The impact of aztreonam–clavulanic acid exposure on gene expression and mutant selection using a multidrug-resistant <i>E. coli</i>

Multidrug-resistant <i>Escherichia coli</i> poses a significant threat to the healthcare system by causing treatment failure in infected patients. The use of a beta-lactam in combination with a beta-lactamase inhibitor has been shown to be an effective strategy to solve this problem. <i>In vitro</i> antimicrobial susceptibility experiments have demonstrated the antimicrobial activity of aztreonam and clavulanate. In this investigation, we conducted a transcriptomic analysis to reveal the downstream differential gene expression in <i>E. coli</i> ymmD45 (a strain newly isolated and found to carry the New Delhi metallo-β-lactamase gene) following exposure to aztreonam and clavulanate separately, as well as their combination. Differential gene expression, pathway enrichment, and gene network analyses demonstrated the polygenic nature of the response to the combination treatment, which suppressed the expression of pivotal virulence genes, disrupted two-component regulatory systems for bacteria to resist external stress, and interfered with the formation of the cellular membrane. Results from single-step mutant selection combined with deep whole-genome sequencing also revealed the spontaneous origin of the resistance mutations and confirmed action mechanisms during the combination treatment. Our study contributes valuable insights into the impact of antibiotic exposure on gene expression, laying the groundwork for understanding antibiotic resistance development in the treatment of multi-drug resistant infections through <i>in vitro</i> studies.IMPORTANCEMultidrug-resistant <i>Escherichia coli</i> is a major challenge in treating infections effectively. Aztreonam and clavulanate combination is promising in combating these resistant bacteria. By investigating the antimicrobial activity of aztreonam and clavulanate using transcriptomic analysis and mutant selection, this research sheds light on the mechanisms underlying antibiotic resistance and the effectiveness of combination therapies. The findings highlight how this particular antibiotic combination suppresses virulence genes, disrupts bacterial regulatory systems, and interferes with cellular functions critical for resistance. Moreover, the study lays the groundwork for understanding antibiotic resistance development in the treatment of multi-drug resistant infections through <i>in vitro</i> studies, offering insights that could inform future strategies in clinical settings. Ultimately, our findings could guide the development of better treatment strategies for multidrug-resistant infections, improving patient outcomes and helping to manage antibiotic resistance in healthcare.