Evaluation ability of adhesion and invasion of Caco-2 cells by Salmonella Enteritidis from biofilms treated with essential oil of Lemongrass (Cymbopogon citratus)

• Grant number: 14/09364-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2014
• End date: June 30, 2015
• Field of knowledge: Agronomical Sciences - Food Science and Technology - Food Science
• Principal Investigator: Elaine Cristina Pereira de Martinis
• Grantee: Gabriela Satti Lameu
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The use of sanitizers in food processing surfaces can lead to long-term corrosion of these surfaces and equipments. The project # 2013/01069-4 was developed granted by this Agency and carried out by this candidate from Mar. 2013 to Feb. 2014, in which was investigated the effect of essential oils from Cymbopogon citratus, Illicium verum, Zingiber officinale and Eucalyptus globulus in biofilms of Salmonella serotype Enteritidis on stainless steel cupons. According to those results, the essential oils of Cymbopogon citratus, Zingiber officinale and Eucalyptus at concentrations of 4% (v/v) were effective for inhibiting Salmonella Enteritidis. Salmonella Enteritidis did not grow on BHI agar plates after treatment of biofilms with essential oils, and the minimum bactericidal concentrations of essential oils (CBM) were determined, according to the literature. However, the coupons were further evaluated by fluorescence microscopy with live/dead staining and viable cells of Salmonella Enteritidis were present even after the treatment with essential oils, despite the absence of growth in BHI agar plates. To further investigate this, the coupons with biofilms of Salmonella Enteritidis treated with essential oils in bactericidal concentrations were washed in phosphate buffered saline (PBS), transferred to test tubes containing BHI broth, and incubated for 24 hours at 37°C. Growth in BHI broth was observed, indicating the recovery of some cells remaining after the treatment with essential oils. The evaluation of CBM by seeding plates is recommended in the literature for these studies, but our results indicated the need for further experimental approaches for evaluating the efficacy of essential oils against Salmonella sp. in biofilms. Thus, this project should be complementary to the project previously developed. Here, we propose to test the pathogenicity of the Salmonella Enteritidis culture incubated after immersion in the solutions of essential oil of Cymbopgon citrates in assays with human epitelial line Caco-2 cell. For the adhesion and invasion test, Caco-2 cells will be grown and transferred to 24 wells- plates and incubated for 24 hours. The results will be observed as bacterial adhesion and internalization efficiency of invasion (EI). The number of internalized bacteria will be calculated in percentage, with reference to the initial inoculums. The results will be important to understand factors involved in the survival of Salmonella in biofilms and to evaluate the pathogenic potential of the cells treated with the essential oils.