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Biomedical subjects

G W Tannock

Publications and source records attributed to G W Tannock.

At least 19 recordsLinked to original sources
PubMed2002

Analysis of the intestinal microflora using molecular methods.

A large and complex bacterial community inhabits the distal intestinal tract of humans. This collection, known as the intestinal microflora, is dominated numerically by obligately anaerobic bacterial species. Many of these species have never been cultivated under laboratory conditions. Nucleic acid-based techniques now permit, however, the analysis of even the non-cultivable members of the bacterial community. Polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) provides a useful technique for comparisons of the composition of faecal or intestinal microfloras. PCR/DGGE has been shown to be useful in demonstrating changes that occur in the composition of the faecal microflora of infants administered antibacterial drugs. This research is important because treatment with oral antibiotics during the first 2 y of life has been identified as a predictor of subsequent atopic disease. The treatment of young children with broad spectrum oral antibiotics might produce perturbations in the composition of the intestinal microflora such that bacteria important in promoting Th1 mechanisms are depleted at a crucial age. This could result in Th2 dominance over Th1 immune responses to environmental antigens and an increased incidence of atopic disorders. PCR/DGGE provides a useful screening method to determine the impact of antibiotic treatment on the composition of the intestinal microflora of children and to identify the bacterial groups that are most affected.

Adult↗
PubMed2002

Molecular methods for exploring the intestinal ecosystem.

Molecular methods have provided renewed impetus for the analysis of the composition of the intestinal microflora in health and disease. The polymerase chain reaction coupled with denaturing gradient gel electrophoresis provides a method whereby the bacterial communities in large numbers of samples can be compared efficiently and effectively. Altered bacterial populations associated with disease states can then be targeted for further investigation. In the long-term, an 'abnormal microflora' might be rectified by the use of probiotics or prebiotics.

Bacteria↗
PubMed2001

Molecular assessment of intestinal microflora.

The application of molecular methodologies to intestinal microflora analysis should enable the development of a detailed knowledge of the microbial ecology of the human colon. This knowledge is essential to derive scientifically valid probiotics. Molecular typing (genetic fingerprinting) methods, eg, ribotyping and pulsed field gel electrophoresis of DNA digests, provide a means of distinguishing bacterial strains inhabiting the intestinal tract. Analysis of lactobacillus, bifidobacterial, and enterobacterial populations with the use of these methods has shown that human and porcine subjects harbor a characteristic collection of bacterial strains. Additionally, perturbations and transitions that occur in these populations and are caused by antibiotic administration or by autogenic or allogenic factors can be detected by molecular analysis of the intestinal microflora. In future studies, molecular typing methods could be used to analyze the composition of bacterial populations before, during, and after the administration of the probiotic product. This experimental approach would provide information on the effect of the probiotic on indigenous strains inhabiting the intestinal tract of humans and other animals.

Animals↗
PubMed2001

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.

Adult↗
PubMed2000

The intestinal microflora: potentially fertile ground for microbial physiologists.

The intestinal microflora provides opportunities for microbial physiological research. The metabolic interactions of bacterial inhabitants of the intestinal community, bacterial bioenergetics, preferential utilization of substrates as energy sources by specific bacterial species, and intercellular signalling are among the topics of challenging research awaiting the attention of microbial physiologists.

Animals↗
PubMed2000

Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.

Animals↗
PubMed2000

Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20.

The composition of the fecal microflora of 10 healthy subjects was monitored before (6-month control period), during (6-month test period), and after (3-month posttest period) the administration of a milk product containing Lactobacillus rhamnosus DR20 (daily dose, 1.6 x 10(9) lactobacilli). Monthly fecal samples were examined by a variety of methods, including bacteriological culture analysis, fluorescent in situ hybridization with group-specific DNA probes, denaturing gradient gel electrophoresis of the V2-V3 region of 16S rRNA genes amplified by PCR, gas-liquid chromatography, and bacterial enzyme activity analysis. The composition of the Lactobacillus population of each subject was analyzed by pulsed-field gel electrophoresis of bacterial DNA digests in order to differentiate between DR20 and other strains present in the samples. Representative isolates of lactobacilli were identified to the species level by sequencing the V2-V3 region of their 16S rRNA genes and comparing the sequences obtained (BLAST search) to sequences in the GenBank database. DR20 was detected in the feces of all of the subjects during the test period, but at different frequencies. The presence of DR20 among the numerically predominant strains was related to the presence or absence of a stable indigenous population of lactobacilli during the control period. Strain DR20 did not persist at levels of >10(2) cells per g in the feces of most of the subjects after consumption of the product ceased; the only exception was one subject in which this strain was detected for 2 months during the posttest period. We concluded that consumption of the DR20-containing milk product transiently altered the Lactobacillus and enterococcal contents of the feces of the majority of consumers without markedly affecting biochemical or other bacteriological factors.

Administration, Oral↗
PubMed2000

Cholic acid is accumulated spontaneously, driven by membrane deltapH, in many lactobacilli.

Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity. Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient. Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids and is different from extrusion and modification, which have been described previously.

Biological Transport↗
PubMed1999

The bowel microflora: an important source of urinary tract pathogens.

The large bowel is home to a complex microbial community that is present throughout the life of the human host. Relatively few microbial species detected in faeces in relatively low numbers have been implicated as major aetiological agents of urinary tract infections. The impact of these few species on human health is considerable, especially when recurrent urinary tract infections are considered, and ways must be found to reduce their pathogenic activities. One approach may be to learn about the ecology of the bowel ecosystem and devise ways by which the numbers of enterobacteria, in particular, can be restricted. This, in turn, would decrease the dose of potential urinary tract pathogens present in the faeces.

Animals↗
PubMed1999

Influence of different functional elements of plasmid pGT232 on maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro and in vivo.

Plasmid pGT232 (5.1 kb), an indigenous plasmid of Lactobacillus reuteri 100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein, repA. An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an L. reuteri population under laboratory growth conditions but was poorly maintained (<33% resistant cells) in the L. reuteri population inhabiting the murine gastrointestinal tract. Stable maintenance (>90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner.

Amino Acid Sequence↗
PubMed1999

Identification of Lactobacillus isolates from the gastrointestinal tract, silage, and yoghurt by 16S-23S rRNA gene intergenic spacer region sequence comparisons.

Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification of Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of Lactobacillus casei and Lactobacillus rhamnosus.

Bacterial Typing Techniques↗
PubMed1999

Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties.

It has been shown previously that inactivation of the cshA gene, encoding a major cell surface polypeptide (259 kDa) in the oral bacterium Streptococcus gordonii, generates mutants that are markedly reduced in hydrophobicity, deficient in binding to oral Actinomyces species and to human fibronectin, and unable to colonize the oral cavities of mice. We now show further that surface fibrils 60.7 +/- 14.5 nm long, which are present on wild-type S. gordonii DL1 (Challis) cells, bind CshA-specific antibodies and are absent from the cell surfaces of cshA mutants. To more precisely determine the structural and functional properties of CshA, already inferred from insertional-mutagenesis experiments, we have cloned the entire cshA gene into the replicative plasmid pAM401 and expressed full-length CshA polypeptide on the cell surface of heterologous Enterococcus faecalis JH2-2. Enterococci expressing CshA exhibited a 30-fold increase in cell surface hydrophobicity over E. faecalis JH2-2 carrying the pAM401 vector alone and 2.4-fold-increased adhesion to human fibronectin. CshA expression in E. faecalis also promoted cell-cell aggregation and increased the ability of enterococci to bind Actinomyces naeslundii cells. Electron micrographs of negatively stained E. faecalis cells expressing CshA showed peritrichous surface fibrils 70.3 +/- 9.1 nm long that were absent from control E. faecalis JH2-2(pAM401) cells. The fibrils bound CshA-specific antibodies, as detected by immunoelectron microscopy, and the antibodies inhibited the adhesion of E. faecalis cells to fibronectin. The results demonstrate that the CshA polypeptide is the structural and functional component of S. gordonii adhesive fibrils, and they provide a molecular basis for past correlations of surface fibril production, cell surface hydrophobicity, and adhesion in species of oral "sanguis-like" streptococci.

Actinomyces↗
PubMed1999

Identification of lactobacilli and bifidobacteria.

Selective culture media and phenotypic tests enable lactobacilli to be differentiated from morphologically similar bacteria. The accurate identification of Lactobacillus species can be accomplished by reference to 16S rRNA gene sequences. Species-specific, PCR primers that target the 16S-23S rRNA spacer region are available for a limited number of Lactobacillus species. Molecular methods for the comprehensive identification of Bifidobacterium species are not yet available. Only DNA-DNA reassociation provides a reliable means of species identification for this genus at present. Bifidobacteria can be differentiated from morphologically similar bacteria by the use of genus-specific, PCR primers or oligonucleotide probes.

Bacterial Typing Techniques↗
PubMed1997

Probiotic properties of lactic-acid bacteria: plenty of scope for fundamental R & D.

Probiotic products are marketed widely throughout the world. This is especially true of yogurts that contain strains of lactic-acid bacteria of intestinal origin. Consumption of these products is aimed at promoting the wellbeing of the consumer by impacting on the collection of microorganisms that normally inhabit the intestinal tract. The development of scientifically valid probiotics requires more detailed knowledge of this intestinal microflora than is currently available.

Bifidobacterium↗
PubMed1997

Cloning and expression of an endo-1,3-1,4-beta-glucanase gene from Bacillus macerans in Lactobacillus reuteri.

Strains of the gastrointestinal species Lactobacillus reuteri were electrotransformed with plasmid constructs containing the endo-1,3-1,4-beta-glucanase gene (bglM) of Bacillus macerans. The enzyme was expressed and secreted by the lactobacilli. A plasmid construct containing the bglM gene lacking its promoter was derived and was demonstrated to be useful as a promoter probe vector.

Bacillus↗
PubMed1997

Analysis of fecal populations of bifidobacteria and lactobacilli and investigation of the immunological responses of their human hosts to the predominant strains.

The bifidobacterial and lactobacillus populations of fecal samples collected from 10 human subjects were studied. The numbers of bifidobacteria were similar in the fecal samples of all of the subjects, but lactobacillus numbers varied, even between samples collected from the same individual. Analysis of the composition of the bacterial populations by ribotyping and pulsed-field gel electrophoresis to differentiate between strains showed that, at least for the numerically predominant strains, each subject harbored a unique collection of bifidobacteria and lactobacilli. Predominant bifidobacterial and lactobacillus strains detected in the feces of each subject were used in immunological assays (lymphocyte transformation, serum antibody titers) to determine the influence of the bacteria on the immune system of their host. Immunoglobulin G antibodies reactive with lactobacilli were detected at high concentrations; antibodies reactive with bifidobacteria were present at lower concentrations. The antibodies appeared to be genus specific rather than strain specific. The results of the study emphasized the complexity of the relationship that exists between the intestinal microflora and the human host.

Adult↗
PubMed1997

Properties of porcine and yogurt lactobacilli in relation to lactose intolerance.

Lactobacilli that had been isolated from the stomach of piglets were tested for properties relevant to the production of fermented milk products for consumption by lactose-intolerant humans. The strains were characterized for beta-galactosidase activity, the ability to reduce the lactose concentration of milk, viability, and pH of the fermented milk over a 30-d period. Strains that had favorable attributes were studied further, and the optimal pH for beta-galactosidase activity, ability to grow in the presence of bile salts, and ability to deconjugate bile salts were determined. Commercial yogurts were also examined to determine whether products varied in characteristics that might affect tolerance of milk products by lactose-intolerant subjects. The Lactobacillus sp. isolated from pigs had lower beta-galactosidase activity than did Lactobacillus delbrueckii ssp. bulgaricus strains ATCC 11842 and NCDO 1489 and strains of lactobacilli isolated from yogurt. The beta-galactosidase activity of all strains decreased rapidly once the fermented milk was stored at 4 degrees C. Strain JB10, originating in the stomach contents of the piglets, had properties that were useful for the manufacture of fermented milk products for lactose-intolerant humans. Milk fermented by this strain had a lactose concentration of about 4.0% and contained 6.6 x 10(6) cfu/ml after storage at 4 degrees C for 20 d. Strain JB10 produced a beta-galactosidase that was active at pH 5.5 (35% of the activity at pH 7.0) and was not inhibited by the presence of bile acids in the culture medium. Beta-Galactosidase activity and lactose concentration varied among yogurts.

Animals↗