Probiotics are a potential strategy for salmonellosis control. A defined pig microbiota (DPM) mixture of nine bacterial strains previously exhibited probiotic and anti-Salmonella properties in vitro. Therefore, we evaluated its gut colonization ability and protection effect against S. typhimurium LT2-induced infection in the gnotobiotic piglet model. The DPM mixture successfully colonized the piglet gut and was stable and safe until the end of the experiment. The colon was inhabited by about 9 log CFU g-1 with a significant representation of bifidobacteria and lactobacilli compared to ileal levels around 7-8 log CFU g-1. Spore-forming clostridia and bacilli seemed to inhabit the environment only temporarily. The bacterial consortium contributed to the colonization of the gut at an entire length. The amplicon profile analysis supported the cultivation trend with a considerable representation of lactobacilli with bacilli in the ileum and bifidobacteria with clostridia in the colon. Although there was no significant Salmonella-positive elimination, it seems that the administered bacteria conferred the protection of infected piglets because of the slowed delayed infection manifestation without translocations of Salmonella cells to the blood circulation. Due to its colonization stability and potential protective anti-Salmonella traits, the DPM mixture has promising potential in pig production applications. However, advanced immunological tests are needed.

• Keywords
• Salmonella typhimurium, bacilli, bacterial consortium, bifidobacteria, clostridia, lactobacilli,
• Publication type
• Journal Article MeSH

Gnotobiotic (GN) animals with simple and defined microbiota can help to elucidate host-pathogen interferences. Hysterectomy-derived germ-free (GF) minipigs were associated at 4 and 24 h post-hysterectomy with porcine commensal mucinolytic Bifidobacterium boum RP36 (RP36) strain or non-mucinolytic strain RP37 (RP37) or at 4 h post-hysterectomy with Lactobacillus amylovorus (LA). One-week-old GN minipigs were infected with Salmonella Typhimurium LT2 strain (LT2). We monitored histological changes in the ileum, mRNA expression of Toll-like receptors (TLRs) 2, 4, and 9 and their related molecules lipopolysaccharide-binding protein (LBP), coreceptors MD-2 and CD14, adaptor proteins MyD88 and TRIF, and receptor for advanced glycation end products (RAGE) in the ileum and colon. LT2 significantly induced expression of TLR2, TLR4, MyD88, LBP, MD-2, and CD14 in the ileum and TLR4, MyD88, TRIF, LBP, and CD14 in the colon. The LT2 infection also significantly increased plasmatic levels of inflammatory markers interleukin (IL)-6 and IL-12/23p40. The previous colonization with RP37 alleviated damage of the ileum caused by the Salmonella infection, and RP37 and LA downregulated plasmatic levels of IL-6. A defined oligo-microbiota composed of bacterial species with selected properties should probably be more effective in downregulating inflammatory response than single bacteria.

• Keywords
• Bifidobacterium, Lactobacillus, Salmonella Typhimurium, Toll-like receptor, cytokines, gnotobiotic minipig, lipopolysaccharide,
• Publication type
• Journal Article MeSH

A balanced microbiota is a main prerequisite for the host's health. The aim of the present work was to develop defined pig microbiota (DPM) with the potential ability to protect piglets against infection with Salmonella Typhimurium, which causes enterocolitis. A total of 284 bacterial strains were isolated from the colon and fecal samples of wild and domestic pigs or piglets using selective and nonselective cultivation media. Isolates belonging to 47 species from 11 different genera were identified by MALDI-TOF mass spectrometry (MALDI-TOF MS). The bacterial strains for the DPM were selected for anti-Salmonella activity, ability to aggregate, adherence to epithelial cells, and to be bile and acid tolerant. The selected combination of 9 strains was identified by sequencing of the 16S rRNA gene as Bacillus sp., Bifidobacterium animalis subsp. lactis, B. porcinum, Clostridium sporogenes, Lactobacillus amylovorus, L. paracasei subsp. tolerans, Limosilactobacillus reuteri subsp. suis, and Limosilactobacillus reuteri (two strains) did not show mutual inhibition, and the mixture was stable under freezing for at least 6 months. Moreover, strains were classified as safe without pathogenic phenotype and resistance to antibiotics. Future experiments with Salmonella-infected piglets are needed to test the protective effect of the developed DPM.

• Keywords
• bacterial consortium, gnotobiotic piglets, intestinal pathogens, pig intestinal bacteria, probiotic properties testing,
• Publication type
• Journal Article MeSH

Preterm germ-free piglets were monoassociated with probiotic Bifidobacterium animalis subsp. lactis BB-12 (BB12) to verify its safety and to investigate possible protection against subsequent infection with Salmonella Typhimurium strain LT2 (LT2). Clinical signs of salmonellosis, bacterial colonization in the intestine, bacterial translocation to mesenteric lymph nodes (MLN), blood, liver, spleen, and lungs, histopathological changes in the ileum, claudin-1 and occludin mRNA expression in the ileum and colon, intestinal and plasma concentrations of IL-8, TNF-α, and IL-10 were evaluated. Both BB12 and LT2 colonized the intestine of the monoassociated piglets. BB12 did not translocate in the BB12-monoassociated piglets. BB12 was detected in some cases in the MLN of piglets, consequently infected with LT2, but reduced LT2 counts in the ileum and liver of these piglets. LT2 damaged the luminal structure of the ileum, but a previous association with BB12 mildly alleviated these changes. LT2 infection upregulated claudin-1 mRNA in the ileum and colon and downregulated occludin mRNA in the colon. Infection with LT2 increased levels of IL-8, TNF-α, and IL-10 in the intestine and plasma, and BB12 mildly downregulated them compared to LT2 alone. Despite reductions in bacterial translocation and inflammatory cytokines, clinical signs of LT2 infection were not significantly affected by the probiotic BB12. Thus, we hypothesize that multistrain bacterial colonization of preterm gnotobiotic piglets may be needed to enhance the protective effect against the infection with S. Typhimurium LT2.

• Keywords
• Bifidobacterium animalis subsp. lactis BB-12, Salmonella Typhimurium, immunocompromised, inflammatory cytokines, intestinal barrier, preterm host,
• Publication type
• Journal Article MeSH

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can be actively secreted by immune cells after different immune stimuli or passively released from cells undergoing necrosis. HMGB1 amplifies inflammation, and its hypersecretion contributes to multiple organ dysfunction syndrome and death. We tested possible immunomodulatory effect of commensal Lactobacillus amylovorus (LA), Lactobacillus mucosae (LM) or probiotic Escherichia coli Nissle 1917 (EcN) in infection of gnotobiotic piglets with Salmonella Typhimurium (ST). Transcription of HMGB1 and Toll-like receptors (TLR) 2, 4, and 9 and receptor for advanced glycation end products (RAGE), TLR4-related molecules (MD-2, CD14, and LBP), and adaptor proteins (MyD88 and TRIF) in the ileum and colon were measured by RT-qPCR. Expression of TLR4 and its related molecules were highly upregulated in the ST-infected intestine, which was suppressed by EcN, but not LA nor LM. In contrast, HMGB1 expression was unaffected by ST infection or commensal/probiotic administration. HMGB1 protein levels in the intestine measured by ELISA were increased in ST-infected piglets, but they were decreased by previous colonization with E. coli Nissle 1917 only. We conclude that the stability of HMGB1 mRNA expression in all piglet groups could show its importance for DNA transcription and physiological cell functions. The presence of HMGB1 protein in the intestinal lumen probably indicates cellular damage.

• Keywords
• Escherichia coli Nissle 1917 (EcN), Lactobacillus amylovorus (LA), Lactobacillus mucosae (LM), Salmonella Typhimurium (ST), Toll-like receptor 4 (TLR4), gnotobiotic piglet, high mobility group box 1 (HMGB1), intestine,
• MeSH
• Escherichia coli immunology   MeSH
• Germ-Free Life immunology   MeSH
• Lactobacillus acidophilus immunology   MeSH
• Swine * immunology   microbiology   MeSH
• Probiotics * MeSH
• HMGB1 Protein immunology   MeSH
• Salmonella typhimurium immunology   MeSH
• Signal Transduction immunology   MeSH
• Intestines immunology   microbiology   MeSH
• Toll-Like Receptor 4 immunology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• HMGB1 Protein MeSH
• Toll-Like Receptor 4 MeSH

Salmonella Typhimurium is an enteric pathogen that causes acute and chronic infections in humans and animals. One-week-old germ-free piglets were orally colonized/infected with the Salmonella Typhimurium LT2 strain or its isogenic rough ΔrfaL, ΔrfaG or ΔrfaC mutants with exactly defined lipopolysaccharide (LPS) defects. After 24 h, the piglets were euthanized and the colonization of the small intestine, translocations into the mesenteric lymph nodes, liver, spleen, lungs, and bacteremia, along with changes in the ileum histology, and transcription levels of the tight junction proteins claudin-1, claudin-2, and occludin were all assessed. Additionally, transcription levels of IL-8, TNF-α, and IL-10 in the terminal ileum, and their local and systemic protein levels were evaluated. Wild-type Salmonella Typhimurium showed the highest translocation, histopathological changes, upregulation of claudins and downregulation of occludin, transcription of the cytokines, intestinal IL-8 and TNF-α levels, and systemic TNF-α and IL-10 levels. Depending on the extent of the incompleteness of the LPS, the levels of the respective elements decreased, or no changes were observed at all in the piglets colonized/infected with Δrfa mutants. Intestinal IL-10 and systemic IL-8 levels were not detected in any piglet groups. This study provided foundational data on the gnotobiotic piglet response to colonization/infection with the exactly defined rough Salmonella Typhimurium LT2 isogenic mutants.

• Keywords
• Salmonella Typhimurium, cytokines, germ-free piglet, gnotobiotic, lipopolysaccharide, tight junction proteins, Δrfa mutant,
• MeSH
• Cytokines immunology   MeSH
• Germ-Free Life * MeSH
• Liver microbiology   MeSH
• Lipopolysaccharides toxicity   MeSH
• Lymph Nodes microbiology   MeSH
• Mutation MeSH
• Lung microbiology   MeSH
• Swine MeSH
• Salmonella typhimurium genetics   physiology   MeSH
• Salmonella Infections immunology   microbiology   pathology   MeSH
• Spleen microbiology   MeSH
• Intestine, Small immunology   microbiology   pathology   MeSH
• Virulence * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• Lipopolysaccharides MeSH

Non-typhoid Salmonellae are worldwide spread food-borne pathogens that cause diarrhea in humans and animals. Their multi-drug resistances require alternative ways to combat this enteric pathogen. Mono-colonization of a gnotobiotic piglet gastrointestinal tract with commensal lactobacilli Lactobacillus amylovorus and Lactobacillus mucosae and with probiotic E. coli Nissle 1917 and their interference with S. Typhimurium infection was compared. The impact of bacteria and possible protection against infection with Salmonella were evaluated by clinical signs, bacterial translocation, intestinal histology, mRNA expression of villin, claudin-1, claudin-2, and occludin in the ileum and colon, and local intestinal and systemic levels of inflammatory cytokines IL-8, TNF-α, and IL-10. Both lactobacilli colonized the gastrointestinal tract in approximately 100× lower density compare to E. coli Nissle and S. Typhimurium. Neither L. amylovorus nor L. mucosae suppressed the inflammatory reaction caused by the 24 h infection with S. Typhimurium. In contrast, probiotic E. coli Nissle 1917 was able to suppress clinical signs, histopathological changes, the transcriptions of the proteins, and the inductions of the inflammatory cytokines. Future studies are needed to determine whether prebiotic support of the growth of lactobacilli and multistrain lactobacilli inoculum could show higher protective effects.

• Keywords
• E. coli Nissle 1917, Lactobacillus amylovorus, Lactobacillus mucosae, Salmonella Typhimurium, cytokine, food-borne pathogen, gnotobiotic piglet, intestine,
• Publication type
• Journal Article MeSH