The heat inactivation of microbial spores and the mortality of vegetative cells exposed to heat or a hostile environment have been traditionally assumed to be governed by first-order reaction kinetics. The concept of thermal death time and the standard methods of calculating the safety of commercial heat preservation processes are also based on this assumption. On closer scrutiny, however, at least some of the semilogarithmic survival curves, which have been considered linear are in fact slightly curved. This curvature can have a significant effect on the thermal death time, which is determined by extrapolation. The latter can be considerably smaller or larger depending on whether the semilogarithmic survival curve has downward or an upward concavity and how the experimenter chooses to calculate decimal reduction time. There are also numerous reports of organisms whose semilogarithmic survival curves are clearly and characteristically nonlinear, and it is unlikely that these observations are all due to a mixed population or experimental artifacts, as the traditional explanation implies. An alternative explanation is that the survival curve is the cumulative form of a temporal distribution of lethal events. According to this concept each individual organism, or spore, dies, or is inactivated, at a specific time. Because there is a spectrum of heat resistance in the population--some organism or spores are destroyed sooner, or later, than others--the shape of the survival curve is determined by its distributions properties. Thus, semilogarithmic survival curves whether linear or with an upward or a downward concavity are only reflections of heat resistance distributions having a different, mode variance, and skewness, and not of mortality kinetics of different orders. The concept is demonstrated with published data on the lethal effect of heat on pathogens and spores alone and in combination with other factors such as pH or high pressure. Their different survival patterns are all described in terms of different Weibull distribution of resistances as a first approximation, although alternative distribution functions can also be used. Changes in growing or environmental condition shift the resistances distribution's mode and can also affect its spread and skewness. The presented concept does not take into account the specific mechanisms that are the cause of mortality or inactivation--it only describes their manifestation in a given microbial population. However, it is consistent with the notion that the actual destruction of a critical system or target is a probabilistic process that is due, at least in part, to the natural variability that exists in microbial populations.
A novel ,8-lactamase inhibitor has been isolated from Streptomyces clavuligerus ATCC 27064 and given the name clavulanic acid. Conditions for the cultivation of the organism and detection and isolation of clavulanic acid are described. This compound resembles the nucleus of a penicillin but differs in having no acylamino side chain, having oxygen instead of sulfur, and containing a (3-hydroxyethylidine substituent in the oxazolidine ring. Clavulanic acid is a potent inhibitor of many 8-lactamases, including those found inEscherichia coli (plasmid mediated), Klebsiella aerogenes, Proteus mirabilis, and Staphylococcus aureus, the inhibition being ofa progressive type. The cephalosporinase type of /8-lactamase found in Pseudomonas aeruginosa and Enterobacter cloacae P99 and the chromosomally mediated (8-lactamase ofE. coli are less well inhibited. The minimum inhibitory concentrations of ampicillin and cephaloridine against ,8-lactamase-producing, penicillin-resistant strains of S. aureus, K. aerogenes, P. mirabilis, and E. coli have been shown to be considerably reduced by the addition of low concentrations of clavulanic acid.Streptomyces clavuligerus ATCC 27064 (NRRL 3585) has been described as producing several antibiotics structurally related to cephalosporin C, namely, the 3-carbamoyloxymethyl analogue, the 7-methoxy derivative of the latter compound (cephamycin C), and deacetoxy cephalosporin C, as well as penicillin N (8, 9, 12, 16; M. Gorman, M. M. Hoehn, R. Nagarajan, L. D. Boeck, E. A. Presti, J. G. Whitney, and R. L. Hamill, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 11th, Atlantic City, N.J., abstr. 14, p. 7, 1971). During an investigation of the metabolites produced by this culture, a pronounced /8-lactamase inhibitory activity was detected in the culture filtrate using a special bioassay procedure based on /8-lactamase inhibition (4). The substance responsible for the /3-lactamase inhibitory activity was named clavulanic acid (4) and has been shown to have the structure given in Fig. 1 (10). We describe below the detection, isolation, and preliminary information on the /3-lactamase-inhibitory properties of clavulanic acid.MATERIALS AND METHODSCultural conditions for S. clavuligerus. S. clavuligerus ATCC 27064 (NRRL 3585) was grown at 26°C on agar slopes containing 1% Yeatex yeast extract, 1% glucose, and 2% Oxoid agar no. 3, pH 6.8. Mycelium and spores from the slope were used to inoculate flasks containing a seed stage medium consisting of (wt/vol) 1% malt extract (Oxoid), 1% bacteriological peptone (Oxoid), and 2% glycerol. The medium was made up using tap water and adjusted to pH 7.0 with sodium hydroxide solution. Inoculated flasks were shaken for 3 days at 26°C.Production stage flasks containing the DAS medium were inoculated with 5% vegetative inoculum from the seed flasks. The DAS medium consisted of 2% dextrin, 1% Arkasoy 50 soyabean flour (British Arkady Co., Manchester, U.K.), 0.1% Scotasol dried distillers solubles (Thomas Borthwick Ltd, Glasgow, U.K.), and 0.01% FeSO4 7H2O...
Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10(4) cells were 4.66 at 30 degrees C, 4.36 at 10 degrees C and 4.19 at 5 degrees C. These limits were salt dependent, low (4-6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4.66 at 30 degrees C but this was increased to 4.83 at 10 degrees C. At 5 degrees C growth occurred at pH 7.0 but not at pH 5.13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10 degrees C and 30 degrees C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.
Saccharomyces cerevisiae has a single integral plasma membrane heat shock protein (Hsp). This Hsp30 is induced by several stresses, including heat shock, ethanol exposure, severe osmostress, weak organic acid exposure and glucose limitation. Plasma membrane H(+)-ATPase activities of heat shocked and weak acid-adapted, hsp30 mutant and wild-type cells, revealed that Hsp30 induction leads to a downregulation of the stress-stimulation of this H(+)-ATPase. Plasma membrane H(+)-ATPase activity consumes a substantial fraction of the ATP generated by the cell, a usage that will be increased by the H(+)-ATPase stimulation occurring with several Hsp30-inducing stresses. Hsp30 might therefore provide an energy conservation role, limiting excessive ATP consumption by plasma membrane H(+)-ATPase during prolonged stress exposure or glucose limitation. Consistent with the role of Hsp30 being energy conservation, Hsp30 null cultures give lower final biomass yields. They also have lower ATP levels, consistent with higher H(+)-ATPase activity, at the glucose exhaustion stage of batch fermentations (diauxic lag), when Hsp30 is normally induced. Loss of Hsp30 does not affect several stress tolerances but it extends the time needed for cells to adapt to growth under several stressful conditions where the maintenance of homeostasis will demand an unusually high usage of energy, hsp30 is the first yeast gene identified as both weak organic acid-inducible and assisting the adaptation to growth in the presence of these acids.
The weak acid sorbic acid transiently inhibited the growth of Saccharomyces cerevisiae in media at low pH. During a lag period, the length of which depended on the severity of this weak-acid stress, yeast cells appeared to adapt to this stress, eventually recovering and growing normally. This adaptation to weak-acid stress was not due to metabolism and removal of the sorbic acid. A pma1-205 mutant, with about half the normal membrane H ؉-ATPase activity, was shown to be more sensitive to sorbic acid than its parent. Sorbic acid appeared to stimulate plasma membrane H ؉-ATPase activity in both PMA1 and pma1-205. Consistent with this, cellular ATP levels showed drastic reductions, the extent of which depended on the severity of weak-acid stress. The weak acid did not appear to affect the synthesis of ATP because CO 2 production and O 2 consumption were not affected significantly in PMA1 and pma1-205 cells. However, a glycolytic mutant, with about one-third the normal pyruvate kinase and phosphofructokinase activity and hence a reduced capacity to generate ATP, was more sensitive to sorbic acid than its isogenic parent. These data are consistent with the idea that adaptation by yeast cells to sorbic acid is dependent on (i) the restoration of internal pH via the export of protons by the membrane H ؉-ATPase in an energy-demanding process and (ii) the generation of sufficient ATP to drive this process and still allow growth.
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