Antimicrobial resistance (AMR) driven by genetic mutation of antibiotic resistance genes is a major global health concern. Clinically, the TEM-1 β-lactamase encoded by blaTEM-1 is among the most frequently occurring resistance determinants. Understanding which TEM-1 mutations—and which combinations of mutations—confer high-level resistance is important for surveillance, mechanistic insight, and guiding the development of next-generation antibiotics.
The reported work applies gene editing and in vitro evolution techniques to map high-resistance TEM-1 genotypes and to observe combinatorial effects when multiple mutations are present.
The authors describe adapting continuous directed evolution (CDE) together with base editor (BE) gene editing as a strategy to discover TEM-1 mutants. CDE is used to generate and select variants iteratively, while BE tools introduce targeted nucleotide changes without double-strand DNA breaks. By coupling these approaches, the study aimed to accelerate the generation and identification of clinically relevant resistance mutations in a controlled laboratory setting.
The combination of CDE and base editing provides a platform to produce mutation diversity focused on the gene of interest and to select for functional phenotypes under antibiotic pressure.
Initial discovery of TEM-1 mutants was performed with the gene present in a single-copy bacterial artificial chromosome (BAC) vector, using BE-mediated CDE to generate variants and select for resistant phenotypes. Identifying mutants in a single-copy context approximates chromosomal expression levels and can reduce artefacts associated with high plasmid copy number during selection.
After candidate mutants were identified in the BAC context, each mutant genotype was verified in a high-copy-number pUC plasmid. Moving validated genotypes into pUC plasmids enabled confirmation of sequence identities and assessment of phenotypes under a different gene-dosage regime.
The researchers examined combinatorial antibiotic resistance by integrating TEM-1 phenotypes produced during CDE with previously reported TEM-1 variants. This approach tests whether mutations discovered de novo interact synergistically, additively, or antagonistically with known resistance-associated substitutions.
Combinatorial analysis is critical because clinical resistance often arises from multiple concurrent mutations, and interactions among substitutions can substantially alter substrate specificity and inhibitor susceptibility.
As a functional readout, the study measured minimum inhibitory concentrations (MICs) to the cephalosporin cefotaxime. The authors report that an Escherichia coli strain harboring a vector encoding 10 TEM-1 mutations exhibited a cefotaxime MIC of 1280 μg/mL. The abstract states this is the highest such MIC reported thus far by the authors.
This finding illustrates that combinations of multiple TEM-1 substitutions can produce extreme levels of resistance in an E. coli model under the tested conditions.
The work demonstrates the potential of integrating gene-editing technologies with directed evolution to systematically explore the mutational landscape of clinically relevant resistance genes. Using base editors with CDE can accelerate identification of high-risk genotypes, help map molecular mechanisms underlying substrate expansion or increased catalytic activity, and inform surveillance priorities.
Results such as high MICs for cefotaxime when multiple substitutions are combined underscore the importance of monitoring combinatorial genotypes in clinical isolates and considering multidimensional resistance evolution when developing antimicrobial strategies.
The abstract provides a concise overview but does not report several details needed for full evaluation, including the precise identities and positions of the TEM-1 mutations discovered, the CDE experimental parameters (selection conditions, duration, mutation spectra), quantitative details of mutant verification, replicates and statistical analysis, or potential fitness costs associated with the mutations in the absence of antibiotic. Information on whether resistance phenotypes were examined against other β-lactams or inhibitors is also not provided in the abstract.
For comprehensive methods, full genotype lists, and complete datasets, consult the full published article.