Any Future for Faecal Microbiota Transplantation as a Novel Strategy for Gut Microbiota Modulation in Human and Veterinary Medicine?

Alterations in the composition of the intestinal microbiome, also known as dysbiosis, are the result of many factors such as diet, antibiotics, stress, diseases, etc. There are currently several ways to modulate intestinal microbiome such as dietary modulation, the use of antimicrobials, prebiotics, probiotics, postbiotics, and synbiotics. Faecal microbiota transplantation (FMT) represents one new method of gut microbiota modulation in humans with the aim of reconstructing the intestinal microbiome of the recipient. In human medicine, this form of bacteriotherapy is successfully used in cases of recurrent Clostridium difficile infection (CDI). FMT has been known in large animal medicine for several years. In small animal medicine, the use of FMT is not part of normal practice.

Toxicological evaluation of fluorescent 11-mercaptoundecanoic gold nanoclusters as promising label-free bioimaging probes in different cancer cell lines.

Due to advancement in nanomaterials and increasing use of functionalized gold nanoclusters (AuNCs) in different biomedical applications, better understanding of their potential cytotoxicity is necessary. Interactions of ultra-small fluorescent AuNCs with mammalian cells remains up to this day poorly understood, therefore, cytotoxic evaluation of thoroughly characterized ca. 2.5 nm spherical water-soluble 11-mercaptoundecanoic acid coated AuNCs (AuNC@M) with diverse fluorescent properties in variety of mammalian cancer cell lines was performed. Cell viability was assessed by traditional MTT assay and xCELLigence real time cell analyzer. Cell apoptosis was evaluated via an Annexin V-FITC/propidium iodide (PI) assay. Confocal fluorescence imaging confirmed that tested AuNC@M entered live cells and were homogeneously distributed in their cytoplasm. The results suggested that the cytotoxicity of tested nanoclusters was very low, or near the control level at concentrations 0.1 and 0.5 mg/mL in the cell lines after 24 h exposition. The purity of tested AuNC@M had no relevant effect on cell viability and no differences were observed after 24 h in our study. The low toxicity toward cancer cells further strengthens our view that AuNC@M are promising label-free fluorescent probes for bio-labelling and bio-imaging, or they can even serve as platforms for antitumor drug delivery systems.

Innovative Animal Model of DSS-Induced Ulcerative Colitis in Pseudo Germ-Free Mice.

The aim of this study was to investigate the use of a standardized animal model subjected to antibiotic treatment, and the effects of this treatment on the course of dextran sodium sulphate (DSS)-induced colitis in mice. By decontamination with selective antibiotics and observation of pathogenesis of ulcerative colitis (UC) induced chemically by exposure of mice to various concentrations of DSS, we obtained an optimum animal PGF model of acute UC manifested by mucin depletion, epithelial degeneration and necrosis, leading to the disappearance of epithelial cells, infiltration of lamina propria and submucosa with neutrophils, cryptitis, and accompanied by decreased viability of intestinal microbiota, loss of body weight, dehydration, moderate rectal bleeding, and a decrease in the selected markers of cellular proliferation and apoptosis. The obtained PGF model did not exhibit changes that could contribute to inflammation by means of alteration of the metabolic status and the induced dysbiosis did not serve as a bearer of pathogenic microorganisms participating in development of ulcerative colitis. The inflammatory process was induced particularly by exposure to DSS and its toxic action on compactness and integrity of mucosal barrier in the large intestine. This offers new possibilities of the use of this animal model in studies with or without participation of pathogenic microbiota in IBD pathogenesis.

Dietary Lactobacillus plantarum LS/07 and inulin in the management of chronic disease risk factors.

The aim of this study was to investigate the possibilities of modification of chronic disease risk factors with probiotic strain Lactobacillus plantarum LS/07 and prebiotic inulin in rats with western high fat diet. The Sprague-Dawley rats were divided into four groups: control group (CG group), group with high fat diet (HFD group), group receiving high fat diet in combination with Lactobacillus plantarum LS/07 (HFD+PRO group), and group receiving high fat diet in combination with oligofructose enriched inulin (HFD+PRE group). The activity of ß-glucuronidase, lipid parameters, bile acids, oxLDL, short chain fatty acids, and counts of coliforms and lactobacilli were determined. High fat diet as a key risk factor of chronic diseases had adverse effect on expression of metabolic and biochemical parameters. Dietary intake of Lactobacillus plantarum LS/07 (HFD+PRO group) and inulin (HFD+PRE group) suppressed weight gain of rats. In HFD+PRO group, the level of total cholesterol (P<0.001), LDL-CH (P<0.05), oxLDL (P<0.001), total bile acids (P<0.001) were statistically significantly decreased, while the production of short chain fatty acids was enhanced. Changes in the selected parameters exhibited a similar tendency also in the HFD+PRE group. Activity of ß-glucuronidase was statistically significantly decreased (P<0.001) in the HFD+PRE group. Lactobacillus plantarum LS/07 and inulin caused a statistically significant increase in the count of lactobacilli (P<0.001) and a decrease in the number of coliforms (P<0.001). These results indicate Lactobacillus plantarum LS/07 and inulin could be used in diet for human and animals as an important nutritional supplement or in medicinal products.

Cytokine production in vitro and in rat model of colitis in response to Lactobacillus plantarum LS/07.

Over the past decade, it has become clear that specific probiotic lactobacilli are valuable in the prevention and treatment of infectious and inflammatory diseases of gastrointestinal tract but their successful application would benefit greatly from a better understanding of the mechanisms of individual strains. Hence, each probiotic strain should be characterized for their immune activity before being proposed for clinical applications. The aim of the study was to characterize the immunomodulatory activity of the strain Lactobacillus (L.) plantarum LS/07 in vitro using functional gut model and to study its anti-inflammatory potential in dextran sulphate sodium (DSS)-induced colitis in rats. We showed that L. plantarum LS/07 induced production of IL-10 in macrophages derived from blood monocytes as well as monocyte/macrophages cell line stimulated indirectly via enterocytes in vitro. In rat model of colitis, L. plantarum LS/07 attenuated the DSS-induced signs of inflammatory process in colon such as weight loss, diarrhoea, infiltration of inflammatory cells associated with decreased colon weight/length ratio, inhibited gut mucosa destruction and depletion of goblet cells. Moreover, the strain increased the concentration of anti-inflammatory cytokine IL-10 in mucosal tissue. In conclusion, the protective effects of L. plantarum LS/07 in the DSS-induced colitis model seem to be related to the stimulation of IL-10 and the restoration of goblet cells and indicate it as a good candidate to prevent and treat diseases associated with inflammation.

Ability of Lactobacillus plantarum LS/07 to modify intestinal enzymes activity in chronic diseases prevention.

The ability of probiotic strain Lactobacillus plantarum LS/07 to modify the activity of intestinal bacterial enzymes - ß-glucuronidase (ß-GLUCUR), ß-galactosidase (ß-GAL), and ß-glucosidase (ß-GLU) in prevention of chronic diseases - cancer, atherosclerosis and dysbiosis was investigated. The male Sprague-Dawley rats were randomly divided into 12 experimental groups: controls groups - C (control), AT (atherosclerotic), CC (carcinogenic), dysbiotic groups - each group in combination with antibiotics (ATB), probiotics groups - in combinatioan with probiotic (PRO) alone, and each group with combination of antibiotic and probiotic (ATB+PRO). In the control group the ß-glucuronidase activity did not change throughout the experiment. High fat diet in atherosclerotic group significantly increased the activity of ß-glucuronidase (P<0.001) and ß-glucosidase (P<0.01). Azoxymethane application in carcinogenic group significantly increased ß-glucuronidase (P<0.01), but reduced ß-glucosidase (P<0.01) activity. Daily application of probiotics alone and in combination with antibiotic increased ß-galactosidase, of ß-glucosidase, and decreased ß-glucuronidase activity. In control antibiotic group we observed significant increase in ß-glucuronidase (P<0.05) and decreased ß-glucosidase (P<0.01) activity which can be caused by the change of microflora in favor of coliform bacteria. These findings indicate the positive effects of probiotic Lactobacillus plantarum LS/07 and suggest its use in disease prevention in human medicine and some animal species.

Anticancer and Immunomodulatory Effects of Lactobacillus plantarum LS/07, Inulin and Melatonin in NMU-induced Rat Model of Breast Cancer.

BACKGROUND/AIM: Chemopreventive activity of a new probiotic strain Lactobacillus plantarum LS/07 (PRO) and prebiotic oligofructose-enriched inulin (PRE) in rat mammary carcinogenesis induced by procarcinogen 7,12-dimethylbenz[a]anthracene has been reported before. This study evaluated the anticancer and immunomodulatory efficacy of PRO, PRE, PRO+PRE (PRO/PRE) and combination with melatonin (PRO+PRE+MEL) in a rat model, when breast cancer was induced by a direct-acting carcinogen N-nitroso-N-methylurea (NMU). MATERIALS AND METHODS: Daily administration of PRO (at a dose of 8.4×10(8) colony-forming units (c.f.u.)/rat), PRE (in the diet, 20 g/kg) and MEL (in tap water, 20 mg/l) started 14 days before the first NMU dose and lasted for 16 weeks. RESULTS: Although tumor growth was not altered, a marked decrease in the ratio of high-/low-grade carcinomas and in tumoral Ki-67 expression was found after PRO+PRE treatment; melatonin augmented these effects. PRO+PRE+MEL combination enhanced CD4(+) and CD8(+) T-cell tumor infiltration induced by PRO/PRE and increased CD25(+)FoxP3(+) regulatory T-cells in tumors. CONCLUSION: In mammary carcinogenesis, Lactobacillus plantarum LS/07 and inulin exert prodifferentiating, antiproliferative and immunomodulatory activities, which are significantly amplified by melatonin co-administration.

Preventive use of Lactobacillus plantarum LS/07 and inulin to relieve symptoms of acute colitis.

The aim of presented study was to investigate the influence of Lactobacillus plantarum LS/07 and inulin on the activity of ß-glucuronidase enzyme, and counts of coliform and lactobacilli in fresh caecal digesta, cytokine levels (IL-6, IL-8), and trancription nuclear factor kappa beta (NFκB) activities in colon tissue and blood samples of rats with dextran sulphate sodium (DSS) induced acute colitis. The rats were randomly divided into four groups - CG, AC, AC+PRE and AC+PRO. Colitis was induced using of 5% DSS in drinking water for 7d. DSS application increased activity of ß-glucuronidase (P < 0.001), increased counts of coliforms, and decreased lactobacilli counts (P < 0.05) in comparison to control group. Serum and tissue levels of IL-6 and IL-8 as well as tissue NFκB activities showed increased expression in acute colitis group. Inulin diet modified counts of microorganims and decreased ß-glucuronidase activity, suppressed NFκB activities (P < 0.001) and down regulate synthesis of IL-6 (P < 0.01) in serum and colon tissue and tissue IL-8 (P < 0.05). Lactobacillus plantarum LS/07 decreased ß-glucuronidase activity (P < 0.05), levels of IL-6 and IL-8 (P < 0.001). These results were consistent with the addition of histological findings. Our results indicate that dietary intake of Lactobacillus plantarum LS/07 and inulin suppressed expression observed markers, which play an important role in the inflammatory process, which predisposes their use in prevention or treatment of acute colitis.

Probiotics and immunity: provisional role for personalized diets and disease prevention.

There is great interest in the interaction between diet and immune system and concomitantly in the potential of probiotic bacteria, especially given recent advances in understanding of gut microbiota effects on health in the context of microbiome research. Following our recent study on bacterial wall elasticity as a predictive measure of phagocytic cellular reactions and related outcomes, a question was raised regarding the scope of the application of these findings in various medical conditions in the context of predictive, preventive, and personalized medicine (PPPM). This summarizing review of the data describes the contributions, both observed and potential, of probiotics to the gut-brain axis and various medical conditions, including immune and atopic states, metabolic and inflammatory diseases-including liver disease and diabetes mellitus-cancer, and more. It also suggests novel insights for a number of beneficial applications of probiotics and advances in development of novel probiotic-based treatments and personalized diets, as well as application of sophisticated imaging techniques and nanobiotechnologies that can be adopted in the near future by innovative medical experts, warranting further research and practical translation.

Co-administration of a probiotic strain Lactobacillus plantarum LS/07 CCM7766 with prebiotic inulin alleviates the intestinal inflammation in rats exposed to N,N-dimethylhydrazine.

The aim of this study was to determine the anti-inflammatory effects of preventive administration of a probiotic strain Lactobacillus plantarum LS/07 CCM7766 alone or in combination with prebiotic inulin or with flax-seed oil in the gut of rats, which developed chronic inflammation following administration of the pro-carcinogen N,N-dimethylhydrazine (DMH). After 28weeks administration of probiotic/prebiotic-containing diet, rats were killed and their colons were examined by immunohistological criteria, whereas cytokines were determined in the jejunal mucosa. Application of DMH triggered the production of pro-inflammatory cytokines IL-2, IL-6, IL-17, and TNF-α, expression of pro-inflammatory mediators NF-κB, COX-2 and iNOS and caused depletion of goblet cells. Supplementing the diet with L. plantarum and its combination with the prebiotic abolished DMH-induced inflammatory process in the jejunal mucosa by inhibiting the production of pro-inflammatory cytokines and by stimulation of anti-inflammatory IL-10 cytokine synthesis, whereas concentration of TGF-ß1 was not influenced significantly. Diet prevented a decrease in goblet cell numbers but numbers of mast cells were lowered only moderately. However, combined treatment of rats with L. plantarum and flax-seed oil had no significant effect on the parameters examined, except for decreased expression of NF-κB, in comparison with the negative control. Results indicate that the preventive administration of probiotic L. plantarum LS/07 CCM7766 alone or in combination with prebiotic inulin to rats with DMH-induced chronic inflammation can reduce inflammatory process in the jejunal and colon mucosa, probably indirectly, and involves down-regulation of synthesis of pro-inflammatory cytokines and suppression of NF-κB activity in mucosal cells.

Preventive effects of probiotic bacteria Lactobacillus plantarum and dietary fiber in chemically-induced mammary carcinogenesis.

AIM: The purpose of the present study was to evaluate the chemopreventive efficacy of a new probiotic bacterial strain, Lactobacillus plantarum LS/07 (PRO), prebiotic oligofructose-enriched inulin (PRE) and PRO-PRE combination in a rat model of breast cancer. MATERIALS AND METHODS: Mammary carcinogenesis was induced by 7,12-dimethylbenz[a]anthracene (DMBA). Daily oral administration of PRO (at a dose of 8.4×10(8) c.f.u./rat) and PRE (in the diet, 20 g/kg) started two weeks before the first DMBA dose and lasted until the end of the experiment (16 weeks). RESULTS: Administration of PRO, PRE and PRO-PRE combination significantly suppressed the tumor frequency, increased Cd4(+) T-cells in tumor tissue and reduced serum tumor necrosis factor-α concentration. In PRO and PRO-PRE groups, the decline of Cd8(+) T-cells in blood and their increase in tumor tissue was observed. CONCLUSION: Long-term administration of Lactobacillus plantarum LS/07 with and without inulin is effective against breast cancer, at least partially, through immunomodulatory mechanisms.

Early diagnosis of colorectal cancer in rats with DMH induced carcinogenesis by means of urine autofluorescence analysis.

Cancer is one of the most highlighted topics of current research. Early detection of this disease allows more effective therapy, hence higher chance of cure. Application of fluorescence spectral techniques into oncological diagnostic is one of the potential alternatives. Chemically induced carcinogenesis in rats is widely used model for exploration of various aspects of colorectal cancer. This study shows value of discriminate analysis of urine fluorescent fingerprint between healthy control group of rats and those with dimethylhydrazine induced early lesions of colorectal cancer. Using fluorescence spectroscopy, significant difference (P < 0.05) between both of group was achieved.

The effects of two Lactobacillus plantarum strains on rat lipid metabolism receiving a high fat diet.

The aim of our study was to evaluate the effects of the different probiotic strains, Lactobacillus plantarum LS/07 and Lactobacillus plantarum Biocenol LP96, on lipid metabolism and body weight in rats fed a high fat diet. Compared with the high fat diet group, the results showed that Lactobacillus plantarum LS/07 reduced serum cholesterol and LDL cholesterol, but Lactobacillus plantarum Biocenol LP96 decreased triglycerides and VLDL, while there was no change in the serum HDL level and liver lipids. Both probiotic strains lowered total bile acids in serum. Our strains have no significant change in body weight, gain weight, and body fat. These findings indicate that the effect of lactobacilli on lipid metabolism may differ among strains and that the Lactobacillus plantarum LS/07 and Lactobacillus plantarum Biocenol LP96 can be used to improve lipid profile and can contribute to a healthier bowel microbial balance.

Prebiotics and bioactive natural substances induce changes of composition and metabolic activities of the colonic microflora in cancerous rats.

Prebiotics are defined as selectively fermented food ingredients that induce specific changes in the composition and/or activity in the gastrointestinal microbiota beneficial to the host well-being and health. The aim of the presented experiment was to investigate the effect of a prebiotic applied alone or in combination with Hyppocastani extractum siccum, and Lini oleum virginale in rats with dimethylhydrazine induced colon cancer. Wistar albino rats were fed high fat diet supplemented with the prebiotic alone or in combination with Horse chestnut and flaxseed oil. The activity of faecal glycolytic enzymes, lipid parameters, bile acids, short chain fatty acids and counts of coliforms and lactobacilli were determined. Treatment with the prebiotic alone and in combination with selected substances significantly decreased the activity of glycolytic bacterial enzyme ß-glucuronidase (P<0.001) and increased activities of ß-galactosidase and ß-glucosidase. Bile acids concentration was significantly decreased (P<0.01) except for the combination of the prebiotic with Horse chestnut. The prebiotic alone decreased the lipid parameters (P<0.001) and enhanced production of short chain fatty acids. Application of prebiotic and bioactive natural substances significantly reduced number of coliforms (P<0.05). Prebiotic alone significantly increased the count of lactobacilli (P<0.05). These results show that prebiotics have a protective effect and may be the useful for colon cancer prevention and treatment.

Beneficial modification of functional renal parameters in 5/6 nephrectomized rats by nutraceutical. In view of a kidney-protective intervention.

The objective of this study is to ascertain the potential beneficial effects of a novel phytoterapeutic formula (DTS, Kyotsu Jigyo, Japan) on renal function and morphological structure in 5/6 nephrectomized rats. Male Spraque-Dawley rats, 240-280 g, were divided into sham control (Group A) and nephrectomized (Group B and Group C) groups. The 5/6 nephrectomy was performed by removal of the right kidney and 2/3 ligation of left renal artery. After surgery, the animals were kept in individual cage for 6 weeks. Rats in Group A and Group B were fed with a normal protein diet only while those in Group C were fed normal protein diet added with DTS (10 mg/rat/day). The DTS supplementation was started a day after surgery. After 5 weeks, all rats were subjected to renal function study and then their left kidneys were isolated for morphological study. There were no significant differences in body weight, blood pressure, and heart rate among groups. DTS supplementation significantly increased (p<0.05) plasma creatinine concentration, glomerular filtration rate, effective renal plasma flow, and urine flow rate in nephrectomized rats when compared to sham control (Group A) and untreated nephrectomized (Group B) controls. In contrast, plasma urea concentration and morphological structure were not significantly modified by DTS supplementation in nephrectomized animals. These data suggest that feeding with a normal protein diet and DTS supplementation improves renal function without any morphological effect in 5/6 nephrectomized rats if not a slight preservation.(www.actabiomedica.it).

Effect on the immune system of germ-free piglets of probiotics potentiated with polyunsaturated fatty acids.

The present study investigated the influence of polyunsaturated fatty acids (PUFA) on the immune system of germ-free piglets. Oil with increased content of omega-3 PUFA was administered to piglets from the experimental group (EG) for four weeks. Piglets from the control group (CG) received identical volumes of saline solution. At the age of 21 days both groups of germ-free piglets were inoculated perorally with Lactobacillus casei subsp. casei at a dose of 2 ml (1x10(8) mli). At the age of 28 days, i.e. after one-week colonisation of germ-free piglets with Lactobacillus casei subsp. casei, significant differences were recorded in phagocytic activity of neutrophils (PANe) and phagocytic activity of potentially phagocytizing cells (PA) (P < 0.05). Between EG and CG there have been observed no significant differences in absolute numbers of CD4+ and CD8+ T lymphocytes and numbers of IgM cells and in additional investigated parameters - number of CD2+ T lymphocytes, index of phagocytic activity of neutrophils (IPANe) and index of phagocytic activity (IPA). The total number of leukocytes (Le) in EG was also higher. Of the parameters determined in blood serum we observed a significant increase in concentration of alpha linolenic, eicosapentaenoic and docosahexaenoic acids and a parallel decrease in the level of arachidonic acid.

HlyA knock out yields a safer Escherichia coli A0 34/86 variant with unaffected colonization capacity in piglets.

Escherichia coli A0 34/86 (O83:K24:H31) has been successfully used for prophylactic and therapeutic intestinal colonization of premature and newborn infants, with the aim of preventing nosocomial infections. Although E. coli A0 34/86 was described as a nonpathogenic commensal, partial sequencing revealed that its genome harbours gene clusters highly homologous to virulence determinants of different types of E. coli, including closely linked genes of the alpha-haemolysin operon (hlyCABD) and for the cytotoxic necrotizing factor (cnf1). A haemolysin-deficient mutant (Delta hlyA) of E. coli A0 34/86 was generated and its colonization capacity was determined. The results show that a single dose of the A0 34/86 wild-type or Delta hlyA strains resulted in efficient intestinal colonization of newborn conventional piglets, and that this was still considerable after several weeks. No difference was observed between the wild-type and the mutant strains, showing that haemolysin expression does not contribute to intestinal colonization capacity of E. coli A0 34/86. Safety experiments revealed that survival of colostrum-deprived gnotobiotic newborn piglets was substantially higher upon colonization by the nonhaemolytic strain than following inoculation by its wild-type ancestor. We suggest that the E. coli A0 34/86 Delta hlyA mutant may represent a safer prophylactic and/or immunomodulatory tool with unaffected colonization capacity.

Characterization of the flexible genome complement of the commensal Escherichia coli strain A0 34/86 (O83 : K24 : H31).

Colonization by the commensal Escherichia coli strain A0 34/86 (O83 : K24 : H31) has proved to be safe and efficient in the prophylaxis and treatment of nosocomial infections and diarrhoea of preterm and newborn infants in Czech paediatric clinics over the past three decades. In searching for traits contributing to this beneficial effect related to the gut colonization capacity of the strain, the authors have analysed its genome by DNA-DNA hybridization to E. coli K-12 (MG1655) genomic DNA arrays and to 'Pathoarrays', as well as by multiplex PCR, bacterial artificial chromosome (BAC) library cloning and shotgun sequencing. Four hundred and ten E. coli K-12 ORFs were absent from A0 34/86, while 72 out of 456 genes associated with pathogenicity islands of E. coli and Shigella were also detected in E. coli A0 34/86. Furthermore, extraintestinal pathogenic E. coli-related genes involved in iron uptake and adhesion were detected by multiplex PCR, and genes encoding the HlyA and cytotoxic necrotizing factor toxins, together with 21 genes of the uropathogenic E. coli 536 pathogenicity island II, were identified by analysis of 2304 shotgun and 1344 BAC clone sequences of A0 34/86 DNA. Multiple sequence comparisons identified 31 kb of DNA specific for E. coli A0 34/86; some of the genes carried by this DNA may prove to be implicated in the colonization capacity of the strain, enabling it to outcompete pathogens. Among 100 examined BAC clones roughly covering the A0 34/86 genome, one reproducibly conferred on the laboratory strain DH10B an enhanced capacity to persist in the intestine of newborn piglets. Sequencing revealed that this BAC clone carried gene clusters encoding gluconate and mannonate metabolism, adhesion (fim), invasion (ibe) and restriction/modification functions. Hence, the genome of this clinically safe and highly efficient colonizer strain appears to harbour many 'virulence-associated' genes. These results highlight the thin line between bacterial 'virulence' and 'fitness' or 'colonization' factors, and question the definition of enterobacterial virulence factors.

Testing two Lactobacillus plantarum and Lactobacillus acidophilus strains for their suitability as a lipoid probiotic.

Two strains of lactobacilli (Lactobacillus acidophilus T-135 and Lactobacillus plantarum 4/97) were selected in order to study their inhibitory properties against frequent udder pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Salmonella enteritidis and Bacillus pumilus), their production of organic acids as well as their ability to survive on the teat skin, the teat duct mucosa and in a lipoid emulsion. Both strains inhibited the tested pathogenic microbes and survived on the investigated surfaces and in an emulsion for more than 6 hours and 11 days, respectively.