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SILVA, E. P. Influence of temperature, DNA degrading enzymes and staphylococcal culture supernatant on biofilm formation by Listeria monocytogenes on abiotic surface. 2012. 110f. Dissertation (Master). Faculdade de Ciências Farmacêuticas de Ribeirão Preto – Universidade de São Paulo, Ribeirão Preto, 2012. Listeriosis is a rare and serious mainly food-borne infection. It is caused by the bacterium Listeria monocytogenes that primarily affects pregnant women and immunologically compromised individuals. This pathogen is recognized as a problem for the food industry due to its ability to form biofilms. Biofilms are microbial communities adhered to biotic or abiotic surfaces embebed by self produced extracellular polymers. These structures are resistant to cleaning and disinfection procedures, allowing the survival and persistence of L. monocytogenes in food processing environments. Thus, several strategies have been adopted to prevent and control the formation of biofilms on food contact surfaces. The present study investigated biofilm formation by L. monocytogenes on abiotic surface at different temperatures, in the presence of DNA degrading enzymes (DNAses) and in the presence of culture supernatant with and without staphylococcal thermonuclease activity. For this purpose, we used culture techniques and microscopy. The results showed that L. monocytogenes was able to adhere on stainless steel surface on average 10-10 CFU per cm. The different incubation temperatures affected the adhesion of L. monocytogenes on stainless steel surface, although other factors in combination were also involved, such as the bacterial strain and the type of system used for assay. By fluorescence microscopy, we noticed the formation of mature biofilms by L. monocytogenes, with channels likely involved in flow of nutrients and holes typical of cell dispersion. Furthermore, we found a predominance of metabolically active cells surrounded by extracellular matrix polymer containing extracellular DNA (eDNA) stained with acridin orange. By laser confocal microscopy, we observed the formation of microcolonies containing eDNA and homogeneous thin layer of viable cells stained with SYTO 9 and DDAO. The eDNA was also observed in locations with absence of cells characteristics of cell dispersion. The culture supernatant of S. aureus with thermonuclease activity was able to inhibit L. monocytogenes free on culture medium and interfered with the formation of biofilm by the pathogen for up to 24h at 37°C. The commercial DNAses did not affect biofilm formation by L. monocytogenes, possibly excluding the action of thermonuclease of S. aureus on the inhibition of planktonic or sessile forms of L. monocytogenes. It has been found a production of a thermostable protein by S. aureus and it was able to inhibit L. monocytogenes in agar antagonism assays but further studies are needed to characterize such inhibitory substance.

Influência da temperatura, de enzimas degradantes de DNA e do sobrenadante de cultura de estafilococos na formação de biofilme por Listeria monocytogenes em superfície abiótica