Rapid identification of Robinsoniella peoriensis using specific 16S rRNA gene PCR primers.

Robinsoniella peoriensis is a Gram-positive, spore-forming anaerobic bacterium initially isolated and characterized from swine manure and feces. Since then strains of this species have been identified from a variety of mammalian and other GI tracts. More recently strains of this species have been isolated from a plethora of human infections. Therefore, it is of great interest to develop methods to rapidly identify this microorganism in the medical and other laboratories. This report describes the use of PCR primers targeting the 16S rRNA gene of R. peoriensis to identify strains of this bacterium.

Examination of the Aerobic Microflora of Swine Feces and Stored Swine Manure.

Understanding antibiotic resistance in agricultural ecosystems is critical for determining the effects of subtherapeutic and therapeutic uses of antibiotics for domestic animals. This study was conducted to ascertain the relative levels of antibiotic resistance in the aerobic bacterial population to tetracycline, tylosin, and erythromycin. Swine feces and manure samples were plated onto various agar media with and without antibiotics and incubated at 37°C. Colonies were counted daily. Randomly selected colonies were isolated and characterized by 16S rRNA sequence analyses and additional antibiotic resistance and biochemical analyses. Colonies were recovered at levels of 10 to 10 CFU mL for swine slurry and 10 to 10 CFU g swine feces, approximately 100-fold lower than numbers obtained under anaerobic conditions. Addition of antibiotics to the media resulted in counts that were 60 to 80% of those in control media without added antibiotics. Polymerase chain reaction analyses for antibiotic resistance genes demonstrated the presence of a number of different resistance genes from the isolates. The recoverable aerobic microflora of swine feces and manure contain high percentages of antibiotic-resistant bacteria, which include both known and novel genera and species, and a variety of antibiotic resistance genes. Further analyses of these and additional isolates should provide additional information on these organisms as potential reservoirs of antibiotic resistance genes in these ecosystems.

Sodium Tetraborate Decahydrate Treatment Reduces Hydrogen Sulfide and the Sulfate-Reducing Bacteria Population of Swine Manure.

Emission of odorous and toxic gases from stored livestock manure is well documented and poses a serious health risk to farmers and livestock. Hydrogen sulfide emissions have been sharply rising with increasingly intensive livestock production and are of particular concern because of the acute toxicity of this gas. Numerous strategies, technologies, and chemical treatments have been used to control hydrogen sulfide emissions, but none have worked particularly well because they are neither cost-effective nor environmentally sustainable, or they are too toxic for animals. The inhibitory effect of the sodium tetraborate decahydrate (i.e., borax) treatment to reduce hydrogen sulfide production using sulfate-reducing bacteria was examined in shallow manure pits in a starter-grower swine facility. Monitoring of air emissions and DNA analysis revealed that treatment of stored swine manure effectively reduced hydrogen sulfide production, and the reduction correlated to a decrease in the sulfate-reducing bacteria population in the stored swine manure.

Savagea faecisuis gen. nov., sp. nov., a tylosin- and tetracycline-resistant bacterium isolated from a swine-manure storage pit.

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular methods was performed on three strains of a Gram-stain positive, non-sporeforming, motile aerobic rod-shaped bacterium resistant to tylosin and tetracycline isolated from a swine-manure storage pit. On the basis of 16S rRNA gene sequence analyses, it was confirmed that these isolates are highly related to each other and form a hitherto unknown lineage within the Planococcaceae. In particular, pairwise analysis of the 16S rRNA gene sequence demonstrated that the novel organism is closely related to members of the genus Sporosarcina (92.8-94.5 %), Pyschrobacillus (93.5-93.9 %) and Paenisporosarcina (93.3-94.5 %). The predominant fatty acids were found to consist of iso-C15:0 and iso-C17:1 ω10c and the G+C mol% was determined to be 41.8. Based on biochemical, chemotaxonomic, and phylogenetic evidence, it is proposed that these novel strains be classified as a novel genus and species, Savagea faecisuis gen nov., sp. nov. The type strain is Con12(T) (=CCUG 63563(T) = NRRL B-59945(T) = NBRC 109956(T)).

Peptoniphilus stercorisuis sp. nov., isolated from a swine manure storage tank and description of Peptoniphilaceae fam. nov.

A species of a previously unknown Gram-positive-staining, anaerobic, coccus-shaped bacterium recovered from a swine manure storage tank was characterized using phenotypic, chemotaxonomic, and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies and biochemical characteristics demonstrated that this organism is genotypically and phenotypically distinct, and represents a previously unknown sub-line within the order Clostridiales, within the phylum Firmicutes. Pairwise sequence analysis demonstrated that the novel organism clustered within the genus Peptoniphilus, most closely related to Peptoniphilus methioninivorax sharing a 16S rRNA gene sequence similarity of 95.5%. The major long-chain fatty acids were found to be C14:0 (22.4%), C16:0 (15.6%), C16:1ω7c (11.3%) and C16 : 0 ALDE (10.1%) and the DNA G +C content was 31.8 mol%. Based upon the phenotypic and phylogenetic findings presented, a novel species Peptoniphilus stercorisuis sp. nov. is proposed. The type strain is SF-S1(T) ( = DSM 27563(T) = NBRC 109839(T)). In addition, it is proposed to accommodate the genera Peptoniphilus, Anaerococcus, Anaerosphaera, Finegoldia, Gallicola, Helcococcus, Murdochiella and Parvimonas in a new family of the order Clostridiales, for which the name Peptoniphilaceae fam. nov. is proposed; the type genus of the family is Peptoniphilus.

Inhibition of hydrogen sulfide, methane, and total gas production and sulfate-reducing bacteria in in vitro swine manure by tannins, with focus on condensed quebracho tannins.

Management practices from large-scale swine production facilities have resulted in the increased collection and storage of manure for off-season fertilization use. Odor and emissions produced during storage have increased the tension among rural neighbors and among urban and rural residents. Production of these compounds from stored manure is the result of microbial activity of the anaerobic bacteria populations during storage. In the current study, the inhibitory effects of condensed quebracho tannins on in vitro swine manure for reduction of microbial activity and reduced production of gaseous emissions, including the toxic odorant hydrogen sulfide produced by sulfate-reducing bacteria (SRB), was examined. Swine manure was collected from a local swine facility, diluted in anaerobic buffer, and mixed with 1 % w/v fresh feces. This slurry was combined with quebracho tannins, and total gas and hydrogen sulfide production was monitored over time. Aliquots were removed periodically for isolation of DNA to measure the SRB populations using quantitative PCR. Addition of tannins reduced overall gas, hydrogen sulfide, and methane production by greater than 90 % after 7 days of treatment and continued to at least 28 days. SRB population was also significantly decreased by tannin addition. qRT-PCR of 16S rDNA bacteria genes showed that the total bacterial population was also decreased in these incubations. These results indicate that the tannins elicited a collective effect on the bacterial population and also suggest a reduction in the population of methanogenic microorganisms as demonstrated by reduced methane production in these experiments. Such a generalized effect could be extrapolated to a reduction in other odor-associated emissions during manure storage.

Two novel species Enterococcus lemanii sp. nov. and Enterococcus eurekensis sp. nov., isolated from a swine-manure storage pit.

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular genetic methods was performed on six strains of unknown Gram-positive, nonspore-forming, facultative anaerobic coccus-shaped bacteria isolated from a swine-manure storage pit. On the basis of the 16S rRNA, RNA polymerase α-subunit (rpoA) and 60 kDa chaperonin (cpn60) gene sequence analyses, it was shown that all the isolates were enterococci but formed two separate lines of descent. Pairwise 16S rRNA gene sequence comparisons demonstrated that the two novel organisms were most closely related to each other (97.9 %) and to Enterococcus aquimarinus (97.8 %). Both organisms contained major amounts of C(16:0), C(16:1) ω7c, C(16:1) ω7c, and C(18:1) ω7c/12t/9t as the major cellular fatty acids. Based on biochemical, chemotaxonomic and phylogenetic evidence, the names Enterococcus lemanii sp. nov. (type strain PC32(T) = CCUG 61260(T) = NRRL B-59661(T)) PPC27A = CCUG 61369; PPC38 = CCUG 61261 [corrected] and Enterococcus eurekensis sp. nov. (type strain PC4B(T) = CCUG 61259(T) = NRRL B-59662(T)) PPC15 = CCUG 61368; PPC107 = CCUG 61372 [corrected] are proposed for these hitherto undescribed species.

Erratum to: Two novel species Enterococcus lemanii sp. nov. and Enterococcus eurekensis sp. nov., isolated from a swine-manure storage pit.

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular genetic methods was performed on six strains of an unknown Gram-positive, nonspore-forming, facultative anaerobic coccus-shaped bacterium isolated from a swine-manure storage pit. On the basis of 16S rRNA, RNA polymerase-subunit (rpoA), and the 60-kilodalton chaperonin (cpn60) gene sequence analyses, it was shown that all the isolates were enterococci but formed two separate lines of descent. Pairwise 16S rRNA sequence comparisons demonstrated that the two novel organisms were most closely related to each other (97.9 %) and to Enterococcus aquimarinus (97.8 %). Both organisms contained major amounts of C16:0, C16:1 ω7c, and C18:1 ω7c/12t/9t as the major cellular fatty acids. Based on biochemical, chemotaxonomic, and phylogenetic evidence, the names Enterococcus lemanii sp. nov. (type strain PC32(T) = CCUG 61260(T) = NRRL B-59661(T)) and Enterococcus eurekensis sp. nov. (type strain PC4B(T) = CCUG 61259(T) = NRRL B-59662(T)) are proposed for the hitherto undescribed species.

Peptostreptococcus russellii sp. nov., isolated from a swine-manure storage pit.

Using a polyphasic approach, a taxonomic study was performed on seven strains of an unknown Gram-reaction-positive, non-spore-forming, obligately anaerobic coccus-shaped bacterium, isolated from a swine-manure storage pit. Comparative 16S rRNA gene sequencing confirmed that all seven isolates were highly related to each other and formed a hitherto unknown lineage within the clostridial rRNA XI cluster of organisms. Pairwise analysis demonstrated that the novel organism was most closely related to Peptostreptococcus anaerobius CCUG 7835(T) and Peptostreptococcus stomatis CCUG 51858(T) with 16S rRNA gene sequence similarities of 95.5 and 93.0 %, respectively. The peptidoglycan type of the cell wall was determined to be A4α l-Lys-d-Asp and glucose, xylose and traces of mannose were detected as the cell-wall sugars. Based on biochemical, chemotaxonomic and phylogenetic evidence the unknown bacterium represents a new species of the genus Peptostreptococcus, for which the name Peptostreptococcus russellii sp. nov, is proposed. The type strain is RT-10B(T) ( = CCUG 58235(T)  = NRRL B-59380(T)  = DSM 23041(T)).

A newly developed Escherichia coli isolate panel from a cross section of U.S. animal production systems reveals geographic and commodity-based differences in antibiotic resistance gene carriage.

There are limited numbers of Escherichia coli isolate panels that represent United States food animal production. The majority of existing Escherichia coli isolate panels are typically designed: (i) to optimize genetic and/or phenotypic diversity; or (ii) focus on human isolates. To address this shortfall in agriculturally-related resources, we have assembled a publicly-available isolate panel (AgEc) from the four major animal production commodities in the United States, including beef, dairy, poultry, and swine, as well as isolates from agriculturally-impacted environments, and other commodity groups. Diversity analyses by phylotyping and Pulsed-field Gel Electrophoresis revealed a highly diverse composition, with the 300 isolates clustered into 71 PFGE sub-types based upon an 80% similarity cutoff. To demonstrate the panel's utility, tetracycline and sulfonamide resistance genes were assayed, which identified 131 isolates harboring genes involved in tetracycline resistance, and 41 isolates containing sulfonamide resistance genes. There was strong overlap in the two pools of isolates, 38 of the 41 isolates harboring sulfonamide resistance genes also contained tetracycline resistance genes. Analysis of antimicrobial resistance gene patterns revealed significant differences along commodity and geographical lines. This panel therefore provides the research community an E. coli isolate panel for study of issues pertinent to U.S. food animal production.

Catabolic pathway for the production of skatole and indoleacetic acid by the acetogen Clostridium drakei, Clostridium scatologenes, and swine manure.

Skatole (3-methylindole) is a malodorous chemical in stored swine manure and is implicated as a component of foul-tasting pork. Definitive evidence for the skatole pathway is lacking. Deuterium-labeled substrates were employed to resolve this pathway in the acetogenic bacterium Clostridium drakei and Clostridium scatologenes and to determine if a similar pathway is used by microorganisms present in stored swine manure. Indoleacetic acid (IAA) was synthesized from tryptophan by both bacteria, and skatole was synthesized from both IAA and tryptophan. Microorganisms in swine manure produced skatole and other oxidation products from tryptophan, but IAA yielded only skatole. A catabolic mechanism for the synthesis of skatole is proposed.

Genome Sequence of a Novel Multiple-Antibiotic-Resistant Member of the Erysipelotrichaceae Family Isolated from a Swine Manure Storage Pit.

The swine gastrointestinal tract and stored swine manure may serve as reservoirs of antibiotic resistance genes, as well as sources of novel bacteria. Here, we report the draft genome sequence of a novel taxon in the Erysipelotrichaceae family, isolated from a swine manure storage pit that is resistant to multiple antibiotics.

Miscanthus×giganteus xylooligosaccharides: Purification and fermentation.

A procedure was developed to recover xylooligosaccharides (XOS) from Miscanthus×giganteus (M×G) hydrolyzate. M×G hydrolyzate was prepared using autohydrolysis, and XOS rich fractions were acquired using activated carbon adsorption and stepwise ethanol elution. The combined XOS fractions were purified using a series of ion exchange resin treatments. The end product, M×G XOS, had 89.1% (w/w) total substituted oligosaccharides (TSOS) composed of arabinose, glucose, xylose and acetyl group. Bifidobacterium adolescentis and Bifidobacterium catenulatum (health promoting bacteria) were cultured in vitro on M×G XOS and a commercial XOS source, which was used as a comparison. B. adolescentis grew to a higher cell density than B. catenulatum in both XOS cultures. Total xylose consumption for B. adolescentis was 84.1 and 84.8%, respectively for M×G and commercial XOS cultures; and for B. catenulatum was 76.6 and 73.6%, respectively. The xylobiose (X2), xylotriose (X3) and xylotetraose (X4) were almost utilized for both strains. Acetic and lactic acids were the major fermentation products of the XOS cultures.

Development and application of a polymerase chain reaction assay for the detection and enumeration of bile acid 7alpha-dehydroxylating bacteria in human feces.

BACKGROUND: Secondary bile acids are synthesized in the human colon from the bacterial 7alpha-dehydroxylation of primary bile acids. Increased levels of secondary bile acids have been correlated with an increased risk of colon cancer and cholesterol gallstone disease. Based on 16s rDNA sequence analysis, stock cultures of bacterial strains with bile acid 7alpha-dehydroxylation all belong to the genus Clostridium. METHODS: We developed a polymerase chain reaction (PCR)-based assay that can specifically amplify the baiCD gene, specific to bile acid 7alpha-dehydroxylation pathway. The PCR primer pair was designed using known nucleotide sequences from two different baiCD genes described for Clostridium scindens VPI 12708 and Clostridium hiranonis TO931. Although the DNA sequences of these genes were <70% identical, several regions were conserved enough to design primers with little or no redundancy. RESULTS: The PCR assay was effective in detecting the baiCD gene in several strains known to exhibit bile acid 7alpha-dehydroxylation activity. The PCR assay also detected the baiCD gene in DNA extracted from fecal dilution series and correlated with the levels of cholic acid 7alpha-dehydroxylating bacteria detected by activity assays. The PCR assay was sensitive enough to detect the baiCD gene in DNA samples extracted directly from as little as 0.5 mg feces. CONCLUSIONS: This new method should be useful for the monitoring of levels of bile acid 7alpha-dehydroxylating bacteria in human fecal samples.

Draft Genome Sequences of Streptococcus bovis Strains ATCC 33317 and JB1.

We report the draft genome sequences of Streptococcus bovis strain ATCC 33317 (CVM42251) isolated from cow dung and strain JB1 (CVM42252) isolated from a cow rumen in 1977. The strains were sequenced using the Genome Sequencer FLX 454 system. The genome sizes are approximately 2 Mb and 2.2 Mb, respectively.

Treatment of colitis with a commensal gut bacterium engineered to secrete human TGF-ß1 under the control of dietary xylan 1.

BACKGROUND: While cytokine therapy and the use of immunosuppressive cytokines such as transforming growth factor-ß (TGF-ß) offer great potential for the treatment of inflammatory bowel disease (IBD), issues concerning formulation, stability in vivo, delivery to target tissues, and potential toxicity need to be addressed. In consideration of these problems we engineered the human commensal bacterium Bacteroides ovatus for the controlled in situ delivery of TGF-ß(1) and treatment of colitis. METHODS: Sequence encoding the human tgf-ß1 gene was cloned downstream of the xylanase promoter in the xylan operon of B. ovatus by homologous recombination. Resulting recombinants (BO-TGF) were tested for TGF-ß production in the presence and absence of polysaccharide xylan in vitro and in vivo, and used to treat experimental murine colitis. Clinical and pathological scores were used to assess the effectiveness of therapy. Colonic inflammatory markers including inflammatory cytokine expression were assessed by colorimetric assay and real-time polymerase chain reaction (PCR). RESULTS: BO-TGF secreted high levels of biologically active dimeric TGF-ß in vitro and in vivo in a xylan-controlled manner. Administration of xylan in drinking water to BO-TGF-treated mice resulted in a significant clinical improvement of colitis, accelerating healing of damaged colonic epithelium, reducing inflammatory cell infiltration, reducing expression of proinflammatory cytokines, and promoting production of mucin-rich goblet cells in colonic crypts. These beneficial effects are comparable and in most cases superior to that achieved by conventional steroid therapy. CONCLUSIONS: This novel drug delivery system has potential for the targeted and controlled delivery of TGF-ß(1) and other immunotherapeutic agents for the long-term management of various bowel disorders.

Robinsoniella peoriensis gen. nov., sp. nov., isolated from a swine-manure storage pit and a human clinical source.

A polyphasic taxonomic study was performed on six strains of an unknown Gram-positive, non-motile, spore-forming, short oval to rod-shaped bacterium isolated from a swine-manure storage pit. In addition to these strains, an isolate deposited in the Culture Collection of the University of Göteborg (Sweden) was found to be biochemically related to the manure strains. The major end products of metabolism included acetate and succinate but not butyrate. Comparative 16S rRNA gene sequencing confirmed that all these isolates were closely related to each other and formed a hitherto unknown lineage within the clostridial rRNA XIVa cluster of organisms. On the basis of phylogenetic, biochemical and phenotypic evidence, it is proposed that the unknown bacterium represents a novel genus and species, for which the name Robinsoniella peoriensis gen. nov., sp. nov. is proposed. The type strain of Robinsoniella peoriensis is PPC31T (=CCUG 48729T =NRRL B-23985T).

Evaluation of the sulfate-reducing bacterial population associated with stored swine slurry.

Hydrogen sulfide, produced by sulfate-reducing bacteria (SRB), is one of the most potent malodors emitted from anaerobic swine waste storage systems. However, little is known about the prevalence and diversity of SRB in those systems. The goals of this study were to evaluate the SRB population in swine manure storage systems and to develop quantitative, real-time PCR (QRT-PCR) assays to target four of the SRB groups. Dissimilatory sulfite reductase (DSR) gene sequences were obtained from swine slurry stored in underground pits (43 clones) or in lagoons (34 clones). QRT-PCR assays were designed to target the dsrA gene of four novel groups of SRB. Sequences of dsrA clones from slurry samples grouped with those from three different cultured SRB: Desulfobulbus sp. (46 clones), Desulfovibrio sp. (24 clones and 5 isolates), and Desulfobacterium sp. (7 clones). However, DsrA sequences from swine slurry clones were generally less than 85% similar to those of cultured organisms. SRB from all four targeted SRB groups were detected in underground waste storage pits (6.6 x 10(3)-8.5 x 10(7) dsrA copies mL(-1) slurry), while only two groups of SRB were detected in lagoons (3.2 x 10(5)-2.5 x 10(6) dsrA copies mL(-1) slurry). To date, this is the only study to evaluate the phylogeny and concentration of SRB in any livestock waste storage system. The new QRT-PCR assays should facilitate sensitive, specific detection of the four novel groups of SRB in livestock waste storage systems.

Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins.

The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5' region of the corresponding mRNA was determined by 5'RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.

Structure-function relationships of a catalytically efficient beta-D-xylosidase.

Beta-D-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (kcat, kcat/Km) for promoting hydrolysis of 1,4-beta-D-xylooligosaccharides. 1H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl- beta-D-xylopyranoside (4NPX) and 1,4-beta-D-xylobiose (X2). Progress curves of 4-nitrophenyl-beta-D-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of kcat and kcat/Km decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites -1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites beyond -1 are blocked by protein and subsites beyond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode kcat and kcat/Km by <103 and to a similar extent for substrates 4NPX and 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs kcat/Km with pKa values of 5.0 and 7.0 assigned to D14 and E186, respectively. kcat(4NPX) has a pKa value of 7.0 and kcat(4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, "dianionic" enzyme form (D14-E187-) binds 4NPX but not 4NPA.