Avaliação do papel de óxido nítrico, de óleos essenciais e de sanitizantes na dispersão de biofilmes de Listeria monocytogenes em superfície abiótica

Abstract

Avaliação do papel de óxido nítrico, de óleos essenciais e de sanitizantes na dispersão de biofilmes de Listeria monocytogenes em superfície abiótica Fernanda Barbosa dos Reis Teixeira Ribeirão Preto 2014 Palavras-chave: Listeria monocytogenes, biofilme, dispersão, óxido nítrico, óleos essenciais, sanitizantes. iii ABSTRACT REIS-TEIXEIRA, F. B. Evaluation of the role of nitric oxide, essential oils and sanitizers in biofilm dispersion of Listeria monocytogenes on abiotic surface. 2014. 131f. Thesis (Doctoral). Faculdade de Ciências Farmacêuticas de Ribeirão Preto -Universidade de São Paulo, Ribeirão Preto (Brazil), 2014.Biofilms of Listeria monocytogenes are potential sources of contamination of processed foods, and may interfere in the effectiveness of cleaning and sanitizing procedures in food industry. In the present study the structures and dispersion of biofilms formed by two strains of L. monocytogenes on different abiotic surfaces, such as stainless steel, glass and polystyrene were evaluated. Different cultivation systems such as polystyrene and stainless steel 96-wells microplates, 24-wells microplates containing round stainless steel or glass slides, and 8-wells polystyrene chambers with borosilicate bottom were used. The experiments were done for 1, 4 and 8 days of incubation at 25°C. Biofilm formation was observed in 96-wells microplates of polystyrene and stainless steel by the quantification of biofilm biomass by staining with crystal violet, and also in 24-wells microplates system with circular slides of stainless steel or glass, with enumeration of adhered cells on agar plates. The structure of biofilms were observed by fluorescence microscopy (for the system of 24-wells microplates containing circular slides of stainless steel) and by confocal laser scanning microscopy (for the system of glass bottom chambers). For this, biofilms were stained with the LIVE/DEAD ® bacterial viability kit, in order to differentiate viable cells (stained with Syto 9) from dead cells (stained with propidium iodide). The estimative of the number of viable cells was done by measurement of fluorescence. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the essential oils plants of Cymbopogon citratus (lemon grass) and Zingiber officinale (ginger) and two commercial sanitizers (babassu coconut oil-based sanitizer and chlorine dioxide-based sanitizer) against L. monocytogenes were also determined. Subsequently, the antimicrobials were evaluated against L. monocytogenes biofilms formed by 4 and 8 days at 25°C on the stainless steel and glass surfaces by enumeration on agar plates of adhered cells and microscopic observations. The presence of molecules related to oxidative and nitrosative stresses in mature biofilms of two strains of L. monocytogenes formed on glass and stainless steel surfaces was also evaluated, and the possible role of exogenous nitric oxide (NO) and inhibitors of NO were studied for induction of biofilm dispersal cells and changed of genic expression levels of L. mon...