Biomedical subjects

R J Forster

Publications and source records attributed to R J Forster.

At least 19 recordsLinked to original sources

PubMed2005

Characterization of culturable anaerobic bacteria from the forestomach of an eastern grey kangaroo, Macropus giganteus.

AIM: To determine the culturable biodiversity of anaerobic bacteria isolated from the forestomach contents of an eastern grey kangaroo, Macropus giganteus, using phenotypic characterization and 16S rDNA sequence analysis. METHODS AND RESULTS: Bacteria from forestomach contents of an eastern grey kangaroo were isolated using anaerobic media containing milled curly Mitchell grass (Astrebla lappacea). DNA was extracted and the 16S rDNA sequenced for phylogenetic analysis. Forty bacterial isolates were obtained and placed in 17 groups based on phenotypic characteristics and restriction enzyme digestion of 16S rDNA PCR products. DNA sequencing revealed that the 17 groups comprised five known species (Clostridium butyricum, Streptococcus bovis, Clostridium sporogenes, Clostridium paraputrificum and Enterococcus avium) and 12 groups apparently representing new species, all within the phylum Firmicutes. CONCLUSIONS: Foregut contents from Australian macropod marsupials contain a microbial ecosystem with a novel bacterial biodiversity comprising a high percentage of previously unrecognized species. SIGNIFICANCE AND IMPACT OF THE STUDY: This study adds to knowledge of Australia's unique biodiversity, which may provide a future bioresource of genetic information and bacterial species of benefit to agriculture.

Animals↗

PubMed2004

Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67.

AIM: To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus. METHODS: Four phages infecting R. albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique. The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics. Two phages, phiRa02 and phiRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Podoviridae [corrected] The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae. SIGNIFICANCE OF THE STUDY: Viruses of the family Inoviridae [corrected] have not previously been isolated from rumen bacteria. The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen. This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal.

Anaerobiosis↗

PubMed2003

Rapid electrochemical genosensor assay using a streptavidin carbon-polymer biocomposite electrode.

A sensor capable of detecting a specific DNA sequence was designed by bulk modification of a graphite epoxy composite electrode with streptavidin (2% w/w). Streptavidin is used to immobilise a biotinylated capture DNA probe to the surface of the electrode. Simultaneous hybridisation occurs between the biotin DNA capture probe and the target-DNA and between the target-DNA and a digoxigenin modified probe. The rapid binding kinetic of streptavidin-biotin allows a one step immobilisation/hybridisation procedure. Secondly, enzyme labelling of the DNA duplex occurs via an antigen-antibody reaction between the Dig-dsDNA and an anti-Dig-HRP. Finally, electrochemical detection is achieved through a suitable substrate (H2O2) for the enzyme-labelled duplex. Optimisation of the sensor design, the modifier content and the immobilisation and hybridisation times was attained using a simple nucleotide sequence. Regeneration of the surface is achieved with a simple polishing procedure that shows good reproducibility. The generic use of a modified streptavidin carbon-polymer biocomposite electrode capable of surface regeneration and a one step hybridisation/immobilisation procedure are the main advantages of this approach. In DNA analysis, this procedure, if combined with the polymerase chain reaction, would represent certain advantages with respect to classical techniques, which prove to be time consuming in situations where a simple and rapid detection is required. This innovative developed material may be used for the detection of any analyte that can be coupled to the biotin-streptavidin reaction, as is the case of immunoassays.

Biosensing Techniques↗

PubMed2003

Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets.

AIM: To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. METHODS AND RESULTS: Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 10(7) cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 10(6) CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 10(8) CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. CONCLUSION: Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7-10 days earlier than in uninoculated cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR.

Animal Feed↗

PubMed2002

Enumeration of Megasphaera elsdenii in rumen contents by real-time Taq nuclease assay.

AIMS: To develop a real-time Taq nuclease assay (TNA) to enable the in vivo enumeration of Megasphaera elsdenii. METHODS AND RESULTS: Megasphaera elsdenii YE34 was phenotypically characteristic of the species and had 16S rDNA sequence similarity of 98% to previously described isolates. Calibration of the number of cells of M. elsdenii against the cycle threshold of fluorescent dye release gave a straight-line relationship with a correlation coefficient approximating unity. The specificity of the assay for M. elsdenii was confirmed by performing it against a panel of 24 heterogeneous, mainly ruminal bacteria. Megasphaera elsdenii was not detected in ruminal contents from a pasture-fed steer but was readily detected 2 and 50 h after the probiotic introduction of the bacterium into the rumen. CONCLUSIONS: Real-time TNA has provided a sensitive and specific means of enumerating the M. elsdenii population in rumen contents. SIGNIFICANCE AND IMPACT OF THE STUDY: Megasphaera elsdenii is an important lactate-degrading ruminal bacterium that has been selected for probiotic use to prevent acidosis and enhance starch utilization in grain-fed cattle. The assay developed in this study provides a tool for determining the ability of probiotically-introduced M. elsdenii to establish useful populations in the rumen.

Animals↗

PubMed2001

Phylogenetic analysis of methanogens from the bovine rumen.

BACKGROUND: Interest in methanogens from ruminants has resulted from the role of methane in global warming and from the fact that cattle typically lose 6 % of ingested energy as methane. Several species of methanogens have been isolated from ruminants. However they are difficult to culture, few have been consistently found in high numbers, and it is likely that major species of rumen methanogens are yet to be identified. RESULTS: Total DNA from clarified bovine rumen fluid was amplified using primers specific for Archaeal 16S rRNA gene sequences (rDNA). Phylogenetic analysis of 41 rDNA sequences identified three clusters of methanogens. The largest cluster contained two distinct subclusters with rDNA sequences similar to Methanobrevibacter ruminantium 16S rDNA. A second cluster contained sequences related to 16S rDNA from Methanosphaera stadtmanae, an organism not previously described in the rumen. The third cluster contained rDNA sequences that may form a novel group of rumen methanogens. CONCLUSIONS: The current set of 16S rRNA hybridization probes targeting methanogenic Archaea does not cover the phylogenetic diversity present in the rumen and possibly other gastro-intestinal tract environments. New probes and quantitative PCR assays are needed to determine the distribution of the newly identified methanogen clusters in rumen microbial communities.

Animals↗

PubMed2001

Electron self-exchange in the solid-state: cocrystals of hydroquinone and bipyridyl triazole.

Solid-state voltammetry, spectroscopy, and microscopy studies have been used to probe the proton and electron conductivity within a self-assembled cocrystal, HQBpt. This crystallographically defined material contains 3,5-bis(pyridin-2-yl)-1,2,4-triazole, HBpt, dimers that are pi-stacked and hydrogen bonded to 1,4-hydroquinone, H(2)Q, in a herringbone arrangement. When deposited onto platinum microelectrodes, the cocrystal exhibits a well-defined voltammetric response corresponding to oxidation of H(2)Q to the quinone, Q, across a wide range of voltammetric time scales, electrolyte compositions, and pH values. Scanning electron microscopy reveals that redox cycling in aqueous perchlorate solutions in which the pH is systematically varied from 1 to 7 triggers electrocrystallization and the extensive formation of rodlike crystals. Fast scan rate voltammetry reveals that the homogeneous charge transport diffusion coefficient, D(app), is independent of the perchlorate concentration for 0.1 < [ClO(4)(-)] < 1.0 M (pH 6.6) at 3.14 +/- 0.11 x 10(-)(9) cm(2) s(-)(1). Moreover, D(app) is independent of the perchloric acid concentration for concentrations greater than approximately 2.0 M, maintaining a value of 4.81 +/- 0.07 x 10(-)(8) cm(2) s(-)(1). The observation that D(app) is independent of the supporting electrolyte suggests that the rate-determining step for homogeneous charge transport is not the availability of charge-compensating counterions or protons, but the dynamics of electron self-exchange between H(2)Q and Q. We have used the Dahms-Ruff formalism to determine electron self-exchange rate constants which are 2.84 +/- 0.22 x 10(9) and 9.69 +/- 0.73 x 10(10) M(-)(1) s(-)(1) for pH values greater than approximately 2.0 and less than -0.3, respectively. Significantly, these values are more than 2 orders of magnitude larger that those found for benzoquinone self-exchange reactions in aqueous solution. These results indicate that hydrogen bonds play an important role in supporting rapid electron transfer. The increase in D(app) between pH 1.0 and -0.3 is associated with protonation of the HBpt moieties, which triggers a reversible change in the material's structure.

Journal Article↗

PubMed2001

Lachnobacterium bovis gen. nov., sp. nov., a novel bacterium isolated from the rumen and faeces of cattle.

Phenotypic and phylogenetic analysis was performed on four strains of a previously undescribed Gram-positive, rod-shaped, anaerobic bacterium isolated from the rumen and faeces of cattle. This bacterium fermented glucose primarily to lactic acid along with minor amounts of acetic and butyric acids. The four strains produced a temperature-sensitive bacteriocin-like inhibitory substance. Comparative 16S rRNA gene sequence analysis indicated that the bacterium was a member of the clostridial XIVa cluster of the low-G+C content Gram-positive bacteria. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be assigned to a new genus, Lachnobacterium, as Lachnobacterium bovis gen. nov., sp. nov. The type strain is YZ 87T (= ATCC BAA-151T = DSM 14045T = LRC 5382T). Its G+C content is 33.9 mol %.

Anaerobiosis↗

PubMed2001

Identification of bacteriocin-like inhibitors from rumen Streptococcus spp. and isolation and characterization of bovicin 255.

Streptococci obtained from rumen sources were tested for the production of antibacterial compounds using a deferred-antagonism plating assay. Of 35 isolates tested, 7 were identified that inhibited the growth of other streptococci. None of the inhibitory activity was due to bacteriophage. Three isolates, LRC0253, LRC0255, and LRC0476, were selected for further characterization. Analysis of 16S ribosomal DNA indicated that LRC0476 was a strain of Streptococcus bovis, while isolates LRC0253 and LRC0255 are likely strains of Streptococcus gallolyticus. The antibacterial compounds produced by these bacteria were protease sensitive, remained active in a pH range from 1 to 12, and did not lose activity after heating at 100 degrees C for 15 min. The inhibitory peptide from strain LRC0255 was purified using pH-dependent adsorption and desorption to bacterial cells, followed by ammonium sulfate precipitation and reversed-phase chromatography and gel filtration. The peptide was 6 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An oligonucleotide probe based on the N-terminal sequence of the purified peptide was used to identify the gene encoding the inhibitory peptide. The antibacterial peptide has characteristics that are very similar to those described for class II bacteriocins of gram-positive bacteria.

Amino Acid Sequence↗

PubMed2000

Electrochemiluminescent metallopolymer coatings: combined light and current detection in flow injection analysis.

The application of thin films of the metallopolymer [Ru(bpy)2PVP10]2+ for the electrochemiluminescent (ECL) detection of oxalate in a flow injection analysis system is reported, where bpy is 2,2'-bipyridyl and PVP is poly(4-vinylpyridine). Immobilization of the ECL reagent means that it can be regenerated in situ, eliminating the need to constantly deliver it to the reaction zone. Electrochemically generated Ru3+ reacts with the analyte to form the excited-state [Ru2+]*, which luminesces at 610 nm. The reaction is optimal at low pH, where the layer is swollen and homogeneous charge transport through the layer is more facile. Unlike traditional approaches, we simultaneously monitor both the amperometric and luminescent response of the modified electrode. The precision of both signals is similar at approximately 2% (n = 10). However, the ECL response has a larger dynamic range extending from the low-micromolar to high-millimolar range and a lower limit of detection, approximately 0.2 microM or 4 pmol of oxalate injected. The ECL approach displays excellent selectivity for oxalate over a wide range of potential interferences including oxygen, amines, iron sulfate, ammonium nitrate, urea, and glucose. Ascorbic acid represents the most significant ECL interference. However, the signal observed for a 1 mM solution of ascorbic acid is still only 2.6% of the response observed for the injection of a similar concentration of oxalate.

Journal Article↗

PubMed2000

Development of a competitive polymerase chain reaction assay for the ruminal bacterium Butyrivibrio fibrisolvens OB156 and its use for tracking an OB156-derived recombinant.

A competitive polymerase chain reaction assay targeting the 16S rDNA was developed for quantitating the rumen bacterium Butyrivibrio fibrisolvens OB156. A competitor DNA, serving as an internal control in the competitive polymerase chain reaction reaction, was constructed by polymerase chain reaction using a looped oligo longer than the normal primer. Coamplification of the target DNA with known amounts of the competitor DNA allowed quantitation of the target DNA in both pure culture and mixed culture systems, where minimum quantifiable level of OB156 was 1.7x10(2) and 5.6x10(4) cells, respectively. When an erythromycin-resistant recombinant derived from OB156 was inoculated into a rumen fluid culture, its numbers decreased with time. The rate of decrease measured by the competitive polymerase chain reaction assay was much slower than the rate determined by culture enumeration using erythromycin selection. The competitive polymerase chain reaction assay also showed 48 h persistence of the recombinant at 10(4) ml(-1) even after disappearance of culturable recombinant, suggesting maintenance of the target DNA from uncultivable cells. In an in vivo tracking trial, the recombinant became undetectable within 72 h with either assay, indicating rapid hydrolysis and/or outflow of the cells from the rumen.

Animals↗

PubMed1998

Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes.

Comparative DNA sequence analysis of 16S rRNA genes (rDNA) was undertaken to further our understanding of the make-up of bacterial communities in the rumen fluid of dairy cattle. Total DNA was extracted from the rumen fluid of 10 cattle fed haylage/corn silage/concentrate rations at two different times. Rumen samples were collected on two separate occasions from five cows each. In experiment 1, 31 cloned rDNA sequences were analysed. In experiment 2, DNA extractions were amplified using either 12 or 30 cycles of PCR in order to examine biases introduced during the reactions. A set of 53 sequences were analysed in experiment 2 from DNA amplified using 12 cycles and 49 sequences from PCR using 30 cycles. Sequences from the 5' end of 16S rRNA gene were compared with existing sequences in the Ribosomal Database Project. Clones from experiment 1 produced a data set in which 55% of the sequences were similar to low G+C Gram-positive bacteria related to the genus Clostridia, the majority of which were closely related to bacteria in Cluster XIV. Approximately 30% of the cloned sequences were related to bacteria in the Prevotella-Bacteroides group. Clones from experiment 2 produced a data set in which the majority of sequences were related to the Prevotella-Bacteroides group, regardless of the number of cycles of PCR. The remaining sequences clustered with members of the genus Clostridia. The majority of rDNA sequences analysed in this study represent novel rumen bacteria which have not yet been isolated.

Journal Article↗

PubMed1997

Group-specific 16S rRNA hybridization probes for determinative and community structure studies of Butyrivibrio fibrisolvens in the rumen.

Oligonucleotide probes covering three phylogenetically defined groups of Butyrivibrio spp. were successfully designed and tested. The specificity of each probe was examined by hybridization to rRNAs from an assortment of B. fibrisolvens isolates as well as additional ruminal and nonruminal bacteria. The sensitivity of the hybridization method was determined by using one of the probes (probe 156). When RNA was extracted from a culture of OB156, the probe was able to detect target cells at densities as low as 10(4) cells/ml. When 10(4) or more target cells/ml were added to cattle rumen samples, detectable hybridization signals were obtained with 1,000 ng of total RNA loaded onto the nylon membrane. In contrast, the sensitivity was reduced to 10(6) target cells/ml at 100 ng of RNA per slot. The probes were used to type 19 novel Butyrivibrio isolates. The phylogenetic placement was confirmed by partial 16S rRNA gene sequencing. The use of the probes in community-based studies indicated that the Butyrivibrio groups examined in this paper did not represent a significant portion of the bacterial 16S rRNA pool in the rumen of the cattle, sheep, and deer examined.

Animals↗

PubMed1997

Sequence analysis and characterization of pOM1, a small cryptic plasmid from Butyrivibrio fibrisolvens, and its use in construction of a new family of cloning vectors for Butyrivibrios.

As a preliminary step in the development of vector systems, we have isolated and begun to characterize small, cryptic plasmids from several strains of the rumen bacterium Butyrivibrio fibrisolvens. We present here the complete nucleotide sequence of Butyrivibrio plasmid pOM1, which was isolated from B. fibrisolvens Bu49. While it is very similar in size to the previously characterized Butyrivibrio plasmids pRJF1 and pRJF2, pOM1 exhibits a restriction pattern which is quite distinct. Analysis of sequence data reveals that pOM1 contains only two open reading frames of significant length (ORF1 and ORF2), both of which are required for self-replication and maintenance. The protein encoded in ORF1 shows homologies with Pre (plasmid recombination enzyme) proteins encoded in plasmids from gram-positive organisms such as Staphylococcus aureus, Streptococcus agalactiae, Lactobacillus plantarum, and Bacillus thuringiensis. The putative translation product of ORF2, on the other hand, resembles Rep (replication) proteins of a different group of gram-positive plasmids, for which the Staphylococcus plasmid pSN2 is a prototype. Unlike the other characterized-Butyrivibrio plasmids, pOM1 appears to replicate via a rolling-circle mechanism. Experimental evidence showing the presence of a single-stranded replication intermediate consistent with this mechanism is presented. pOM1 has been used in the construction of a new Escherichia coli-B. fibrisolvens shuttle vector, pSMerm1, which has been successfully used to introduce a cloned gene into B. fibrisolvens harboring the pRJF1 plasmid.

Amino Acid Sequence↗

PubMed1996

16S rDNA analysis of Butyrivibrio fibrisolvens: phylogenetic position and relation to butyrate-producing anaerobic bacteria from the rumen of white-tailed deer.

Complete 16S rDNA sequences of six strains of Butyrivibrio fibrisolvens, including the type strain (ATCC 19171), were determined. The type strain was found to have less than 89% sequence similarity to the other isolates that were examined. The five plasmid-bearing strains formed a closely related cluster and three of these strains (OB156, OB157 and OB192) were very highly related (> 99%), indicating that they are isolates of the same genomic species. The phylogenetic position of Butyrivibrio was found to be within the subphylum Clostridium, of Gram-positive bacteria. The closest relatives to the type strain were Eubacterium cellulosolvens and Cl. xylanolyticum and the closest relatives to the separately clustered strains were Roseburia ceciola, Lachnospira pectinoschiza and Eubacterium rectale.

Animals↗

PubMed1995

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: comparison with other Butyrivibrio plasmids and application in the development of cloning vector.

A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79, bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM beta 1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens.

Amino Acid Sequence↗

PubMed1995

A stable and efficient transformation system for Butyrivibrio fibrisolvens OB156.

A 9.5-kb shuttle vector capable of replication and selection in both Escherichia coli and Butyrivibrio fibrisolvens was constructed. Plasmid pUC118 provided replication functions and ampicillin resistance selection in E. coli. In B. fibrisolvens, replication was controlled by the native plasmid pRJF1 from strain OB156, and selectability was provided by a 3.5-kb fragment of plasmid pAM beta 1 containing the erythromycin resistance gene. Optimum conditions for transformation were 15 kV/cm, 2 h recovery, and plating in an agar overlay on medium containing 10 micrograms erythromycin/ml. Maximum efficiency was 1.1 x 10(5) transformants per micrograms plasmid DNA (average 3 x 10(4)), and restriction mechanisms reduced efficiency by a factor of 2 x 10(2). Nonselective growth for 200 generations gave no measurable loss of plasmid.

Escherichia coli↗

PubMed1993

The complete nucleotide sequence of a small cryptic plasmid from a rumen bacterium of the genus Butyrivibrio.

The complete nucleotide sequence of a plasmid, designated pRJF1, isolated from a rumen bacterium of the genus Butyrivibrio, has been determined. pRJF1 is a small plasmid (2631 bp) which shows the high AT content (64%) typical of plasmids from gram-positive organisms. Computer analysis of sequence data revealed two major open reading frames encoded on the same strand but in different frames. The smaller, ORF1 (435 bp), is preceded by Shine Dalgarno (SD) and Escherichia coli-10, -35 sequences and is predicted to encode an acidic polypeptide of 145 aa. The putative protein shows little homology to known bacterial proteins except in a small region similar to part of the Pre protein (plasmid recombination enzyme) of Bacillus plasmid pTB913. Pre proteins of the pTB913 family, however, show conservation of several amino acids in the amino terminal region of the protein that may be related to function. This pattern of amino acid conservation is not observed in ORF1. The predicted product of ORF2 (849 bp) is a basic protein of 283 as that shows significant similarity to repA gene products of plasmids from gram-negative (but not gram-positive) bacteria. No typical Shine Dalgarno sequence or -10, -35 sequences precede this coding region. While sequence homologous to nick sites of plasmids from gram-positive organisms that replicate via ssDNA intermediates were also detected in pRJF1, the order of the three consecutive inverted repeats typical of nick sites is not conserved. pRJF1, however, does contain an extended region of directly and inversely repeated sequence motifs reminiscent of theta replication origins.

Amino Acid Sequence↗