Caracterización fenotípica y genotípica de "Listeria monocytogenes"

Resumo

Listeria monocytogenes is a Gram-positive and food-borne pathogen that causes infections in animals and humans. L. monocytogenes is able to survive in different environments of food industry so, it is very difficult to remove from plants. Due to the severity of listeriosis is important to go in-depth on the characteristics of the strains that appear in along the food chain and food products. The aim of this study is to characterize L. monocytogenes strains with genotyping and phenotyping methods for helping the industries to prevent the pathogen in food industry. During this PhD thesis, L. monocytogenes strains from different food industries will be characterized: 176 from a slaughterhouse, 45 from a cheese factory and 26 from a shrimps company. All of them have been isolated from environmental and final product samples. For genotyping characterization molecular biology tools, such as PCR methods will be used to identify and classify different genes of L. monocytogenes: real time PCR, conventional PCR and electrophoresis, stress survival islet- 1, Tn 6188 (radC, quacH) and bcrABC. Moreover, PFGE (Pulse-field gel electrophoresis), MLST (Multi-Locus sequence typing) and MvLST (Multi virulence locus sequence typing) will be used to perform strains typification. Furthermore, a better knowledge about transcriptome expressions in stress situations is necessary. Thus, for phenotyping characterization different experiments will be performed, such as growth curves, test of resistance / tolerance to detergent / disinfectant, stress oxidative, antibiotic resistances, cell culture and in vitro studies in different food products. The presumptive L. monocytogenes colonies ffrom the different samples were identified by real-time PCR and then classified by their serogroups. From 176 isolates in slaughterhouse, 27.84% belonged to 1/2a; 3a serotype; 68.75 % to 1/2c, 3c serotype; 1.14 % to 1/2b, 3b, 7 serotype and 2.27 % to 4b,4c,4e serotype. The 45 isolates from the cheese factory were classified as follows: 72.73 % in 1/2a, 3a serotype; 11.36 % in 1/2b, 3b, 7 serotype; 11.36 % in 4b, 4d, 4e serotype and 4.55 % in 1/2c, 3c serotype. One of them was a mixed serotype. Strains from the shrimps company were classified as follows: 92.31 % in 1/2a, 3a serotype and 7.69 % as 4b, 4d, 4e serotype. Thirteen different PFGE-types were obtained among the slaughterhouse strains, 7 PFGE-types among the cheese factory strains and only 2 in the case of shrimps company strains. Regarding MLST results, 8 different profiles were obtained from the slauthgerhouse strains, 7 profiles in the case of the cheese factory strains and 2 profiles among the shrimps company strains. With respect to phenotyping experiments, all of the tested strains presented similar growth curves. In addition, the majority of strains were sensitive to high concentration of disinfectant and had a better tolerance to the detergents tested. Oxidative stress studies are currently in process. The rest of the experiments listed will be planned further on when the protocols will be optimized.