Prevalence and characterization of salmonella strains of the poultry industryDetermining factors in its survival and biofilm formation

Abstract

pathogen was responsible for 94,530 confirmed cases of salmonellosis in the European Union, of which 9,818 occurred in Spain. The main source of salmonellosis in humans are poultry products. Therefore, one of the great challenges of food safety is to reduce the prevalence of this pathogen in the poultry production chain. For this, it is important to know in detail the mechanisms of resistance of this pathogen. One of the main factors contributing to the persistence of Salmonella in the food chain is the ability to resist stressors through the biofilm formation. Therefore, the objectives of this thesis were on the one hand to know the prevalence and antimicrobial resistance of Salmonella strains isolated from chicken farms and the importance of the different subspecies of Salmonella enterica in public health. The other objective was to know the capacity of the strains of Salmonella isolated from the poultry industry to produce biofilms in different environmental conditions similar to those of the food industry. The results showed that the prevalence of Salmonella in poultry farms in northwestern Spain was lower than the prevalence reported by the European Union. It was remarkable the high prevalence of Salmonella enterica subspecies arizonae, normally linked to cold-blooded animals. In addition, the resistances present in the isolated strains were low being the tetracyclines, sulphonamides and penicillins the groups with greater resistance. Resistance and presence of virulence genes varied among subspecies of Salmonella. Due to the high prevalence of Salmonella enterica subspecies arizonae, an exhaustive review of the non-enteric subspecies of Salmonella enterica was carried out. The data collected from the main studies that evaluated these subspecies show genetic variations in the main islands of pathogenicity of Salmonella that can affect its virulence. In addition, cases of infections in humans by non-enteric subspecies are linked to people with a depressed immune system. These data, together with their low antibiotic resistance and colonization capacity, require studies that confirm that these subspecies should be considered only opportunistic pathogens. The results of the biofilm studies showed that Salmonella strains isolated from poultry production are capable of forming biofilms under different environmental conditions. Nutrient levels influenced the formation of biofilms. In the same sense, the temperature significantly influenced the amount of biofilm formed, the production of cellulose and the morphotype produced. However, strains of Salmonella enterica subspecies arizonae were only able to produce a morphotype. Likewise, oxygen levels affected the formation of biofilms in Salmonella. Anaerobic conditions reduce the ability of Salmonella to produce biofilms, affect its morphotype and block the production of cellulose, one of the main components of Salmonella biofilms. The analysis of gene expression confirmed the results of the phenotypic assays since important genes in the formation of biofilms were negatively regulated in anaerobiosis. Salmonella enterica strains were able to produce biofilms in polystyrene and stainless steel in the presence of food residues such as chicken juice, tomato and milk at both 22ºC and at refrigeration temperatures. The combination of milk and chicken residues with different atmospheres also influenced the formation of biofilms in stainless steel. Especially the chicken residues reduced the expression of genes and small RNAs related to the formation of biofilms under anaerobic conditions while the virulence genes were positively regulated under these conditions. Another factor that affects the survival of pathogens in the food chain is the presence of food preservatives. While in vitro studies showed that the minimum inhibitory concentrations of sulfites and nitrites in Salmonella strains were much higher than the values allowed by legislation, studies conducted in a food matrix showed that those concentrations allowed by legislation were sufficient to inhibit the growth of this pathogen. Another substance, such as sodium acetate in combination with citric acid, proved to be a good alternative to nitrites and sulphites, having lower in vitro inhibitory concentrations lower than those preservatives and inhibiting the growth of Salmonella in the food matrix. Both nitrites, sulphites and sodium acetate affected the formation of Salmonella biofilms in polystyrene and stainless steel. Above all, the sulfites caused a reduction in the formation of biofilms on both surfaces and the sodium acetate in stainless steel. They also caused a reduction in the transcription of genes and small RNAs that contribute to the formation of biofilms. Finally, a literature review work was carried out to know the current status of the Salmonella biofilm studies along the food chain. The evaluation of the studies carried out in this field show the need to reproduce conditions similar to those of the food industry, using for it both sources of nutrients and temperatures as close as possible to the real conditions. For this, the use of food waste will be key. It also highlights the importance of extending biofilm assays to wild Salmonella strains and not using only reference strains to obtain data closer to reality. In addition, phenotypic studies must be accompanied by transcriptomic and proteomic studies that unravel the metabolic pathways used by Salmonella to produce biofilm in different environmental conditions. Finally, the need to develop standard laboratory protocols in the study of biofilms that allow reproducible results between laboratories and studies carried out.