Biofilms in water industry: case study of Salmonella enterica serovar Typhimurium - biofilm formation and control in vitro and in situ.

Abstract

Biofilm formation is an important issue in food industries constituting an important factor of cross contamination. On the other hand, Salmonella is recognized as one of the most significant enteric foodborne pathogens. In recent years, the resistance of biofilms to antimicrobials has led to the search of novel antimicrobial compounds. To this direction, the aim of the present work was 1) to monitor biofilm formation in mono- and multi-species cultures of Salmonella Typhimurium (ST) with bacteria isolated from a bottling plant by fluorescence-based bioreporters and 2) evaluate the disinfection activity of Thymbra capitata hydrosol against ST biofilm cells, in comparison with the commonly used benzalkonium chloride (BC). In order to achieve this, a set of 16 samples were collected from a bottling plant. After bacterial identification, they left to form biofilm on stainless steel (SS) for 6 days at 20°C. In parallel, ST biofilm development with mixed communities was examined by using ten different ST bioreporters by fluorescence microscopy. Furthermore, the disinfectant efficacy of hydrosol and BC was comparatively tested against 24 h ST planktonic and biofilm cells following 6 min of treatment. Additionally, their bactericidal effect inside biofilm was evaluated by real-time visualization of the disinfection activity using time-lapse confocal laser scanning microscopy (CLSM). Regarding the obtained results, mixed bacterial communities recovered from bottling plant consist of Citrobacter, Staphylococcus, Pseudomonas, Bacillus, Exiguobacterium species. The presence of indigenous bacteria neither inhibited nor enhanced biofilm formation of ST. Furthermore, the csrA-based bioreporter was shown to be induced in multispecies biofilms with Citrobacter. The role of CsrA, which is a key global regulator during multispecies biofilm formation needs to be further studied. Moreover, regarding the hydrosol resistance coefficient (Rc=1.56) was significantly lower compared to the one of BC (Rc=208.33), showing that hydrosol was almost equally active against both planktonic and biofilm cells, whereas 200 times higher concentration of BC was needed to achieve the same effect against biofilm cells, as planktonic ones. Furthermore, CLSM revealed a significant advantage of the hydrosol as its great ability to penetrate quickly the biofilm structure and act effectively.