Salmonella enterica subsp. enterica serovar Typhimurium ( S . Typhimurium) is a ubiquitous Salmonella serovar that causes enterocolitis in humans, livestock, poultry, and some pets. Gnotobiotic animals are suitable for studying the development of initial colonization in the gastrointestinal tract, host-microbiota cross-talk, and interactions among microbiota strains. To clarify the impact of pre-colonization of the germ-free piglets with defined pig microbiota (DPM) on the subsequent infection with S . Typhimurium LT2 (LT2), we focused on the acute host immune response. Colonization with DPM did not cause signs of enterocolitis (sleepiness, anorexia, fever, and diarrhea), histological changes in the intestine, the density of acid mucin-producing cells, expression of villin, claudin-1, occludin, TLR4, MD-2, CD14, LBP, MyD88, and TRIF, induction of inflammatory cytokines IL-1 β , IL-6, IL-8, IL-10, IL-12/23p40, TNF- α , IFN- γ , and HMGB1. LT2 translocated into the mesenteric lymph nodes, liver, spleen, and blood of the LT2 group, as well as damaged the intestinal structure, and upregulated intestinal inflammatory cytokine levels. The pre-colonization with DPM ameliorated the LT2-induced signs of enterocolitis, intestinal damage, and inflammatory changes. Changes in intestinal levels of soluble TLR4 and TLR2 indicate the possible inclusion of soluble TLRs in the regulation of the inflammatory process. The controlled initial colonization of the newborn gastrointestinal tract with a defined microbial consortium can increase newborn resistance to enteric pathogens and promote thriving of young animals.

Probiotics and prebiotics are increasingly added to infant formulas and supplements, potentially shaping early gut colonization. Their effects depend heavily on the existing microbiota, underscoring the need for individual, longitudinal, and polyphasic approaches.This study comprehensively analyzed microbial changes in an infant's stool over the first three years of life, capturing dietary transitions and interventions. The vaginally born, partially breastfed male infant received continuously formula with Limosilactobacillus reuteri DSM 17938 and 2´FL, and a two-week probiotic drop intervention with Bifidobacterium breve BR03 and Lactiplantibacillus plantarum LP01 to ease colic discomfort associated with clostridial dominance. Eighty-six fecal samples were analyzed using amplicon sequencing, complemented by qPCR and extensive culture-based throughput quantification of selected taxa identified by MALDI-TOF MS (>2000 isolates).Clostridia, enterobacteria and enterococci dominated the first month alongside supplemented Li. reuteri. Probiotic drops reduced clostridia and increased bifidobacteria, when B. breve persisted, while Lp. plantarum did not. During the liquid food period, supplemented B. breve and naturally occurring Lacticaseibacillus rhamnosus increased together, while Escherichia coli decreased in favor of Enterobacter cloacae. Later shifts reflected dietary transitions, including solid-food introduction and breastfeeding cessation.This longitudinal polyphasic study lies in its high-resolution design, which captures transient microecological successions and strain persistence that are typically masked in larger, less-frequently sampled cohorts. By combining molecular methods with extensive cultivation, we successfully bridge the gap between community-level sequencing and viable, strain-level absolute quantification. Ultimately, this work provides a rare micro-ecological view into real-time microbiota resilience and contributes an extensive repository of physiologically contextualized infant-derived strains.

Listeria monocytogenes (LM) is one of the most emerging pathogens responsible for the serious foodborne disease listeriosis. The risk of disease outbreaks can be reduced by suppressing the adherence of LM to the intestinal epithelial cells. This effect can be achieved by prebiotic supplementation. The aim of this work was to determine the effect of prebiotics beta-(1,3)-D-glucan, inulin, fructooligosaccharides, galactooligosaccharides, lactulose, raffinose, stachyose, human milk oligosaccharides (HMOs), and 2’-fucosyllactose on the ability of LM to adhere to the human adenocarcinoma Caco-2 cell line. Despite strain-specific variability, a statistically significant reduction in LM adhesion to intestinal epithelial cells was observed in the presence of beta-(1,3)-D-glucan (~60% reduction), inulin (~46%), and HMOs (~44%). In contrast, the remaining tested prebiotics did not show a significant impact on LM adhesion. These findings highlight the potential of specific prebiotics, especially beta-glucans, to limit LM adherence, suggesting a protective effect for the host.

Listeria monocytognes is an emerging pathogen responsible for the serious foodborne disease, listeriosis. The commensal gut microbiota is the first line of defense against pathogen internalization. The gut microbiome can be modified by prebiotic substrates, which are frequently added to food products and dietary supplements. Prebiotics should selectively support the growth of beneficial microbes and thus improve host health. Nevertheless, little is known about their effect on the growth of L. monocytogenes. The aim of this study was to evaluate the growth ability of four L. monocytogenes strains, representing the most common serotypes, on prebiotic oligosaccharides (beta-(1,3)-D-glucan, inulin, fructooligosaccharides, galactooligosaccharides, lactulose, raffinose, stachyose and 2′-fucosyllactose and a mixture of human milk oligosaccharides) as a sole carbon source. The results showed that only beta-(1,3)-D-glucan was metabolized by L. monocytogenes. These cell culture data suggest that beta-(1,3)-D-glucan may not be selectively utilized by healthy commensal bacteria, and its role in intestinal pathogen growth warrants further exploration in vivo.

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.

Lactulose is commonly used in pharmacy for constipation and hepatic encephalopathy treatment. The prebiotic effect of lactulose is also often mentioned. However, its cryoprotective effect in combination with lecithin on the main representatives of probiotics has not been tested yet. The 12 taxa of bifidobacteria and Lactobacillaceae members were used for the purpose. These were mixed in a ratio of 1:1 with lactulose + lecithin (finally 5.0% and 1.25%, respectively; LL). The 25% glycerol (G+) solution and cultures themselves were applied as positive and negative controls, respectively. Bacterial suspensions were stored at a mild freezing temperature (-20 degrees C) until the end of the experiment (210th day). The LL solution had a comparable (insignificant difference at the P-value = 0.05) cryoprotective effect as the positive control in five of six bifidobacteria and in three of six representatives of Lactobacillaceae. The better cryoprotective effect was revealed in other Lactobacillaceae. At the end of the experiment, the generally accepted therapeutic minimum (>10(7) Colony Forming Units/mL) was determined in LL solution in five bifidobacteria and four Lactobacillaceae strains. The presented results improve knowledge about long-term mild cryopreservation of the most commonly used probiotics and could contribute to developing new forms of (nutri)synbiotics.

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.

Sarcina spp. has been isolated from the gastrointestinal tracts of diverse mammalian hosts. Their presence is often associated with host health complications, as is evident from many previously published medical case reports. However, only a handful of studies have made proper identification. Most other identifications were solely based on typical Sarcina-like morphology without genotyping. Therefore, the aim of this work was culture detection and the taxonomic classification of Sarcina isolates originating from different mammalian hosts. Sarcina-like colonies were isolated and collected during cultivation analyses of animal fecal samples (n = 197) from primates, dogs, calves of domestic cattle, elephants, and rhinoceroses. The study was carried out on apparently healthy animals kept in zoos or by breeders in the Czech Republic and Slovakia. Selected isolates were identified and compared using 16S rRNA gene sequencing and multi-locus sequence analysis (MLSA; Iles, pheT, pyrG, rplB, rplC, and rpsC). The results indicate the taxonomic variability of Sarcina isolates. S. ventriculi appears to be a common gut microorganism in various captive primates. In contrast, a random occurrence was also recorded in dogs. However, dog isolate N13/4e could represent the next potential novel Sarcina taxonomic unit. Also, a potentially novel Sarcina species was found in elephants, with occurrences in all tested hosts. S. maxima isolates were detected rarely, only in rhinoceroses. Although Sarcina bacteria are often linked to lethal diseases, our results indicate that Sarcina spp. appear to be a common member of the gut microbiota and seem to be an opportunistic pathogen. Further characterization and pathogenic analyses are required.

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.

Microwave (MW) irradiation is a non-destructive method that can be applied as an alternative method to inhibit the growth of microorganisms. The present study evaluated the effect of MW irradiation on the occurrence of moulds in nuts and almonds. Samples of unshelled natural almonds, pistachios, and in-shell peanuts were treated with different doses of MW irradiation (2400–4000 W). The effect of MW irradiation on mould counts was evaluated by cultivating immediately after irradiation and after 3 and 6 months of storage. The most represented genera in all analysed samples were Aspergillus (68%), Penicillium (21%), and a small amount of Cladosporium (3%). Mould numbers significantly decreased after MW treatment. The treatments with MW irradiations at 3000 and 4000 W significantly reduced the mould colony counts, and their effect persisted during storage; irradiation at 2400 W was partially effective. The strongest effect of MW irradiation was observed in in-shell peanuts. MW irradiation seems to be a promising method for maintaining the microbiological quality of nuts.

Five Bifidobacterium strains, VB23T, VB24T, VB25T, VB26T and VB31T, were isolated from chimpanzee (Pan troglodytes), cotton-top tamarin (Saguinus oedipus), Goeldi's marmoset (Callimico goeldii), moustached tamarin (Saguinus mystax) and patas monkey (Erythrocebus patas), respectively, which were kept in two Czech zoos. These strains were isolated from faecal samples and were Gram-positive, non-motile, non-sporulating, anaerobic and fructose-6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA revealed close relatedness between VB23T and Bifidobacterium angulatum LMG 11039T (96.0 %), VB24T and Bifidobacterium pullorum subsp. pullorum DSM 20433T (96.1 %), VB25T and Bifidobacterium goeldii LMG 30939T (96.5 %), VB26T and Bifidobacterium imperatoris LMG 30297T (98.1 %), and VB31T and B. angulatum LMG 11039T (99.40 %). Internal transcribed spacer profiling revealed that VB23T, VB24T, VB25T, VB26T and VB31T had highest similarity to Bifidobacterium breve LMG 13208T (77.2 %), Bifidobacterium longum subsp. infantis ATCC 15697T (85.8 %), Bifidobacterium biavatii DSM 23969T (76.9 %), B. breve LMG 13208T (81.2 %) and B. angulatum LMG 11039T (88.2 %), respectively. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analyses with their closest neighbours supported the independent phylogenetic positions of the strains with values between 86.3 and 94.3 % for ANI and 25.8 and 54.9 % for dDDH. These genomic and phylogenetic analyses suggested that the evaluated strains were novel Bifidobacterium species named Bifidobacterium erythrocebi sp. nov. (VB31T=DSM 109960T=CCUG 73843T), Bifidobacterium moraviense sp. nov. (VB25T=DSM 109958T=CCUG 73842T), Bifidobacterium oedipodis sp. nov. (VB24T=DSM 109957T=CCUG 73932T), Bifidobacterium olomucense sp. nov. (VB26T=DSM 109959T=CCUG 73845T) and Bifidobacterium panos sp. nov. (VB23T=DSM 109963T=CCUG 73840T).

Enteral nutrition (EN) formulas of polymeric type ordinarily have similar content of intact macronutrients but may vary in prebiotic saccharides and micronutrients. These components can play an important role in the intestinal microbiota modulation. The aim of this study was to investigate microbial changes of faecal samples after their in vitro anaerobic cultivation in four polymeric EN formulas using plate technique method, metabolite analysis, and microbiota profiling using 16S rRNA sequencing. Detected cultivable commensal groups (bifidobacteria, lactobacilli, Escherichia coli) in faecal samples of donors were able to grow in EN formulas. However, their counts varied depending on the individual donor and the type of EN formula. Similar trend was found in detected metabolites such as acetate, lactate, and butyrate. Also, taxonomic composition and diversity of original and cultivated faecal microbiota of one individual on different EN formula indicate a possible effect of the prebiotics and micronutrients to modulate gut microbiota.

Pectinatella magnificais an invasive freshwater bryozoan that has expanded in many localities worldwide, including fishing areas. It contains microbial communities, predominantly consisting ofAeromonasbacteria that are frequently associated with fish infections. The objective of this study was to investigate the potential pathogenicity ofAeromonasspp. associated withP. magnificaand evaluate the health risks for fish.Aeromonasstrains were isolated fromP. magnifica(101 strains) and from surrounding water (29 strains) in the South Bohemian region and investigated for the presence of 14 virulence-associated genes using PCR. We demonstrated high prevalence of phospholipase GCAT, polar flagellin, enolase, DNAse, aerolysin/cytotoxic enterotoxin, serine protease and heat-stable cytotonic enterotoxin-coding genes. Further, all twelve isolates that were analysed for cytotoxicity against intestinal epithelial cells were found to be cytotoxic. Six of the isolates were also tested as co-cultures composed of pairs. Enhanced cytotoxicity was observed when the pair was composed of strains from different species. In conclusion,P. magnificais colonized byAeromonasstrains that have a relatively high prevalence of virulence-associated genes and the ability to provoke disease. Results also suggest a possibly increased risk arising from mixed infections.

Non-typhoidal Salmonella serovars are worldwide spread foodborne pathogens that cause diarrhea in humans and animals. Colonization of gnotobiotic piglet intestine with porcine indigenous mucinolytic Bifidobacterium boum RP36 strain and non-mucinolytic strain RP37 and their interference with Salmonella Typhimurium infection were compared. Bacterial interferences and impact on the host were evaluated by clinical signs of salmonellosis, bacterial translocation, goblet cell count, mRNA expression of mucin 2, villin, claudin-1, claudin-2, and occludin in the ileum and colon, and plasmatic levels of inflammatory cytokines IL-8, TNF-α, and IL-10. Both bifidobacterial strains colonized the intestine comparably. Neither RP36 nor RP37 B. boum strains effectively suppressed signs of salmonellosis. Both B. boum strains suppressed the growth of S. Typhimurium in the ileum and colon. The mucinolytic RP36 strain increased the translocation of S. Typhimurium into the blood, liver, and spleen.

Dietary plant glucosides are phytochemicals whose bioactivity and bioavailability can be modified by glucoside hydrolase activity of intestinal microbiota through the release of acylglycones. Bifidobacteria are gut commensals whose genomic potential indicates host-adaption as they possess a diverse set of glycosyl hydrolases giving access to a variety of dietary glycans. We hypothesized bifidobacteria with β-glucosidase activity could use plant glucosides as fermentation substrate and tested 115 strains assigned to eight different species and from different hosts for their potential to express β-glucosidases and ability to grow in the presence of esculin, amygdalin, and arbutin. Concurrently, the antibacterial activity of arbutin and its acylglycone hydroquinone was investigated. Beta-glucosidase activity of bifidobacteria was species specific and most prevalent in species occurring in human adults and animal hosts. Utilization and fermentation profiles of plant glucosides differed between strains and might provide a competitive benefit enabling the intestinal use of dietary plant glucosides as energy sources. Bifidobacterial β-glucosidase activity can increase the bioactivity of plant glucosides through the release of acylglycone.

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.

Current studies indicate a link between the intake of exclusive enteral nutrition (EEN) and the induction of complex changes in the intestinal microbiota, as well as the clinical improvement of Crohn's disease (CD). The first aim of this study was to test the ability of various commensal bacterial strains (n = 19) such as bifidobacteria, lactobacilli, and Escherichia coli to grow on three different polymeric EN in vitro. Tested EN formulas were found to be suitable growth media for tested commensals. Furthermore, the counts of these bacteria and total counts of anaerobic bacteria in the fecal samples of children with CD (n = 15) before and after 6 weeks of EEN diet administration were determined using cultivation on selective media. The counts of cultivable commensal bacteria in the fecal samples of CD children were not significantly affected by EEN. However, tested bacteria showed some individual shifts in counts before and after EEN therapy. Moreover, cultured bifidobacteria were found to be in reduced counts in CD children. Therefore, the application of bifidogenic prebiotic compounds to EN for CD patients might be considered.

Bifidobacteria are widely known for their probiotic potential; however, little is known regarding the ecological significance and potential probiotic effects of the phylogenetically related 'scardovial' genera (Aeriscardovia, Alloscardovia, Bombiscardovia, Galliscardovia, Neoscardovia, Parascardovia, Pseudoscardovia and Scardovia) and Gardnerella classified with bifidobacteria within the Bifidobacteriaceae family. Accurate classification and genotyping of bacteria using certain housekeeping genes is possible, whilst current phylogenomic analyses allow for extremely precise classification. Studies of applicable genetic markers may provide results comparable to those obtained from phylogenomic analyses of the family Bifidobacteriaceae. Segments of the glyS (624 nucleotides), pheS (555 nucleotides), rpsA (630 nucleotides), and rpsB (432 nucleotides) genes and their concatenated sequence were explored. The mean glyS, pheS, rpsB and rpsA gene sequence similarities calculated for Bifidobacterium taxa were 84.8, 85.2, 90.2 and 86.8%, respectively. Interestingly, the average value of the Average Nucleotide Identity among 67 type strains of the family Bifidobacteriaceae (84.70%) calculated based on values published recently was in agreement with the average pairwise similarity (84.6%) among 75 type strains of Bifidobacteriaceae family computed in this study using the concatenated sequences of four gene fragments. Similar to phylogenomic analyses, several gene sequence and phylogenetic analyses revealed that concatenated gene regions allow for classification of Bifidobacteriaceae strains into particular phylogenetic clusters and groups. Phylogeny reconstructed from the concatenated sequences assisted in defining two novel phylogenetic groups, the Bifidobacterium psychraerophilum group consisting of B. psychraerophilum, Bifidobacterium crudilactis and Bifidobacterium aquikefiri species and the Bifidobacterium bombi group consisting of B. bombi, Bifidobacterium bohemicum and Bifidobacterium commune.

Complex polysaccharide polymers of natural origin are widely used as natural food thickeners. They are useful for their technological properties, and at the same time they are biodegradable and safe for consumers. In addition, natural food thickeners, such as natural gums (NGs) and starch, may also represent suitable potentially prebiotic substrates for probiotic genera such as the genus Bifidobacterium. Therefore, 204 bifidobacterial strains of 60 species and subspecies were tested for their ability to utilize 6 NGs (locust bean, guar, tragacanth, arabic, xanthan, and karaya gums) and starch.