Biofilm-driven LPS remodeling and colanic acid overproduction mediate biocide adaptation and antibiotic cross-resistance in E. coli.

The identification of the determinants driving antimicrobial resistance is a prerequisite for improving the control of resistance emergence and dissemination. Disinfectant biocides, daily used in food-processing industries, have already been associated with the cross-selection of antibiotic-resistant bacterial populations. However, very few studies have addressed this issue using a biofilm model, the predominant bacterial lifestyle in food-processing environments. In this work, we examined the adaptation of Escherichia coli biofilms to four biocidal active substances over one month, and assessed their subsequent effects on antibiotic resistance. Exposure to N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (TMN) and benzalkonium chloride significantly increased the emergence of rifampicin-resistant (RifR) variants in biofilms. Genomic analyses revealed that the RifR variants selected upon TMN exposure recurrently harboured mutations in genes related to lipopolysaccharide (LPS) biosynthesis that conferred low-level rifampicin resistance in biofilm. These variants displayed altered LPS profiles, a more negative surface charge, and reduced membrane permeability. Proteomic and phenotypic analyses supported a metabolic reorientation of envelope sugar precursors, with decreased modulation of LPS synthesis and a marked induction of the colanic acid biosynthetic pathway in TMN-selected variants. This shift resulted in increased matrix production and reinforced biofilm-associated tolerance. Together, these data identify outer membrane reprogramming, linking LPS modulation with colanic acid overproduction, as a previously unknown mechanism of TMN adaptation that simultaneously promotes antibiotic cross-resistance in E. coli biofilms.