Biofilm Adaptation to Biocides and Impact on Antibioresistance – BAoBAb

The BAoBAb project relied on a microplate biofilm model and a set of ten Escherichia coli strains that were isolated from the food chain. During one-month adaptive evolution experiments, the biofilms were exposed to recurrent cycles of biocide treatments (triamines, QACs, PHMB and hydrogen peroxide) to mimic industrial practices. After each cycle, the exposed biofilm cells were plated on antibiotic-containing media to quantify the emergence of cross-resistance and isolate resistant variants.

Structural modifications of the biofilms were analysed using confocal laser scanning microscopy (CLSM) to reveal architectural and spatial organisation changes under biocide pressure. A comparative genomics approach (variant calling) was used to identify mutations that emerged during evolution, based on whole-genome sequencing of resistant variants and their parental strains. Targeted RT-qPCR analyses were conducted to compare the expression of these genes between parental strains and variants.

In addition, comparative proteomic analysis in the presence and absence of biocide was used to identify changes in protein expression between parental strains and resistant variants under biocidal stress, and to better assess the metabolic impact of the detected mutations. By combining experimental evolution, phenotypic analyses and multi-scale molecular characterisation, the BAoBAb project shed light on the adaptive trajectories of biofilms subjected to biocide pressure and identified recurrent pathways involved in adaptation to specific biocides.

First, evolutionary trajectories in biofilms are specific to this lifestyle and depend on the genetic background of the strains, even in the absence of biocidal pressure. Biofilm evolution promotes the emergence of resistance to gentamicin, and more broadly to aminoglycosides, through contrasting genetic strategies: some strains acquire point mutations affecting central metabolism or respiration (e.g., atpG, aceE, cydA), whereas the reference strain E. coli MG1655 evolves predominantly through large genomic deletions including sbmA, which is involved in aminoglycoside uptake, at the cost of a higher fitness penalty. These results highlight the role of the biofilm as a driver of evolutionary adaptation, shaping distinct resistance trajectories depending on the genetic context of the strains.

The impact of different biocidal active substances was then evaluated. Exposure to PHMB (a biguanide) stimulates curli production and the formation of dense zones within the biofilm, creating microenvironments that favor stress responses and transient metabolic adaptations. These changes induce temporary cross-resistance to gentamicin. Stable resistances, linked to mutations in respiration-related genes (cydA, atpG), appear more rarely. Thus, PHMB primarily acts by remodeling biofilm structure, thereby promoting bacterial adaptation and tolerance (Charron et al., 2023).

Particular focus was placed on N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (Triamine, TMN), a biocidal molecule increasingly used in the agri-food industry. Repeated exposure to TMN was observed to recurrently promote, across different strains, the emergence of rifampicin-resistant variants. This effect is specifically associated with the biofilm lifestyle and was not observed in planktonic or supernatant populations. Genomic analyses revealed recurrent mutations in genes associated with lipopolysaccharide (LPS) biosynthesis in resistant variants selected in the presence of TMN. These alterations are likely to modify membrane permeability and influence bacterial survival under stress conditions. In addition, proteomic analyses revealed modulation of the expression of proteins involved in exopolysaccharide synthesis (notably a redirection of sugars used for LPS synthesis toward colanic acid production), in biofilm structuring, and in the response to environmental stress (manuscript submitted to NPJ Antimicrobial Resistance in December 2025).

These data underscore the importance of integrating the biofilm lifestyle into assessments of the risk of selecting for antibiotic resistance associated with biocide use, within an integrated One Health approach.

The BAoBAb project provides new insights into how Escherichia coli biofilms adapt to biocide exposure and how such adaptations may promote the emergence of antimicrobial resistance. A major strength of the project lies in its multi-scale approach, combining experimental evolution, confocal microscopy, genomics, transcriptomics and proteomics. This integrative framework revealed that evolutionary trajectories in biofilms are strongly shaped by both the biofilm lifestyle and the genetic background of the strains, even in the absence of biocides. BAoBAb also uncovered biocide-specific adaptive responses, such as PHMB-induced biofilm reinforcement and transient cross-resistance to gentamicin, or the repeated emergence of rifampicin-resistant variants under TMN pressure. These variants consistently carried mutations in LPS biosynthesis genes and showed metabolic rerouting towards colanic acid production, a mechanism not previously described for TMN adaptation.

These findings highlight the importance of considering biofilms in risk assessments related to biocide use, as adaptive dynamics differ markedly from planktonic populations. They also point to the need for deeper investigation of how industrial disinfection practices may influence both the emergence and dissemination of antimicrobial resistance.

Future research should extend these analyses to a broader diversity of bacterial species relevant to food-processing environments, as well as to mixed-species biofilms that better reflect real industrial microbiomes. Comparative multi-omics approaches will be essential to identify conserved or species-specific signatures of biocide-driven adaptation. Another key prospect concerns the stability, reversibility and potential horizontal transfer of the mutations selected during biofilm evolution. Finally, the project opens possibilities for developing surveillance tools based on genomic or proteomic biomarkers, and for evaluating improved disinfection strategies—such as biocide alternation or combination—to limit adaptive potential in biofilms. BAoBAb thus provides a solid foundation for future One Health-oriented risk mitigation strategies.

The objectives of the BAoBAb project are to better understand bacterial adaptation strategies to disinfectant biocides used in the food industry, to evaluate the impact of this collective adaptation on the development of resistance to antibiotics and to identify the underlying mechanisms. The role of biocides in antimicrobial resistance (AMR) dissemination will also be addressed by investigating the recolonization abilities of AMR variants selected in biofilm during biocide exposure and the impact of biocide residues on horizontal gene transfer in biofilms. For this purpose, an approche combining methods of experimental evolution in biofilm, comparative genomics and advanced fluorescence imaging will be applied. The results will be used to better assess the risk associated with the use of biocides in relation to AMR and ultimately identify molecular and phenotypic markers of adaptation and development of cross-resistance that can be used to develop surveillance tool on the food chain.