Effect of sub-inhibitory tigecycline (½-MIC) on AcrAB-TolC and mar/ram/sox regulatory genes in Enterobacter cloacae complex isolates

The global rise of antimicrobial resistance (AMR) in <i>Enterobacterales</i>, including the <i>Enterobacter cloacae</i> complex, is narrowing treatment options. Tigecycline, a last-resort antibiotic for the treatment of multidrug-resistant (MDR) Gram-negative pathogens, is increasingly compromised by emerging resistance mechanisms, notably efflux pump overexpression and regulatory network adaptation. In this study, sixty clinical isolates of <i>Enterobacter cloacae</i> (thirty-eight tigecycline-resistant [TGC-R], twenty-two tigecycline-susceptible [TGC-S]) were analyzed to investigate gene expression changes in efflux pumps and regulatory genes under tigecycline pressure (1/2 minimum inhibitory concentration [MIC]) and standard conditions. Tigecycline exposure markedly increased <i>tolC</i> and <i>acrA</i> together with the regulators <i>marA</i> and <i>ramA</i>, while <i>acrB</i> increased only modestly. This indicates a strong regulatory component to the tigecycline response. In contrast, TGC-S isolates exhibited significant induction of <i>marA, marB</i> without corresponding activation of efflux pumps. Δlog₂FC analysis highlighted distinct transcriptional shifts between exposed and unexposed groups, with resistant strains displaying greater divergence. Heatmaps and boxplot visualizations, supported by Wilcoxon test statistics, underscored the regulatory responses associated with tigecycline pressure. These findings indicate that, alongside AcrAB-TolC upregulation, stress-responsive regulators (<i>marA</i>, <i>ramA</i>) are strongly induced by sub-inhibitory tigecycline, underscoring the multifactorial regulation of tigecycline response in the <i>E</i>. <i>cloacae</i> complex.