Biomedical subjects
Publications and source records attributed to A E Beezer.
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Microcalorimetric investigation of the interaction of polyene antibiotics with mid-exponential cells of a growing culture of Saccharomyces cerevisiae has been used as the basis of a bioassay procedure. The assay is rapid, sensitive and reproducible. The results are compared to classical assays and potency ranking orders.
Microcalorimetric bioassay procedures have been applied to the determination of phenol coefficients and dilution coefficients. The derived values are compared with those from a standard AOAC test procedure. This microcalorimetric results indicate close agreement with AOAC values, good reproducibility (+/- 2%), rapidity (30 min per test), potential for automation and the determination of in-use dilutions of disinfectants.
The uptake of nystatin by sensitive Saccharomyces cerevisiae cells has been studied as a function of temperature and nystatin concentration. Following analysis of the data, kinetic rate constants and a monolayer capacity are calculated. This shows that c 13 X more nystatin is taken up than can be supported by the cell surface. The process is accompanied by an activation energy of 41.6 kJ mol-1. It is suggested that these data support a rapid uptake process onto the cell wall followed by a slower diffusion process through the wall to the membrane.
Uptake of nystatin by sensitive cells of Saccharomyces cerevisiae was studied as a function of pH and ionic strength. The pH was shown to affect radically uptake kinetics and extent, whilst ionic strength variation appeared not to modify uptake at all. The effects of variation in the sterol concentration of the cells employed showed a decrease in nystatin uptake as sterol concentration increased. This was not the anticipated observation. Calcium ions were shown to "protect" the yeast cells by associating with the cell and not the antibiotic. Possible explanations are discussed.
The electrophoretic behaviour of fresh and liquid nitrogen stored inocula of Saccharomyces cerevisiae has been studied as a function of pH, ionic strength, buffer composition, growth temperature of the inoculum, number of washings of the inoculum prior to electrophoretic investigation, growth medium used in the preparation of the inoculum, interaction of the inoculum with nystatin, and interaction of the inoculum with calcium ions. The results provide a basis for a discussion of the role of the yeast cell surface in the mode of action of nystatin (a polyene antibiotic), on interaction with sensitive Saccharomyces cerevisiae cells. They indicate the profound importance of the cell surface in the interaction with the antibiotic. Moreover, the results of the temperature growth/electrophoresis experiments support the view that a sharp change in membrane fluidity occurs in the yeast membrane. The results are also discussed in the light of those reported in previous papers on the interaction of yeast cells with nystatin.
A rapid, sensitive microcalorimetric bioassay of good reproducibility has been developed for the antibiotics penicillin-G, carbenicillin, ampicillin and cefoxitin. The use of growing organisms as responding agents allows discussion of the results for systems which may involve biological fluids.
The partition coefficients of resorcinol and some resorcinol monoalkyl-ethers from water to n-octanol have been measured as a function of temperature. These data are used to evaluate delta G, delta H and delta S for the solute transfer process. The data suggest direct calorimetric measurement of these parameters should be performed. Correlation with calorimetrically determined 'biological response' will permit a more detailed and precise commentary upon the quantitative structure-activity relationships for these compounds.
The effects of pH, metal ions (calcium and magnesium) and sterols (cholesterol and ergosterol), on the interaction of polyene antibiotics with respiring Saccharomyces cerevisiae (NCYC 239) cells have been studied by flow microcalorimetry. The bioactivity of nystatin decreases as the pH of the medium increases toward neutral. Calcium and magnesium ions attenuate the biological effects of nystatin, amphotericin B and candicidin. The activities of filipin and pimaracin (natamycin) are unaffected by the presence of these ions. Ergosterol and cholesterol show similar effects. Some possible explanations for these observations are suggested.
A rapid flow microcalorimetric method for the bioassay of phenol disinfectants is described. The assay may be used for phenol within the range 50-80 mM with a reproducibility of +/- 2%. Assay results on other phenols examined indicate the range of the assay. Commercial disinfectants may be assayed directly (the flow microcalorimetric technique makes no demands for optical clarity nor absence of suspended material on the solutions used) with the same reproducibility. An in-use concentration of the chosen disinfectant may be selected from the experimental data since the flow microcalorimetric output permits "time-to-death' of the test organism to be observed directly.
The proposed flow microcalorimetric method for the diagnosis of bacteriuria has been extended to include antibiotic sensitivity testing. Sensitive organisms rapidly (4-8 min) show thermal responses to the added antibiotics over the normal range of concentrations (1 x, 2x, MIC value).
The use of microcalorimetry in the routine identification of microorganisms is critically discussed and assessed. By use of flow microcalorimetric studies on Saccharomyces cerevisiae and Kluyveromyces fragilis the role of physical parameters and that of oxygen tension are discussed. The conclusion reached is that identification of microorganisms by microcalorimetry and subsequent discussion of metabolic events revealed by the thermogram, except under restrictive conditions, is inappropriate. However flow microcalorimetry, in contrast to batch microcalorimetry which has been used in the published material on microorganism identification, may allow characterization of yeasts suitable for particular industrial processes.
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Improvements in the reproducibility of nystatin agar diffusion assays have been achieved by the use of liquid nitrogen stored inocula and deep frozen standard stock solutions. The overall percentage variability of the assay has been reduced from over 5% with daily prepared standards and inocula to around 1% with a frozen inocula and to 0.6% with a combination of frozen inocula and standards. The implications of these improvements in the standardization of nystatin assays, and microbiological assays generally are discussed.
The classical agar diffusion and turbidimetric methods of assay for nystatin are compared with the more recently documented assays for this antibiotic which depend upon physicochemical measurement of the response of micro-organisms. Liquid nitrogen stored inocula were used throughout. It is concluded that the newer methods of assay are as reproducible and reliable as the agar diffusion and turbidimetric methods and that they are generally more sensitive. The choice between the assay methods compared can thus be based on speed, cost and sample through-put.
The clinically important polyene antibiotic amphotericin B, in combination with two antifungal imidazole compounds, clotrimazole and miconazole, was studied in vitro. With use of results of cytoplasmic leakage, metabolic heat output, and minimal inhibitory concentration studies, a definite antagonistic response was demonstrated. It is suggested that, if combined antifungal drug therapy is clinically indicated, the drug combination be tested against the isolate by the simple technique of measuring cytoplasmic leakage or by the more elaborate method of flow microcalorimetry.
The growth of Kluyveromyces fragilis in a complex medium under anaerobic and aerobic growth conditions has been conducted using a flow microcalorimeter. Growth under defined conditions is characterized by an initial exponential rise in heat output rate to a maximum, followed by a decline to a baseline deflection. Inadequate oxygenation can result in a more structured thermogram. The results are used to illustrate the deficiencies of some calorimetric incubations, and observations reported by other authors are critically discussed.
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