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Antimicrobial resistance is a critical public health issue that requires a thorough understanding of the factors that influence the selection and spread of antibiotic-resistant bacteria. Biocides, which are widely used in cleaning and disinfection procedures in a variety of settings, may contribute to this resistance by inducing similar defense mechanisms in bacteria against both biocides and antibiotics. However, the strategies used by bacteria to adapt and develop cross-resistance remain poorly understood, particularly within biofilms –a widespread bacterial habitat that significantly influences bacterial tolerance and adaptive strategies. Using a combination of adaptive laboratory evolution experiments, genomic and RT-qPCR analyses, and biofilm structural characterization using confocal microscopy, we investigated in this study how Escherichia coli biofilms adapted after 28 days of exposure to three biocidal active substances and the effects on cross-resistance to antibiotics. Interestingly, polyhexamethylene biguanide (PHMB) exposure led to an increase of gentamicin resistance (GenR) phenotypes in biofilms formed by most of the seven E. coli strains tested. Nevertheless, most variants that emerged under biocidal conditions did not retain the GenR phenotype after removal of antimicrobial stress, suggesting a transient adaptation (adaptive resistance). The whole genome sequencing of variants with stable GenR phenotypes revealed recurrent mutations in genes associated with cellular respiration, including cytochrome oxidase (cydA, cyoC) and ATP synthase (atpG). RT-qPCR analysis revealed an induction of gene expression associated with biofilm matrix production (especially curli synthesis), stress responses, active and passive transport and cell respiration during PHMB exposure, providing insight into potential physiological responses associated with adaptive crossresistance. In addition, confocal laser scanning microscopy (CLSM) observations demonstrated a global effect of PHMB on biofilm architectures and compositions formed by most E. coli strains, with the appearance of dense cellular clusters after a 24h-exposure. In conclusion, our results showed that the PHMB exposure stimulated the emergence of an adaptive cross-resistance to gentamicin in biofilms, likely induced through the activation of physiological responses and biofilm structural modulations altering gradients and microenvironmental conditions in the biological edifice.

Measurement(s) whole genome sequencing Technology Type(s) Illumina Sequencing Factor Type(s) Multi-locus sequence types • Geographic location • Animal associated environment isolates • Food product and food production environment isolates Sample Characteristic - Organism Listeria monocytogenes Sample Characteristic - Environment Farm • Ruminant • Agricultural soil • Wild animals • food processing building • dairy food product • meat or meat product (from mammal) (us cfr) • chicken meat food product • fish food product • vegetable or vegetable product (us cfr) Sample Characteristic - Location Europe

The foodborne pathogen, Listeria monocytogenes, (Lm), frequently undergoes selection pressure associated with the extensive use of disinfectants, such as quaternary ammonium compounds, which are widely used in food processing plants. The repeated exposure to sub-inhibitory biocide concentrations can induce increased tolerance to these compounds, but can also trigger the development of antibiotic resistance, and both increase the risk of food contamination and persistence in food production environments. Although the acquisition of genes can explain biocide tolerance, the genetic mechanisms underlying the adaptive cross-resistance to antibiotics remain unclear. We previously showed that repeated exposure to benzalkonium chloride (BC) and didecyldimethyl ammonium chloride (DDAC) led to reduced susceptibility to ciprofloxacin in Lm strains from diverse sources. Here, we compared the genomes of 16 biocide-adapted and 10 parental strains to identify the molecular mechanisms of fluoroquinolone cross-resistance. A core genome SNP analysis identified various mutations in the transcriptional regulator fepR (lmo2088) for 94% of the adapted strains and mutations in other effectors at a lower frequency. FepR is a local repressor of the MATE fluoroquinolone efflux pump FepA. The impact of the mutations on the structure and function of the protein was assessed by performing in silico prediction and protein homology modeling. Our results show that 75% of the missense mutations observed in fepR are located in the HTH domain of the protein, within the DNA interaction site. These mutations are predicted to reduce the activity of the regulator, leading to the overexpression of the efflux pump responsible for the ciprofloxacin-enhanced resistance.