Loss of intestinal epithelial barrier function in Salmonella Enteritidis infection

Intestinal infection with Salmonella enterica serotype Enteritidis, a food-borne infection spread to humans especially through contaminated eggs and egg-products as well as undercooked contaminated fresh meat, is the most common cause of intestinal inflammation in the European Union. Enteritis caused by Salmonella Enteritidis is characterized by fever, diarrhoea and abdominal pain. The disruption of the intestinal epithelial barrier function contributes to diarrhoea and is responsible for the perpetuation of the inflammatory process. In this sense, oxidative stress and the proinflammatory cytokines TNF-α, IFN-γ and IL-1β are described to induce the disorganization of the tight junctions (TJ), the most apical epithelial intercellular junctions and responsible for the paracellular permeability. The interest of this chapter relies not only in the investigation dealing with the mechanisms of TJ regulation but also in the contribution to the development of new tools for the prevention of epithelial barrier disruption in enteritis caused by Salmonella Enteritidis.

Efecto de los contenidos de C, Mn y Si en el comportamiento a fluencia en caliente de aceros de construcción al carbono y microaleados: aplicación a la obtención de grano ultrafino en productos largos laminados

Després d’aplicar alguns tractaments d’elaboració i conservació als aliments, queden bacteris lesionats. Aquests bacteris perden la capacitat de créixer en els medis de cultiu selectiu convencionals, de manera que se’n subestima el recompte. Malgrat això, poden recuperar-se als aliments i suposar un risc per la salut, ja que alguns encara poden mantenir activitat metabòlica i integritat estructural. En aquest projecte, es van optimitzar protocols de preparació de mostres per citometria de flux (CF) per avaluar l’estat fisiològic de patògens alimentaris (Escherichia coli O157:H7, Salmonella Enteritidis i Listeria monocytogenes) sotmesos a estrès. Es van estudiar principalment dos paràmetres fisiològics: la integritat de membrana, mitjançant iodur de propidi i fluorocroms de la família SYTO; i l’activitat respiratòria, per la reducció intracel•lular d’una sal de tetrazole, el CTC. En primer lloc, es van avaluar variables de protocol, com la concentració de colorant, la ràtio entre colorants, la solució de tinció i el temps d’incubació, en mostres control (cèl•lules sanes i mortes). A continuació, els protocols optimitzats es van aplicar a suspensions bacterianes en medi de cultiu que prèviament havien estat sotmeses a estressos físics i fisicoquímics. Durant l’etapa final del projecte, els coneixements adquirits sobre la preparació de mostres per CF es van aplicar a l’anàlisi de mostres de matriu complexa: amanides comercials inoculades amb E. coli O157:H7. Als assajos amb indicadors d’integritat de membrana en suspensions bacterianes sotmeses a estrès, es van poder quantificar cèl•lules amb la membrana parcialment danyada (presumptes cèl•lules lesionades). El recompte de cèl•lules que mantingueren l’activitat respiratòria després de ser sotmeses a estrès va ser superior al que es va obtenir mitjançant recompte en placa convencional, cosa que va evidenciar la presència de cèl•lules actives però no cultivables. La introducció d’estratègies per reduir les interferències provocades per les partícules alimentàries i l’ús d’un anticòs amb marcatge fluorescent va permetre detectar selectivament les cèl•lules d’E. coli O157:H7 i avaluar-ne la integritat de membrana simultàniament. L’anàlisi de cèl•lules bacterianes per CF requereix de la exhaustiva optimització dels protocols, que són específics per cada soca i matriu. Malgrat això, i a diferència del mètode convencional per recompte en placa, ofereix la possibilitat d’obtenir una gran quantitat d’informació sobre el sovint complex estat fisiològic d’una mostra.

Effect of cattle on Salmonella carriage, diversity and antimicrobial resistance in free-ranging wild boar (Sus scrofa) in northeastern Spain

Salmonella is distributed worldwide and is a pathogen of economic and public health importance. As a multi-host pathogen with a long environmental persistence, it is a suitable model for the study of wildlife-livestock interactions. In this work, we aim to explore the spill-over of Salmonella between free-ranging wild boar and livestock in a protected natural area in NE Spain and the presence of antimicrobial resistance. Salmonella prevalence, serotypes and diversity were compared between wild boars, sympatric cattle and wild boars from cattle-free areas. The effect of age, sex, cattle presence and cattle herd size on Salmonella probability of infection in wild boars was explored by means of Generalized Linear Models and a model selection based on the Akaike’s Information Criterion. Prevalence was higher in wild boars co-habiting with cattle (35.67%, CI 95% 28.19–43.70) than in wild boar from cattle-free areas (17.54%, CI 95% 8.74–29.91). Probability of a wild boar being a Salmonella carrier increased with cattle herd size but decreased with the host age. Serotypes Meleagridis, Anatum and Othmarschen were isolated concurrently from cattle and sympatric wild boars. Apart from serotypes shared with cattle, wild boars appear to have their own serotypes, which are also found in wild boars from cattle-free areas (Enteritidis, Mikawasima, 4:b:- and 35:r:z35). Serotype richness (diversity) was higher in wild boars co-habiting with cattle, but evenness was not altered by the introduction of serotypes from cattle. The finding of a S. Mbandaka strain resistant to sulfamethoxazole, streptomycin and chloramphenicol and a S. Enteritidis strain resistant to ciprofloxacin and nalidixic acid in wild boars is cause for public health concern.

Seguridad alimentaria en productos cárnicos: aplicación de sistemas emergentes de conservación y evaluación de patógenos mediante el desarrollo de nuevas técnicas moleculares

Durante el desarrollo del proyecto se ha podido constatar la relevancia de la utilización de métodos alternativos de conservación tales como la alta presión hidrostática; así como el efecto añadido de los bioconservantes naturales aplicados en masa o envasado activo en la seguridad alimentaria de los productos cárnicos curado cocidos. Asimismo el desarrollo y la identificación, cuantificación de los patógenos alimentarios a estudio (Listeria monocytogenes y Salmonella spp) mediante técnicas de biología molecular, PCR convencional, PCR cuantitativa y NASBA, además de suponer un importante logro científico, ha significado una enorme ventaja en los ensayos tipo “challenge test” tanto por rapidez en la analítica realizada como para la evaluación simultánea de un número elevado de muestras por ensayo. Cabe destacar la importancia del NASBA en la detección selectiva de células viables.

Understanding the two-component sensing System of virulent bacteria

Report for the scientific sojourn carried out at the l’ Institute for Computational Molecular Science of the Temple University, United States, from 2010 to 2012. Two-component systems (TCS) are used by pathogenic bacteria to sense the environment within a host and activate mechanisms related to virulence and antimicrobial resistance. A prototypical example is the PhoQ/PhoP system, which is the major regulator of virulence in Salmonella. Hence, PhoQ is an attractive target for the design of new antibiotics against foodborne diseases. Inhibition of the PhoQ-mediated bacterial virulence does not result in growth inhibition, presenting less selective pressure for the generation of antibiotic resistance. Moreover, PhoQ is a histidine kinase (HK) and it is absent in animals. Nevertheless, the design of satisfactory HK inhibitors has been proven to be a challenge. To compete with the intracellular ATP concentrations, the affinity of a HK inhibidor must be in the micromolar-nanomolar range, whereas the current lead compounds have at best millimolar affinities. Moreover, the drug selectivity depends on the conformation of a highly variable loop, referred to as the “ATP-lid, which is difficult to study by X-Ray crystallography due to its flexibility. I have investigated the binding of different HK inhibitors to PhoQ. In particular, all-atom molecular dynamics simulations have been combined with enhanced sampling techniques in order to provide structural and dynamic information of the conformation of the ATP-lid. Transient interactions between these drugs and the ATP-lid have been identified and the free energy of the different binding modes has been estimated. The results obtained pinpoint the importance of protein flexibility in the HK-inhibitor binding, and constitute a first step in developing more potent and selective drugs. The computational resources of the hosting institution as well as the experience of the members of the group in drug binding and free energy methods have been crucial to carry out this work.

Qualitat microbiològica dels productes vegetals frescos mínimament processats i desenvolupament de tecnologies de bioconservació mitjançant bacteris de l'àcid làctic

Food safety today is conditiones by the implementation of new Tecnologies for food preservation based on mild treatments and mínimum process, Novel foods, severe restrictions in the toxicològic profile of chemical preservatives, And the drastic limitation /prohibition of antibiotics, and as a consequence, by the New emergint pathogens or by the increase of classical food-borne pathogens. Biopreservatives appear within this context with strong expectations, because thei are safe microorganisms of ten isolated from foods, chemical compounds of natural origin -antimicrobial peptides and proteins, botanical extracts, enzymes- and sintetic compounds based on natural structures, but less toxic and more eficients, like the amtimicrobial peptides. Among the microbial biopreservatives, lactic acid bacteria have shown great possibilities in the preservation of cured meat products, ready to eat fresh fruit and vegetables, as well as to decrease microbial spoilage in food by products before processing for valorization. Our laboratory has performed an extense survey of the microbiological quality of fresh fruit and vegetables, and of ready-to-eat products, and have detect low levels, but significant of Salmonella sp., E. coli and Listeria spp., including L.monocytogenes, in retail markets and Supermarkets of Catalonia. Due to this reason, we started a project consisting of Developing application of lactic acid bacteria (LAB) obtained from these products, as biopreservatives. LAB were abundant in ready-to-eat fresh fruits and vegetables, specially in germinated seeds. From these products we obtained strains of Leuconostoc, Lactobacillus and Weissella, producing bacteriocins and with a Significant activity of control of L.monocytogenes in fresh apple and cut salad. Ather strains were efective in the inhibition of fungal rot during postharvest caused by Penicillium expansum

Epidemiology of foodborne Norovirus outbreaks in Catalonia, Spain

Background: Noroviruses are one of the principal biological agents associated with the consumption of contaminated food. The objective of this study was to analyse the size and epidemiological characteristics of foodborne outbreaks of gastroenteritis in Catalonia, a region in the northeast of Spain.Methods: In all reported outbreaks of gastroenteritis associated with food consumption, faecal samples of persons affected were analysed for bacteria and viruses and selectively for parasites. Study variables included the setting, the number of people exposed, age, sex, clinical signs and hospital admissions. The study was carried out from October 2004 to October 2005.Results: Of the 181 outbreaks reported during the study period, 72 were caused by Salmonella and 30 by norovirus (NoV); the incidence rates were 14.5 and 9.9 per 100,000 person-years, respectively. In 50% of the NoV outbreaks and 27% of the bacterial outbreaks (p = 0.03) the number of persons affected was ¿10; 66.7% of NoV outbreaks occurred in restaurants; no differences in the attack rates were observed according to the etiology. Hospitalizations were more common (p = 0.03) in bacterial outbreaks (8.6%) than in NoV outbreaks (0.15%). Secondary cases accounted for 4% of cases in NoV outbreaks compared with 0.3% of cases in bacterial outbreaks (p < 0.001)Conclusion: Norovirus outbreaks were larger but less frequent than bacterial outbreaks, suggesting that underreporting is greater for NoV outbreaks. Food handlers should receive training on the transmission of infections in diverse situations. Very strict control measures on handwashing and environmental disinfection should be adopted in closed or partially-closed institutions.

The effect of recombination in Aeromonas

Although several approaches have been attempted, the estimation of recombination frequencies in natural populations ofbacteria remains challenging. Previous studies have demonstrated awide variety of situations among bacterial species, ranging from theclonal diversification of Salmonella or Escherichia coli, which aremainly due to mutation, to the frequent recombination found inNeisseria gonorrhoeae or Helicobacter pylori. Most of the populationstudies done with bacterial species suggest that recombination occursin nature but that it is infrequent compared to mutation. Consequently,bacterial populations consist largely of independent clonal lineages.Our research suggests little or null influence of recombination in thegenetic structure of "Aeromonas hydrophila Species Complex", despite the presence of some strains with recombinant gene fragments.

The effect of recombination in Aeromonas

Although several approaches have been attempted, the estimation of recombination frequencies in natural populations ofbacteria remains challenging. Previous studies have demonstrated awide variety of situations among bacterial species, ranging from theclonal diversification of Salmonella or Escherichia coli, which aremainly due to mutation, to the frequent recombination found inNeisseria gonorrhoeae or Helicobacter pylori. Most of the populationstudies done with bacterial species suggest that recombination occursin nature but that it is infrequent compared to mutation. Consequently,bacterial populations consist largely of independent clonal lineages.Our research suggests little or null influence of recombination in thegenetic structure of "Aeromonas hydrophila Species Complex", despite the presence of some strains with recombinant gene fragments.

Epidemiology of foodborne Norovirus outbreaks in Catalonia, Spain

Background: Noroviruses are one of the principal biological agents associated with the consumption of contaminated food. The objective of this study was to analyse the size and epidemiological characteristics of foodborne outbreaks of gastroenteritis in Catalonia, a region in the northeast of Spain. Methods: In all reported outbreaks of gastroenteritis associated with food consumption, faecal samples of persons affected were analysed for bacteria and viruses and selectively for parasites. Study variables included the setting, the number of people exposed, age, sex, clinical signs and hospital admissions. The study was carried out from October 2004 to October 2005. Results: Of the 181 outbreaks reported during the study period, 72 were caused by Salmonella and 30 by norovirus (NoV); the incidence rates were 14.5 and 9.9 per 100,000 person-years, respectively. In 50% of the NoV outbreaks and 27% of the bacterial outbreaks (p = 0.03) the number of persons affected was ≥10; 66.7% of NoV outbreaks occurred in restaurants; no differences in the attack rates were observed according to the etiology. Hospitalizations were more common (p = 0.03) in bacterial outbreaks (8.6%) than in NoV outbreaks (0.15%). Secondary cases accounted for 4% of cases in NoV outbreaks compared with 0.3% of cases in bacterial outbreaks (p < 0.001) Conclusion: Norovirus outbreaks were larger but less frequent than bacterial outbreaks, suggesting that underreporting is greater for NoV outbreaks. Food handlers should receive training on the transmission of infections in diverse situations. Very strict control measures on handwashing and environmental disinfection should be adopted in closed or partially-closed institutions.

Mouthrinses: a comparative microbiological study

This study was performed in order to evaluate the efficacy of different mouthrinses whose use is extended in Spain. Six different antiseptic mouthrinses were studied by means of determination of Minimal Inhibitory Concentration (MIC) values against Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Bacillus subtilis, Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. Also in vivo experiments were carried out in volunteers by the use of mouthrinses and evaluation of bacterial populations before and after the treatment. Finally, the kinetics of bacterial death was determined. Results suggested that the determination of MIC values is not a reliable method to evaluate the antibacterial effect of such products. On the other hand those rinsing solutions based on the effect of oxygen, such as those containing carbamide peroxide have a greater efficacy against anaerobic bacteria compared with rinses whose active molecule is a disinfectant. Finally, the kinetics of bacterial death demonstrates that the essential oil rinse kills bacteria much faster. All tested mouthrinses were active as antibacterial although those based on oxygen production or essential oils were more active than solutions based on chlorhexidine and Triclosan