G. M. Wyatt

Intestinal microflora and gastrointestinal adaptation in the rat in response to non-digestible dietary polysaccharides

1. A comparison was made of the effect of a fibre-free diet and diets containing non-digestible polysaccharides on rat caecal and colonic physiology and microflora. 2. All polysaccharide-containing diets led to enlargement of the caecum and colon, associated with increased weight of contents, and of tissue. Carboxymethylcellulose (CMC) had the most marked effect and animals given this also had watery faeces. 3. The density of bacteria in the caecum and colon varied significantly with diet and the proportion of aerobic bacteria in the flora was increased by the CMC diet. 4. In vitro, CMC and hydroxypropylmethylcellulose were poorly fermented. 5. There was a high correlation (caecum r 0.93; colon r 0.94) between tissue weight and wet weight of organ contents but no correlation with bacterial density, number of bacteria per organ, moisture content or short-chain fatty acid content. 6. It is concluded that caecal and colonic enlargement is due to tissue hypertrophy in response to increased bulk of contents, irrespective of the nature of that bulk which varies with diet; it is unlikely that short-chain fatty acids or other microbial metabolites are the stimulus for the trophic response seen when non-digestible dietary polysaccharides are fed to rats.

Instability in the faecal flora of a patient suffering from food-related irritable bowel syndrome

The faecal microbial flora of a patient with severe irritable bowel syndrome related to multiple food intolerances was very variable and contained a high proportion of facultative bacteria and an unusual incidence of Clostridium species.

Development of a surface plasmon resonance-based immunoassay for Listeria monocytogenes

A polyclonal antibody was produced against Internalin B (InlB)-enriched extract and used to develop an inhibition assay to detect Listeria monocytogenes cells in solution using surface plasmon resonance. The gene sequence encoding for the InlB protein was cloned into a Qiagen pQE-60 vector, expressed in Escherichia coli, and purified by immobilized metal affinity chromatography. Protein G-purified anti-InlB-enriched extract polyclonal antibody was incubated with various concentrations of L. monocytogenes cells and subsequently injected over a purified-recombinant InlB (rInlB)-immobilized CM5 sensor chip surface. A decrease in antibody binding response was observed with increasing L. monocytogenes cell concentrations. Intraday and interday assay variability studies were carried out to evaluate precision and reproducibility. The assay had a limit of detection of less than 2 x 10(5) cells per ml and could be successfully reproduced with coefficients of variation of between 2.5 and 7.7%.

The effect of redox potential, and its interaction with sodium chloride concentration, on the probability of growth of Clostridium botulinum type E from spore inocula

Abstract A most probable number (mpn) count has been used to assess the effect of redox potential and of redox potential combined with increasing concentrations of sodium chloride on the probability of growth of spores of C. botulinum type E strain Beluga. In the majority of cases the maximum count developed within five days. In medium at pH 6·8 – 7·0, containing 0·1% w/v NaCl, incubated at 20°C, the probability of growth of spores in five days was not significantly lower at an E h of approx +60 mV, adjusted by the introduction of air, than in strictly anaerobic conditions at E h −400 mV. Increasing the E h above +60 mV resulted in a decrease in the probability of growth of spores, and at a redox potential between approx +122 mV and +164 mV the probability of growth was decreased by a factor of over 10 5 compared with that at the lowest E h . The inclusion of 3·25% w/v and 4% w/v NaCl in medium at a redox potential of −400 mV decreased the probability of growth by factors of 10 2 and 10 4 respectively, while the combination of 3·25% w/v NaCl and an E h between +62 mV and +122 mV decreased the probability of growth by a factor of 10 6 .

Intestinal microflora, morphology and enzyme activity in zinc-deficient and Zn-supplemented rats

1. Immature, male Wistar rats were given a low-zinc diet (2 mg/kg) for 22-24 d. Control groups received a similar diet supplemented with 58 mg Zn/kg either ad lib., or in amounts matched to the consumption of the Zn-deficient group. Food consumption, rate of growth and food conversion efficiency were markedly lower in the Zn-deficient group of rats compared with controls. Appetite, growth rate and food utilization improved dramatically over a subsequent 4 d period of Zn supplementation. 2. Morphological examination of samples of jejunum and ileum confirmed that Zn deficiency in the rat is accompanied by a reduction in villous dimensions and increase in villous density. After a short period of Zn supplementation, villous density and the basal width and maximum height of individual villi in the jejunum returned to normal. Similar changes occurred in the ileum but to a lesser extent. 3. Mucosal alkaline phosphatase (EC 3.1.3.1) activity was significantly lower in the small intestine of Zn-deficient rats compared with Zn-supplemented rats. Disaccharidase activities were lower in the Zn-deficient group, compared with their feed-restricted counterparts, but were similar to values for ad lib.-fed controls. Tissue alkaline phosphatase and disaccharidase activities were consistently higher after a 4 d period of Zn supplementation, compared with non-supplemented animals, but this increase was only significant for alkaline phosphatase. 4. Although there were striking similarities in the mucosal characteristics of gnotobiotic and Zn-deficient rats, there was no indication that even severe dietary Zn depletion reduced the numbers of viable bacteria present in either the small or large intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

The combined effect of low temperature and low pH on survival of, and growth and toxin formation from, spores of Clostridium botulinum

An inoculum of 7 × 104 spores consisting of approximately equal numbers of spores of two strains each of Clostridium botulinum type A, proteolytic type B, non-proteolytic type B and type E was added to 20 ml volumes of culture medium at pH 7 or adjusted to pHs 5·1, 4·8 or 4·5 with citric acid, which were then incubated under strictly anaerobic conditions, at redox potentials equivalent to approximately −200 mV at pH 7, at 20°C, 16°C, 12°C or 8°C for 8 weeks. Germination, multiplication and formation of toxin occurred rapidly in medium at pH 7 incubated at all temperatures; the toxin detected was type E. In media at pH 5·1, 4·8 or 4·5 no significant change in viable count occurred at any temperature and no toxin formation was detected; counts of viable micro-organisms in heated samples showed that a high number of spores had failed to germinate. From this experiment the probability of growth from single spores in these conditions was calculated to be When inoculated media at pH 5·1, 4·8 and 4·5 that had been incubated at 8°C for 8 weeks were transfered to 30°C, growth and toxin formation were detected at pH 5·1 and pH 4·8 within 23–52 days and 43–61 days respectively, but not at pH 4·5 within 76 days. The toxins detected in these conditions were types A and B.

The faecal flora of two patients with food-related irritable bowel syndrome during challenge with symptom-provoking foods

The faecal microbial flora of two patients with food-related irritable bowel syndrome was examined while they were on a diet excluding symptom-provoking foods, and then on a diet including such a food. The patients reacted differently to the challenge diet but some changes in faecal output, flora and short chain fatty acid content were seen.The faecal microbial flora of two patients with food-related irritable bowel syndrome was examined while they were on a diet excluding symptom-provoking foods, and then on a diet including such a food. The patients reacted differently to the challenge diet but some changes in faecal output, flora and short chain fatty acid content were seen.

Rapid enzyme‐linked immunosorbent assays for the detection of salmonella enteritidis in eggs

Rapid antibody‐based assays have been developed for the detection of Salmonella enteritidis in eggs. The assays consist of an initial non‐selective enrichment step in a chemically defined medium followed by either an indirect competitive enzyme‐linked immunosorbent assay or an antibody‐capture enzyme‐linked immunosorbent assay. The limits of detection are 6 × 105 cells and 5×104 cells/ml respectively, and both assays are highly reproducible. A positive result is obtained from an egg inoculated with 10 S. enteritidis cells in 18 h using either assay. The potential for further improvements is discussed.

The Nature of Reducing Compounds Formed from Sucrose by Erwinia carotovora var. atroseptica

SUMMARY: When grown in a medium containing 2 to 4% sucrose, Erwinia carotovora var. atroseptica formed two reducing compounds which accumulated in the culture medium. These compounds had the properties of the disaccharides 6-O-α-d-glucopyranosyl-d-fructofuranose (palatinose, isomaltulose) and 1-O -α-d-glucosyl-fructose.

Alteration of the Binding Characteristics of a Recombinant scFv Anti-parathion Antibody - 1. Mutagenesis Targeted at the V H CDR 3 Domain

A single-chain variable fragment (scFv) recombinant antibody against the organophosphorous insecticide parathion has been used to generate a set of mutants. Single point mutations were introduced into positions 97-100b of the third complementarity-determining region (CDR) of the heavy chain using oligonucleotide-directed mutagenesis. The mutant scFvs were characterised in an ELISA for parathion and related pesticides. Three groups of clones with altered properties were identified: those which lost all ability to bind to the solid-phase pesticide conjugate in the ELISA; those with reduced binding to the solid phase conjugate, and those with changed ELISA sensitivity to free pesticide. supstitutions into positions 100a and 100b were particularly represented in the first group, indicating that these are either important residues for binding of parathion or that they adversely affect folding of the scFv. The mutation tyrosine to histidine had very different effects depending on the position of the tyrosine in...