BACKGROUND: Only a few studies dealt with the occurrence of endospore-forming clostridia in the microbiota of infants without obvious health complications. METHODS: A methodology pipeline was developed to determine the occurrence of endospore formers in infant feces. Twenty-four fecal samples (FS) were collected from one infant in monthly intervals and were subjected to variable chemical and heat treatment in combination with culture-dependent analysis. Isolates were identified by MALDI-TOF mass spectrometry, 16S rRNA gene sequencing, and characterized with biochemical assays. RESULTS: More than 800 isolates were obtained, and a total of 21 Eubacteriales taxa belonging to the Clostridiaceae, Lachnospiraceae, Oscillospiraceae, and Peptostreptococcaceae families were detected. Clostridium perfringens, C. paraputrificum, C. tertium, C. symbiosum, C. butyricum, and C. ramosum were the most frequently identified species compared to the rarely detected Enterocloster bolteae, C. baratii, and C. jeddahense. Furthermore, the methodology enabled the subsequent cultivation of less frequently detectable gut taxa such as Flavonifractor plautii, Intestinibacter bartlettii, Eisenbergiella tayi, and Eubacterium tenue. The isolates showed phenotypic variability regarding enzymatic activity, fermentation profiles, and butyrate production. CONCLUSIONS: Taken together, this approach suggests and challenges a cultivation-based pipeline that allows the investigation of the population of endospore formers in complex ecosystems such as the human gastrointestinal tract.
- MeSH
- Clostridium * genetika MeSH
- feces mikrobiologie MeSH
- Firmicutes genetika MeSH
- kojenec MeSH
- lidé MeSH
- mikrobiota * MeSH
- RNA ribozomální 16S genetika MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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°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 (>107 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.
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.
- Publikační typ
- časopisecké články MeSH
Bifidobacteria, which commonly inhabit the primate gut, are beneficial contributors to host wellbeing. Anatomical differences and natural habitat allow an arrangement of primates into two main parvorders; New World monkeys (NWM) and Old World monkeys (OWM). The number of newly described bifidobacterial species is clearly elevated in NWM. This corresponds to our finding that bifidobacteria were the dominant group of cultivated gut anaerobes in NWM, while their numbers halved in OWM and were often replaced by Clostridiaceae with sarcina morphology. We examined an extended MALDI-TOF MS database as a potential identification tool for rapid screening of bifidobacterial distribution in captive primates. Bifidobacterial isolates of NWM were assigned mainly to species of primate origin, while OWM possessed typically multi-host bifidobacteria. Moreover, bifidobacterial counts reflected the feed specialization of captive primates decreasing from frugivore-insectivores, gummivore-insectivores, frugivore-folivores to frugivore-omnivores. Amplicon sequencing analysis supported this trend with regards to the inverse ratio of Actinobacteria and Firmicutes. In addition, a significantly higher diversity of the bacterial population in OWM was found. The evolution specialization of primates seems to be responsible for Bifidobacterium abundance and species occurrence. Balanced microbiota of captive primates could be supported by optimized prebiotic and probiotic stimulation based on the primate host.
- MeSH
- Bifidobacterium genetika izolace a purifikace MeSH
- feces mikrobiologie MeSH
- mikrobiota * MeSH
- primáti mikrobiologie MeSH
- probiotika MeSH
- RNA ribozomální 16S genetika MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The family Bifidobacteriaceae constitutes an important phylogenetic group that particularly includes bifidobacterial taxa demonstrating proven or debated positive effects on host health. The increasingly widespread application of probiotic cultures in the twenty-first century requires detailed classification to the level of particular strains. This study aimed to apply the glutamine synthetase class I (glnAI) gene region (717 bp representing approximately 50% of the entire gene sequence) using specific PCR primers for the classification, typing, and phylogenetic analysis of bifidobacteria and closely related scardovial genera. In the family Bifidobacteriaceae, this is the first report on the use of this gene for such purposes. To achieve high-value results, almost all valid Bifidobacteriaceae type strains (75) and 15 strains isolated from various environments were evaluated. The threshold value of the glnAI gene identity among Bifidobacterium species (86.9%) was comparable to that of other phylogenetic/identification markers proposed for bifidobacteria and was much lower compared to the 16S rRNA gene. Further statistical and phylogenetic analyses suggest that the glnAI gene can be applied as a novel genetic marker in the classification, genotyping, and phylogenetic analysis of isolates belonging to the family Bifidobacteriaceae.
- MeSH
- bakteriální geny MeSH
- Bifidobacterium klasifikace enzymologie MeSH
- DNA bakterií genetika MeSH
- DNA primery MeSH
- fylogeneze * MeSH
- genetické markery MeSH
- genotyp MeSH
- glutaminsynthetasa genetika MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- techniky typizace bakterií MeSH
- Publikační typ
- časopisecké články MeSH
A fructose-6-phosphate phosphoketolase-positive strain (GSD1FST) was isolated from a faecal sample of a 3 weeks old German Shepherd dog. The closest related taxa to isolate GSD1FST based on results from the EZBioCloud database were Bifidobacterium animalis subsp. animalis ATCC 25527T, Bifidobacterium animalis subsp. lactis DSM 10140T and Bifidobacterium anseris LMG 30189T, belonging to the Bifidobacterium pseudolongum phylogenetic group. The resulting 16S rRNA gene identities (compared length of 1454 nucleotides) towards these taxa were 97.30, 97.23 and 97.09 %, respectively. The pairwise similarities of strain GSD1FST using argS, atpA, fusA, hsp60, pyrG, rpsC, thrS and xfp gene fragments to all valid representatives of the B. pseudolongum phylogenetic group were in the concatenated range of 83.08-88.34 %. Phylogenomic analysis based on whole-genome methods such as average nucleotide identity revealed that bifidobacterial strain GSD1FST exhibits close phylogenetic relatedness (88.17 %) to Bifidobacetrium cuniculi LMG 10738T. Genotypic characteristics and phylogenetic analyses based on nine molecular markers, as well as genomic and comparative phenotypic analyses, clearly proved that the evaluated strain should be considered as representing a novel species within the B. pseudolongum phylogenetic group named as Bifidobacterium canis sp. nov. (GSD1FST=DSM 105923T=LMG 30345T=CCM 8806T).
- MeSH
- aldehydlyasy MeSH
- bakteriální geny MeSH
- Bifidobacterium klasifikace MeSH
- DNA bakterií genetika MeSH
- feces mikrobiologie MeSH
- fylogeneze * MeSH
- psi mikrobiologie MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- techniky typizace bakterií MeSH
- zastoupení bazí MeSH
- zvířata MeSH
- Check Tag
- psi mikrobiologie MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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.
- MeSH
- Escherichia coli imunologie MeSH
- gnotobiologické modely imunologie MeSH
- Lactobacillus acidophilus imunologie MeSH
- prasata * imunologie mikrobiologie MeSH
- probiotika * MeSH
- protein HMGB1 imunologie MeSH
- Salmonella typhimurium imunologie MeSH
- signální transdukce imunologie MeSH
- střeva imunologie mikrobiologie MeSH
- toll-like receptor 4 imunologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články 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.
- Publikační typ
- časopisecké články MeSH
