Global Microscopic Description of Nucleon-Nucleus Scattering with Quantified Uncertainties.

We develop for the first time a microscopic global nucleon-nucleus optical potential with quantified uncertainties suitable for analyzing nuclear reaction experiments at next-generation rare-isotope beam facilities. Within the improved local density approximation and without any adjustable parameters, we begin by computing proton-nucleus and neutron-nucleus optical potentials from a set of five nuclear forces from chiral effective field theory for 1800 target nuclei in the mass range 12≤A≤242 for energies between 0 MeV

Stored swine manure and swine faeces as reservoirs of antibiotic resistance genes.

Inclusion of low levels of antibiotics in the feed of domestic food animals promotes improved growth, animal performance and overall health benefits. However, this practice has come under scrutiny due to concerns over such feeding on bacterial antibiotic resistance (AR) and potential impact on human health. There is a paucity of data on the types and levels of AR genes that may be present in agricultural practices. Using PCR detection of AR genes, this study demonstrates that both stored swine manure and swine faeces harbour a variety of AR genes, and bacterial members of these communities contain genes that may move between micro-organisms. Thus, both ecosystems may serve as reservoirs of AR genes.

Microbial production of volatile sulphur compounds in the large intestine of pigs fed two different diets.

AIMS: To investigate the production of volatile sulphur compounds (VSC) in the segments of the large intestine of pigs and to assess the impact of diet on this production. METHODS AND RESULTS: Pigs were fed two diets based on either wheat and barley (STD) or wheat and dried distillers grains with solubles (DDGS). Net production of VSC and potential sulphate reduction rate (SRR) (sulphate saturated) along the large intestine were determined by means of in vitro incubations. The net production rate of hydrogen sulphide and potential SRR increased from caecum towards distal colon and were significantly higher in the STD group. Conversely, the net methanethiol production rate was significantly higher in the DDGS group, while no difference was observed for dimethyl sulphide. The number of sulphate-reducing bacteria and total bacteria were determined by quantitative PCR and showed a significant increase along the large intestine, whereas no diet-related differences were observed. CONCLUSION: VSC net production varies widely throughout the large intestine of pigs and the microbial processes involved in this production can be affected by diet. SIGNIFICANCE AND IMPACT OF THE STUDY: This first report on intestinal production of all VSC shows both spatial and dietary effects, which are relevant to both bowel disease- and odour mitigation research.

Effect of dietary inorganic sulfur level on growth performance, fecal composition, and measures of inflammation and sulfate-reducing bacteria in the intestine of growing pigs.

Two experiments investigated the impact of dietary inorganic S on growth performance, intestinal inflammation, fecal composition, and the presence of sulfate-reducing bacteria (SRB). In Exp. 1, individually housed pigs (n = 42; 13.8 kg) were fed diets containing 2,300 or 2,100 mg/kg of S for 24 d. Decreasing dietary S had no effect on ADG, ADFI, or G:F. In Exp. 2, pigs (n = 64; 13.3 kg) were fed diets containing 0, 0.625, 1.25, 2.5, or 5.0% CaSO(4), thereby increasing dietary S from 2,900 to 12,100 mg/kg. Two additional diets were fed to confirm the lack of an impact due to feeding low dietary S on pig performance and to determine if the increased Ca and P content in the diets containing CaSO(4) had an impact on growth performance. Pigs were fed for 35 d. Ileal tissue, ileal mucosa, and colon tissue were harvested from pigs fed the 0 and 5% CaSO(4) diets (low-S and high-S, respectively) to determine the impact of dietary S on inflammation-related mRNA, activity of mucosal alkaline phosphatase and sucrase, and pathways of inflammatory activation. Real-time PCR was used to quantify SRB in ileal and colon digesta samples and feces. Fecal pH, sulfide, and ammonia concentrations were also determined. There was no impact on growth performance in pigs fed the diet reduced in dietary S or by the increase of dietary Ca and P. Increasing dietary S from 2,900 to 12,100 mg/kg had a linear (P < 0.01) effect on ADG and a cubic effect (P < 0.05) on ADFI and G:F. Real-time reverse-transcription PCR analysis revealed that pigs fed high-S increased (P < 0.05) the relative abundance of intracellular adhesion molecule-1, tumor necrosis factor-α, and suppressor of cytokine signaling-3 mRNA, and tended (P = 0.09) to increase the relative abundance of IL-6 mRNA in ileal tissue. Likewise, pigs fed high-S had reduced (P < 0.05) abundance of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor-α and increased (P < 0.05) phospho-p44/p42 mitogen-activated protein kinase in ileal tissue, but there was no effect of dietary S on mucosal alkaline phosphatase or sucrase activity. Pigs fed the high-S diet had decreased (P < 0.05) total bacteria in ileal digesta, but increased (P < 0.05) prevalence of SRB in colon contents. Fecal sulfide was increased (P < 0.05) and fecal pH was deceased (P < 0.05) in pigs fed high-S. The data indicate that growing pigs can tolerate relatively high amounts of dietary inorganic S, but high dietary S content alters inflammatory mediators and intestinal bacteria.

The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome.

Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.

Development and comparison of SYBR Green quantitative real-time PCR assays for detection and enumeration of sulfate-reducing bacteria in stored swine manure.

AIMS: To develop and evaluate primer sets targeted to the dissimilatory sulfite reductase gene (dsrA) for use in quantitative real-time PCR detection of sulfate-reducing bacteria (SRB) in stored swine manure. METHODS AND RESULTS: Degenerate primer sets were developed to detect SRB in stored swine manure. These were compared with a previously reported primer set, DSR1F+ and DSR-R, for their coverage and ability to detect SRB communities in stored swine manure. Sequenced clones were most similar to Desulfovibrio sp. and Desulfobulbus sp., and these SRB populations differed within different manure ecosystems. Sulfur content of swine diets was shown to affect the population of Desulfobulbus-like Group 1 SRB in manure. CONCLUSIONS: The newly developed assays were able to enumerate and discern different groups of SRB, and suggest a richly diverse and as yet undescribed population of SRB in swine manure. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR assays described here provide improved and efficient molecular tools for quantitative detection of SRB populations. This is the first study to show population shifts of SRB in swine manure, which are a result of either the effects of swine diets or the maturity of the manure ecosystem.

Engineering of the gut commensal bacterium Bacteroides ovatus to produce and secrete biologically active murine interleukin-2 in response to xylan.

AIMS: The aim of this work was to engineer a gut commensal bacterium, Bacteroidesovatus, to produce and secrete a biologically active cytokine in a regulated manner as a basis for novel immunotherapies for chronic gut disorders. METHODS AND RESULTS: Bacteroides ovatus was engineered to produce murine interleukin-2 (MuIL2) intracellularly in response to xylan in culture media by inserting the MuIL2 gene into the xylanase operon of the organism. A second strain was engineered to secrete MuIL2 by adding Bacteroides fragilis enterotoxin secretion signal sequence to the protein. The recombinant strains produced MuIL2 only in the presence of xylan as determined by ELISA of cell lysates and culture supernatants. The IL2-dependent cell line CTLL-2 was used to demonstrate that MuIL2 produced by both B. ovatus strains was biologically active. This activity could be blocked by an anti-IL2 neutralizing antibody. The xylan-inducible nature of this system was demonstrated by RT-PCR. CONCLUSIONS: Bacteroides ovatus was successfully engineered to produce and secrete biologically active MuIL2 in a xylan-inducible manner. SIGNIFICANCE AND IMPACT OF THE STUDY: The production and secretion of a biologically active mammalian protein by a member of the gut microflora could lead to the development of new long-term immunotherapies for inflammatory gut diseases.

Application of group specific amplified rDNA restriction analysis to characterize swine fecal and manure storage pit samples.

Group specific amplified ribosomal-DNA restriction analysis was evaluated as a method to rapidly assess microbial community structure in swine fecal and manure storage pit samples. PCR primer sequences were evaluated for their specificity to ribosomal DNA from selected bacterial groups by optimizing annealing temperatures and determining specificity using a set of primer target and non-target organisms. A number of primer sets were identified targeting the following groups: Bacteroides-Prevotella, clostridial clusters I and II, clostridial clusters IX and XI, clostridial clusters XIVa and XIVb, Lactobacillus, Desulfovibrionaceae and Streptococcus-Lactococcus, as well as an universal primer set to represent total populations. Each bacterial group was digested with at least three restriction enzymes. We applied the group specific amplified ribosomal-DNA restriction analysis to swine fecal and manure storage pit samples obtained on two separate occasions. Fecal and manure storage pit samples obtained on the same day were more similar to each other than to any other samples. Results were consistent with 16S ribosomal DNA sequencing data from bacterial isolates and clones obtained from swine feces and manure storage pit. The group specific amplified ribosomal-DNA restriction analysis technique was able to rapid detect gross bacterial community differences among swine fecal and manure storage pit samples and determine groups of interest for more detailed examination.

Persistence and functional impact of a microbial inoculant on native microbial community structure, nutrient digestion and fermentation characteristics in a rumen model.

Small sub-unit (SSU) rRNA-targeted oligonucleotide probes were used to monitor the persistence of a genetically engineered bacterium inoculated in model rumens. Eight dual flow continuous culture fermenters were operated with either standard artificial saliva buffer or buffer with chondroitin sulfate (0.5 g/l) added. After 168 h of operation, fermenters were inoculated with Bacteroides thetaiotaomicron BTX (BTX), at approximately 1% of total bacteria. B. thetaiotaomicron was quantified using a species-specific probe and shown to persist in fermenters 144 h after inoculation (relative abundance 0.48% and 1.42% of total SSU rRNA with standard and chondroitin sulfate buffers, respectively). No B. thetaiotaomicron SSU rRNA was detected in fermenter samples prior to inoculation with strain BTX. Relative abundances of Bacteria, Eucarya and Archaea were not affected by either inoculation or buffer type. Fiber digestion, in particular the hemicellulose fraction, increased after strain BTX addition. Chondroitin sulfate addition to the buffer increased bacterial nitrogen flow in fermenters, but did not alter fiber digestion. Neither inoculum nor buffer type altered total short chain fatty acid (VFA) concentrations but proportions of individual VFA differed. In model rumens, B. thetaiotaomicron BTX increased fiber digestion when added to mixed ruminal microbes, independent of chondroitin sulfate addition; but further study is needed to determine effects on other fiber-digesting bacteria.

Identification of a broad-specificity xylosidase/arabinosidase important for xylooligosaccharide fermentation by the ruminal anaerobe Selenomonas ruminantium GA192.

Strains of Selenomonas ruminantium vary considerably in their capacity to ferment xylooligosaccharides. This ability ranges from strain GA192, which completely utilized xylose through xylotetraose and was able to ferment considerable quantities of larger oligosaccharides, to strain HD4, which used only the simple sugars present in the hydrolysate. The ability of S. ruminantium GA192 to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidase activities. The production of these activities appears to be regulated in response to carbon source used for growth. Both arabinosidase and xylosidase were induced by growth on xylose or xylooligosaccharides, but no activity was detected in glucose-or arabinose-grown cultures. A genetic locus from S. ruminantium GA192 was cloned into Escherichia coli JM83 that produced both xylosidase and arabinosidase activities. Analyses of crude extracts from the E. coli clone and S. ruminantium GA192 by using native polyacrylamide gel electrophoresis and methylumbelliferyl substrates indicated that a single protein was responsible for both activities. The enzyme expressed in E. coli was capable of degrading xylooligosaccharides derived from xylan. DNA sequencing of the locus demonstrated the presence of an open reading frame that encodes for a protein of 61,174 molecular weight.

Sequence analyses of a broad host-range plasmid containing ermT from a tylosin-resistant Lactobacillus sp. Isolated from swine feces.

Anaerobic bacteria resistant to the macrolide antibiotics tylosin and erythromycin were isolated from the feces of swine. One of the strains, 121B, was initially identified by 16S rDNA sequence analysis as an unknown Lactobacillus sp. The strain was found to contain at least two plasmids, one of which was capable of replicating and providing erythromycin and tylosin resistance to Bacillus subtilis, Streptococcus gordonii, and Escherichia coli. DNA sequence analyses of the 4,232-bp plasmid, p121BS, identified one open reading frame encoding a methylase gene highly similar (> 98% amino acid identity, > 99% DNA sequence identity) to the ermT gene from the Lactobacillus reuteri plasmid pGT633. This is only the second ermT gene to be reported. p121BS also contains two additional open reading frames with significant amino acid similarities to replication proteins from Lactobacillus and other Gram-positive bacteria.

Development of molecular methods for identification of Streptococcus bovis from human and ruminal origins.

Streptococcus bovis has been identified as a causative agent in humans for a variety of diseases, including endocarditis, meningitis, and septicemia. Identification of S. bovis strains of human origin in clinical settings has been problematic due to variations in biochemical tests as compared to ruminal strains of S. bovis, and other streptococcal species. DNA-DNA hybridization with chromosomal DNA from various S. bovis strains indicates that strains of human origin are different from those of ruminal origin. Specific probes have been designed from S. bovis 16S rDNA gene sequences that differentiate strains of human and ruminal origin by direct hybridization and PCR analyses. These techniques now allow for rapid identification of S. bovis strains for clinical and other scientific investigations.

Streptococcus bovis meningitis in an infant.

Streptococcus bovis is a nonenterococcal, group D streptococcus which has been identified as a causative agent for serious human infections, including endocarditis, bacteremia, and septic arthritis. Several cases of adult S. bovis meningitis have been reported, usually in association with underlying disease. In the neonatal period, it is an uncommon agent of meningitis. We report, to our knowledge, the third documented case of neonatal S. bovis meningitis in the English language literature. As in the previous cases, this neonate showed no anatomical or congenital immunologic lesion which might be expected to predispose the patient to meningitis. Sequencing of the 16S ribosomal DNA gene was performed and a new PCR test was used to secure a more reliable identification of the strain.

Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes.

A model rumen system, dual-flow continuous culture fermenters, was evaluated by two comparative criteria in two experiments using ribosomal (r)RNA-targeted DNA probes to compare key microbial groups in samples. The initial experiment measured temporal changes in population structure during adaptation of ruminal microbial populations in fermenters over 240 h. The fermenter inoculum contained 34.9% Bacteria, 60.1% Eukarya and 6.8% Archaea measured as a fraction of total small subunit (SSU) rRNA quantified using a universal probe. The cellulolytic bacterial genus Fibrobacter comprised 9.5% of total SSU rRNA in the inoculum. After 240 h of fermenter operation, the average abundance was 80.9% Bacteria, 6.1% Eukarya, 5.1% Archaea and Fibrobacter genus accounted for 6.6% of the total SSU rRNA. Divergence between ruminal and fermenter population structure was evaluated in the second experiment and samples were classified as ruminal, inoculum or fermenter (96, 120, 144 and 168 h of fermenter operation). Fermenter samples had higher relative abundances of Bacteria (84.5%) and Archaea (2.1%) and lower relative abundances of Eukarya (1.8%) than ruminal samples (average 48.0% Bacteria, 1.3% Archaea and 61.5% Eukarya). The relative abundance of Fibrobacter was similar in all samples, averaging 2.5%. The ruminal and fermenter samples had similar proportions of F. succinogenes and F. succinogenes subgroup 3 (as a percentage of Fibrobacter SSU rRNA). Fibrobacter succinogenes subgroup 1 and F. intestinalis proportions of Fibrobacter were lower in fermenter samples (8.2% and 0.7% respectively) than in ruminal samples (28.4% and 2.2% respectively). Fermenters were able to maintain a core prokaryotic community structure similar to the native microbial community in the rumen. Although protozoa populations were lost, maintenance of Fibrobacter and archaeal populations indicated that the model system supported a functional community structure similar to the rumen. This model rumen system may serve as a suitable tool for studying aspects of ruminal microbial ecology and may resolve some of the relationships between microbial community structure and function by providing control of experimental conditions.

Phylogenetic diversity of methanogenic archaea in swine waste storage pits.

Total DNA was isolated from swine feces and a swine waste storage pit and used as templates for PCR amplification of archaeal 16 rDNA using specific primers. Only the sample from the center of the waste pit produced a PCR product. DNA sequence analyses of random clones demonstrated a variety of methanogenic archaea. Six groups of sequences were identified, including those similar to Methanobrevibacter sp., Methanocorpusculum sp., and Methanoculleus sp. Three groups of sequences represented unidentified organisms. These data suggest that swine waste storage pits may represent an untapped source of novel methanogenic archaea.

Identification of a novel adenylate cyclase in the ruminal anaerobe, Prevotella ruminicola D31d.

Our previous evaluation of ruminal and other anaerobic bacteria showed only Prevotella ruminicola D31d produced detectable concentrations of cyclic AMP. In order to investigate the synthesis of this important metabolic regulator, the gene for adenylate cyclase (cya), which produces cyclic AMP, was cloned and expressed in a cyaA mutant of Escherichia coli. The cloned P. ruminicola D31d gene was able to complement the cyaA mutation and permitted fermentation of lactose on MacConkey Lactose agar plates. Analysis of the DNA sequence of the 2.5-kilobase pair insert revealed an open reading frame encoding for a 67-kDa protein. This protein was novel in that no amino acid similarity was observed with other procaryotic or eucaryotic adenylate cyclases in the GenBank database. Production of cyclic AMP in the E. coli clone was confirmed with a radioimmunoassay technique. This is the first example of an adenylate cyclase gene identified from an anaerobic bacterium.

Xylooligosaccharide utilization by the ruminal anaerobic bacterium Selenomonas ruminantium.

Fermentation of xylooligosaccharides by 11 strains of Selenomonas ruminantium was examined. Xylooligosaccharides were prepared by the partial hydrolysis of oat spelt xylan in dilute phosphoric acid (50 mM, 121 degrees C, 15 min) and were added to a complex, yeast extract-Trypticase-containing medium. Strains of S. ruminantium varied considerably in their capacity to ferment xylooligosaccharides. Strains GA192, GA31, H18, and D used arabinose, xylose, and the oligosaccharides xylobiose through xylopentaose, as well as considerable quantities of larger, unidentified oligosaccharides. Other strains of S. ruminantium (HD4, HD1, 20-21a, H6a, W-21, S23, 5-1) were able to use only the simple sugars present in the substrate mixture. The ability of S. ruminantium strains to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidase activities. Both enzyme activities were induced by growth on xylooligosaccharides, but no activity was detected in glucose- or arabinose-grown cultures. Xylooligosaccharide-fermenting strains of S. ruminantium exhibited considerable variation in substrate utilization patterns, and the assimilation of individual carbohydrate species also appeared to be regulated. Lactic, acetic, and propionic acids were the major fermentation end products detected.

Cloning, sequence, and expression of the L-(+) lactate dehydrogenase of Streptococcus bovis.

The ldh gene encoding the fructose-1,6-diphosphate-dependent L-(+) lactate dehydrogenase from the ruminal bacterium Streptococcus bovis was cloned and sequenced. A genomic library of S. bovis JB1 DNA was constructed in lambda ZAP II and screened by use of a heterologous probe derived from the cloned Streptococcus mutans ldh gene. Several clones were isolated that contained a common 2.9-kb fragment as determined by restriction analysis. Nucleotide sequence analysis revealed a 987-bp open reading frame with extensive homology to Streptococcus thermophilus and S. mutans ldh nucleic acid and amino acid sequences. Expression of the cloned S. bovis ldh gene in Escherichia coli was confirmed by the ability to complement the ldh mutation of E. coli FMJ39, by using an in-gel activity screen and by enzymatic assay. Increased LDH activity was observed in S. bovis JB1 containing the cloned ldh genes on a multicopy plasmid.

Survival of the recombinant Bacteroides thetaiotaomicron strain BTX in in vitro rumen incubations.

The survival of Bacteroides thetaiotaomicron strain BTX under rumen-simulating conditions was studied. Strain BTX is a recombinant variant of strain 5482 engineered for the production of high levels of xylanase, an enzyme important in the degradation of hemicellulose. Strain BTX was not inhibited by compounds present in rumen fluid and it grew well in media containing rumen fluid (up to 75%) or high concentrations of volatile fatty acids (total concentration, 100 mmol l-1). The ability of strain BTX to compete with other microorganisms under rumen-like conditions was studied in in vitro incubations of rumen contents. These experiments employed a consecutive batch culture (CBC) system consisting of alfalfa suspended in a rum flid buffer inoculated with blended rumen contents and maintained by transfers (10%, v/v) at 48 h intervals. CBC cultures contained a diversity of microbial morphotypes and accumulated fermentation products in rumen-like proportions. WHen added alone, the numbers of BTX cells were maintained for only a few hours, and then declined precipitously until undetectable after 48 h. If CBC cultures were also supplemented with chondroitin sulphate (a mucopolysaccharide used by Bact. thetaiotaomicron), strain BTX grew and the pattern of its population generally followed that of the total population of ruminal bacteria in these cultures. When transferred into fresh CBC cultures containing chondroitin sulphate, BTX was again able to grow and increase in numbers, but to a diminished degree. Although BTX was able to survive and maintain itself in chondroitin sulphate supplemented cultures, this was at a very low level (10(5) ml-1). The potential for manipulation of rumen function by inoculation with recombinant bacteria is discussed.