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Chronic kidney disease is an independent risk factor for the development of coronary artery disease and overlaps with other risk factors such as hypertension and diabetes. Percutaneous coronary intervention is a cornerstone of therapy for coronary artery disease and requires contrast media, which can lead to renal injury. Identifying patients at risk for contrast-associated acute kidney injury (CA-AKI) is critical for preventing kidney damage, which is associated with both short- and long-term mortality. Determination of the potential risk for CA-AKI and a new need for dialysis using validated risk prediction tools identifies patients at high risk for this complication. Identification of patients at risk for renal injury after contrast exposure is the first critical step in prevention. Contrast media volume, age and sex of the patient, a history of chronic kidney disease and/or diabetes, clinical presentation, and hemodynamic and volume status are factors known to predict incident contrast-induced nephropathy. Recognition of at-risk patient subpopulations allows for targeted, efficient, and cost-effective strategies to reduce the risk of renal complications resulting from contrast media exposure., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background Ectonucleotidases maintain vascular homeostasis by metabolizing extracellular nucleotides, modulating inflammation and thrombosis, and potentially, myocardial flow through adenosine generation. Evidence implicates dysfunction or deficiency of ectonucleotidases CD39 or CD73 in human disease; the utility of measuring levels of circulating ectonucleotidases as plasma biomarkers of coronary artery dysfunction or disease has not been previously reported. Methods and Results A total of 529 individuals undergoing clinically indicated positron emission tomography stress testing between 2015 and 2019 were enrolled in this single-center retrospective analysis. Baseline demographics, clinical data, nuclear stress test, and coronary artery calcium score variables were collected, as well as a blood sample. CD39 and CD73 levels were assessed as binary (detectable, undetectable) or continuous variables using ELISAs. Plasma CD39 was detectable in 24% of White and 8% of Black study participants ( P =0.02). Of the clinical history variables examined, ectonucleotidase levels were most strongly associated with underlying liver disease and not other traditional coronary artery disease risk factors. Intriguingly, detection of circulating ectonucleotidase was inversely associated with stress myocardial blood flow (2.3±0.8 mL/min per g versus 2.7 mL/min per g±1.1 for detectable versus undetectable CD39 levels, P <0.001) and global myocardial flow reserve (Pearson correlation between myocardial flow reserve and log(CD73) -0.19, P <0.001). A subanalysis showed these differences held true independent of liver disease. Conclusions Vasodilatory adenosine is the expected product of local ectonucleotidase activity, yet these data support an inverse relationship between plasma ectonucleotidases, stress myocardial blood flow (CD39), and myocardial flow reserve (CD73). These findings support the conclusion that plasma levels of ectonucleotidases, which may be shed from the endothelial surface, contribute to reduced stress myocardial blood flow and myocardial flow reserve.

Chronic kidney disease is a major risk factor for developing coronary artery disease, serving as an independent risk factor while overlapping with other risk factors. Percutaneous coronary intervention is a cornerstone of therapy for coronary artery disease and requires contrast media, which can contribute to renal injury. Identifying patients at risk for contrast-induced nephropathy is critical for preventing renal injury, which is associated with short- and long-term mortality. Determination of the potential risk for contrast-induced nephropathy and a new need for dialysis using validated risk prediction tools is a method of identifying patients at high risk for this complication., Competing Interests: Disclosure The authors have no disclosures., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The effect of growth temperature and growth rate on the susceptibility to heat and pH stress were investigated in Listeria monocytogenes grown in continuous culture where these two growth variables could be varied independently of each other, and in batch culture. After growth at 30 degrees C or 10 degrees C at constant growth rate, or at 30 degrees C at different growth rates, cells did not differ in their resistance to heat at 55 degrees C. Cells grown at 30 degrees C were more resistant to acid stress at pH 2.5 than cells grown at the same growth rates at 10 degrees C. Cells grown at low growth rate at 30 degrees C gave greater resistance to acid stress than those grown at high growth rate. Growth temperature and growth rate had independent effects on the susceptibility of L. monocytogenes to acid stress conditions. This may have implications for the survival of L. monocytogenes in acidic foods.

Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods.

Using a novel capillary thermal cycler, randomly amplified polymorphic DNA (RAPD) generated DNA fingerprints were obtained in 3 h. The RAPD profiles were produced using a random 10-mer primer (5'-ACCGCCTGCT-3') which discriminated between different Listeria spp. Unique fragment profiles of Listeria monocytogenes serotypes were produced from serotypes 1a, 2, 3a, 4ab, 4a and 4c but serotypes, 1/2a, 4b, 4d and 7 had similar profiles.

The polymerase chain reaction was used to amplify specifically a 448 base pair internal fragment of the flaA gene from bacteria belonging to the genus Listeria. This gene encodes for the flagellin protein and allowed specific genus discrimination between other commonly found Gram-positive and Gram-negative bacteria and all Listeria spp. with the exception of Jonesia denitrificans.

Uptake of [14C]glycine-betaine by Listeria monocytogenes was stimulated by NaCl with optimal stimulation at 0.4-0.5 M. The glycine-betaine transport system had a Km of 22 microM and a Vmax of 11.7 nmol-1 min-1 mg-1 protein when grown in the absence of NaCl. When grown in the presence of 0.8 M NaCl the Vmax increased to 27.0 nmol-1 min-1 mg-1 protein in 0.8 M NaCl. At NaCl concentrations above 0.5 M the uptake rate of glycine-betaine was reduced. Measurement of intracellular K+ concentrations and fluorescent dye quenching indicated that higher NaCl concentrations also led to a decrease in the electrochemical potential difference across the cytoplasmic membrane. Uptake of glycine was also observed, but this was not stimulated by NaCl.

Listeria monocytogenes survived and, under most conditions, multiplied when inoculated directly into the cheese milk of laboratory made Camembert cheeses. The rate and extent of growth was reduced at lower storage temperatures. Significantly higher rates of growth occurred at the surface compared with the centre of the cheeses, and these were probably associated with increased pH and proteolysis at the cheese surface due to the mould ripening process. Similar results were obtained with Camenbert cheeses surface inoculated after manufacture. There was also temperature-dependent growth of List. monocytogenes on a range of inoculated commercially manufactured soft cheeses. Significant growth occurred in Cambazola, French and English Brie, blue and white Lymeswold, French Camembert and Brie with garlic. Little if any growth occurred in blue and white Stilton, Mycella, Chaume and full fat soft cheese with garlic and herbs at the temperatures examined.

The primary habitat of the intracellular pathogen Listeria monocytogenes is considered to be soil and decaying vegetation. As an opportunistic pathogen it must be able to recognize its entry into host tissue and, in response, co-ordinately induce the expression of virulence factors. No signature molecule, which facilitates this regulation, has been identified for any human pathogen. Our studies have demonstrated for the first time that the expression of major virulence determinants in L. monocytogenes can be repressed by an environmentally ubiquitous molecule. Transcriptional hlyA and plcA fusions to luxAB were used to monitor virulent gene expression in the presence of various disaccharides. These studies revealed that the expression of listeriolysin O and phosphatidylinositol-specific phospholipase C is repressed specifically by the plant-derived disaccharide, cellobiose.

Oligonucleotide primers were designed against rRNA sequences to give a DNA-based PCR assay for the rapid identification/detection of Brochothrix spp. The PCR products could be confirmed by hybridization to an internal oligonucleotide probe. The method successfully and sensitively detected/identified these organisms in pure cultures but was of limited value as a detection method because the detection sensitivity, in relation to conventional plate counts, varied and the assay sensitivity was reduced in the presence of staphylococci. Furthermore, sensitivity was also lost when the assay was applied directly to meat samples. However, a separation step using a lectin (from Agaricus bisporus) immobilized on magnetic beads prior to the PCR assay, allowed the direct detection of low numbers (> 10(2) cfu g-1) of Brochothrix in meat samples within a working day.

The agglutination of a selection of Gram-positive and Gram-negative bacteria by the haemolymph and coelomic fluid from several invertebrates was studied. The haemolymph from Lumbricus terrestris and Limulus polyphemus caused the strongest agglutination of most of the bacteria studied. When the agglutinating fraction of Lim. Polyphemus was liganded to magnetic microspheres 53% of the cells in pure cultures of Listeria monocytogenes C200, 15% of Salmonella enteritidis 37782, 92% of Staphylococcus aureus NCDO 949, 19% of Escherichia coli E4936/76 and 65% of E. coli W2-2 were adsorbed to the beads. The immobilized haemolymph from Lumb. terrestris adsorbed 42% of Salm. enteritidis 37782, 64% of E. coli 4936/76 and 27% of Staph. aureus NCDO 1499 cells and the coelomic fluid from Haemopsis sanguisuga adsorbed 42, 48 and 50% of these cultures respectively. With immobilized Haem. sanguisuga agglutinins, 21-27% of Staph. aureus NCDO 2044 cells were recovered from full-fat pasteurized milk and 20-51% from braising steak. Immobilized Lim. polyphemus agglutinins recovered 17-34% of Staph. aureus cells from raw egg. The potential of agglutinins isolated from invertebrates for enhancing rapid microbiological assays of foods is discussed.

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.

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.

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).

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.

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.

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%).

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.

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.

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.

The agglutination of a selection of bacteria by some lectins was examined. The lectin from Codium fragile agglutinated seven strains of Salmonella typhimurium. The lectin from Helix pomatia agglutinated eight of 12 strains of Listeria monocytogenes and a further two strains gave a weak agglutination reaction. Helix pomatia lectin conjugated to magnetic microspheres enabled the adsorption of L. monocytogenes from suspension with subsequent elution by the competing ligand N-acetyl galactosamine. Affinity chromatography of a suspension of L. monocytogenes through a column of H. pomatia lectin immobilized on agarose, also adsorbed cells and enabled subsequent elution with N-acetyl galactosamine. The column technique enabled the more rapid adsorption of bacteria perhaps because of improved interactions between bacteria and immobilized lectin.

Some factors affecting the fluorescence of bacteria stained with acridine orange and the direct epifluorescent filter technique (DEFT) were studied. When bacterial cells from a chemostat operated at dilution rates between 0.1 and 0.7/h were used the differential fluorescence observed in the DEFT related to cell 'activity' and the orange fluorescence, which was predominant at high growth rates, may be related to an increase in the RNA content of the cells. Heat affected the colour of cell fluorescence and this was dependent on the cell type and, in particular, age. Uptake of acridine orange into the cells was also found to be an important factor determining the colour of fluorescence. However, with heat-treated cells there was no correlation between the amount of uptake and colour of fluorescence. The relative amounts and degree of denaturation of the different types of nucleic acids remaining in the cells after heat treatment appeared primarily to determine the colour of fluorescence.

A modification to the enzyme amplification system described by Self (1984), has been developed in which the addition of semicarbazide hydrochloride increased the sensitivity of detection of protein A-bearing Staphylococcus aureus in foods from 4-6 X 10(3) to 20 colony forming units (c.f.u.) g-1 or ml-1. This may be due to the removal of acetaldehyde produced as a by-product of the amplification cycle thereby permitting further cycling to proceed.

Listeria monocytogenes possessed glucose oxidase and NADH oxidase activities in whole cells and lysed protoplasts respectively. The NADH oxidase activity sedimented with the membrane fraction and was inhibited by the respiratory inhibitors rotenone, 2-heptyl-4-hydroxy-quinoline-N-oxide and cyanide, suggesting the presence of a membrane associated respiratory chain.

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.

An amperometric electrochemical immunoassay specific for protein A-bearing Staphylococcus aureus was developed. The method was based on a sandwich immunosorbent assay and incorporated an enzyme amplification step, using a NAD-specific redox cycle generating NADH (C. H. Stanley, A. Johannsson, and C. H. Self, J. Immunol. Methods 83:89-95, 1985). Reduction of the mediator, ferricyanide, was dependent on the initial concentration of antigen. The final potential was measured by using a Pt disk electrode polarized at +0.8 V to the Ag/AgCl reference electrode. The assay was rapid (4 h) and generated protein A- and cell (S. aureus)-dependent signals. The system was highly sensitive and could detect 10 pg of protein A ml-1 and less than 100 CFU of S. aureus ml-1. Similar sensitivities were observed with S. aureus cultures inoculated into beef and milk, but the sensitivity was reduced slightly (ca. 10(3) g-1) with samples of Cheddar cheese.

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.

An amperometric enzyme-linked immunosensor was developed to detect and quantify levels of Staphylococcus aureus electrically in pure cultures and in foods. The assay was a modification of a 'sandwich' ELISA for the protein A of Staph. aureus, employing catalase-labelled anti-protein A antibody. On addition of hydrogen peroxide to the assay system the catalase released O2 which was monitored using an amperometric oxygen electrode. The rate of current increase was proportional to the antigen concentration (protein A or Staph. aureus). Protein A was detected reliably at 0.1 ng/ml representing a 20-fold increase in sensitivity over the conventional ELISA that used horseradish peroxidase. Pure cultures of Staph. aureus were detected at 10(-3)-10(-4) cfu/ml with the amperometric electrode (cf greater than 10(5)/ml for conventional ELISA). The same level of sensitivity was achieved for inoculated food samples. Low levels of contamination (1 cfu/g) of Staph. aureus were detected after incubation at 37 degrees C for 18 h, and the immunosensor could from the basis of a test for screening and identification of protein A-bearing Staph. aureus in 24 h, although natural variations in protein A content between different strains could make the system unreliable in accurate quantification of cell numbers.

A fluorescent antibody-microcolony technique was developed for the rapid detection of salmonellas in pure cultures. Examination of microcolonies made the detection of salmonellas by epifluorescence microscopy easier and more reliable than using fluorescent antibody and single cells. After a study of the most effective selective enrichment media for increasing the number of salmonellas, the technique was examined with various samples of raw meats. It was able to detect salmonellas in 24 h and appeared to be as sensitive as conventional cultural techniques. Of the 101 samples studied, complete agreement was obtained with conventional methods for 94 but six apparently false positive results and one false negative result occurred.

Heat treatment at 80 degrees C for 10 min effectively destroyed all vegetative cells (except for Gram-positive cocci) and made easier the counting of bacterial spores, which stained orange, green or rarely transparent/black with a dull green halo, in the direct epifluorescent filter technique. The numbers of both orange- or green-staining spores were lower than the plate count. A variety of physiological conditions were used to investigate the relationship of the different staining patterns with germination status. It was concluded that orange-staining spores had germinated and their number agreed with the plate count after incubation in yeast glucose broth at 30 degrees C for 4 h. This observation was unreliable, however, but it was found that a total spore count in the DEFT gave a good agreement with the plate count.

The capacity of E. coli cells to regulate intracellular pH (pHi) during net potassium uptake has been investigated. The data show: (a) that cells sense their intracellular pH; (b) that the pH gradient (delta pH) exerts a feedback regulation on pHi; (c) that a mechanism of regulation of pHi exists which may be independent of Na+ [Zilberstein, Agmon, Schuldiner & Padan (1982) J. Biol. Chem. 257, 3687-3691]; and (d) that cells have a limited capacity to raise their intracellular pH in the absence of net K+ transport.

Effects of the addition of a proteinase (Neutrase 1-5S) and a peptidase (aminopeptidase DP-102) as agents for accelerating the ripening of Cheddar cheese on the survival of some non-starter bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and a Salmonella sp.) were studied throughout a 4-month ripening period. The enzymes were found to have no significant effect on the survival of the Gram-positive bacteria but some significant effects were observed, at some stages of the ripening period, with the Gram-negative bacteria in that lower levels were recovered from cheeses treated with the enzyme system.

The ability of the direct epifluorescent filter technique (DEFT) to enumerate the viable numbers of various species of yeasts was evaluated. A DEFT count could be made in less than 10 min and the DEFT counts of non-heat-treated samples agreed well with plate counts. The DEFT was unsuitable for the enumeration of yeasts in heat-treated samples because non-viable cells fluoresced orange. A double staining technique using Janus Green B and acridine orange was developed to overcome this problem. The modified DEFT enabled viable and non-viable yeasts to be differentiated in heat-treated samples of pure cultures and improved the relationship between the DEFT count and plate count. The method proved to be of little value, however, for use with beverage products because of unreliable staining patterns.

The effects of cinnamic, propionic, benzoic and sorbic acids on the growth and intracellular pH of Escherichia coli were investigated. The data suggest that the potency of weak acids as food preservatives is related to their capacity to reduce specifically the intracellular pH. The data also suggest that although both the undissociated forms of the acid cause the intracellular pH to fall, growth inhibition is due predominantly to the undissociated acid.

The growth patterns of microcolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0.89), pseudomonads (r = 0.93) and staphylococci (r = 0.92) after incubation at 30 degrees C for 3 or 6 h (staphylococci) at contamination levels of above 10(3) bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.

The phs mutation of Escherichia coli has been suggested to affect the Na+/H+ antiport (D. Zilberstein, E. Padan, and S. Schuldiner, FEBS Lett. 168:327-330, 1980). We have recently shown that the mutation affects the rpoA gene and thus affects transcription. The extent of the pleiotropy of the phs mutation was investigated. In addition to the previously reported growth defect on L-glutamate and melibiose, the mutation also affects at least two other metabolic systems. The transport and metabolism of arabinose is impaired and the transport of sulfate is reduced. The extent to which the effects of the phs mutation on metabolism are due to a defect in the Na+/H+ antiport was investigated, and no causal role for this transport system in the metabolic defects was found.

A rapid (less than 6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3- to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.

A two electrode system mounted as a single probe was developed to measure electrochemically the rate of reduction of a redox mediator (thionine) by bacteria. The system gave a rapid (2 min) bacterial-dependent current above 2.5 x 10(5) cfu/ml with pure cultures of bacteria, but when applied to the measurement of the bacterial contamination in samples of meat and milk it was unable to detect or quantify the contamination reliably. Incubation of samples for a few hours before examination enabled the system to detect bacteria in excess of 10(6) cfu/ml.

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk (r = 0.94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0.1 M, pH 3.0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better (r = 0.90) than that using standard 2 ml samples (r = 0.88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.

The role of K+ transport in the generation of a pH gradient in Escherichia coli has been investigated. In K+-depleted cells, net K+ uptake dissipated delta psi (membrane potential) and led to an increase in delta pH (pH gradient). The magnitude of the delta pH formed bore a simple relationship to the net K+ uptake and was substantially independent of the respiratory rate. In K+-replete cells, generation of a pH gradient was again K+-dependent, although no net uptake of this cation occurred. The results are discussed in terms of K+ cycling, and it is suggested that delta pH is in part a function of the rate of cycling and independent of the respiratory rate.