Development of a stable low-copy mini F plasmid derivative for evaluating β-lactamase substrate specificity through antimicrobial susceptibility testing

transformants expressing 22 β-lactamase genes (Ambler classes A-D), including carbapenemases that were cloned into pMiniF-1. The resulting antimicrobial susceptibility profiles and MIC differences did not contradict the known substrate specificities of the enzymes. Meropenem and imipenem MICs for the IMP-6 producer were fourfold higher and eightfold lower, respectively, than IMP-1. Aztreonam MICs were similar between transformants lacking β-lactamase genes and those with class B β-lactamases. Imipenem MIC for the OXA-48 producer was ≥8-fold higher than for OXA-1, whereas cefepime MICs were 16-fold lower. We demonstrate that pMiniF-1 enables the comparison of β-lactamase substrate specificity via AST. The pMiniF-1 plasmid (Catalog No. RDB21122) is available from the RIKEN BioResource Research Center (Tsukuba, Ibaraki, Japan) (https://web.brc.riken.jp/en/).IMPORTANCEβ-lactamases play a central role in bacterial resistance to β-lactam antibiotics, and novel variants continue to emerge. However, the impact of individual β-lactamase variants on β-lactam resistance across diverse enzyme-substrate combinations remains insufficiently characterized. Traditional kinetic analyses require enzyme purification and multiple reaction assays, which hinder comprehensive experimental evaluation. Expression-based approaches have also been used to assess β-lactamase function, but conventional systems depend on unstable high-copy plasmids, raising concerns about reproducibility due to variable plasmid copy numbers. In this study, we developed pMiniF-1, a stable, low-copy plasmid that enables consistent gene expression without antibiotic selection, thereby maintaining stable copy numbers under drug-free conditions. Using 22 β-lactamase genes, we demonstrated that susceptibility testing with pMiniF-1 adequately reflects their known substrate specificity profiles. This system provides a reliable method for assessing β-lactamase substrate specificity and will facilitate comparative studies on the functional diversity and evolution of emerging β-lactamase variants.