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

Effect of LCFM on intestinal IL-10 and TGF-β.

<p>Cytokines expression was analyzed by qPCR. <i>L. casei</i> only group: mice fed for a week with LCFM. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: untreated mice. <i>L. casei</i> + Strep group: animals were fed with the probiotic for a week and then received streptomycin. Results are expressed as mean +/- SD. Four animals per group were analyzed. (NS): no significant differences. Representative data from 3 independent experiments.</p

Effect of LCFM consumption on joint during <i>Salmonella</i> enterocolitis.

<p><b>A</b>. TNF-α was measured in joint homogenates by ELISA 5 days after oral infection with the pathogen. Five animals per group were analyzed. <b>B</b>. IL-17 expression was analyzed by qPCR at day 5 post infection in joint draining lymph nodes. Seven animals per group were analyzed. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: untreated mice. <i>L. casei</i> + Strep group: animals were fed with the probiotic for a week and then received streptomycin. Results are expressed as mean +/- SD. (NS): no significant differences. Data were collected from 3 independent experiments. </p

Effect of LCFM consumption on joint pathology during <i>Salmonella</i> enterocolitis.

<p>A to C: Histology of knee joints 5 days after oral inoculation with the pathogen. D: Synovial inflammation scores. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: untreated mice. (A) EC group: moderate hyperplasia, with 3 to 5 layers of synoviocytes (arrows). (B) <i>L. casei</i> + EC group: normal synovial capsule (arrows), undistinguishable from Control group. (C) Control animals: normal synovial capsule (arrows). HE; 40x. Data were collected from 3 independent experiments. </p

Effect of LCFM consumption on intestinal pathology during <i>Salmonella</i> enterocolitis.

<p>A to C: Histology of the intestine 2 days after oral infection with the pathogen. D: Intestinal inflammation scores. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: untreated mice. (A) EC group: moderate enterocolitis, loss of normal villus architecture and height. (B) <i>L. casei</i> + EC group: regular display of intestinal villus, undistinguishable from Control group (C). HE; 40x. (NS): no significant differences. Data were collected from 3 independent experiments. </p

Effect of LCFM on mesenteric cytokines after <i>Salmonella</i> enterocolitis.

<p>Cytokine expression was analyzed by qPCR 5 days after oral inoculation with 3-4 x 10³ UFC of the pathogen. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: untreated mice. <i>L. casei</i> + Strep group: animals were fed with the probiotic for a week and then received streptomycin. Results are expressed as mean +/- SD. Seven animals per group were analyzed. (NS): no significant differences. Representative data from 3 independent experiments. </p

Effect of LCFM on intestinal permeability after <i>Salmonella</i> enterocolitis.

<p>A single dose of FITC–dextran was administered by gavage to the animals at day 2 post infection and the intensity of fluorescence was measured in serum 5 h later. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: uninfected mice. <i>L. casei</i> + Strep group: animals were fed with the probiotic for a week and then received streptomycin. Results are expressed as mean +/- SD. Seven animals per group were analyzed. (NS): no significant differences. Representative data from 3 independent experiments. </p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Synthesis of RcsB (A) and PmrA (B) protein in <i>S.</i> Enteritidis <i>dam</i> mutant.

<p>Western blot analysis of total proteins from <i>S.</i> Enteritidis #5694 wild type strain and <i>dam</i> mutant strains harboring an <i>rcsB</i>::3×FLAG (A) or <i>pmrA::3×</i>FLAG (B) transcriptional fusion in the chromosome grown in LB medium and harvested at an OD₆₀₀ of 0.6. Protein loading was normalized to 10⁶ CFU. Blots were probed with anti-FLAG antibodies. Band intensity was determined by densitometry; relative intensities are presented in arbitrary units (a.u.). <b>Panel A.</b> wt: wild type strain #5694 SE<i>rcsB</i>::3×FLAG; <i>dam</i>: <i>dam</i> mutant strain SE<i>rcsB</i>::3×FLAG. <b>Panel B.</b> wt: wild type strain #5694 SE<i>pmrA</i>::3×FLAG; <i>dam</i>: <i>dam</i> mutant strain SE<i>pmrA</i>::3×FLAG. Plasmid pIZ833 bears the <i>dam</i> gene. * Significant difference p<0.05. Data are expressed as means ± SD of percent change in band intensity relative to wild type of five independent experiments performed in duplicates.</p