Commensal Bacteria Impact on Intestinal Toll-like Receptor Signaling in Salmonella-Challenged Gnotobiotic Piglets.

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.

Species and Strain Variability among Sarcina Isolates from Diverse Mammalian Hosts.

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.

Defined Pig Microbiota with a Potential Protective Effect against Infection with Salmonella Typhimurium.

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.

Lactulose in combination with soybean lecithin has a cryoprotective effect on probiotic taxa of bifidobacteria and Lactobacillaceae.

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.

The Effect of Microwave Irradiation on the Representation and Growth of Moulds in Nuts and Almonds.

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 novel bifidobacterial species isolated from faeces of primates in two Czech zoos: Bifidobacterium erythrocebi sp. nov., Bifidobacterium moraviense sp. nov., Bifidobacterium oedipodis sp. nov., Bifidobacterium olomucense sp. nov. and Bifidobacterium panos sp. nov.

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).

Colonization of Germ-Free Piglets with Mucinolytic and Non-Mucinolytic Bifidobacterium boum Strains Isolated from the Intestine of Wild Boar and Their Interference with Salmonella Typhimurium.

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.

Pathogenic profile and cytotoxic activity of Aeromonas spp. isolated from Pectinatella magnifica and surrounding water in the South Bohemian aquaculture region.

Pectinatella magnifica is an invasive freshwater bryozoan that has expanded in many localities worldwide, including fishing areas. It contains microbial communities, predominantly consisting of Aeromonas bacteria that are frequently associated with fish infections. The objective of this study was to investigate the potential pathogenicity of Aeromonas spp. associated with P. magnifica and evaluate the health risks for fish. Aeromonas strains were isolated from P. 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. magnifica is colonized by Aeromonas strains 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.

Sight-threatening Complication of Cicatricial Ectropion in a Patient with Lamellar Ichthyosis - Case Report.

We report a case of lamellar ichthyosis and sight-threatening complications of cicatricial ectropion in an adult male patient which was surgically managed with tectonic penetrating keratoplasty. We present a case of autosomal-recessive lamellar ichthyosis in a 47-year-old man who was referred to our outpatient eye clinic for treatment of primary keratouveitis of the right eye with keratolysis and exudation in the anterior chamber. A diagnosis of cicatricial ectropion with serious lagophthalmos was established on examination. The patient underwent tectonic penetrating keratoplasty, cataract extraction, and intra-ocular lens placement with no perioperative complications. The patient was subsequently treated with oral fluconazole 200 mg once daily for 12 days due to a positive fungal culture for Candida albicans and systemic oral acyclovir 250 mg 3 times per day for 12 days as prophylaxis for a labial herpetic infection. Post-operative complications included corneal rejection and nonhealing neurotropic epithelial defect of the graft. Long-term treatment with topical cyclosporine (Ikervis®) and dexamethasone led to resolution of the corneal rejection. Lubrication with artificial tears containing hyaluronic acid, perfluorohexyl octane (Evotears®), and vitamin A ointment led to symptomatic relief of dry eye disease. The patient was referred to a dermatologist and was started on systemic retinoid acitretin at a dose of 0.5 mg/kg per day. Ten months after surgery, the patient's visual acuity was 0.1 based on the Snellen chart and the corneal graft was stable. Infection in the cornea can rapidly progress to corneal melting in patients with severe cicatricial ectropion. A good patient outcome depends on the interdisciplinary approach to patient management by the ophthalmologist, dermatologist, and plastic surgeon.

Bifidobacterium ß-Glucosidase Activity and Fermentation of Dietary Plant Glucosides Is Species and Strain Specific.

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 ß-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 ß-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 ß-glucosidase activity can increase the bioactivity of plant glucosides through the release of acylglycone.

An association of neovascular age-related macular degeneration with polymorphisms of CFH, ARMS2, HTRA1 and C3 genes in Czech population.

PURPOSE: We investigated associations between neovascular age-related macular degeneration (AMD) and rs10490924 polymorphism of ARMS2 gene (age-related maculopathy susceptibility 2), rs1061170 polymorphism of gene for complement factor H (CFH), rs2230199 polymorphism of gene for complement component C3 and rs11200638 polymorphism of gene for serine protease high-temperature requirement A1 (HTRA1) in the Czech population. METHODS: We analysed samples of DNA from 307 patients diagnosed with neovascular form of late AMD (average age: 73.7 ± 7.7 years) and 191 control subjects, recruited from patients awaiting cataract surgery (average age, 73.6 ± 8.7 years). RESULTS: HTRA1, CFH and ARMS2 genes polymorphisms were found to be related to neovascular AMD in the Czech population. All analysed polymorphisms were statistically significantly associated with neovascular AMD, with stronger associations in females than in males. In whole group, CC genotype of CFH gene polymorphism, TT genotype of ARMS2 gene polymorphism and AA genotype of HTRA1 gene polymorphism showed the greatest risk for neovascular AMD with odds ratios equal to 8.43, 10.07, 9.83, respectively (p < 0.0001). Only CG polymorphism of C3 gene showed statistically significant risk for neovascular AMD. In addition, we observed an association between waist circumference and neovascular AMD in both sexes, which further suggests the significance of excessive abdominal fat as a risk factor of AMD. We found a statistically significant association between polymorphisms in HTRA1, CFH and ARMS2 genes and neovascular AMS in the Czech population. The association was stronger in females than in males. CONCLUSION: We demonstrated a relationship between neovascular AMD and genes for HTRA1, CFH, ARMS2 and C3 in Czech population. To our knowledge, the relationship between these polymorphisms and neovascular AMD in Czech population has never been investigated before.

Uveal melanoma - testing of abnormalities of chromosome 3 and 8 in the Czech Republic.

AIM: The aim of this cytogenetic study is to confirm the significance of chromosome 3 loss (monosomy 3) and of the gain of chromosome 8 as prognostic markers in histopathological samples of enucleated eyes with uveal melanoma in the Czech population. METHODS: This is a retrospective study of 52 enucleated eyes. Chromosome 3 and 8 status were tested by CISH, and in a few samples FISH was used. The correlation between monosomy 3 and gain of chromosome 8 and clinical features (histopathological type, size of the tumour) were evaluated. A follow up for the detection of metastases was conducted in all patients. The statistical significance of chromosomal abnormalities as a prognostic factor for the development of metastases was calculated. RESULTS: There were 52 patients, 27 men (51.9%) and 25 women (48.1%) enrolled in our study group. The mean age was 63 ± 14 years. Loss of the one copy of chromosome 3 (monosomy 3) was detected in 26 (50.0%) patients, monosomy 8 was present in 34.6% of patients with monosomy 3. After 5 years there were no metastases in 82% of patients without monosomy 3 as opposed to 40% of patients with monosomy 3. We confirmed a statistically significant association between progression free survival (PFS) and the presence of monosomy 3 (P=0.017). The association between PFS and gain of chromosome 8 was significant as well (0.010). CONCLUSIONS: Our data showed the association of progression-free survival with the presence of monosomy 3 in uveal melanomas. We provided a good prognostic value of monosomy 3 in uveal melanoma.

Glutamine synthetase type I (glnAI) represents a rewarding molecular marker in the classification of bifidobacteria and related genera.

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.

High Mobility Group Box 1 and TLR4 Signaling Pathway in Gnotobiotic Piglets Colonized/Infected with L. amylovorus, L. mucosae, E. coli Nissle 1917 and S. Typhimurium.

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.

Colonization of Germ-Free Piglets with Commensal Lactobacillus amylovorus, Lactobacillus mucosae, and Probiotic E. coli Nissle 1917 and Their Interference with Salmonella Typhimurium.

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.

Genetic marker-based multi-locus sequence analysis for classification, genotyping, and phylogenetics of the family Bifidobacteriaceae as an alternative approach to phylogenomics.

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.

Enteral Nutrition as a Growth Medium for Cultivable Commensal Bacteria and Its Effect on Their Quantity in the Stool of Children with Crohn's Disease.

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.

The threonine-tRNA ligase gene region is applicable in classification, typing, and phylogenetic analysis of bifidobacteria.

In the modern era, molecular genetic techniques are crucial in ecological studies, as well as in the classification, typing, and phylogenetic analysis of prokaryotes. These techniques are mainly aimed at whole genome comparisons and PCR-derived experiments, including amplifying the 16S rRNA and other various housekeeping genes used in taxonomy, as well as MLST (multilocus sequence typing) and MLSA (multilocus sequence analysis) of different taxonomic bacterial groups. The gene encoding threonine-tRNA ligase (thrS) is a gene potentially applicable as an identification and phylogenetic marker in bacteria. It is widely distributed in bacterial genomes and is subject to evolutionary selection pressure due to its important function in protein synthesis. In this study, specific primers were used to amplify a thrS gene fragment (~740 bp) in 36 type and 30 wild strains classified under family Bifidobacteriaceae. The full-length gene has not yet been considered as a possible identification, classification, and phylogenetic marker in bifidobacteria. The thrS sequences revealed higher sequence variability (82.7% of pairwise identities) among members of the family than that shown by 16S rRNA gene sequences (96.0%). Although discrepancies were found between the thrS-derived and previously reported whole genome phylogenetic analyses, the main phylogenetic groups of bifidobacteria were properly assigned. Most wild strains of bifidobacteria were better differentiated based on their thrS sequences than on their 16S rRNA gene identities. Phylogenetic confidence of the evaluated gene with respect to other alternative genetic markers widely used in taxonomy of bifidobacteria (fusA, GroELhsp60, pyrG, and rplB genes) was confirmed using the localized incongruence difference - Templeton analysis.

Cultivable bacteria from Pectinatella magnifica and the surrounding water in South Bohemia indicate potential new Gammaproteobacterial, Betaproteobacterial and Firmicutes taxa.

Pectinatella magnifica is a freshwater bryozoan, which has become a subject of scientific interest because of its invasive expansion worldwide. To obtain a comprehensive overview of its influence on environments, information on associated bacteria is needed. In this study, cultivable bacteria associated with P. magnifica were investigated. In total, 253 isolates were selected for preliminary identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and clustered based on repetitive extragenic palindromic-PCR profiles. Among these, 169 strains were selected and identified using 16S rRNA gene comparative analyses. The sequences were grouped into 76 phylotypes and affiliated with 67 species. The majority of isolated bacteria belonged to Gammaproteobacteria, followed by Betaproteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. Most strains within the Betaproteobacteria were isolated exclusively from bryozoan colonies. Aeromonas was the genus predominantly isolated from both P. magnifica and the water samples. Based on 16S rDNA similarity values, 15 putative new species belonging to the genera Aeromonas, Aquitalea, Clostridium, Herbaspirillum, Chromobacterium, Chryseobacterium, Morganella, Paludibacterium, Pectobacterium, Rahnella, Rhodoferax and Serratia, and putative new genera belonging to families Clostridiaceae and Sporomusaceae were revealed. The majority of the detected bacteria were species widely distributed in the environments; nevertheless, a possible symbiotic association of two new putative species with P. magnifica cannot be excluded.

Gene encoding the CTP synthetase as an appropriate molecular tool for identification and phylogenetic study of the family Bifidobacteriaceae.

An alternative molecular marker with respect to the 16S rRNA gene demonstrating better identification and phylogenetic parameters has not been designed for the whole Bifidobacteriaceae family, which includes the genus Bifidobacterium and scardovial genera. Therefore, the aim of the study was to find such a gene in available genomic sequences, suggest appropriate means and conditions for asmplification and sequencing of the desired region of the selected gene in various strains of the bacterial family and verify the importance in classification and phylogeny. Specific primers flanking the variable region (~800 pb) within the pyrG gene encoding the CTP synthetase were designed by means of gene sequences retrieved from the genomes of strains belonging to the family Bifidobacteriaceae. The functionality and specificity of the primers were subsequently tested on the wild (7) and type strains of bifidobacteria (36) and scardovia (7). Comparative and phylogenetic studies based on obtained sequences revealed actual significance in classification and phylogeny of the Bifidobacteriaceae family. Gene statistics (percentages of mean sequence similarities and identical sites, mean number of nucleotide differences, P- and K-distances) and phylogenetic analyses (congruence between tree topologies, percentages of bootstrap values >50 and 70%) indicate that the pyrG gene represents an alternative identification and phylogenetic marker exhibiting higher discriminatory power among strains, (sub)species, and genera than the 16S rRNA gene. Sequences of the particular gene fragment, simply achieved through specific primers, enable more precisely to classify and evaluate phylogeny of the family Bifidobacteriaceae including, with some exceptions, health-promoting probiotic bacteria.