Study of formation and dispersal of Listeria monocytogenes biofilms on abiotic sur...
| Grant number: | 10/10051-3 |
| Support type: | Scholarships in Brazil - Doctorate |
| Effective date (Start): | September 01, 2010 |
| Effective date (End): | July 31, 2014 |
| Field of knowledge: | Agronomical Sciences - Food Science and Technology |
| Principal Investigator: | Elaine Cristina Pereira de Martinis |
| Grantee: | Fernanda Barbosa dos Reis Teixeira |
| Home Institution: | Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil |
Abstract Listeria monocytogenes biofilms are potential sources of contamination of processed foods, and may interfere in the effectiveness of procedures for cleaning and sanitizing in food industry. In the present study the structure and dispersion of biofilms formed by two strains of L. monocytogenes on different abiotic surfaces, such as stainless steel, glass and polystyrene was evaluated. Different cultivation systems such as polystyrene and stainless steel 96-wells microplates, 24-wells microplates containing circular slides of stainless steel or glass, 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, by enumerating in agar plates the adhered cells. 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 quantification of the number of viable cells estimated was done through quantitation 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. Subsequently, it was tested for anti-biofilm effect to removal L. monocytogenes biofilms formed by 4 and 8 days at 25°C on the stainless steel and glass surfaces by enumeration in agar plates of adhered cells and microscopic observations. The presence of molecules related to oxidative and nitrosative stress 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 was studied in inducing biofilm dispersal cells and changed of genic expression levels of L. monocytogenes related to oxidative and nitrosative stress genes (Lmo0990, Lmo0807 and Lmo1485), as well as the regulation of virulence and biofilm formation gene (PrfA). The presence of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) was observed in biofilms of L. monocytogenes with 4 and 8 days of incubation at 25°C formed on stainless steel and glass surfaces, using specific fluorescent dyes and microscopic views. In order to indirectly confirm the presence of nitric oxide (NO) in cultures of L. monocytogenes incubated for 4 and 8 days at 25°C, the dosage of nitrite was carried out by reaction with Griess reagent. The minimum inhibitory concentration (MIC) of nitric oxide donor sodium nitroprusside (SNP) and two NO inhibitors such as N-nitro-L-arginine methyl ester (L-NAME) and 2-(4-carboxyphenyl)-4,4,5,5-tetrametilimidazoline-1-oxy-3-oxide (Carboxy-potassium salt, C-PTIO), were performed against L. monocytogenes. It was evaluated the effectiveness of SNP and of the two inhibitors of NO to eliminate biofilms of L. monocytogenes preformed for 4 and 8 days on stainless steel surfaces. (AU) | |