The initial hypothesis is that the application of enzymes might produce the disruption of the biofilm matrix that acts as a barrier to antimicrobials, facilitating the subsequent effect of the disinfectant. The specificity is achieved based on the previous characterisation of the L. monocytogenes-carrying biofilms present in industry that permits the enzyme selection, the dose adjustment and the study of the possible tolerance development. The experimental work was development in the following stages: - Characterisation of the L. monocytogenes-carrying communities present in fish, meat and dairy industry. This allowed detecting the presence and subtypes of L. monocytogenes, to characterise the accompanying microbiota and to study the adhesion dynamics of L. monocytogenes isolates on stainless steel (SS) as well as the association capacity and biofilm formation in mixed culture with the accompanying species. - Effectiveness of the enzyme-BAC combination to remove early-stage L. monocytogenes-carrying biofilms. The effects of different enzymes alone and combined with BAC against early-stage L. monocytogenes mixed biofilms grown on SS was assessed. Results obtained demonstrated the efficacy of the enzyme-BAC combined application to remove L. monocytogenes mixed biofilms and highlighted that this efficacy varies with the composition and age of the biofilm, pointing out the importance of designing strain-specific cleaning and disinfection strategies.

Listeria monocytogenes is considered one of the major foodborne pathogenic bacteria in Europe. In nature, it is found forming part of multispecies biofilms, resistance structures constituted by an extracellular matrix acting as a protective barrier against external agents, hindering their action and generating sublethal concentrations inside the biofilm. In industrial environments, biofilms are usually exposed to sublethal concentrations of biocides, due to the fact that the biofilm is located in inaccessible locations or because of inefficient application. This can favour the appearance of resistant and persistent bacteria in industrial plants, which lead to an excessive biocide deployment with a subsequent higher environmental impact. Is hence necessary, to propose more effective and efficient cleaning and disinfection systems, able to ensure pathogen control, generate less resistance while maintaining the main environmental impact standards. In the present dissertation, the design of a specific cleaning and disinfection system based on the combined application of enzymes and benzalkonium chloride (BAC) against L. monocytogenes mixed biofilms present in the food industry is proposed

- Quantification of the effects of pronase-BAC combined application against L. monocytogenes-E. coli late-stage dual-species biofilms. The individual and combined effects on the occupied surface, and the number of viable adhered and released cells after the application of pronase and BAC against late-stage L. monocytogenes-E. coli dual-species biofilms were assessed. Results demonstrated a synergistic effect of pronase-BAC application against L. monocytogenes-E. coli dual-species biofilms, a higher efficacy against L. monocytogenes, and the need to use high BAC doses to ensure the absence of adhered and released viable cells. - Tolerance development to pronase-BAC combined treatments in L. monocytogenes-E. coli mixed biofilms. The effects of the frequency and duration of consecutive sublethal exposures to pronase-BAC on the development of tolerance in L. monocytogenes-E. coli mixed biofilms was assessed. Results showed that only when sublethal exposures are alternated with recovery periods, a tolerance development to the application of pronase-BAC combined treatments takes place.

Peer reviewed

192 páginas