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Gene expression heterogeneity has an impact on human bacterial pathogens responses, including antimicrobial resistance genes expression. Promoter region variability in resistance genes may include different regulatory boxes involved in metabolic processes essential for bacterial survival. In this study, a molecular tool was developed to characterize promoter region variability and resistance gene expression. For these, 46 bacterial clinical isolates were used. Sequence analysis of resistance genes promoter region showed the existence of different variants for each promoter and identified gene-specific regulatory boxes. Using a fluorescent transcriptional reporter, we analysed the most common promoter variants of the most prevalent acquired resistance genes in clinical isolates (qnrA, qnrB, blaOXA-48, blaKPC-3 and blaVIM-1, aac(6')-Ib-cr, and fosA). Gene expression fluctuations were found to be dependent on culture medium used. Specific gene promoter variants showed lower expression levels in poor (M9) medium (p