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Biomedical subjects

R G Kroll

Publications and source records attributed to R G Kroll.

15 recordsLinked to original sources
PubMed1992

Rapid identification of Aerococcus viridans using the polymerase chain reaction and an oligonucleotide probe.

A polymerase chain reaction/oligonucleotide probe method was developed for the specific identification of the Gram-positive bacterium Aerococcus viridans. Primers for the enzymatic amplification reaction were designed from specific sequences within the 16S rRNA. The method was also highly sensitive and 10 cfu of A. viridans could be detected in 5 h although the reliability of detection was poor in mixed cultures with Escherichia coli.

Base Sequence↗
PubMed1992

The cytochrome composition of the meat spoilage bacterium Brochothrix thermosphacta: identification of cytochrome a3-and d-type terminal oxidases under various conditions.

Brochothrix thermosphacta, grown in batch culture in a yeast-dextrose broth, at temperatures from 30 degrees C to 10 degrees C, contained diverse membrane-bound respiratory cytochromes. Under conditions of moderate aeration, cytochromes of the a-, b- and d-type were detected at all growth temperatures, but the proportions changed as a function of temperature, with the spectra of cells grown at 10 or 15 degrees C being dominated by a-type cytochrome(s). Cytochrome a3 was detected by its reactions with CO and cyanide in cells from all growth conditions. An additional cytochrome a, which was not cyanide-reactive, was also detected, suggesting the presence of an aa3 oxidase complex. Cytochrome d was cyanide- and CO-reactive, but not detectable in photodissociation spectra, presumably because of the very rapid recombination of CO at the sub-zero temperatures used. Decreasing the oxygen transfer rates to batch cultures resulted in enhanced expression of cytochrome d and changed the proportion of the aa3-type oxidase that could be attributed to ligand-binding cytochrome a3; at the lowest oxygen transfer rates, no cytochrome a was detected, suggesting the presence of a cytochrome ba3 terminal oxidase complex. Intact cells showed no evidence of a c-type cytochrome and no haem C was detected in membrane preparations. After growth at 10 degrees C, the cytochrome composition of B. campestris was essentially identical to that of B. thermosphacta. The multiplicity of putative terminal oxidases in B. thermosphacta is discussed.

Carbon Monoxide↗
PubMed1992

Direct application to dairy foods of a Listeria-specific oligonucleotide probe to 16S rRNA.

A potentially Listeria-specific 28 base oligonucleotide probe was designed from 16S rRNA sequence data. Using either 32P or non-radioactive (alkaline phosphatase) labels, the probe was shown to be highly specific as it hybridised to RNA extracted from all of the species of Listeria but not to any of the other bacteria tested. Both probe methods were highly sensitive and ca 10(2) cfu/ml Listeria could be detected in pure cultures. A rapid procedure for extracting RNA from milk, Camembert and cottage cheese was developed. This allowed the direct application of the probe to these foods and gave a rapid and specific method of detecting > 10(2) cfu/g or ml Listeria in these foods.

Base Sequence↗
PubMed1992

The use of bacterial luciferase for monitoring the environmental regulation of expression of genes encoding virulence factors in Listeria monocytogenes.

A promoter probe vector, which utilized the lux AB genes from Vibrio fischeri as reporters of gene expression, was constructed for use in Listeria monocytogenes. Using this system gene expression can be monitored non-destructively and in real-time, simply by measuring cellular bioluminescence. Derivatives of the promoter probe were constructed that contained the cloned promoters from the hlyA and plcA genes of L. monocytogenes. The activity of these promoters was dependent on the transcriptional activator PrfA. Accordingly, in a strain containing an intact copy of the prfA gene, expression from both the hlyA and plcA promoters was 25-45-fold higher than in prfA mutants. Heat shock was identified as an environmental signal which induced expression of hlyA and plcA. Conversely, oxidative stress had no effect upon the expression of the virulence factors. In addition, the composition of the growth media was found to have a dramatic effect upon the expression of hlyA and plcA, suggesting the presence of an unidentified signal which may regulate induction of expression of virulence genes in L. monocytogenes.

Bacterial Proteins↗
PubMed1992

Use of the polymerase chain reaction and oligonucleotide probes for the rapid detection and identification of Carnobacterium species from meat.

The polymerase chain reaction (PCR) was used selectively to amplify specific rDNA sequences of Carnobacterium divergens, C. mobile, C. piscicola and C. gallinarum in purified DNA extracts, crude cell lysates and food samples. The PCR products were visualized by agarose gel electrophoresis and identified, at species level, by hybridization reactions with three specific oligonucleotide probes for C. divergens, C. mobile and C. piscicola/C. gallinarum designed from 16S rRNA sequence data. The PCR was sufficiently sensitive to amplify DNA from a single bacterium to detectable levels after 30 cycles of amplification. Both radioactive (32P) and non-radioactive alkaline phosphatase labelled probes was able to detect the PCR products. Detection was highly specific and the probes did not hybridize with DNA samples from any other of the bacterial species tested. These methods enabled the rapid and specific detection and identification of carnobacteria from pure cultures and samples of meat.

Base Sequence↗
PubMed1992

An investigation of dye reduction by food-borne bacteria.

The rates of reduction of seven redox dyes by 13 bacterial strains were measured and found to vary greatly between different bacterium/dye combinations. Phenazine ethosulphate and toluidine blue were the most rapidly reduced dyes by the majority of bacteria and resorufin and 2-hydroxy-1,4-naphthoquinone were reduced slowly, if at all. There was also considerable variation in the rates of reduction with any single dye/organism combination. Glucose stimulated the rates of endogenous dye reduction in about half of the organisms. For Bacillus cereus, Pseudomonas fluorescens and Escherichia coli, dye reduction was stimulated by a range of exogenous substrates but lactose, the primary available carbon and energy source in milk, had little effect. In Lactococcus lactis, dye reduction was stimulated by sugars but not by organic acids. Oxygen successfully competed with dye reduction in organisms containing respiratory chains, but with membrane fractions, dye reduction was more rapid than oxygen consumption. All the organisms showed little cytosolic dye reduction, except L. lactis which showed substantial rates of reduction of some dyes by this fraction. With the membrane fraction of E. coli and Ps. fluorescens, cyanide inhibited NADH and succinate-dependent dye reduction, Antimycin A inhibited lactate and succinate and rotenone had no significant effect, but inhibition was not always observed with membrane from both organisms.

Animals↗
PubMed1992

Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods.

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10(4) cfu/ml (phenyl phosphate system) or 10(5) cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1-5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).

Biosensing Techniques↗
PubMed1992

The use of immobilized lectins in the separation of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. from pure cultures and foods.

Lectins from Helix pomatia, Canavalia ensiformis, Agaricus bisporus and Triticum vulgaris agglutinated cultures of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. This agglutination was specific as it was inhibited (except with A. bisporus lectin) by the competing sugar substrates. The ability of three of these lectins, immobilized on a variety of supports, to separate these micro-organisms from pure cultures was investigated. Immobilization of the lectins on magnetic microspheres was the most effective method. Immobilized T. vulgaris lectin bound 87-100% of cells from cultures of L. monocytogenes, 80-100% of Staph. aureus, 33-45% of Salmonella spp. and 42-77% of E. coli. The A. bisporus lectin bound 31-63% of cells in cultures of L. monocytogenes, 83% of Staph. aureus but only 3-5% of the salmonella cells. Similarly H. pomatia lectin bound greater than 92% of Staph. aureus and 64% of L. monocytogenes cells but was poor at binding the Gram-negative organisms. This preference for binding Gram-positive organisms was confirmed when mixed cultures were studied. The T. vulgaris lectin was effective in removing L. monocytogenes (43%) and Staph. aureus (26%) from diluted milk and Salmonella (31-54%) from raw egg. Agaricus bisporus lectin removed L. monocytogenes from undiluted milk (10-47%) or ground beef (32-50%).

Animals↗
PubMed1992

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.

Alanine↗
PubMed1990

A semi-homogeneous amperometric immunosensor for protein A-bearing Staphylococcus aureus in foods.

A semi-homogeneous amperometric immunosensor specific to the protein A of Staphylococcus aureus was developed using direct electrochemical detection of phenol produced by alkaline phosphatase from phenyl phosphate. The immunosensor could reliably detect strains of protein A-bearing S. aureus in pure cultures at ca. 10(4) cfu/ml, and at ca. 10(5) cfu/g or ml in various food samples. Due to its semi-homogeneous nature, the system was very simple, easy to operate, and labour-saving. The good correlation between the amperometric current generated by the immunosensor and plate counts illustrated the potential usefulness of this simple system. It proved to be a reliable 24-h detection method for food samples containing very low numbers of protein A-bearing S. aureus after pre-enrichment, as it was able to detect cells that could not directly be enumerated by plate counts.

Biosensing Techniques↗
PubMed1990

An improved amperometric immunosensor for the detection and enumeration of protein A-bearing Staphylococcus aureus.

An amperometric immunosensor specific to the protein A of Staphylococcus aureus, was developed using the direct electrochemical detection of phenol produced by alkaline phosphatase from phenylphosphate. The immunosensor could detect protein A at 0.01 ng/ml and could reliably detect and quantify pure cultures of protein A-bearing Staph. aureus above 10(3) cfu/ml. A similar sensitivity of detection was obtained with samples of beef and milk.

Animals↗