In Vitro Fermentation of NUTRIOSE® FB06, a wheat dextrin soluble fibre, in a continuous culture human colonic model system

Wheat dextrin soluble fibre may have metabolic and health benefits, potentially acting via mechanisms governed by the selective modulation of the human gut microbiota. Our aim was to examine the impact of wheat dextrin on the composition and metabolic activity of the gut microbiota. We used a validated in vitro three-stage continuous culture human colonic model (gut model) system comprised of vessels simulating anatomical regions of the human colon. To mimic human ingestion, 7 g of wheat dextrin (NUTRIOSE® FB06) was administered to three gut models, twice daily at 10.00 and 15.00, for a total of 18 days. Samples were collected and analysed for microbial composition and organic acid concentrations by 16S rRNA-based fluorescence in situ hybridisation and gas chromatography approaches, respectively. Wheat dextrin mediated a significant increase in total bacteria in vessels simulating the transverse and distal colon, and a significant increase in key butyrate-producing bacteria Clostridium cluster XIVa and Roseburia genus in all vessels of the gut model. The production of principal short-chain fatty acids, acetate, propionate and butyrate, which have been purported to have protective, trophic and metabolic host benefits, were increased. Specifically, wheat dextrin fermentation had a significant butyrogenic effect in all vessels of the gut model and significantly increased production of acetate (vessels 2 and 3) and propionate (vessel 3), simulating the transverse and distal regions of the human colon, respectively. In conclusion, wheat dextrin NUTRIOSE® FB06 is selectively fermented in vitro by Clostridium cluster XIVa and Roseburia genus and beneficially alters the metabolic profile of the human gut microbiota.

An assessment of the prebiotic potential of single and blended substrates in anaerobic in vitro batch culture fermentations using canine faecal samples as inocula

Previously, using an in vitro static batch culture system, it was found that rice bran (RB), inulin, fibersol, mannanoligosaccharides (MOS), larch arabinogalactan and citrus pectin elicited prebiotic effects (in terms of increased numbers of bifidobacteria and lactic acid bacteria) on the faecal microbiota of a dog. The aim of the present study was to confirm the prebiotic potential of each individual substrate using multiple faecal donors, as well as assessing the prebiotic potential of 15 substrate blends made from them. Anaerobic static and stirred, pH-controlled batch culture systems inoculated with faecal samples from healthy dogs were used for this purpose. Fluorescence in situ hybridization (FISH) analysis using seven oligonucleotide probes targeting selected bacterial groups and DAPI (total bacteria) was used to monitor bacterial populations during fermentation runs. High-performance liquid chromatography was used to measure butyrate produced as a result of bacterial fermentation of the substrates. RB and a MOS/RB blend (1:1, w/w) were shown to elicit prebiotic and butyrogenic effects on the canine microbiota in static batch culture fermentations. Further testing of these substrates in stirred, pH-controlled batch culture fermentation systems confirmed the prebiotic and butyrogenic effects of MOS/RB, with no enhancement of Clostridium clusters I and II and Escherichia coli populations.

Metaproteomics analysis reveals the adaptation process for the chicken gut microbiota

The animal gastrointestinal tract houses a large microbial community, the gut microbiota, that confers many benefits to its host, such as protection from pathogens and provision of essential metabolites. Metagenomic approaches have defined the chicken fecal microbiota in other studies, but here, we wished to assess the correlation between the metagenome and the bacterial proteome in order to better understand the healthy chicken gut microbiota. Here, we performed high-throughput sequencing of 16S rRNA gene amplicons and metaproteomics analysis of fecal samples to determine microbial gut composition and protein expression. 16 rRNA gene sequencing analysis identified Clostridiales, Bacteroidaceae, and Lactobacillaceae species as the most abundant species in the gut. For metaproteomics analysis, peptides were generated by using the Fasp method and subsequently fractionated by strong anion exchanges. Metaproteomics analysis identified 3,673 proteins. Among the most frequently identified proteins, 380 proteins belonged to Lactobacillus spp., 155 belonged to Clostridium spp., and 66 belonged to Streptococcus spp. The most frequently identified proteins were heat shock chaperones, including 349 GroEL proteins, from many bacterial species, whereas the most abundant enzymes were pyruvate kinases, as judged by the number of peptides identified per protein (spectral counting). Gene ontology and KEGG pathway analyses revealed the functions and locations of the identified proteins. The findings of both metaproteomics and 16S rRNA sequencing analyses are discussed.

In vitro colonic metabolism of coffee and chlorogenic acid results in selective changes in human faecal microbiota growth

Coffee is a relatively rich source of chlorogenic acids (CGA), which, like other polyphenols are postulated to exert preventative effects against cardiovascular disease and type-2 diabetes. As a considerable proportion of ingested CGA reaches the large intestine, CGA may be capable of exerting beneficial effects in the large gut. Here we utilise a stirred, anaerobic, pH controlled, batch culture fermentation model of the distal region of the colon in order to investigate the impact of coffee and CGA on the growth of the human faecal microbiota. Incubation of the coffee with the human faecal microbiota led to the rapid metabolism of CGA (4h) and the production of dihydrocaffeic acid and dihydroferulic acid, whilst caffeine remained un-metabolised. The coffee with the highest levels of CGA (p<0.05, relative to the other coffees) induced a significant increase in Bifidobacterium spp. relative to the control at 10 hours post exposure (p<0.05). Similarly, an equivalent quantity of CGA (80.8mg; matched with that in high CGA coffee) induced a significant increase in Bifidobacterium spp. (p<0.05). CGA alone also induced a significant increase in the Clostridium coccoides-Eubacterium rectale group (p<0.05). This selective metabolism and subsequent amplification of specific bacterial populations could be beneficial to host health.

Impact of polysialylated CD56 on natural killer cell cytotoxicity

Background Siglec-7, a sialic acid binding inhibitory receptor expressed by NK cells is masked in vivo by a so far unknown ligand. It shows a strong binding prevalence for α-2,8-linked disialic acids in vitro. Results Here we describe the expression of PSA-NCAM (α-2,8-linked polysialic acid modified NCAM) on functional adult peripheral blood natural killer cells and examine its possible role in masking Siglec-7. Unmasking of Siglec-7 using Clostridium perfringens neuraminidase massively reduces NK cell cytotoxicity. By contrast a specific removal of PSA using Endo-NF does not lead to a reduction of NK cell cytotoxicity. Conclusion The results presented here therefore indicate that PSA-NCAM is not involved in masking Siglec-7.

Effect of prebiotics on the fecal microbiota of elderly volunteers after dietary supplementation of Bacillus coagulans GBI-30, 6086

In advancing age, gut populations of beneficial microbes, notably Bifidobacterium spp., show a marked decline. This contributes to an environment less capable of maintaining homoeostasis. This in vitro investigation studied the possible synergistic effects of probiotic supplementation in modulating the gut microbiota enabling prebiotic therapy to in elderly persons. Single stage batch culture anaerobic fermenters were used and inoculated with fecal microbiota obtained from volunteers after taking a 28 day treatment of Bacillus coagulans GBI-30, 6086 (GanedenBC30 (BC30)) or a placebo. The response to prebiotic supplements fructooligosaccharides (FOS) and galactooligosaccharides (GOS) in the fermenters was assessed. Bacterial enumeration was carried out using fluorescent in situ hybridisation and organic acids measured by gas chromatography. Baseline populations of Faecalibacterium prausnitzii, Clostridium lituseburense and Bacillus spp. were significantly higher in those having consumed BC30 compared to the placebo. Both prebiotics increased populations of several purportedly beneficial bacterial groups in both sets of volunteers. Samples from volunteers having ingested the BC30 also increased populations of C. lituseburense, Eubacterium rectale and F. prausnitzii more so than in persons who had consumed the placebo, this also resulted in significantly higher concentrations of butyrate, acetate and propionate. This shows that consumption of BC30 and subsequent use of prebiotics resulted in elevated populations of beneficial genres of bacteria as well as organic acid production

Maternal weaning modulates emotional behavior and regulates the gut-brain axis

Evidence shows that nutritional and environmental stress stimuli during postnatal period influence brain development and interactions between gut and brain. In this study we show that in rats, prevention of weaning from maternal milk results in depressive-like behavior, which is accompanied by changes in the gut bacteria and host metabolism. Depressive-like behavior was studied using the forced-swim test on postnatal day (PND) 25 in rats either weaned on PND 21, or left with their mother until PND 25 (non-weaned). Non-weaned rats showed an increased immobility time consistent with a depressive phenotype. Fluorescence in situ hybridization showed non-weaned rats to harbor significantly lowered Clostridium histolyticum bacterial groups but exhibit marked stress-induced increases. Metabonomic analysis of urine from these animals revealed significant differences in the metabolic profiles, with biochemical phenotypes indicative of depression in the non-weaned animals. In addition, non-weaned rats showed resistance to stress-induced modulation of oxytocin receptors in amygdala nuclei, which is indicative of passive stress-coping mechanism. We conclude that delaying weaning results in alterations to the gut microbiota and global metabolic profiles which may contribute to a depressive phenotype and raise the issue that mood disorders at early developmental ages may reflect interplay between mammalian host and resident bacteria.

Biorefinery development through utilization of biodiesel industry by-products as sole fermentation feedstock for 1,3-propanediol production

Rapeseed meal (RSM) hydrolysate was evaluated as substitute for commercial nutrient supplements in 1,3-propanediol (PDO) fermentation using the strain Clostridium butyricum VPI 1718. RSM was enzymatically converted into a generic fermentation feedstock, enriched in amino acids, peptides and various micro-nutrients, using crude enzyme consortia produced via solid state fermentation by a fungal strain of Aspergillus oryzae. Initial free amino nitrogen concentration influenced PDO production in batch cultures. RSM hydrolysates were compared with commercial nutrient supplements regarding PDO production in fed-batch cultures carried out in a bench-scale bioreactor. The utilization of RSM hydrolysates in repeated batch cultivation resulted in a PDO concentration of 65.5 g/L with an overall productivity of 1.15 g/L/h that was almost 2 times higher than the productivity achieved when yeast extract was used as nutrient supplement.

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved.

One-step purification and immobilization of his-tagged rhamnosidase for naringin hydrolysis

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca²⁺ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.

Clostridial spores for cancer therapy : targeting solid tumour microenvironment

Solid tumour accounts for 90% of all cancers. The current treatment approach for most solid tumours is surgery, however it is limited to early stage tumours. Other treatment options such as chemotherapy and radiotherapy are non-selective, thus causing damage to both healthy and cancerous tissue. Past research has focused on understanding tumour cells themselves, and conventional wisdom has aimed at targeting these cells directly. Recent research has shifted towards understanding the tumour microenvironment and it’s differences from that of healthy cells/tissues in the body and then to exploit these differences for treatmeat of the tumour. One such approach is utilizing anaerobic bacteria. Several strains of bacteria have been shown to selectively colonize in solid tumours, making them valuable tools for selective tumour targeting and destruction. Amongst them, the anaerobic Clostridium has shown great potential in penetration and colonization of the hypoxic and necrotic areas of the tumour microenvironment, causing significant oncolysis as well as enabling the delivery of therapeutics directly to the tumour in situ. Various strategies utilizing Clostridium are currently being investigated, and represent a novel area of emerging cancer therapy. This review provides an update review of tumour microenvironment as well as summary of the progresses and current status of Clostridial spore-based cancer therapies.

Meropenem versus piperacillin-tazobactam for definitive treatment of bloodstream infections due to ceftriaxone non-susceptible Escherichia coli and Klebsiella spp (the MERINO trial): study protocol for a randomised controlled trial

BACKGROUND: Gram-negative bacteria such as Escherichia coli or Klebsiella spp. frequently cause bloodstream infections. There has been a worldwide increase in resistance in these species to antibiotics such as third generation cephalosporins, largely driven by the acquisition of extended-spectrum beta-lactamase or plasmid-mediated AmpC enzymes. Carbapenems have been considered the most effective therapy for serious infections caused by such resistant bacteria; however, increased use creates selection pressure for carbapenem resistance, an emerging threat arising predominantly from the dissemination of genes encoding carbapenemases. Recent retrospective data suggest that beta-lactam/beta-lactamase inhibitor combinations, such as piperacillin-tazobactam, may be non-inferior to carbapenems for the treatment of bloodstream infection caused by extended-spectrum beta-lactamase-producers, if susceptible in vitro. This study aims to test this hypothesis in an effort to define carbapenem-sparing alternatives for these infections.

METHODS/DESIGN: The study will use a multicentre randomised controlled open-label non-inferiority trial design comparing two treatments, meropenem (standard arm) and piperacillin-tazobactam (carbapenem-sparing arm) in adult patients with bacteraemia caused by E. coli or Klebsiella spp. demonstrating non-susceptibility to third generation cephalosporins. Recruitment is planned to occur in sites across three countries (Australia, New Zealand and Singapore). A total sample size of 454 patients will be required to achieve 80% power to determine non-inferiority with a margin of 5%. Once randomised, definitive treatment will be for a minimum of 4 days, but up to 14 days with total duration determined by treating clinicians. Data describing demographic information, antibiotic use, co-morbid conditions, illness severity, source of infection and other risk factors will be collected. Vital signs, white cell count, use of vasopressors and days to bacteraemia clearance will be recorded up to day 7. The primary outcome measure will be mortality at 30 days, with secondary outcomes including days to clinical and microbiological resolution, microbiological failure or relapse, isolation of a multi-resistant organism or Clostridium difficile infection.

Purificação e caracterização da bacteriocina cereína 8A, produzida por uma linhagem de Bacillus cereus

Foi estudada uma bacteriocina produzida por uma linhagem de B. cereus 8A, isolado de solo da região Sul do Brasil. Na primeira etapa de estudo determinaram-se as condições básicas de produção de bacteriocina com amplo espectro de ação denominada de Cereína 8A. Observou-se que durante a fase estacionária ocorre o máximo da sua produção, iniciando sua síntese no final da fase exponencial. As condições de maior produção foram a 30º C, agitação e contínua e numa faixa de pH de 7,0-8,5. A bacteriocina bruta inibiu várias bactérias indicadoras, como Listeria monocytogenes, Clostridium perfringens e Bacillus cereus. O teste de termoestabilidade mostrou a perda de atividade quando submetida a uma temperatura a partir de 87º C. Verificou-se a resistência da bacteriocina bruta frente à tripsina e papaína, mas não frente à proteinase K e pronase E. B. cereus e L. monocytogenes foram utilizadas como bactérias indicadoras para a determinação do modo de ação, após a determinação da dose bactericida de 200 UA mL-1 e 400 UA mL-1 respectivamente. A Cereína 8A demonstrou uma ação inibidora em culturas de Escherichia coli e Salmonella Enteritidis, quando tratadas com EDTA. A atividade esporicida foi observada contra esporos de B. cereus após tratamento com 400 UA ml -1. A análise da biomassa de L. monocytogenes e B. cereus após tratamento com a Cereína 8A, através da espectrofotometria de infravermelho determinou alteração no perfil, correspondente à fração dos ácidos graxos da membrana celular bacteriana. A substância peptídica foi separada por meio da precipitação com sulfato de amônio, extração com 1-butanol e aplicação em coluna de cromatografia por troca iônica tipo Q-Sepharose. A Cereína 8A purificada mostrou maior sensibilidade a proteases e ao calor e um peso molecular de aproximadamente 26 kDa. O espectro ultravioleta foi típico de um polipeptídeo e o espectro de infravermelho indica presença de grupamentos NH, acil e ligações peptídicas na sua estrutura. Uma hipótese do mecanismo de ação seria a desestruturação da membrana celular pela abertura de poros.

Efeitos do tripolifosfato de sódio sobre as características microbiológicas, físico-químicas e vida-de-prateleira em lingüiça frescal de frango.

Neste trabalho foram analisados os efeitos da adição de 0,125%, 0,25%, 0,375% e 0,5% de tripolifosfato de sódio sobre as contagens microbiológicas de Coliformes Totais, Escherichia coli, Salmonella Spp., Staphylococcus aureus, Clostridium sulfito redutores, microrganismos mesófilos aeróbios, psicrotróficos aeróbios, nas características físico-químicas de oxidação lipídica e pH em lingüiças frescais de carne de frango, armazenadas sob refrigeração a 5ºC nos dias 0, 8, 15 e 22. O tripolifosfato de sódio não aumentou a segurança microbiológica das lingüiças de frango, pois verificou-se aumento progressivo nas contagens das bactérias presentes, como mesófilos aeróbios e psicrotróficos aeróbios não demonstrando diferença significativa com relação ao tratamento aplicado. Para os coliformes totais evidenciou-se variação nas contagens dos mesmos, no decorrer do experimento, não apresentando correlação com as concentrações de tripolifosfato de sódio adicionadas nas lingüiças de frango. Para os demais microrganismos pesquisados os resultados encontrados nas amostras no dia 0 foram: para Salmonella Spp. ausência em 25g das amostras, Staphylococcus aureus <1,0x103 UFC/g, Clostridium sulfito redutor <1,0x103 UFC/g, portanto, apresentaram-se dentro do padrão exigido pela legislação para todas as amostras de lingüiças. As lingüiças frescais de frango adicionadas de 0,5% de tripolifosfato de sódio apresentaram valores de TBA mais baixos que os demais tratamentos e que o grupo controle, porém este resultado não foi significativamente diferente Com relação aos dias de análises, estes revelaram diferença significativa para os números de TBA. Os valores foram de 0,93 = dia 0; 1,39 = dia 8; 4,09 = dia 15 e de 3,18 = dia 22, inicialmente os números de TBA foram baixos, os quais, apresentaram aumento progressivo ao longo da vida-de-prateleira das lingüiças, exceto nos tratamentos com 0,25% e 0,375% de tripolifosfato de sódio que apresentaram decréscimo destes números do dia 15 para o dia 22. Os valores do pH apresentaram diferença significativa para os dias de análise, tais como: dia 0 = 6,09; dia 8 = 6,25; dia 15 = 6,30; dia 22 = 6,47, estes valores demonstraram aumento do pH durante a vida-de-prateleira das amostras. Não houve diferença significativa entre os distintos tratamentos, porém evidenciou-se que os valores do pH dos tratamentos com adição de 0,5% de tripolifosfato de sódio apresentaram-se mais elevados do que o do grupo controle e também dos demais tratamentos. O tripolifosfato de sódio em diferentes concentrações não agiu prolongando a vida-de-prateleira das lingüiças frescais de frango.