Salmonella enterica serovar Enteritidis biofilm lifestyle induces lower pathogenicity and reduces inflammatory response in a murine model compared to planktonic bacteria.

Salmonellaenterica serovar Enteritidis (S. Enteritidis) is the most frequent serovar involved in human salmonellosis. It has been demonstrated that about 80% of infections are related to biofilm formation. There is scant information about the pathogenicity of S. Enteritidis and its relationship to biofilm production. In this regard, this study aimed to investigate the differential host response induced by S. Enteritidis biofilm and planktonic lifestyle. To this purpose, biofilm and planktonic bacteria were inoculated to BALB/c mice and epithelial cell culture. Survival studies revealed that biofilm is less virulent than planktonic cells. Reduced signs of intestinal inflammation and lower bacterial translocation were observed in animals inoculated with Salmonella biofilm compared to the planktonic group. Results showed that Salmonella biofilm was impaired for invasion of non-phagocytic cells and induces a lower inflammatory response in vivo and in vitro compared to that of planktonic bacteria. Taken together, the outcome of Salmonella-host interaction varies depending on the bacterial lifestyle.

Dam methylation is required for efficient biofilm production in Salmonella enterica serovar Enteritidis.

The ecological success of Salmonella enterica to survive in different environments is due, in part, to the ability to form biofilms, something which is especially important for food industry. The aim of the current study was to evaluate the involvement of Dam methylation in biofilm production in S. Enteritidis strains. The ability to generate biofilms was analyzed in wild type and dam mutant strains. In S. Enteritidis, the absence of Dam affected the capacity to develop pellicles at the air-liquid interface and reduced the ability to form biofilm on polystyrene surfaces. Curli and cellulose production, determined by Congo red and calcofluor assays, were affected in dam mutant strains. Relative quantitative real-time PCR experiments showed that the expression of csgD and csgA genes is reduced in mutants lacking dam gene with respect to the wild type strains, whereas transcript levels of bcsA are not affected in the absence of Dam. To our knowledge, this is the first report on the participation of Dam methylation on biofilm production in Enteritidis or any other serovar of S. enterica. Results presented here suggest that changes in gene expression required for biofilm production are finely regulated by Dam methylation. Thus, Dam methylation could modulate csgD expression and upregulate the expression of factors related with biofilm production, including curli and cellulose. This study contributes to the understanding of biofilm regulation in Salmonella spp. and to the design of new strategies to prevent food contamination and humans and animals infections.

Salmonella enterica serovar Enteritidis enterocolitis during late stages of gestation induces an adverse pregnancy outcome in the murine model.

Foodborne diseases caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) are a significant health problem. Pregnancy, state of immunological tolerance, is a predisposing condition for the development of infections with intracellular pathogens. Salmonella species can cause pregnancy complications such as chorioamnionitis, transplacental fetal infection, pre term labor, abortions, neonatal and maternal septicemia. However, the specific mechanisms by which Salmonella infections trigger these alterations are not clear. In the present work, using a self-limiting enterocolitis murine model, we show that the ingestion of a low dose of S. Enteritidis at late stages of pregnancy (day 15 of gestation) is sufficient to induce massive maternal infection. We found that Salmonella infection leads to 40% of pre term delivery, 33% of abortion and fetal growth restriction. Placental dysfunction during S. Enteritidis enterocolitis was confirmed through cellular infiltration and hypoxia markers (MPO activity and COX-1 and COX-2 expression, respectively). Apoptosis in placental tissue due to Salmonella infection was also evident at day 18 of gestation when investigated by morphometric procedure, DNA fragmentation and Fas/FasL expression. Also, the expression of IFN-γ, TNF-α, IL-17 and IL-10 was up regulated in response to Salmonella not only in placenta, but also in amniotic fluid and maternal serum. Altogether, our results demonstrate that S. Enteritidis enterocolitis during late stages of gestation causes detrimental effect on pregnancy outcome.

AvrA effector protein of Salmonella enterica serovar Enteritidis is expressed and translocated in mesenteric lymph nodes at late stages of infection in mice.

Salmonellosis is a major health problem worldwide. Salmonella enterica serovar Enteritidis (S. Enteritidis) has been a primary cause of Salmonella outbreaks in many countries. AvrA is an SPI-1 effector protein involved in the enteritis pathway, with critical roles in inhibiting inflammation and apoptosis. In this work, we constructed an AvrA-FLAG-tagged strain of S. Enteritidis to analyse the expression profile of AvrA in vitro, in cell culture and in vivo. AvrA expression and secretion were observed in vitro under culture conditions that mimicked intestinal and intracellular environments. In agreement, bacteria isolated from infected cell monolayers expressed and translocated AvrA for at least 24 h post-inoculation. For in vivo experiments, BALB/c mice were inoculated by the natural route of infection with the AvrA-FLAG strain. Infecting bacteria and infected cells were recovered from mesenteric lymph nodes (MLN). Our results showed that AvrA continues to be synthesized in vivo up to day 8 post-inoculation. Moreover, AvrA translocation was detected in the cytosol of cells isolated from MLN 8 days after infection. Interestingly, we observed that AvrA is secreted by both type three secretion system (T3SS)-1 and T3SS-2. In summary, these findings indicate that AvrA expression is not constrained to the initial host-bacteria encounter in the intestinal environment as defined previously. The AvrA effector may participate also in systemic S. Enteritidis infection.

Consumption of Lactobacillus casei fermented milk prevents Salmonella reactive arthritis by modulating IL-23/IL-17 expression.

Reactive arthritis is the development of sterile joint inflammation as a sequel to a remote infection, often in the gut. We have previously shown that a low dose of S. enteritidis inoculated to streptomycin-pretreated mice generates a self-limiting enterocolitis suitable for studying reactive arthritis. Here we show that consumption of Lactobacillus casei prior to infection abolishes intestinal and joint inflammation triggered by Salmonella. BALB/c mice were sacrificed after infection; intestinal and joint samples were analyzed for histological changes and expression of cytokines. TNF-α was measured by ELISA and the expression of IL-1ß, IL-6, IL-10, IL-17, IL-23 and TGF-ß was assessed by qPCR. L. casei consumption prevented Salmonella-induced synovitis, the increment of TNF-α in knees and the increase of IL-17 expression in popliteal and inguinal lymph nodes. At intestinal level consumption of L. casei drastically diminished S. enteritidis invasiveness and shortened splenic persistence of the pathogen. Bacterial loads recovered at days 2 and 5 from Peyer's patches were 10-fold lower in mice fed with L. casei. In accordance, we found that the augment in gut permeability induced during enterocolitis was decreased in those animals. Consumption of L. casei prior to infection failed to increase anti- inflammatory molecules such as IL-10 and TGF-ß in the intestine. On the other hand, consumption of L. casei abrogated the expression of TNF-α, IL-17, IL-23, IL-1ß and IL-6 in cecum and mesenteric lymph nodes. These cytokines are needed for differentiation of immune cells involved in the development of reactive arthritis such as Th17 and γδ T cells. Trafficking of these inflammatory cells from the gut to the joints has been proposed as a mechanism of generation of reactive arthritis. Our results suggest that L. casei consumption prevents Salmonella-induced synovitis by altering the intestinal milieu necessary for differentiation of cells involved in the generation of joint inflammation.