No abstract

Efficacy of liposomal as compared with free ampicillin in treatment of infection due to Listeria monocytogenes was studied in normal and congenitally athymic (nude) mice. After intravenous injection the multilamellar vesicles used were taken up mainly by liver and spleen, the target organs of L. monocytogenes. Injection of empty liposomes in two doses of 2 mumol of vesicle lipid each, administered at 40 and 112 hr after bacterial inoculation, did not influence the course of listerial infection in normal and nude mice. In normal mice liposomal entrapment of ampicillin resulted in significant improvement in the antibiotic's therapeutic index. The use of liposomal ampicillin at a total dose of 0.54 mg (two doses of 0.27 mg each) resulted in a therapeutic effect similar to that resulting from a total dose of 48 mg of free ampicillin (eight doses of 6 mg each). However, neither ampicillin treatment schedules cured infections in nude mice.

The effect of liposomal encapsulation of ampicillin on the antibacterial activity against intracellular Listeria monocytogenes was studied by comparing survival of L. monocytogenes within peritoneal mouse macrophages in the presence of free ampicillin alone or in combination with liposome-entrapped ampicillin. In the presence of 50 ,ug of free ampicillin per ml of the incubation medium, intracellular growth of L. monocytogenes was still observed, although less as compared with intracellular growth in the absence of ampicillin. At a concentration of 50 ,ug of free ampicillin plus 100 ,ug of liposome-entrapped ampicillin per ml, 99% of the intracellular bacteria were killed. On the other hand, a concentration of 150 ,ug of free ampicillin per ml plus empty liposomes only inhibited intracellular bacterial growth, and the bacteria were not killed. In addition, empty liposomes at a concentration of 1 ,umol of lipid per ml had no effect on intracellular bacterial growth. In broth, liposome-entrapped ampicillin at a concentration of 100 ,ug/mI was not bactericidal for L. monocytogenes, indicating that significant leakage of ampicillin from the liposomes with subsequent killing of the bacteria by the free drug did not occur. Therefore, we concluded that liposomal encapsulation of ampicillin results in an increased availability of the antibiotic for the intracellular bacteria.

In this study, the degree of tolerance was determined in several populations of Staphylococcus aureus isolates. The degree of tolerance of a staphylococcal strain can be established in a reproducible way by exposing the strain to increasing concentrations of a ,-lactam antibiotic and determining the number of surviving bacteria at each concentration. The The efficacy of antimicrobial treatment in infections caused by staphylococci with a high MBC:MIC ratio was studied several times. A negative correlation between tolerance and antimicrobial response was demonstrated in some (3,8,10, 11) but not all (6, 7) cases. The diverse results obtained in these studies may be based on differences in laboratory conditions used to demonstrate the phenomenon. In a previous study, we have demonstrated that the percentage of surviving bacteria exposed to high concentrations of a P-lactam antibiotic is reproducible constantly within certain limits (5). In the present study, we attempt to indicate the threshold value between susceptible and tolerant strains by determining the tolerance percentage of a number of S. aureus strains. To investigate whether the prevalence of tolerance has increased in the last few decades, two collections of older strains were studied as well.

The killing rate induced by ampicillin was determined in 20 strains of Escherichia coli. The apparent generation rate constant for each E. coli showed a characteristic concentration-dependent course. This course can be mathematically described and is determined by four parameters. Three of these parameters determine the speed of the process, and the fourth parameter determines a minimal concentration. The susceptibility of the strains, measured as the minimal inhibitory concentration by an agar dilution method, correlated with the minimal concentration and with a minimal inhibitory concentration calculated from the curve, but not with the rate-determining parameters.The in vitro susceptibility of bacteria to antimicrobial agents is usually estimated by measuring the minimal inhibitory concentration (MIC), the minimal bactericidal concentration or both. Both methods give information about the effect of the antibiotic after a specified time interval. These parameters do not account for the kinetic processes that take place when bacteria are exposed to an antibiotic. This paper describes in mathematical terms the kinetics of the processes involved in the killing of bacteria by a P-lactam antibiotic.From the data of the killing curves of Escherichia coli bacteria in contact with ampicillin, a mathematical description of the change in killing rate with the antibiotic concentration was developed. In addition, the parameters from the derived formula have been compared with the traditional susceptibility parameter (MIC) Determination of the killing rate. Proceeding from an overnight culture of an E. coli strain grown in nutrient broth, the optical density at 700 nm of the suspension was adjusted to 0.1, using nutrient broth. Next, the suspension was diluted 3 x 103 times with nutrient broth and preincubated at 37°C for 2 h. The culture was then divided into several subcultures, and ampicillin was added to all except the controls. Table 1 lists the ampicillin concentrations used with each E. coli strain. The subcultures thus obtained were incubated for 2 h, during which samples were taken every 20 min (starting at time zero, immediately after addition of ampicillin). In the samples, the number of CFU per milliliter was determined with the aid of the spiral plate maker system. Counting was done as described in the instructions for the use of petri dishes with a diameter of 14 cm. This means that a nutrient medium is inoculated in a spiral pattern with a continuously diminishing volume of the bacterial suspension. After incubation, the number of colonies is counted on a specific suitable part of the plate. By dividing this number by the volume of the fraction inoculated on the agar area counted, the number of CFU per milliliter is obtained. To eliminate the influence of the high ampicillin concentrations on counting, inoculation was preceded by penicillinase treatment when these high concentrations were added. Killing curves were obtained by plotting the logarithm of the number of CFU per milliliter at a given ampicillin...