Terence R. Whitehead

Xylan regulated delivery of human keratinocyte growth factor-2 to the inflamed colon by the human anaerobic commensal bacterium Bacteroides ovatus

Background Human growth factors are potential therapeutic agents for various inflammatory disorders affecting the gastrointestinal tract. However, they are unstable when administered orally and systemic administration requires high doses increasing the risk of unwanted side effects. Live microorganism-based delivery systems can overcome these problems although they suffer from the inability to control heterologous protein production and there are concerns regarding biosafety and environmental contamination. Methods To overcome these limitations we have developed a new live bacteria drug-delivery system using the human commensal gut bacterium Bacteroides ovatus engineered to secrete human growth factors in response to dietary xylan. The anaerobic nature of B ovatus provides an inherent biosafety feature. B ovatus strains expressing human keratinocyte growth factor-2, which plays a central role in intestinal epithelial homeostasis and repair (BO-KGF), were generated by homologous recombination and evaluated using the dextran sodium sulfate (DSS)-induced model of intestinal epithelial injury and colitis. Results In response to xylan BO-KGF produced biologically active KGF both in vitro and in vivo. In DSS treated mice administration of xylan and BO-KGF had a significant therapeutic effect in reducing weight loss, improving stool consistency, reducing rectal bleeding, accelerating healing of damaged epithelium, reducing inflammation and neutrophil infiltration, reducing expression of pro-inflammatory cytokines, and accelerating production of goblet cells. BO-KGF and xylan treatment also had a marked prophylactic effect limiting the development of inflammation and disruption of the epithelial barrier. Conclusion This novel, diet-regulated, live bacterial drug delivery system may be applicable to treating 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).

Isolation and identification of hyper-ammonia producing bacteria from swine manure storage pits.

Storage of swine manure is associated with the microbiological production of a variety of odorous compounds, including ammonia, organic acids, and alcohols, phenolics, and sulfides. Until recently, little was known about the microorganisms responsible for their production. Results from our laboratory have demonstrated that the predominant microbial populations of stored swine manure are anaerobic, low (G + C), Gram-positive bacteria. However, studies on pure cultures isolated from manure have found few microorganisms that produce appreciable ammonia concentrations. Therefore, selective and enrichment techniques were employed to isolate ammonia-producing bacteria from stored swine manure by using media containing peptone and amino acids as carbon and energy sources. We now report on the isolation of 40 bacterial cultures, a number of which are capable of producing at least 40 mM ammonia in peptone-amino acid medium, concentrations similar to those produced by hyper-ammonia producing (HAP) bacteria isolated from the rumen of cattle. The manure HAP isolates are phylogenetically distinct from the ruminal isolates and may prove to be intimately involved in the production of ammonia during storage of swine manure.

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.

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.

Catabolic Pathway for the Production of Skatole and Indoleacetic Acid by the Acetogen Clostridium drakei, Clostridium scatologenes, and Swine Manure

ABSTRACT 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.

Analyses of the Gene and Amino Acid Sequence of the Prevotella (Bacteroides) ruminicola 23 Xylanase Reveals Unexpected Homology with Endoglucanases from Other Genera of Bacteria

The DNA sequence for the xylanase gene fromPrevotella (Bacteroides) ruminicola 23 was determined. The xylanase gene encoded for a protein with a molecular weight of 65,740. An apparent leader sequence of 22 amino acids was observed. The promoter region for expression of the xylanase gene inBacteroides species was identified with a promoterless chloramphenicol acetyltransferase gene. A region of high amino acid homology was found with the proposed catalytic domain of endoglucanases from several organisms, includingButyrivibrio fibrisolvens, Ruminococcus flavefaciens, andClostridium thermocellum. The cloned xylanase was found to exhibit endoglucanase activity against carboxymethyl cellulose. Analysis of the codon usage for the xylanase gene found a bias towards G and C in the third position in 16 of 18 amino acids with degenerate codons.

Identification of a New Ribosomal Protection Type of Tetracycline Resistance Gene, tet(36), from Swine Manure Pits

ABSTRACT Previously, only one ribosome protection type of a tetracycline resistance gene, tetQ, had been identified in Bacteroides spp. During an investigation of anaerobic bacteria present in swine feces and manure storage pits, a tetracycline-resistant Bacteroides strain was isolated. Subsequent analysis showed that this new Bacteroides strain, Bacteroides sp. strain 139, did not contain tetQ but contained a previously unidentified tetracycline resistance gene. Sequence analysis showed that the tetracycline resistance gene from Bacteroides sp. strain 139 encoded a protein (designated Tet 36) that defines a new class of ribosome protection types of tetracycline resistance. Tet 36 has 60% amino acid identity over 640 aa to TetQ and between 31 and 49% amino acid identity to the nine other ribosome protection types of tetracycline resistance genes. The tet(36) region was not observed to transfer from Bacteroides sp. strain 139 to another Bacteroides sp. under laboratory conditions. Yet tet(36) was found in other genera of bacteria isolated from the same swine manure pits and from swine feces. Phylogenetic analysis of the tet(36)-containing isolates indicated that tet(36) was present not only in the Cytophaga-Flavobacter-Bacteroides group to which Bacteroides sp. strain 139 belongs but also in gram-positive genera and gram-negative proteobacteria, indicating that horizontal transfer of tet(36) is occurring between these divergent phylogenetic groups in the farm environment.

Treatment of colitis with a commensal gut bacterium engineered to secrete human TGF-β1 under the control of dietary xylan 1.

Background: While cytokine therapy and the use of immunosuppressive cytokines such as transforming growth factor‐&bgr; (TGF‐&bgr;) 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‐&bgr;1 and treatment of colitis. Methods: Sequence encoding the human tgf‐&bgr;1 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‐&bgr; 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‐&bgr; 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‐&bgr;1 and other immunotherapeutic agents for the long‐term management of various bowel disorders. (Inflamm Bowel Dis 2010;)

Development and application of a polymerase chain reaction assay for the detection and enumeration of bile acid 7α-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.