Stimbiotic supplementation and xylose-rich carbohydrates modulate broiler's capacity to ferment fibre.

Stimbiotics are a new category of feed additives that can increase fibre fermentability by stimulating fibre-degrading microbiota in the gut. The aim of this study was to test, ex vivo, if the microbiota of broilers fed a stimbiotic are better able to ferment different xylose-rich substrates in an ileal and a caecal environment. The ileal and caecal contents from broiler chickens fed a stimbiotic or from a control group were used as an inoculum in the ex vivo fermentation experiment. Different xylose-rich substrates including monomeric xylose (XYL), XOS with DP 2 to 6 (XOS), short DP XOS of 2 to 3 (sDP-XOS), long DP XOS of 4 to 6 (lDP-XOS) and de-starched wheat bran (WB), were added to each ileal and caecal inoculum in fermentation vessels. Total gas, short-chain fatty acids (SCFA) production, bacterial quantification, and carbohydrate utilisation were monitored for 9 h post-inoculation. No significant interactions were observed in any of the parameters measured in either the ileal or caecal contents (p > 0.05). Stimbiotic ileal inocula resulted in higher total gas (p < 0.001) and volatile fatty acid (VFA) (p < 0.001) production, increased numbers of Lactobacillus spp. (p < 0.001), and decreased numbers of Enterococcus spp. (p < 0.01) after 9 h regardless of the xylose-rich substrate added. Stimbiotic caecal inocula resulted in a higher ratio of VFA to branched-chain fatty acids (BCFAs) by up to +9% (p < 0.05). Ileal microbiota were found to preferentially metabolise WB, while caecal microbiota favoured XOS substrates, particularly lDP-XOS. These results indicate that stimbiotics can promote the abundance of lactic acid bacteria involved in the establishment of fibre-degrading bacteria and VFA content in the gut, which could have beneficial effects on broiler performance. Further, ileal and caecal microbiota differ in their utilisation of different substrates which may impact the effectiveness of different stimbiotic products.

Effect of high contents of dietary animal-derived protein or carbohydrates on canine faecal microbiota.

BACKGROUND: Considerable evidence suggests that food impacts both the gastro-intestinal (GI) function and the microbial ecology of the canine GI tract. The aim of this study was to evaluate the influence of high-carbohydrate (HC), high-protein (HP) and dry commercial (DC) diets on the canine colonic microbiota in Beagle dogs. Diets were allocated according to the Graeco-Latin square design. For this purpose, microbial DNA was isolated from faecal samples and separated by density gradient centrifugation, resulting in specific profiling based on the guanine-cytosine content (%G+C). In addition, 16 S rRNA gene amplicons were obtained from the most abundant %G + C peaks and analysed by sequence analysis, producing a total of 720 non-redundant sequences (240 sequences per diet). RESULTS: The DC diet sample showed high abundance of representatives of the orders Clostridiales, Lactobacillales, Coriobacteriales and Bacteroidales. Sequence diversity was highest for DC diet samples and included representatives of the orders Lactobacillales and Bacteroidales, which were not detected in samples from the HP and HC diets. These latter two diets also had reduced levels of representatives of the family Lachnospiraceae, specifically Clostridial cluster XIVa. The HC diet favoured representatives of the order Erysipelotrichales, more specifically the Clostridial cluster XVIII, while the HP diet favoured representatives of the order Fusobacteriales. CONCLUSIONS: This study detected Coriobacteriales in dog faeces, possibly due to the non-selective nature of the %G + C profiling method used in combination with sequencing. Moreover, our work demonstrates that the effect of diet on faecal microbiota can be explained based on the metabolic properties of the detected microbial taxa.

Design and validation of a real-time PCR technique for assessing the level of inclusion of fungus- and yeast-based additives in feeds.

A reliable method for quantification of non-viable microbe-based nutritional and zootechnical additives introduced into feed is essential in order to ensure regulatory compliance, feed safety and product authenticity in industrial applications. In the present work, we developed a novel real-time quantitative polymerase chain reaction (qPCR) -based analysis protocol for monitoring two microbial additives in feed matrices. To evaluate the applicability of the method, pelleted wheat- and maize-based broiler chicken diets containing a non-viable phytase-producing strain of Aspergillus niger produced in solid state fermentation (150 or 300 g/t) and a non-viable selenium-enriched Saccharomyces cerevisiae (100 or 200 g/t) as model feed ingredients, were manufactured and subjected to analysis. Power analysis of the qPCR results indicated that 2 to 6 replicate feed samples were required to distinguish the product doses applied, which confirms that the microbial DNA was efficiently recovered and that potential PCR inhibitors present in the feed material were successfully removed in DNA extraction. The analysis concept described here was shown to be an accurate and sensitive tool for monitoring the inclusion levels of non-viable, unculturable microbial supplements in animal diets.

The impact of Bacillus subtilis DSM 32315 on the pathology, performance, and intestinal microbiome of broiler chickens in a necrotic enteritis challenge.

It was hypothesized that dietary inclusion of Bacillus subtilis DSM 32315 could inhibit Clostridium perfringens induced necrotic enteritis (NE), thereby improving broiler performance. Male, d 0 chicks were randomly assigned 14 birds/pen, 11 pens/treatment in 3 treatments: a basal diet (control), a coccidiostat fed control (Narasin), and a direct fed microbial (DFM) B. subtilis DSM 32315 treatment. Necrotic enteritis was induced in all birds by oral inoculation of Eimeria maxima oocysts on d 12 and a virulent C. perfringens on d 16. Mortality was reduced (P < 0.001) in DFM and Narasin compared to control. DFM reduced (P < 0.001) feed conversion ratio (FCR) compared to control. Furthermore, DFM and Narasin reduced (P < 0.001) footpad lesions. The DFM was shown to increase (P < 0.05) Bacillus spp. and decrease (P < 0.05) C. perfringens in the ileum and cecum at several time points. To investigate microbiome changes in the cecum, digesta samples were analyzed with % guanine and cytosine (%G+C) microbial profiling which fractionates bacterial chromosomes based on the %G+C in DNA. The method revealed treatment profile peaks in low (27.0 to 34.5%), mid (40.5 to 54.0%), and high (59.0 to 68.0%) G+C fractions. 16S rRNA gene amplification and high throughput sequencing was conducted on each of these fractions in order to elucidate specific bacterial population differences. In the low and mid %G+C fractions, DFM had greater abundance of Lactobacillaceae family members (P = 0.03 and P = 0.01, respectively) and Lactobacillus salivarius (P = 0.04 and P = 0.01, respectively) than control or Narasin. Lactobacillus johnsonii was also greater in the low %G+C fraction compared to control and Narasin (P = 0.01). Lachnospiraceae (P = 0.04) and Ruminococcaceae (P < 0.01) in the mid %G+C fraction were reduced in the DFM compared to control. Positive alterations to the microbial populations in the gut of broilers may at least be a partial mechanism by which B. subtilis DSM 32315 reduced pathology and improved performance of broilers in the NE challenge.

Effect of Lactobacillus brevis ATCC 8287 as a feeding supplement on the performance and immune function of piglets.

Lactobacillus brevis ATCC 8287, a surface (S-layer) strain, possesses a variety of functional properties that make it both a potential probiotic and a good vaccine vector candidate. With this in mind, our aim was to study the survival of L. brevis in the porcine gut and investigate the effect of this strain on the growth and immune function of recently weaned piglets during a feeding trial. For this, 20 piglets were divided evenly into a treatment and a control group. Piglets in the treatment group were fed L. brevis cells (1×10(10)) daily for three weeks, whereas those in the control group were provided an equivalent amount of probiotic-free placebo. For assessing the impact of L. brevis supplementation during the feeding trial, health status and weight gain of the piglets were monitored, pre- and post-trial samples of serum and feces were obtained, and specimens of the small and large intestinal mucosa and digesta were collected at slaughter. The results we obtained indicated that L. brevis-supplemented feeding induced a non-significant increase in piglet body weight and caused no change in the morphology of the intestinal mucosa. L. brevis cells were found to localize mainly in the large intestine, but they could not be isolated from feces. To a lesser extent, L. brevis was detected in the small intestine, although there was no specific attachment to the Peyer's patches. Changes in total serum IgG and IgA concentrations were not caused by supplemented L. brevis and no measurable rise in L. brevis-specific IgG was observed. However, analysis of cytokine gene expression in intestinal mucosa revealed downregulation of TGF-ß1 in the ileum and upregulation of IL-6 in the cecum in the L. brevis-supplemented group. Based on the results from this study, we conclude that whereas L. brevis appears to have some intestinal immunomodulatory effects, the ability of this strain to survive and colonize within the porcine gut appears to be limited.

Real-time PCR analysis of enteric pathogens from fecal samples of irritable bowel syndrome subjects.

BACKGROUND: Growing amount of scientific evidence suggests that microbes are involved in the pathophysiology of irritable bowel syndrome (IBS). The predominant fecal microbiota composition of IBS subjects has been widely studied with DNA-based techniques but less research has been focused on the intestinal pathogens in this disorder. Here, we optimized a highly sensitive panel of 12 quantitative real-time PCR (qPCR) assays to shed light on the putative presence of intestinal pathogens in IBS sufferers. The panel was used to screen fecal samples from 96 IBS subjects and 23 healthy controls. RESULTS: Fifteen IBS samples (17%) tested positive for Staphylococcus aureus with a thermonuclease (nuc) gene-targeting qPCR assay, whereas none of the healthy controls were positive for S. aureus (p <0.05). The S. aureus -positive IBS samples were confirmed by sequencing of the PCR amplicons. Clostridium perfringens was detected from IBS and control groups with a similar frequency (13% and 17%, respectively) with α-toxin (plc) gene -targeting qPCR assay while none of the samples tested positive for the Cl. perfringens enterotoxin-encoding gene (cpe). CONCLUSIONS: The qPCR panel consisting of 12 assays for an extensive set of pathogenic microorganisms provides an efficient alternative to the conventional detection of gastrointestinal pathogens and could accelerate the initiation of targeted antibiotic therapy reducing the risk of post-infectious IBS (PI-IBS). S. aureus has not been previously reported to be associated with the onset of IBS. Although we discovered significant differences in the prevalence of S. aureus between the study groups, its importance in giving rise to IBS symptoms requires further studies.

Association of symptoms with gastrointestinal microbiota in irritable bowel syndrome.

AIM: To investigate the correlations between self-reported symptoms of irritable bowel syndrome (IBS) and the gastrointestinal (GI) microbiota composition. METHODS: Fecal samples were collected from a total of 44 subjects diagnosed with IBS. Their symptoms were monitored with a validated inflammatory bowel disease questionnaire adjusted for IBS patients. Thirteen quantitative real-time polymerase chain reaction assays were applied to evaluate the GI microbiota composition. Eubacteria and GI bacterial genera (Bifidobacterium, Lactobacillus and Veillonella), groups (Clostridium coccoides/Eubacterium rectale, Desulfovibrio desulfuricans) and distinct bacterial phylotypes [closest 16S rDNA sequence resemblance to species Bifidobacterium catenulatum, Clostridium cocleatum, Collinsella aerofaciens (C. aerofaciens), Coprococcus eutactus (C. eutactus), Ruminococcus torques and Streptococcus bovis] with a suspected association with IBS were quantified. Correlations between quantities or presence/absence data of selected bacterial groups or phylotypes and various IBS-related symptoms were investigated. RESULTS: Associations were observed between subjects' self-reported symptoms and the presence or quantities of certain GI bacteria. A Ruminococcus torques (R. torques)-like (94% similarity in 16S rRNA gene sequence) phylotype was associated with severity of bowel symptoms. Furthermore, among IBS subjects with R. torques 94% detected, the amounts of C. cocleatum 88%, C. aerofaciens-like and C. eutactus 97% phylotypes were significantly reduced. Interesting observations were also made concerning the effect of a subject's weight on GI microbiota with regard to C. aerofaciens-like phylotype, Bifidobacterium spp. and Lactobacillus spp. CONCLUSION: Bacteria seemingly affecting the symptom scores are unlikely to be the underlying cause or cure of IBS, but they may serve as biomarkers of the condition.

Diarrhoea-predominant irritable bowel syndrome distinguishable by 16S rRNA gene phylotype quantification.

AIM: To study whether selected bacterial 16S ribosomal RNA (rRNA) gene phylotypes are capable of distinguishing irritable bowel syndrome (IBS). METHODS: The faecal microbiota of twenty volunteers with IBS, subdivided into eight diarrhoea-predominant (IBS-D), eight constipation-predominant (IBS-C) and four mixed symptom-subtype (IBS-M) IBS patients, and fifteen control subjects, were analysed at three time-points with a set of fourteen quantitative real-time polymerase chain reaction assays. All assays targeted 16S rRNA gene phylotypes putatively associated with IBS, based on 16S rRNA gene library sequence analysis. The target phylotypes were affiliated with Actinobacteria, Bacteroidetes and Firmicutes. Eight of the target phylotypes had less than 95% similarity to cultured bacterial species according to their 16S rRNA gene sequence. The data analyses were made with repeated-measures ANCOVA-type modelling of the data and principle component analysis (PCA) with linear mixed-effects models applied to the principal component scores. RESULTS: Bacterial phylotypes Clostridium cocleatum 88%, Clostridium thermosuccinogenes 85%, Coprobacillus catenaformis 91%, Ruminococcus bromii-like, Ruminococcus torques 91%, and R. torques 93% were detected from all samples analysed. A multivariate analysis of the relative quantities of all 14 bacterial 16S rRNA gene phylotypes suggested that the intestinal microbiota of the IBS-D patients differed from other sample groups. The PCA on the first principal component (PC1), explaining 30.36% of the observed variation in the IBS-D patient group, was significantly altered from all other sample groups (IBS-D vs control, P = 0.01; IBS-D vs IBS-M, P = 0.00; IBS-D vs IBS-C, P = 0.05). Significant differences were also observed in the levels of distinct phylotypes using relative values in proportion to the total amount of bacteria. A phylotype with 85% similarity to C. thermosuccinogenes was quantified in significantly different quantities among the IBS-D and control subjects (-4.08 +/- 0.90 vs -3.33 +/- 1.16, P = 0.04) and IBS-D and IBS-M subjects (-4.08 +/- 0.90 vs -3.08 +/- 1.38, P = 0.05). Furthermore, a phylotype with 94% similarity to R. torques was more prevalent in IBS-D patients' intestinal microbiota than in that of control subjects (-2.43 +/- 1.49 vs -4.02 +/- 1.63, P = 0.01). A phylotype with 93% similarity to R. torques was associated with control samples when compared with IBS-M (-2.41 +/- 0.53 vs -2.92 +/- 0.56, P = 0.00). Additionally, a R. bromii-like phylotype was associated with IBS-C patients in comparison to control subjects (-1.61 +/- 1.83 vs -3.69 +/- 2.42, P = 0.01). All of the above mentioned phylotype specific alterations were independent of the effect of time. CONCLUSION: Significant phylotype level alterations in the intestinal microbiotas of IBS patients were observed, further emphasizing the possible contribution of the gastrointestinal microbiota in IBS.

The fecal microbiota of irritable bowel syndrome patients differs significantly from that of healthy subjects.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) is a significant gastrointestinal disorder with unknown etiology. The symptoms can greatly weaken patients' quality of life and account for notable economical costs for society. Contribution of the gastrointestinal microbiota in IBS has been suggested. Our objective was to characterize putative differences in gastrointestinal microbiota between patients with IBS and control subjects. These differences could potentially have a causal relationship with the syndrome. METHODS: Microbial genomes from fecal samples of 24 patients with IBS and 23 controls were collected, pooled in a groupwise manner, and fractionated according to their guanine cytosine content. Selected fractions were analyzed by extensive high-throughput 16S ribosomal RNA gene cloning and sequencing of 3753 clones. Some of the revealed phylogenetic differences were further confirmed by quantitative polymerase chain reaction assays on individual samples. RESULTS: The coverage of the clone libraries of IBS subtypes and control subjects differed significantly (P < .0253). The samples were also distinguishable by the Bayesian analysis of bacterial population structure. Moreover, significant (P < .05) differences between the clone libraries were found in several bacterial genera, which could be verified by quantitative polymerase chain reaction assays of phylotypes belonging to the genera Coprococcus, Collinsella, and Coprobacillus. CONCLUSIONS: The study showed that fecal microbiota is significantly altered in IBS. Further studies on molecular mechanisms underlying these alterations are needed to elucidate the exact role of intestinal bacteria in IBS.

Analysis of the fecal microbiota of irritable bowel syndrome patients and healthy controls with real-time PCR.

OBJECTIVE: The gut microbiota may contribute to the onset and maintenance of irritable bowel syndrome (IBS). In this study, the microbiotas of patients suffering from IBS were compared with a control group devoid of gastrointestinal (GI) symptoms. METHODS: Fecal microbiota of patients (n = 27) fulfilling the Rome II criteria for IBS was compared with age- and gender-matched control subjects (n = 22). Fecal samples were obtained at 3 months intervals. Total bacterial DNA was analyzed by 20 quantitative real-time PCR assays covering approximately 300 bacterial species. RESULTS: Extensive individual variation was observed in the GI microbiota among both the IBS- and control groups. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. Average results from three fecal samples suggested differences in the Clostridium coccoides subgroup and Bifidobacterium catenulatum group between IBS patients (n = 21) and controls (n = 15). Of the intestinal pathogens earlier associated with IBS, no indications of Helicobacter spp. or Clostridium difficile were found whereas one case of Campylobacter jejuni was identified by sequencing. CONCLUSIONS: With these real-time PCR assays, quantitative alterations in the GI microbiota of IBS patients were found. Increasing microbial DNA sequence information will further allow designing of new real-time PCR assays for a more extensive analysis of intestinal microbes in IBS.

Comparison of real-time PCR with SYBR Green I or 5'-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria.

PCR primers and hybridization probes were designed for the 16S rRNA genes of six bacterial species or groups typically present in human faeces or used in the dairy industry. The primers and probes were applied for quantification of the target bacterial genomes added in artificial DNA mixtures or faecal DNA preparations, using dot-blot hybridization and real-time PCR with SYBR Green I and TaqMan chemistries. Dot-blot hybridization with (33)P-labelled oligonucleotide probes was shown to detect a 10 % target DNA fraction present in mixed DNA samples. Applicability of the rDNA-targeted oligonucleotide probes without pre-enrichment of the 16S gene pool by PCR was thus limited to the detection of the predominant microbial groups. Real-time PCR was performed using a 96-well format and was therefore feasible for straightforward analysis of large sample amounts. Both chemistries tested could detect and quantify a subpopulation of 0.01 % from the estimated number of total bacterial genomes present in a population sample. The linear range of amplification varied between three and five orders of magnitude for the specific target genome while the efficiency of amplification for the individual PCR assays was between 88.3 and 104 %. Use of a thermally activated polymerase was required with the SYBR Green I chemistry to obtain a similar sensitivity level to the TaqMan chemistry. In comparison to dot-blot hybridization, real-time PCR was easier and faster to perform and also proved to have a superior sensitivity. The results suggest that real-time PCR has a great potential for analysis of the faecal microflora.