evaluating β-lactamase substrate specificity through
Haruka Nakagawa Kamura1,2, Kotaro Aoki2, Kohji Komori3
1Center for the Planetary Health and Innovation Science (PHIS), Hiroshima University, The IDEC Institute, Higashi-Hiroshima, Japan.
Microbiology Spectrum
|April 16, 2026
Summary
A new plasmid, pMiniF-1, allows for standardized assessment of bacterial β-lactamase activity and β-lactam antibiotic resistance. This tool enables accurate comparisons of enzyme-substrate specificities, aiding the study of emerging antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Bacterial β-lactamases are key to resistance against β-lactam antibiotics, with new variants constantly emerging.
- Assessing β-lactamase substrate specificity traditionally uses antimicrobial susceptibility testing (AST) on transformants with variable gene expression due to non-standardized plasmids.
- Existing expression systems often use high-copy plasmids, leading to inconsistent β-lactamase expression and hindering reproducible research.
Purpose of the Study:
- To develop a standardized and reproducible method for evaluating the impact of β-lactamase substrate specificity on β-lactam resistance.
- To create a stable, low-copy plasmid vector (pMiniF-1) that ensures consistent β-lactamase gene expression for accurate antimicrobial susceptibility testing (AST).
- To validate the utility of pMiniF-1 in reflecting known β-lactamase substrate specificities through AST.
Main Methods:
- Construction of the pMiniF-1 plasmid, a mini F plasmid engineered with a tac promoter, ribosome-binding site, and terminators for controlled gene expression.
- Maintenance of pMiniF-1 at a low, stable copy number (1-2 per cell) without the need for antibiotic selection.
- Antimicrobial susceptibility testing (AST) of *Escherichia coli* transformants expressing 22 different β-lactamase genes (Ambler classes A-D) using the pMiniF-1 vector.
Main Results:
- The pMiniF-1 system demonstrated controlled and consistent β-lactamase expression, enabling accurate AST.
- Antimicrobial susceptibility profiles generated using pMiniF-1 correlated well with the known substrate specificities of the tested β-lactamases.
- Specific examples include differential MICs for IMP-6 vs. IMP-1 against meropenem and imipenem, and OXA-48 vs. OXA-1 against imipenem and cefepime, validating the system's sensitivity.
Conclusions:
- The pMiniF-1 plasmid provides a reliable and reproducible platform for assessing β-lactamase substrate specificity via AST.
- This standardized approach facilitates comparative studies on the functional diversity and evolution of emerging β-lactamase variants.
- The pMiniF-1 plasmid is available for research use, promoting further investigation into antibiotic resistance mechanisms.
