Laboratory Aspects of Combined Antibiotic Treatment

Since Ungar's observation in 1943 that penicillin and sulphonamides enhance each other's action, the subject of synergism and antagonism between antibacterial sub stances has received much attention and has recently been extensively reviewed by Garrod (1953). Tests have been made both in vitro and in vivo, and it is certain that some interaction between antibiotics exists and is likely to be therapeutically important. Clinical experience, though limited, confirms this view; on the one hand, it appears that in the treatment of enterococcal endocarditis a combination of penicillin and streptomycin is more efficient than either drug alone (Hunter, 1950; Robbins and Tompsett, 1951 ; Cates et al., 1951), and, on the other hand, in pneumococcal meningitis, penicillin and aureomycin used together are less effective than penicillin alone (Lepper and Dowling, 1951). Interaction occurs in at least three of the manifesta tions of the antibiotic action: inhibition of growth, lethal effect, and emergence of resistant variants. Growth-inhibition tests are simple to perform, but when used to investigate the combined action of antibiotics often fail to correlate with findings in vivo (Jawetz, Gunnison, Speck, and Coleman, 1951; Chopra and Gupta, 1951 ; Bliss et al., 1952). The estimation of the bactericidal effect of a pair of drugs, as opposed to bac teriostatic effect, is much more laborious, but interaction shown in this way may have more therapeutic significance (Hunter, 1950; Gunnison, Jawetz, and Coleman, 1950; Garrod, 1953). The emergence of resistant variants is also bound up with bactericidal action. The present paper records an investigation -of the lethal action of com binations of antibiotics, using a technique simpler than those previously described. The terms synergism and antagonism are defined as follows: a combination of drugs is synergistic when its lethal effect is greater than that of the more lethal drug alone, and antagonistic when its effect is less. Synergism, defined in this way, includes the term additive described by other authors. There may, of course, be no interaction, in which case the effect of the more lethal drug is dominant. A number of different methods have been used in studying the bactericidal effects of mixtures of antibiotics. Generally, complete viable counts have been made at intervals on cultures containing several different con centrations of antibiotics. In order to carry out testing on a larger scale, some workers have used simplified viable counts (Bigger, 1944; Nichols, 1948; Rantz and Randall, 1952), and others the turbidimetric estimation of growth from surviving bacteria (Thomas and Hayes, 1947; Burnell and Kirby, 1951). All these methods are unsuitable for routine testing in a busy clinical labora tory. The simplified viable count method of Martin et al. (1952), recommended by Garrod (1953), tests the action of a pair of antibiotics at one concentration only. This is likely to be misleading, as there are reports that concentrations are important (Hobby and Dawson, 1946; Bliss et al., 1952). The conditions under which antibiotics act in the body are varied, and it is probable that when a patient receives two drugs their relative con centration fluctuates widely. Agar diffusion methods used to detect inhibitory levels have the great advantage of allowing a range of relative concentrations to be tested easily, and as they are also widely employed for routine sensitivity tests it is not surprising that attempts have been made to show interaction by such methods (Peyre and Velu, 1952; King et al., 1953 ; Garrod, 1953). These attempts have, however, been based on interactions at the inhibitory level, and we believe that the appearances of inhibition on solid media can give misleading information. A further step is required to show whether the growth one sees is alive or whether in fact it has been killed. The technique we have used is an agar diffusion method amplified by a simple form of sampling to reveal the l thal action of the antibiotics.