Comparative assessment of the disinfection effectiveness of thymol and benzalkonium chloride against adapted and non-adapted to thymol biofilm cells of a Salmonella Typhimurium epidemic phage type DT193 strain

Nowadays there is a need to develop cost-effective, safe, and preferably eco-friendly methods to combat the pathogenic and other detrimental robust biofilms. Ιn this work, the disinfection actions of thymol (THY), a phytocompound exhibiting wide antimicrobial action, and benzalkonium chloride (BAC), a synthetic biocide commonly used as surface disinfectant in the food industry and elsewhere, were comparatively evaluated against adapted and non-adapted to THY biofilm cells of a Salmonella enterica ser. Typhimurium epidemic phage type DT193 strain. Initially, the minimum inhibitory and bactericidal concentrations (MICs, MBCs) of each compound were determined against planktonic bacteria, together with their minimum biofilm inhibitory and eradication concentrations (MBICs, MBECs) against biofilms formed on polystyrene (PS). Bacteria were subsequently left to form biofilms on model stainless steel (SS) surfaces incubated in 1/10 diluted tryptone soy broth (dTSB) containing or not a sub-inhibitory THY concentration. Those sessile populations were finally submitted to disinfection (for 15 min) with THY or BAC and the viable biofilm bacteria were quantified by using in parallel agar plating and selective fluorescence staining. The results showed that when the terpenoid was applied at sub-MIC during biofilm formation, it was able to cause significant reductions of the final sessile populations on both surfaces (PS and SS). Disinfection results revealed the significant anti-biofilm action of THY on the non-adapted (control) biofilms (5 log reductions at MBC) and its superiority to that of BAC (<2 log reductions at 2 x MBC). However, the disinfection treatments applied on the THY-adapted biofilms were quite less efficient (achieving 2.6 and 1.3 log reductions, for THY and BAC, respectively). This demonstrates adaptation of Salmonella to THY, conferring co- and cross-disinfection resistance, as for many other antibacterial chemicals. Overall, the results demonstrate that natural compounds extracted from plants may be promising agents in helping to combat biofilms, multicellular microbial structures well-known for their remarkable hardiness against many stresses and antimicrobials.