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...

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