Diversidade genética, resistência a sanitizantes, formação de biofilme e expressão de genes de adesão em Staphylococcus aureus provenientes de leite

Kroning, Isabela Schneid. Genetic diversity, resistance to sanitizers, biofilm formation and expression of adhesion genes in Staphylococcus aureus from milk. 2019. 112f. Thesis (Doctor degree) Programa de Pós-Graduação em Ciência e Tecnologia de Alimentos. Universidade Federal de Pelotas, Pelotas. Staphylococcus aureus is the third microorganism most involved in foodborne diseases in Brazil. The ability to form biofilm on different surfaces of the food industry is a risk factor for the consumer, due to the possibility of contamination of food products. The aims of this study were to verify the genetic diversity, as well as to evaluate the capacity of biofilm formation and the expression of adhesion genes in S. aureus isolates from milk. In addition, it was aimed to determine the susceptibility profile to benzalkonium chloride and chlorhexidine sanitizers and to detect the presence of the main resistance genes to sanitizers in these isolates. The spa typing was used to evaluate genetic diversity. For evaluate the ability of biofilm formation, firstly polystyrene microplates were used to select different biofilm formation profiles. After this, nine isolates from different biofilm formation profiles (three poor biofilm producer, three moderate biofilm producer and three non-biofilm producers) were selected, and these were evaluated for their ability to form biofilm on stainless steel at temperatures of 7, 10 and 37 °C in Tryptone Soy Broth (TSB) and UHT milk. Realtime quantitative PCR (RT-qPCR) was used to evaluate the expression of adhesion genes (cna and ebpS). To verify the susceptibility to sanitizers benzalkonium chloride and chlorhexidine the broth microdilution method was used and the PCR was used to detect the main resistance genes to sanitizers. Of the 31 isolates of S. aureus evaluated, 18 (58%) produced exopolysaccharide. From the polystyrene evaluation, 14 isolates (45%) were able to produce biofilms on this surface. Of these, nine isolates with different biofilm formation profiles (weak biofilm producer, moderate biofilm producer and no biofilm producer) were selected to verify their ability to produce biofilm on stainless steel surface, and all isolates produced biofilms at temperatures of 7, 10 and 37 °C, in TSB and in UHT milk, independent of polystyrene formation profile. Regarding to the presence of the genes of adhesion fnbA, fnbB, clfB, ebpS and cna, were found in 48%, 3%, 52%, 80% and 62% of the isolates, respectively. The real-time quantitative PCR (RT-qPCR) technique showed variation in expression levels of the ebpS and cna genes; however, only in the C2 isolate at a temperature of 10 °C, there was a significantly higher expression of the two genes tested in relation to other multiplication temperatures. The isolates showed a resistance profile to benzalkonium chloride (90.3%) and chlorhexidine (100%); in addition, the mepA, norA and norB genes were the most prevalent, with frequencies of 77.4%, 80.6% and 100%, respectively. The spa type t127 was the most prevalent among the isolates, and this spa type has a very high genetic relation with isolates from human sources, being able to be the source of transmission of S. aureus to milk. The results obtained in the present study highlight the importance of monitoring S. aureus in the food industry environment, since this microorganism is able to form biofilm in different surfaces, temperatures and culture media and harbor phenotypic resistance and genotypic to sanitizers. Key-words: biofilm; benzalkonium chloride; chlorhexidine; RT-qPCR; spa typing