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.

• Klíčová slova
• Bifidobacterium, Lactobacillus, Salmonella Typhimurium, Toll-like receptor, cytokines, gnotobiotic minipig, lipopolysaccharide,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• bacterial consortium, gnotobiotic piglets, intestinal pathogens, pig intestinal bacteria, probiotic properties testing,
• Publikační typ
• časopisecké články MeSH

Gnotobiotic (GN) animals with defined microbiota allow us to study host-microbiota and microbiota-microbiota interferences. Preterm germ-free (GF) piglets were mono-associated with probiotic Bifidobacterium animalis subsp. lactis BB-12 (BB12) to ameliorate/prevent the consequences of infection with the Salmonella Typhimurium strain LT2 (LT2). Goblet cell density; expression of Toll-like receptors (TLRs) 2, 4, and 9; high mobility group box 1 (HMGB1); interleukin (IL)-6; and IL-12/23p40 were analyzed to evaluate the possible modulatory effect of BB12. BB12 prevented an LT2-induced decrease of goblet cell density in the colon. TLRs signaling modified by LT2 was not influenced by the previous association with BB12. The expression of HMGB1, IL-6, and IL12/23p40 in the jejunum, ileum, and colon and their levels in plasma were all decreased by BB12, but these changes were not statistically significant. In the colon, differences in HMGB1 distribution between the GF and LT2 piglet groups were observed. In conclusion, the mono-association of GF piglets with BB12 prior to LT2 infection partially ameliorated the inflammatory response to LT2 infection.

• Klíčová slova
• Bifidobacterium animalis subsp. lactis BB-12, Salmonella Typhimurium, Toll-like receptors, high mobility group box 1, immunodeficient host, inflammatory cytokines, intestinal barrier, mucin, tight junction proteins,
• MeSH
• Bifidobacterium animalis * MeSH
• gnotobiologické modely MeSH
• lidé MeSH
• novorozenec nedonošený MeSH
• novorozenec MeSH
• prasata MeSH
• probiotika * farmakologie   MeSH
• protein HMGB1 * MeSH
• Salmonella typhimurium MeSH
• toll-like receptory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protein HMGB1 * MeSH
• toll-like receptory MeSH

Intra-amniotic infections (IAI) are one of the reasons for preterm birth. High mobility group box 1 (HMGB1) is a nuclear protein with various physiological functions, including tissue healing. Its excessive extracellular release potentiates inflammatory reaction and can revert its action from beneficial to detrimental. We infected the amniotic fluid of a pig on the 80th day of gestation with 1 × 104 colony forming units (CFUs) of E. coli O55 for 10 h, and evaluated the appearance of HMGB1, receptor for glycation endproducts (RAGE), and Toll-like receptor (TLR) 4 in the amniotic membrane and fluid. Sham-infected amniotic fluid served as a control. The expression and release of HMGB1 were evaluated by Real-Time PCR, immunofluorescence, immunohistochemistry, and ELISA. The infection downregulated HMGB1 mRNA expression in the amniotic membrane, changed the distribution of HMGB1 protein in the amniotic membrane, and increased its level in amniotic fluid. All RAGE mRNA, protein expression in the amniotic membrane, and soluble RAGE level in the amniotic fluid were downregulated. TLR4 mRNA and protein expression and soluble TLR4 were all upregulated. HMGB1 is a potential target for therapy to suppress the exaggerated inflammatory response. This controlled expression and release can, in some cases, prevent the preterm birth of vulnerable infants. Studies on suitable animal models can contribute to the development of appropriate therapy.

• Klíčová slova
• Toll-like receptor, amniotic fluid, amniotic membrane, cytokines, high mobility group box 1, intra-amniotic infection, pig, preterm birth, receptor for advanced glycation endproducts,
• MeSH
• amnion imunologie   mikrobiologie   patologie   MeSH
• Escherichia coli růst a vývoj   patogenita   MeSH
• infekce vyvolané Escherichia coli genetika   imunologie   mikrobiologie   veterinární   MeSH
• infekční komplikace v těhotenství genetika   imunologie   mikrobiologie   veterinární   MeSH
• interakce hostitele a patogenu genetika   imunologie   MeSH
• lidé MeSH
• messenger RNA genetika   imunologie   MeSH
• modely nemocí na zvířatech MeSH
• plodová voda imunologie   mikrobiologie   MeSH
• prasata MeSH
• předčasný porod prevence a kontrola   MeSH
• protein HMGB1 genetika   imunologie   MeSH
• receptor pro konečné produkty pokročilé glykace genetika   imunologie   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• těhotenství MeSH
• toll-like receptor 4 genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• protein HMGB1 MeSH
• receptor pro konečné produkty pokročilé glykace MeSH
• toll-like receptor 4 MeSH

Salmonella Typhimurium is a Gram-negative bacterium that causes enterocolitis in humans and pigs. Lipopolysaccharide (LPS) is a component of the outer leaflet of Gram-negative bacteria that provokes endotoxin shock. LPS can be synthesized completely or incompletely and creates S (smooth) or R (rough) chemotypes. Toll-like receptors (TLR) 2, 4, and 9 initiate an inflammatory reaction to combat bacterial infections. We associated/challenged one-week-old gnotobiotic piglets with wild-type S. Typhimurium with S chemotype or its isogenic ∆rfa mutants with R chemotype LPS. The wild-type S. Typhimurium induced TLR2 and TLR4 mRNA expression but not TLR9 mRNA expression in the ileum and colon of one-week-old gnotobiotic piglets 24 h after challenge. The TLR2 and TLR4 stimulatory effects of the S. Typhimurium ∆rfa mutants were related to the completeness of their LPS chain. The transcription of IL-12/23 p40, IFN-γ, and IL-6 in the intestine and the intestinal and plasmatic levels of IL-12/23 p40 and IL-6 but not IFN-γ were related to the activation of TLR2 and TLR4 signaling pathways. The avirulent S. Typhimurium ∆rfa mutants are potentially useful for modulation of the TLR2 and TLR4 signaling pathways to protect the immunocompromised gnotobiotic piglets against subsequent infection with the virulent S. Typhimurium.

• Klíčová slova
• Salmonella Typhimurium, chemotype, endotoxin, germ-free, gnotobiotic, lipopolysaccharide, piglet, toll-like receptor 4, ∆rfa mutant,
• MeSH
• gnotobiologické modely fyziologie   MeSH
• ileum metabolismus   mikrobiologie   MeSH
• kolon metabolismus   mikrobiologie   MeSH
• miniaturní prasata MeSH
• mutace fyziologie   MeSH
• prasata MeSH
• Salmonella typhimurium genetika   izolace a purifikace   MeSH
• salmonelóza genetika   metabolismus   patologie   MeSH
• toll-like receptor 4 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• toll-like receptor 4 MeSH