Efficiency of enzymes and benzalkonium chloride treatments against Listeria monocytogenes dual-species blofllms determined by fluorescence mlcroscopy and image analysis

Abstract

In the present study, three different mixed·species Listeria monocytogenes-carrying blofilms previously isolated from surfaces of seafood, meat and dairies Industrial environments were selected in order to test the efficiency of cleanlng and disinfection procedures based on the combination of an enzymatic solution, followed by a treatment with benzalkonium chloride (BAC). Biofilm samples were cultured onto AISI-316 type stainless steel 1 cm2 coupons In a batch system at 25° C. The effect of sequential exposure to different concentrations of Pronase- BAC, Cellulase - BAC or DNAsel - BAC solutions was then assessed in mature biofilms after 168 hours using an orthogonal first order experimental design. Samples were stained using FilmTracerTM LIVE/DEAD Blofllm viability kit (lnvitrogen} and the efficiency of each treatment was then determined by epifluorescence mlcroscopy and subsequent image anatysls of 25·field mosaics representing a total surface of 1.92 mm2, so as to quantify the total area occupied by living and damaged cells attached to the surface. Significant empirical equations obtained reveals that synergistic effects between Pronase - BAC on biofilms coming from flsh and dairy industries obtaining almost a total cell detachment in the latter case. No significant effect was observed among the Cellulase - BAC procedures in the experimental DNAse - BAC against biofilms coming from fish and dairy industries compared with the other two combinations enzyme- benzalkoniom chloride was also demonstrated even with no effect against the consortium isolated from meat industry These results show how the usage of customised treatments combining commerclally available enzymes with classical chemlcally-based disinfection procedures may represent an attractive and efficient alternative against bacterial blofilms present in the food Industry