Impact of ß2-1 fructan on faecal community change: results from a placebo-controlled, randomised, double-blinded, cross-over study in healthy adults.

Healthy adults (n 30) participated in a placebo-controlled, randomised, double-blinded, cross-over study consisting of two 28 d treatments (ß2-1 fructan or maltodextrin; 3×5 g/d) separated by a 14-d washout. Subjects provided 1 d faecal collections at days 0 and 28 of each treatment. The ability of faecal bacteria to metabolise ß2-1 fructan was common; eighty-seven species (thirty genera, and four phyla) were isolated using anaerobic medium containing ß2-1 fructan as the sole carbohydrate source. ß2-1 fructan altered the faecal community as determined through analysis of terminal restriction fragment length polymorphisms and 16S rRNA genes. Supplementation with ß2-1 fructan reduced faecal community richness, and two patterns of community change were observed. In most subjects, ß2-1 fructan reduced the content of phylotypes aligning within the Bacteroides, whereas increasing those aligning within bifidobacteria, Faecalibacterium and the family Lachnospiraceae. In the remaining subjects, supplementation increased the abundance of Bacteroidetes and to a lesser extent bifidobacteria, accompanied by decreases within the Faecalibacterium and family Lachnospiraceae. ß2-1 Fructan had no impact on the metagenome or glycoside hydrolase profiles in faeces from four subjects. Few relationships were found between the faecal bacterial community and various host parameters; Bacteroidetes content correlated with faecal propionate, subjects whose faecal community contained higher Bacteroidetes produced more caproic acid independent of treatment, and subjects having lower faecal Bacteroidetes exhibited increased concentrations of serum lipopolysaccharide and lipopolysaccharide binding protein independent of treatment. We found no evidence to support a defined health benefit for the use of ß2-1 fructans in healthy subjects.

ß2-1 Fructan supplementation alters host immune responses in a manner consistent with increased exposure to microbial components: results from a double-blinded, randomised, cross-over study in healthy adults.

ß2-1 Fructans are purported to improve health by stimulating growth of colonic bifidobacteria, increasing host resistance to pathogens and stimulating the immune system. However, in healthy adults, the benefits of supplementation remain undefined. Adults (thirteen men, seventeen women) participated in a double-blinded, placebo-controlled, randomised, cross-over study consisting of two 28-d treatments separated by a 14-d washout period. Subjects' regular diets were supplemented with ß2-1 fructan or placebo (maltodextrin) at 3×5 g/d. Fasting blood and 1-d faecal collections were obtained at the beginning and at the end of each phase. Blood was analysed for clinical, biochemical and immunological variables. Determinations of well-being and general health, gastrointestinal (GI) symptoms, regularity, faecal SCFA content, residual faecal ß2-1 fructans and faecal bifidobacteria content were undertaken. ß2-1 Fructan supplementation had no effect on blood lipid or cholesterol concentrations or on circulating lymphocyte and macrophage numbers, but significantly increased serum lipopolysaccharide, faecal SCFA, faecal bifidobacteria and indigestion. With respect to immune function, ß2-1 fructan supplementation increased serum IL-4, circulating percentages of CD282+/TLR2+ myeloid dendritic cells and ex vivo responsiveness to a toll-like receptor 2 agonist. ß2-1 Fructans also decreased serum IL-10, but did not affect C-reactive protein or serum/faecal Ig concentrations. No differences in host well-being were associated with either treatment, although the self-reported incidence of GI symptoms and headaches increased during the ß2-1 fructan phase. Although ß2-1 fructan supplementation increased faecal bifidobacteria, this change was not directly related to any of the determined host parameters.

Effect of antimicrobial growth promoter administration on the intestinal microbiota of beef cattle.

BACKGROUND: Antimicrobial growth promoters (AGPs) are antimicrobial agents administered to livestock in feed for prolonged periods to enhance feed efficiency. Beef cattle are primarily finished in confined feeding operations in Canada and the USA, and the administration of AGPs such as chlortetracycline and sulfamethazine (Aureo S-700 G) is the standard. The impacts of AGPs on the intestinal microbiota of beef cattle are currently uncertain; it is documented that AGPs administered to beef cattle pass through the rumen and enter the intestine. To ascertain the impacts of Aureo S-700 G on the small and large intestinal microbiota of beef cattle (mucosa-associated and within digesta), terminal restriction fragment length polymorphism (T-RFLP) analysis and quantitative PCR (qPCR) for total bacteria were applied. Beef cattle were maintained in an experimental feedlot (five replicate pens per treatment), and AGP treatment cattle were administered Aureo S-700 G in feed, whereas control cattle were administered no antimicrobials. As the intestinal microbiota of beef cattle has not been extensively examined, clone library analysis was applied to ascertain the primary bacterial constituents of the intestinal microbiota. RESULTS: Comparative T-RFLP and qPCR analysis (n = 122 samples) revealed that bacterial community fingerprints and bacterial load within digesta differed from those associated with mucosa. However, the administration of Aureo S-700 G did not affect bacterial community fingerprints or bacterial load within the small and large intestine relative to control cattle. Analysis of >1500 near full length 16S rDNA clones revealed considerably greater bacterial diversity in the large relative to the small intestine of beef cattle. Mucosa-associated bacterial communities in the jejunum were dominated by Proteobacteria, and differed conspicuously from those in the ileum and large intestine. Although the ileum contained bacterial clones that were common to the jejunum as well as the cecum, Firmicutes clones associated with mucosa dominated in the ileum, cecum, and descending colon. In the descending colon, clone library analysis did not reveal a difference in the richness or diversity of bacterial communities within digesta relative to those associated with mucosa. However, T-RFLP analysis indicated a significant difference in T-RF relative abundance (i.e. difference in relative taxon abundance) between mucosa-associated and digesta communities attributed in part to the differential abundance of Bacteriodes, Alistipes, Oscillibacter, and unclassified Clostridiales. CONCLUSIONS: These data demonstrate that there was no significant difference in the composition of the predominant intestinal bacteria constituents within animals administered Aureo S-700 G and those not administered AGPs after a 28 day withdrawal period.

High-throughput species identification of enterococci using pyrosequencing.

Here we report the development and validation of an automated high-throughput pyrosequencing-based method for the reliable identification of isolated Enterococcus species. This method exploits the discrete species-specificity of hypervariable groES-EL spacer region and utilizes a universal dispensation order optimized for a wide range of Enterococcus species.

Antibiotics conspicuously affect community profiles and richness, but not the density of bacterial cells associated with mucosa in the large and small intestines of mice.

The influence of three antibiotics (bacitracin, enrofloxacin, and neomycin sulfate) on the mucosa-associated enteric microbiota and the intestines of mice was examined. Antibiotics caused conspicuous enlargement of ceca and an increase in overall length of the intestine. However, there were no pathologic changes associated with increased cecal size or length of the intestine. Conspicuous reductions in the richness of mucosa-associated bacteria and changes to community profiles within the small (duodenum, proximal jejunum, middle jejunum, distal jejunum, and ileum) and large (cecum, ascending colon, and descending colon) intestine occurred in mice administered antibiotics. Communities in antibiotic-treated mice were dominated by a limited number of Clostridium-like (i.e. clostridial cluster XIVa) and Bacteroides species. The richness of mucosa-associated communities within the small and large intestine increased during the 14-day recovery period. However, community profiles within the large intestine did not return to baseline (i.e. relative to the control). Although antibiotic administration greatly reduced bacterial richness, densities of mucosa-associated bacteria were not reduced correspondingly. These data showed that the antibiotics, bacitracin, enrofloxacin, and neomycin sulfate, administered for 21 days to mice did not sterilize the intestine, but did impart a tremendous and prolonged impact on mucosa-associated bacterial communities throughout the small and large intestine.

Distribution and characterization of ampicillin- and tetracycline-resistant Escherichia coli from feedlot cattle fed subtherapeutic antimicrobials.

BACKGROUND: Feedlot cattle in North America are routinely fed subtherapeutic levels of antimicrobials to prevent disease and improve the efficiency of growth. This practice has been shown to promote antimicrobial resistance (AMR) in subpopulations of intestinal microflora including Escherichia coli. To date, studies of AMR in feedlot production settings have rarely employed selective isolation, therefore yielding too few AMR isolates to enable characterization of the emergence and nature of AMR in E. coli as an indicator bacterium. E. coli isolates (n = 531) were recovered from 140 cattle that were housed (10 animals/pen) in 14 pens and received no dietary antimicrobials (control--5 pens, CON), or were intermittently administered subtherapeutic levels of chlortetracycline (5 pens-T), chlortetracycline + sulfamethazine (4 pens-TS), or virginiamycin (5 pens-V) for two separate periods over a 9-month feeding period. Phenotype and genotype of the isolates were determined by susceptibility testing and pulsed field gel electrophoresis and distribution of characterized isolates among housed cattle reported. It was hypothesized that the feeding of subtherapeutic antibiotics would increase the isolation of distinct genotypes of AMR E. coli from cattle. RESULTS: Overall, patterns of antimicrobial resistance expressed by E. coli isolates did not change among diet groups (CON vs. antibiotic treatments), however; isolates obtained on selective plates (i.e., M(A),M(T)), exhibited multi-resistance to sulfamethoxazole and chloramphenicol more frequently when obtained from TS-fed steers than from other treatments. Antibiograms and PFGE patterns suggested that AMR E. coli were readily transferred among steers within pens. Most M(T) isolates possessed the tet(B) efflux gene (58.2, 53.5, 40.8, and 50.6% of isolates from CON, T, TS, and V steers, respectively) whereas among the M(A) (ampicillin-resistant) isolates, the tem1-like determinant was predominant (occurring in 50, 66.7, 80.3, and 100% of isolates from CON, T, TS, and V steers, respectively). CONCLUSIONS: Factors other than, or in addition to subtherapeutic administration of antibiotics influence the establishment and transmission of AMR E. coli among feedlot cattle.

Characterization of tetracycline- and ampicillin-resistant Escherichia coli isolated from the feces of feedlot cattle over the feeding period.

The objective of this study was to investigate tetracycline and ampicillin resistance in Escherichia coli isolated from the feces of 50 crossbred steers housed in 5 feedlot pens. The steers were not administered antibiotics over a 246-day feeding period. A total of 216 isolates were selected for further characterization. The E. coli isolates were selected on MacConkey agar or on MacConkey agar amended with ampicillin (50 microg/mL) or tetracycline (4 microg/mL). Pulsed-field gel electrophoresis (PFGE) typing (XbaI digestion), screening against 11 antibiotics, and multiplex PCR for 14 tet and 3 beta-lactamase genes were conducted. Prevalence of antimicrobial resistance in E. coli at each sampling day was related both temporally and by pen. Multiplex PCR revealed that tet(B) was most prevalent among tetracycline-resistant isolates, whereas beta-lactamase tem1-like was detected mainly in ampicillin-resistant isolates. Our results suggest that antimicrobial resistance in E. coli populations persists over the duration of the feeding period, even in the absence of in-feed antibiotics. Many of the isolates with the same antibiograms had indistinguishable PFGE patterns. Characterization of the factors that influence the nature of this nonselective resistance could provide important information for consideration in the regulation of in-feed antimicrobials for feedlot cattle.

Selected antimicrobial resistance during composting of manure from cattle administered sub-therapeutic antimicrobials.

Composting is being increasingly employed for the recycling of nutrients in manure from the livestock industry. However, composting manure from animals fed antimicrobials has not been well characterized. In this study, compost windrows were prepared using manure collected from cattle (Bos Taurus L.) fed tylosin (TY), chlortetracycline-sulphamethazine (TS), and control cattle (no antimicrobials). The objectives of the 18-wk trial were to quantitatively assess the survival of total E. coli, E. coli resistant to ampicillin (Amp(r)) and tetracycline (Tet(r)), and select tetracycline (tet) and erythromycin resistance methylase (erm) genes. We found that while compost windrows did not reach the recommended temperature of 55 degrees C for 15 d, composting reduced high initial levels of total, Amp(r), and Tet(r) E. coli as early as Week 2. A significant antimicrobial effect on total (P = 0.04) and Amp(r) (P = 0.03) E. coli was observed. Significant antimicrobial x time interactions were observed from Week 0 to Week 3 (Total E. coli: P = 0.04; Amp(r): P = 0.02; Tet(r): P = <0.001). Low absolute abundance of tet and erm genes (<10(6) copies g(-1)) was found and the resistance genes displayed different dynamics; tet(A,C) and erm(A) increased marginally at Week 11 relative to Week 0 and 5 and the remaining genes (tet(G), RPP tet, erm(B), erm(C), erm(F), erm(T), and erm(X)) decreased for most time points and treatments. These results indicate that even though composting reduces antimicrobial resistant E. coli, tet and erm genes could still be detected. Our experiments reiterate advantages of polymerase chain reaction (PCR)-based quantitative assays over cultivation-based methods for the rapid identification of composting effectiveness in eliminating resistance genes before land application.

Diversity and distribution of commensal fecal Escherichia coli bacteria in beef cattle administered selected subtherapeutic antimicrobials in a feedlot setting.

Escherichia coli strains isolated from fecal samples were screened to examine changes in phenotypic and genotypic characteristics including antimicrobial susceptibility, clonal type, and carriage of resistance determinants. The goal of this 197-day study was to investigate the influence of administration of chlortetracycline alone (T) or in combination with sulfamethazine (TS) on the development of resistance, dissemination of defined strain types, and prevalence of resistance determinants in feedlot cattle. Inherent tetracycline resistance was detected in cattle with no prior antimicrobial exposure. Antimicrobial administration was not found to be essential for the maintenance of inherently ampicillin-resistant and tetracycline-resistant (Tet(r)) E. coli in control animals; however, higher Tet(r) E. coli shedding was observed in animals subjected to the two treatments. At day 0, high tetracycline (26.7%), lower sulfamethoxazole-tetracycline (19.2%), and several other resistances were detected, which by the finishing phase (day 197) were restricted to ampicillin-tetracycline (47.5%), tetracycline (31.7%), and ampicillin-tetracycline-sulfamethoxazole (20.8%) from both treated and untreated cattle. Among the determinants, bla(TEM1), tet(A), and sul2 were prevalent at days 0 and 197. Further, E. coli from day 0 showed diverse antibiogram profiles and strain types, which by the finishing phase were limited to up to three, irrespective of the treatment. Some genetically identical strains expressed different phenotypes and harbored diverse determinants, indicating that mobile genetic elements contribute to resistance dissemination. This was supported by an increased linked inheritance of ampicillin and tetracycline resistance genes and prevalence of specific strains at day 197. Animals in the cohort shed increasingly similar genotypes by the finishing phase due to animal-to-animal strain transmission. Thus, characterizing inherent resistance and propagation of cohort-specific strains is crucial for determining antimicrobial resistance in cattle.

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis.

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.

Bedding and within-pen location effects on feedlot pen runoff quality using a rainfall simulator.

Soluble salts, nutrients, and pathogenic bacteria in feedlot-pen runoff have the potential to cause pollution of the environment. A 2-yr study (1998-1999) was conducted at a beef cattle (Bos taurus) feedlot in southern Alberta, Canada, to determine the effect of bedding material [barley (Hordeum vulgare L.) straw versus wood chips] and within-pen location on the chemical and bacterial properties of pen-floor runoff. Runoff was generated with a portable rainfall simulator and analyzed for chemical content (nitrogen [N], phosphorus [P], soluble salts, electrical conductivity [EC], sodium adsorption ratio [SAR], dissolved oxygen [DO], and pH) and populations of three groups of bacteria (Escherichia coli, total coliforms, total aerobic heterotrophs at 27 degrees C) in 1998 and 1999. Bedding had a significant (P < or = 0.05) effect on NH4-N concentration and load in 1999, SO4 load in 1998, SO4 concentration and load in 1999, and total coliforms in both years; where these three variables were higher in wood than straw pens. Location had a significant effect on EC and concentrations of total Kjeldahl nitrogen (TKN), Na, K, SO4, and Cl in 1998, and total coliforms in both years. These seven variables were higher at the bedding pack than pen floor location, indicating that bedding packs were major reservoirs of TKN, soluble salts, and total coliforms. Significantly higher dissolved reactive phosphorus (DRP), total P, and NH4-N concentrations and loads at the bedding pack location in wood pens in 1998, and a similar trend for TKN concentration in 1999, indicated that this bedding-location treatment was a greater source of nutrients to runoff than the other three bedding-location treatments. Bedding, location, and their interaction may therefore be a potential tool to manage nutrients, soluble salts, and bacteria in feedlot runoff.

Characterization of condensed tannins purified from legume forages: chromophore production, protein precipitation, and inhibitory effects on cellulose digestion.

To identify simple screening tools for selecting condensed tannin (CT)-containing forages as candidate sources for further study, CT were isolated from nine legumes, and their molecular weights (MW), chromophore production, capacity to precipitate bovine serum albumin (BSA) and Fraction 1 protein (Rubisco) isolated from alfalfa, and inhibition of filter paper digestion were compared. Sources were as follows: leaves of sericea lespedeza (Lespedeza cuneata Dum.-Cours.), crown vetch (Coronilla varia L.), and sainfoin (Onobrychis viciifolia Scop.); stems of hedysarum (Hedysarum alpinum L.); seeds of alfalfa (Medicago sativa L.); and whole plants of birdsfoot trefoil (Lotus corniculatus var. corniculatus L.) and three varieties of big trefoil (Lotus pedunculatus Cav.), viz., Lotus uliginosus Schkuhr, L. uliginosus var. glabriusculus, and L. uliginosus var. villosus. Molecular weights and sizes (degrees of polymerization) of the CT varied considerably within and among plant species. Average MW ranged from 3036 Da (crown vetch) to 7143 Da (lespedeza). All CT exhibited greater capacity (w/w basis) to bind alfalfa Rubisco than BSA. Relative astringencies (microg CT required to precipitate 1 mg protein) against BSA ranged from 262.5 for CT from lespedeza to 435.5 for CT from L. corniculatus, and against Rubisco, from 49.6 (sainfoin) to 108.2 (alfalfa seed). Including CT at 300 microg/ml in cultures of Fibrobacter succinogenes reduced digestion of cellulose filter paper by 19.8% (sainfoin) to 92.4% (crown vetch) and increased the specific activity of cell-associated endoglucanase. There were no correlations between inhibitory effects of CT on filter paper digestion and (1) chromophore formation during CT assay by butanol-HCl, vanillin-HCl, or H2SO4; (2) precipitation of BSA or alfalfa Rubisco; and (3) MW of CT. The most inhibitory CT for cellulose digestion included those with broad and with narrow MW distributions. Sainfoin was the most desirable source of CT, as it had the highest capacity to bind alfalfa protein and was least inhibitory to cellulose digestion by F. succinogenes. This study suggests that these properties are not easily defined via chemical means, and that biological assays using rumen bacteria may help identify those CT with properties of nutritional interest.

Quantity and quality of runoff from a beef cattle feedlot in southern Alberta.

Southern Alberta, which has a cold climate dominated by strong chinook winds, has the highest density of feedlot cattle in Canada. However, the quantity and quality of runoff from beef cattle (Bos taurus) feedlots in this unique region has not been investigated. Our objectives were to compare runoff quantity (1998-2002) with catch-basin design criteria; determine concentrations of selected inorganic chemical parameters (1998-2000) in runoff in relation to water quality guidelines and the potential implications of irrigating adjacent crop-land; and determine if total heterotrophs, total coliforms, and Escherichia coli (1998-2000) persisted in the catch-basin water and soil. Runoff (< 0.1 to 42.5 mm) for a 24-h duration that included maximum peak discharge was less than the recommended design criteria of 90 mm based on runoff from 24 h of rainfall with a 30-yr return period. We found that curve numbers between 52 and 96 (mode of 90) were required to match the USDA Natural Resources Conservation Service predicted runoff and actual runoff volumes. Total P posed the greatest threat to water quality guidelines, and K posed the greatest threat for exceeding crop fertilizer requirements if catch-basin effluent was used as irrigation water. Water in the catch basin had continually high populations of E. coli throughout the study, with values ranging between log10 2 and log10 8 100 mL(-1). In contrast, soil in the catch basin generally had low populations of E. coli that were < log10 2 g(-1) wet wt., but at times higher populations between log10 2 and log10 6 g(-1) wet wt. were also found.

Bedding and seasonal effects on chemical and bacterial properties of feedlot cattle manure.

Nutrients, soluble salts, and pathogenic bacteria in feedlot-pen manure have the potential to cause pollution of the environment. A three-year study (1998-2000) was conducted at a beef cattle (Bos taurus) feedlot in southern Alberta, Canada to determine the effect of bedding material [barley (Hordeum vulgare L.) straw versus wood chips] and season on the chemical and bacterial properties of pen-floor manure. Manure was sampled for chemical content (N, P, soluble salts, electrical conductivity, and pH) and populations of four groups of bacteria (Escherichia coli, total coliforms, and total aerobic heterotrophs at 27 and 39 degrees C). More chemical parameters of manure were significantly (P < or = 0.05) affected by season (SO4, Na, Mg, K, Ca, sodium adsorption ratio [SAR], total C, NO3-N, NH4-N, total P, and available P) than by bedding (K, pH, total C, C to N ratio, NH4-N, and available P). Bedding had no significant (P > 0.05) effect on the four bacterial groups whereas season affected all four groups. Numbers of E. coli and total coliforms (TC) were significantly higher by 1.72 to 2.02 log10 units in the summer than the other three seasons, which was consistent with a strong positive correlation of E. coli and TC with air temperature. The low ratio of bedding to manure in the pens was probably the major cause of the lack of significant bedding effects. Bedding material and seasonal timing of cleaning feedlot pens and land application of manure may be a potential tool to manage nutrients, soluble salts, and pathogens in manure.

Fate of coliform bacteria in composted beef cattle feedlot manure.

The link between livestock production, manure management, and human health has received much public attention in recent years. Composting is often promoted as a means of sanitizing manure to ensure that pathogenic bacteria are not spread to a wider environment during land application. In a two-year study (1998 and 1999) in southern Alberta, we examined the fate of coliform bacteria during windrow composting of cattle (Bos taurus) manure from feedlot pens bedded with cereal straw or wood chips. Numbers of total coliforms (TC) and Escherichia coli declined as the composting period progressed. In 1998, TC levels (mean of both bedding types) were log10 7.86 cells g(-1) dry wt. for raw manure on Day 0, log10 3.38 cells g(-1) by Day 7, and log10 1.69 cells g(-1) by Day 14. More than 99.9% of TC and E. coli was eliminated in the first 7 d when average windrow temperatures ranged from 33.5 to 41.5 degrees C. The type of bedding did not influence the numbers of TC or E. coli. Dessication probably played a minor role in coliform elimination, since water loss was low (< 0.07 kg kg(-1)) in the first 7 d of composting. However, total aerobic heterotroph populations remained high (> 7.0 log10 CFU g(-1) dry wt., where CFU is colony forming units) throughout the composting period, possibly causing an antagonistic effect. Land application of compost, with its nondetectable levels of E. coli compared with raw manure, should minimize environmental risk in areas of intensive livestock production.