Oral Administration of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> for Modulating Gastrointestinal Microflora in Mice

This study aimed to evaluate the safety of Clostridium butyricum and to investigate the effect of C. butyricum on mice ecosystem in the intestinal tract by way of examining the population of different microorganisms isolated from caecal contents. We firstly evaluated the safety of C. butyricum using acute toxicity test and Ames test. Then forty male BALB/c mice were divided into the following four treatment groups, each consisting of ten mice: normal group, low-dose group, medium-dose group and high-dose group. Caecal contents were removed aseptically, immediately placed into an anaerobic chamber, and dissolved in sterile pre-reduced PBS. The determination of Enterococcus spp., Enterobacter spp., Lactobacillus spp., Bifidobacterium spp. and Clostridium perfringens was analyzed by the spread plate method, cell morphologies and biochemical profiles. The results showed the oral maximum tolerated dose of C. butyricum was more than 10 g/kg body weight in mice and no mutagenicity judged by negative experimental results of Ames test. And in medium- and high-dose groups, the populations of Bifidobacterium spp. and Lactobacillus spp. increased in caecum, as well as the ratios of Bifidobacterium spp. and Lactobacillus spp. to Clostridium perfringens (P < 0.01) as compared with the normal group. This research showed the intake of C. butyricum significantly improved the ecosystem of the intestinal tract in BALB/c mice by increasing the amount of probiotics and reducing the populations of unwanted bacteria.     

Dietary level of fibre and age at weaning affect the proliferation of Clostridium perfringens in the caecum, the incidence of Epizootic Rabbit Enteropathy and the performance of fattening rabbits

An experiment was conducted to investigate the effects of dietary fibre content and weaning age on Clostridium perfringens proliferation in the caecum and fattening mortality in growing rabbits farmed in a facility having Epizootic Rabbit Enteropathy. The experiment consisted in a 2 × 2 factorial arrangement with two weaning ages (28 days vs. 42 days) and two levels of dietary neutral detergent fibre assayed with a heat stable amylase and expressed exclusive of residual ash (aNDFom; 330 g/kg vs. 425 g/kg). Controls were made during two consecutive experimental periods that differed in hygienic environmental conditions by modifying the intensity of cleaning and disinfection in the farm previous to the trial. An interaction (P<0.001) was detected among the independent variables studied on Cl. perfringens enumeration in the caecal contents, as minimal values for this trait were obtained in non-medicated animals reared in a clean environment, and especially when they were weaned at a later age and fed the diet with the lower fibre content. The treatments studied also led to a variation in fattening mortality (from 4.7% to 34.0%), which was highly and positively correlated (P<0.001) to the average Cl. perfringens caecal counts in each combination of treatments. The results of the current study indicate that high counts of Cl. perfringens in the caecum can be used as an indicator of Epizootic Rabbit Enteropathy, and suggest that strategies designed to control its proliferation in the caecum might help to limit fattening mortality in rabbit fed diets not-medicated with antibiotics.     

Effect of apple intake on fecal microbiota and metabolites in humans

The effects of apple intake on the fecal flora, water content, pH, and metabolic activities in eight healthy volunteers and the utilization of apple pectin in vitro were investigated. Although several isolates of Bifidobacterium, Lactobacillus, Enterococcus, and the Bacteroides fragilis group utilized apple pectin, most isolates of Escherichia coli, Collinsela aerofaciense, Eubacterium limosum, and Clostridium perfringens could not. When fecal samples from healthy adults were incubated in liquid broth with apple pectin present or absent, the numbers of Bifidobacterium and Lactobacillus in the former were higher than those in the later. After the intake of apples (2 apples a day for 2 weeks) by eight healthy adult humans, the number of bifidobacteria in feces increased (p < 0.05 on day 7 and p < 0.01 on day 14 of the intake period), and the numbers of Lactobacillus and Streptococcus including Enterococcus tended to increase. However, lecithinase-positive clostridia, including C. perfringens, decreased (p < 0.05), and Enterobacteriaceae and Pseudomonas tended to decrease. Moreover, the concentrations of fecal acetic acid tended to increase on apple intake. The fecal ammonia concentration showed a tendency to reduce and fecal sulfide decreased (p < 0.05) on apple intake. These findings indicate that apple consumption is related to an improved intestinal environment, and apple pectin is one of the effective apple components improving the fecal environment.     

The enteric toxins of Clostridium perfringens

The Gram-positive pathogen Clostridium perfringens is a major cause of human and veterinary enteric disease largely because this bacterium can produce several toxins when present inside the gastrointestinal tract. The enteric toxins of C. perfringens share two common features: (1) they are all single polypeptides of modest (~25–35 kDa) size, although lacking in sequence homology, and (2) they generally act by forming pores or channels in plasma membranes of host cells. These enteric toxins include C. perfringens enterotoxin (CPE), which is responsible for the symptoms of a common human food poisoning and acts by forming pores after interacting with intestinal tight junction proteins. Two other C. perfringens enteric toxins, -toxin (a bioterrorism select agent) and -toxin, cause veterinary enterotoxemias when absorbed from the intestines; - and -toxins then apparently act by forming oligomeric pores in intestinal or extra-intestinal target tissues. The action of a newly discovered C. perfringens enteric toxin, 2 toxin, has not yet been defined but precedent suggests it might also be a pore-former. Experience with other clostridial toxins certainly warrants continued research on these C. perfringens enteric toxins to develop their potential as therapeutic agents and tools for cellular biology.

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Immunization against <Emphasis Type="Italic">Clostridium perfringens</Emphasis> cells elicits protection against <Emphasis Type="Italic">Clostridium tetani</Emphasis>in mouse model: identification of cross-reactive proteins using proteomic methodologies

Background  

Clostridium tetani and Clostridium perfringens are among the medically important clostridial pathogens causing diseases in man and animals. Several homologous open reading frames (ORFs) have been identified in the genomes of the two pathogens by comparative genomic analysis. We tested a likelihood of extensive sharing of common epitopes between homologous proteins of these two medically important pathogens and the possibility of cross-protection using active immunization.     

Identification of a lambda toxin-negative Clostridium perfringens strain that processes and activates epsilon prototoxin intracellularly

Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which is one of the most potent clostridial toxins and is involved in enteritis and enterotoxemias of domestic animals. ETX is produced initially as an inactive prototoxin that is typically then secreted and processed by intestinal proteases or possibly, for some strains, lambda toxin. During the current work a unique C. perfringens strain was identified that intracellularly processes epsilon prototoxin to an active form capable of killing MDCK cells. This activated toxin is not secreted but instead is apparently released upon lysis of bacterial cells entering stationary phase. These findings broaden understanding of the pathogenesis of type B and D infections by identifying a new mechanism of ETX activation.     

Screening Escherichia coli, Enterococcus faecalis, and Clostridium perfringens as Indicator Organisms in Evaluating Pathogen-Reducing Capacity in Biogas Plants

This study was conducted to identify an indicator organism(s) in evaluating the pathogen-reducing capacity of biogas plants. Fresh cow manure containing 10⁴ to 10⁵ colony forming unit (CFU) per milliliter of Escherichia coli and Enterococcus faecalis along with an inoculated Clostridium perfringens strain were exposed to 37°C for 15 days, 55°C for 48 h, and 70°C for 24 h. C. perfringens was the most heat-resistant organism followed by E. faecalis, while E. coli was the most heat-sensitive organism. E. coli was reduced below detection limit at all temperatures with log₁₀ reductions of 4.94 (10 s), 4.37 (40 min), and 2.6 (5 days) at 70°C, 55°C, and 37°C, respectively. Maximum log₁₀ reductions for E. faecalis were 1.77 at 70°C (1 day), 1.7 at 55°C (2 days) and 3.13 at 37°C (15 days). For C. perfringens, maximum log₁₀ reduction at 37°C was 1.35 log₁₀ units (15 days) compared to less than 1 unit at 55 and 70°C. Modeling results showed that E. faecalis and C. perfringens had higher amount of heat-resistant fraction than E. coli. Thus, E. faecalis and C. perfringens can be used as indicator organisms to evaluate pathogen-reducing capacity in biogas plants at high temperatures of 55°C and 70°C while at 37°C E. coli could also be included as indicator organism.     

Construction and characterization of a clostripain-like protease-deficient mutant of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> as a strain for clostridial gene expression

The inherent difficulty of expressing clostridial AT-rich genes in a heterologous host has limited their biotechnological application. We previously reported a plasmid for high-level expression of clostridial genes in Clostridium perfringens (Takamizawa et al., Protein Expr Purif 36:70–75, 2004). In this study, we examined the extracellular proteases of C. perfringens strain 13. Zymographic analysis and caseinase assaying of a culture supernatant showed that it contained a protease activated by dithiothreitol and Ca²⁺, suggesting that clostripain-like protease (Clp) is the most likely candidate for the major extracellular protease. Disruption of the clp gene by homologous recombination markedly decreased the level of caseinase activity in the culture supernatant. Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Clp⁻ mutant but not the wild type strain increased the levels of many polypeptides in the culture supernatant after the late exponential growth phase. Such polypeptides included both cytoplasmic and secretory proteins, suggesting proteins secreted or released into the medium were degraded by Clp. To assess the effects of Clp on the productivity and stability of recombinant proteins, 74-kDa NanI sialidase was expressed in the two strains. The mutant strain produced a higher level of NanI activity than the wild type strain. Furthermore, under the conditions where Clp was activated, NanI was degraded easily in the latter culture but not in the former one. These results indicate that the Clp⁻ mutant could serve as a useful strain for efficiently expressing and preparing protease-free clostridial proteins.     

Galacto-oligosaccharide production using microbial β-galactosidase: current state and perspectives

A recombinant β-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg⁻¹ by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80oC, and the half-life at 70oC was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-β-d-galactopyranoside (pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-β-d-glucopyranoside (pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of β-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 β-galactosidases. The results indicated that the enzyme was a β-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among β-galactosidases. When 50 g l⁻¹ galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l⁻¹ glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l⁻¹), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l⁻¹) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min.     

Differential proteomic analysis of Clostridium perfringens ATCC13124; identification of dominant, surface and structure associated proteins

Background  

Clostridium perfringens is a medically important clostridial pathogen causing diseases in man and animals. To invade, multiply and colonize tissues of the host, a pathogen must be able to evade host immune system, and obtain nutrients essential for growth. The factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis. Many of the virulence determinants and putative vaccine candidates for bacterial pathogens are known to be surface localized.     

The effect of inclusion of a high lactose supplement in finishing diets on nutrient digestibility,nitrogen excretion,volatile fatty acid concentrations and ammonia emission from boars

An experiment (complete randomised design) was conducted to investigate the use of lactose in finishing pig diets on nitrogen excretion patterns and ammonia emissions. Diets were formulated to have similar digestible energy (13.8 MJ/kg) and total lysine (11.0 g/kg) contents. Thirty boars (58 kg) were assigned to one of the five dietary treatments (six per treatment) as follows: (T1) 0 g Lactofeed/kg (860 g whey permeate/kg, 140 g soya bean meal/kg, Volac International, UK) (LF70); (T2) 40 g LF70/kg; (T3) 80 g LF70/kg; (T4) 120 g LF70/kg; and (T5) 160 g LF70/kg. After a 14-day adaptation period, pigs were housed in metabolism crates and faeces and urine were collected for nitrogen balance and ammonia emission. NH₃–N emission was measured over 10 days using a laboratory scale procedure. Coefficient of total tract apparent digestibility (CTTAD) for dry matter and gross energy digestibility were affected by dietary LF70 inclusion (quadratic, P<0.05). T1 had a higher urinary N and total N excretion (P<0.1), a lower N retention (as a proportion of intake) (P<0.05) than the LF70 supplemented diets. T1 had a higher NH₃–N per gram of N intake from days 0 to 4 (P<0.05) than the LF70 supplemented diets. In the faeces isobutyric acid and isovaleric acid were affected by dietary LF70 level (quadratic, P<0.01). There was a linear decrease in the ratio of acetic acid: propionic acid with increasing LF70 levels (P<0.05). In the caecum and colon lactobacilli concentrations were affected by dietary LF70 inclusion (quadratic, P<0.05). In conclusion, the current study demonstrates that the incorporation of lactofeed in finisher pig diets increases CTTAD for gross energy and the concentration of lactobacilli in the hind-gut and decreases total nitrogen excretion. The inclusion of lactofeed also reduces the quantity of ammonia emitted from fresh manure in the first 4 days of storage.     

Reliability of mCP method for identification of Clostridium perfringens from faecal polluted aquatic environments

Aims: The purpose of the work was to evaluate the mCP method to correctly identify and enumerate Clostridium perfringens that are present in surface waters impacted by a mixture of faecal pollution sources. Methods: Clostridium perfringens were enumerated and isolated from sewage influent, surface water and suspended sediments using the mCP method. Molecular characterization of isolates was performed using species‐specific PCR, along with full‐length sequencing of the 16S rRNA gene for a subset of isolates. Results: The environmental isolates were presumptively identified as C. perfringens based on utilization of sucrose, inability to ferment cellobiose and a positive action for acid phosphatase activity. All isolates (n = 126) were classified as C. perfringens based on positive results with species‐specific PCR with a subset confirmed as C. perfringens based on the 16S rRNA gene identity. Conclusions: The molecular results indicated all of the presumptive positive isolates were C. perfringens regardless of the source, e.g. sewage influent or environmental water samples. Sequencing revealed that C. perfringens obtained from sewage and the aquatic environment were nearly identical (c. 99·5% similarity). Significance and Impact of the Study: From this study we conclude that the mCP method is a robust approach to enumerate and isolate C. perfringens from aquatic environments that receive diverse sources of faecal pollution.     

Efficacy of Probiotic Milk Formula on Blood Lipid and Intestinal Function in Mild Hypercholesterolemic Volunteers: A Placebo-control,Randomized Clinical Trial

Several studies have reported that probiotics could modulate host lipid metabolism via altering the intestinal microbiota. Hence, the current study was aimed to assess the efficacy of a mixture of probiotic-contained milk formula (PMF) with three different bacterial strains [Lactobacillus acidophilus (La5), Lactobacillus casei (TMC), Bifidobacterium lactis (Bb12)] on lipid profile and intestinal function in healthy mild hypercholesterolemic volunteers. Totally, 40 healthy mild hypercholesterolemic subjects (180–220 mg/dL) were randomly assigned into two groups as placebo or experimental group. All the subjects were requested to drink either PMF (experimental) or skimmed milk drink formula-placebo (30 g mixed with 200 mL of water) for 10 weeks and continued by 2 weeks of the follow-up period. Supplementation of PMF for 10 weeks significantly improved (p < 0.05) the fecal weight, fecal movement (decreased fecal gastrointestinal passing time) by improving intestinal microbiota (increasing beneficial bacterial species like Lactobacillus, Bifidobacterium spp.), and lag time of low-density lipoprotein (LDL) oxidation. Also, intake of PMF substantially reduced (p < 0.05) the levels of total cholesterol (TC; 8.1%) and low-density lipoprotein cholesterol (LDL-c; 10.4%) and thus showcasing its cardioprotective efficacy. PMF considerably improves gastrointestinal function by modulating fecal movement, intestinal microbiota, and decrease cholesterol and might be helpful in the management of hypercholesterolemia.

     

Clostridial Bacteremia During the First Year of Life: an Analysis of 53 Patients Including Two New Cases

Anaerobic bacteremias, and more specifically clostridial bacteremias, appear to be quite uncommon in infants. This report includes a detailed analysis of 53 infant pediatric patients with clostridial bacteremia, including 51 previously reported cases and two new cases. There were 54 clostridial cultures from these 53 patients;Clostridium perfringens (50%) and C. butyricum (25.9%) together accounted for nearly 76% of clostridia isolated. Only eight of the 54 blood cultures (14.8%) were also positive for one or more non-clostridial bacteria. Patient age, available for 43 of 53 infants, ranged from 1 to 182 days (mean 21 days, median 5 days). Among patients for whom information on each of multiple clinical parameters was available, the following were identified: male to female ratio 2.0; presence of underlying gastrointestinal disease (usually necrotizing enterocolitis) seen in 66% of infants; administration of antibiotic therapy in 80.4%; and patient survival of 56.9%. No statistically significant differences (P<0.05) were noted when survival was assessed against: gender, age at onset of bacteremia (18 days or less vs. greater than 18 days), and birth weight (less than 1500 g vs. greater than 1500 g). The presence of underlying gastrointestinal disease did have a statistically significant negative impact upon survival (P<0.025). Unexpectedly, the use of antibiotic therapy was associated with a statistically significant negative impact upon survival, which disappeared when the 14 C. butyricum cases were dropped from statistical consideration. The significance of clostridial bacteremia during infancy and its associated antibiotic therapy are reviewed and summarized in this report.     

Molecular identification of intestinal microflora in Takifugu niphobles

We investigated the intestinal microflora of coastal fish including Takifugu niphobles using both culture techniques and library cloning. As a result, the numbers of bacteria appeared on agar media were 1.0 × 10⁴ to 1.4 × 10⁹ CFU/g (colony forming units/gram), whereas those of total bacteria stained with 4′,6-diamidino-2-phenylindole were 4.7 × 10¹⁰ to 1.9 × 10¹¹ cells/gram, irrespective of different fish species. In addition, the culture technique showed that the intestinal microflora in all specimens was mainly composed of the genus Vibrio. In contrast, the direct count method showed that spirochaetes with length of 2.5-4.5 μm were present in the intestinal contents of T. niphobles at high densities, whereas such bacteria could not be detected in those of other fish species. Library cloning yielded the sequences of 16S rRNA genes that were divided into seven taxonomic categories of bacteria including Actinobacteria, Bacilli, Clostridia, Gammaproteobacteria, Mollicutes, Spirochaetes and an unclassified bacterial group. These results demonstrate that the molecular diversity of the intestinal bacteria in T. niphobles based on the clone library method reflects the direct observation by fluorescence microscopy to some extent.     

Clinicopathological and immunological studies on toxoids vaccine as a successful alternative in controlling clostridial infection in broilers

The present study was conducted to evaluate the efficacy and safety of three vaccination regimes of Clostridium perfringens (C. perfringens) type A, C and combined A&C toxoids based on their clinical signs and immunological effects. The vaccines were administered two times at two weeks interval (7 & 21 days old), then the birds were challenged (35 days old) with virulent strains of C. perfringens type A, C and combined A&C. Blood samples were taken one week after the first and second vaccination as well as after challenge. The evaluated parameters in this study included: clinical signs, gross intestinal lesions, complete blood count (CBC), serum protein, liver profiles, and enzyme-linked immunosorbent assay (ELISA) test for detecting serum antibody titers. The results revealed that immunization of broilers with C. perfringens type A, C and combined A&C toxoids resulted in a significant decrease in numbers of chickens with intestinal lesions particularly with the A&C toxoids vaccine. Results of the CBC values were significantly increased in all treated groups and challenged groups. Total leukocytic count decreased in challenged non vaccinated group while increased in challenged vaccinated birds. Results of biochemical assays implicated that there were a significant increase in serum protein and liver profiles. ELISA results explored a significant increase in antibody titers after immunization of broilers with C. perfringens type A, C and combined A&C toxoids particularly after the second dose of vaccination. We concluded that immunization of broilers with toxoid vaccines particularly the combined type A & C is safe, well-tolerated and can protect broiler chickens against necrotic enteritis particularly after the second booster dose of the vaccine.     

16S rRNA gene sequence‐based analysis of clostridia related to conversion of germfree mice to the normal state

Aims: To determine phylogenetic groups of clostridia inhabiting the mouse intestine that are essential for normalization of germfree (GF) mice. Methods and Results: Using both the culture method and cloning, clostridia inhabiting the mouse intestine were isolated, and phylogenetic analysis based on 16S rRNA gene sequences was carried out. As a result, the isolates were found to have novel sequences, and no isolate was determined to be identical to previously known identified clostridia. Although the taxonomy of mouse intestinal clostridia was complex, many of them belonged to Clostridium clusters XIVa and IV in conventional (CV) and limited flora mice and ex‐germfree mice administered chloroform‐treated CV mouse faeces. The clostridia that belonged to cluster XIVa were most often present and showed the highest diversity. Conclusions: Clostridia belonging clusters XIVa and IV are dominant in the mouse intestine as in other gut ecosystems. The novel groups in these clusters are essential for normalization of GF mice. Significance and Impact of the Study: The results of this study can be applied in the strict control of mouse intestinal microbiota and will provide important information for normalization of GF mice and also for research on microbiology of the mouse intestine.     

Comparison of alternatives to in‐feed antimicrobials for the prevention of clinical necrotic enteritis

Aims: The capacity for Lactobacillus johnsonii and an organic acid (OA) blend to prevent Clostridium perfringens‐induced clinical necrotic enteritis (NE) in chickens was studied. Methods and Results: Cobb 500 birds were allocated into six groups (n = 25 birds/pen, eight pens/treatment); Unchallenged, Challenged, Antimicrobial (zinc bacitracin (ZnB)/monensin), OA, probiotic Lact. johnsonii and probiotic sham (Phosphate–buffered saline). All birds were challenged with Eimeria spp. and Cl. perfringens except for unchallenged controls. Birds fed antimicrobials were protected from NE development as indicated by maintenance of body weight, low mortality and clostridium levels, and decreased intestinal macroscopic lesion scores compared to challenged controls (P < 0·05). Lactobacillus johnsonii‐fed birds had reduced lesion scores, whilst OA‐fed birds had decreased Cl. perfringens levels. Both Lact. johnsonii and OA‐fed birds had improved feed efficiency between days 0 and 28 compared to challenged controls; however, mortality and body weights were not improved by either treatment. Microbial profiling indicated that the challenge procedure significantly altered the jejunal microbiota. The microbiota of antimicrobial‐fed birds was significantly different from all other groups. Conclusions: Whilst Lact. johnsonii and OA altered specific intestinal parameters, significant protection against NE was not observed. Significance and Impact of the Study: Lactobacillus johnsonii and OA did not prevent NE; however, some improvements were evident. Other related treatments, or combinations of these two treatments, may provide greater protection.     

Impact of beta-glucan on the faecal microbiota of polypectomized patients: a pilot study

Beta-glucans are polysaccharides present in the cell walls of higher plants, in the seeds of some cereals, and certain yeasts and fungi also produce them. It is suggested that they exhibit, among many other health benefits, protective effects against carcinogenesis in the colon, but there is not enough human data to support this. The aim of the study was to determine the effect of barley-derived beta-glucan in the gut microbiota of polypectomized patients. Subjects were randomly assigned to consume 125 g of bread per day with beta-glucan (3 g/d), or without (placebo group), for 3 months. Thirty-three polypectomized men and women (mean age 57.6 years) were recruited into the study, but only 20 completed. Subjects did not consume any probiotics, prebiotics or antibiotics 2 months prior the intervention, or during the study. Stool samples were collected at baseline, on days 30 and 90 of intervention, as well as 2 weeks after the intervention, for enumeration of total aerobes and anaerobes, coliforms, E. coli, enterococci, Bacteroides spp., Clostridium perfringens, bifidobacteria, lactobacilli and Candida spp. Faecal bacterial enzyme activity (beta-glucuronidase and beta-glucosidase), pH, faecal moisture and the concentration of volatile fatty acids in the faeces were measured. Gastrointestinal symptoms were also recorded. Overall, no significant differences were observed in bacterial viable counts between the two feeding groups. Group specific analysis for β-glucan group revealed significantly decreased total coliform counts on the 30th day of the trial compared to the baseline (p = 0.041). Clostridium perfringens concentration increased without reaching statistical significance, on the 30th day, while it decreased significantly on the 90th day of the intervention compared to the 30th day (p = 0.016). An increase was noted in the molar ratio of acetate on the 90th day of the trial compared to placebo (p = 0.018). The molar ratio of butyrate presented a trend to increase on the 30th day, which decreased (p = 0.013) on the 90th day and then increase 2 weeks after the intervention (p = 0.017) compared to placebo. A decrease was recorded in the β-glucan group in the bloating and abdominal pain score after the 30th day of the intervention (Day 30–37) compared to placebo. During β-glucan administration we did not observe any changes on beta-glucuronidase or beta-glucosidase activity, faecal pH, or on faecal moisture.     

Changes in the cell wall of Clostridium species following passage in animals

Morphological changes in clostridial isolates after animal passage with other flora in mixed infections were studied by utilizing a subcutaneous abscess model in mice. We used 26 isolates of 7 clostridial species, and one isolate each of Bacteroides fragilis and Klebsiella pneumoniae. Abscesses were induced by all 7 Clostridium perfringens and 3 Clostridium butyricum isolates and by some of the other isolates. A thick granular wall prior to animal inoculation was shown only in C. perfringens, C. butyricum, and C. difficile. This structure was observed in other clostridia only following their animal passage alone or when co-inoculated with K. pneumoniae or B. fragilis.