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The effects of streptomycin, tetracycline and chloramphenicol on the growth of Escherichia coli were studied quantitatively in a conduction-type batch calorimeter at pH 6.2 and 37°C. Change in the growth thermograms with increasing drug concentrations in the medium were analyzed with a kinetic model of non-competitive inhibition.The number of drug molecules needed to inactivate a unit viable cell, m, was estimated to be 1.2+0.1, 0.7+0.1 and 1.3+0.1 for streptomycin, tetracycline and chloramphenicol, respectively. With the assumption that the drug binding sites are all identical, the microscopic dissociation constant per binding site, K, for the drugs was found to be 0.19, 0.43 and 0.94/rnioldirT3 for streptomycin, tetracycline and chloramphenicol, respectively. With these mand K{ values, drug potency curves were drawn for the three drugs.Microbial calorimetry techniques are being used to study drug actions, and the calorimetric evaluation of the antimicrobial action of various drugs has been reported.1~10) All of these studies involved observation of the changes in the heat effect of cell suspension caused by the addition of the drugs being studied. However, results obtained in this way have only limited quantitative meaning and do not provide information on the interaction between the bacterialcells and the drug. Heat evolution during the growth of soil microbes is influenced by foreign substances that act as metabolic inhibitors, and the inhibition by these substances can be quantitatively characterized by mathematical analysis of growth thermograms.n) One advantage of this method is that the growth activity of microbes can be estimated during their growth as a function of the drug concentration. The growth activity of microbes is best described by the growth rate,12'13) if it is accurately measured, and so it seems that drugs action can be more quantitatively defined by kinetic analysis of growth behavior observed in cultures in the presence of various amountsof drugs. In the preceding paper,14) we reported that heat evolution during Escherichia coli growth on bouillon medium can be monitored in a batch calorimeter with good reproducibility, and that growth rate constant of the cells can be determined from the growth thermograms by a simple mathematical treatment. In this study, this method has been used with the same kind of culture of E. coli cells in medium containing one of three antibiotics at various concentrations, and their effects on the microbial growth were investigated as to growth kinetics. Kinetic parameters characterizing the drug actions were calculated. MATERIALS AND METHODSMicroorganism and its culture. The bacterial strain used was E. coli K-12 IFO 3301, which was cultured on 1% bouillon mediumas the test mediuma,s described elsewhere.14) The pH of the culture mediumwas adjusted to 6.2 and the temperature for the measurements was kept at 37°C.* To whomcorrespondence should be sent.

Heat evolution during the growth of Escherichia coli in a stationary culture on bouillon mediumwas continuously monitored with a conduction-type batch calorimeter at different pHs and temperatures. The growth thermograms were found to be reproducible within percent errors of +2.1%, ±0.8% and ± 1.5% for the peak time ofa thermogram, the peak height and the total heat evolution, respectively. The meanheat evolution for the formation of a unit E. coli cell was co= (1.69±0.08) x 10~8Jceir1 at pH 6.2 and 37°C. The mean heat evolution rate per unit cell during growth was g^^pwcell"1, which was consistent with values calculated for other microbial cells. The growth rate constant calculated on kinetic analysis of the growth thermograms was /i= 0.532±0.068 hr"1 at pH 6.2 and 37°C. The pH dependence of the growth rate constant showed that the growth activity of E. coli cells on bouillon medium is maximumin the pH range of 5.5 to 6.5. From the temperature-dependent variations in the growth rate constant, the apparent activation energy of E. coli growth was found to be £a=65.3±7.1 kJmol"1.

Calorimetric analysis was performed of the effects of penicillin G, ampicillin and polymyxin B on Escherichia coli grown on bouillon mediumat pH 6.2 and 37°C. The presence of any one of these drugs shifted the patterns of growth thermograms toward longer incubation periods, although the growth rate constant remained almost unchanged. The changes in the thermogram patterns were analyzed by means of a simple mathematical model and parameters needed to characterize the actions of the drugs on the growth ofE. coli were determined. The drug concentrations at which the number of viable cells at the start of incubation decreased by half during the incubation were 6.94, 0.46 and 0.04^moldm~3 for penicillin G, ampicillin and polymyxin B, respectively. Drug potency curves were drawn for the three drugs using the parameters determined. 2855 (1986).

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