J Clin Pathol: first published as 10.1136/jcp.15.4.328 on 1 July 1962. Downloaded from J. clin. Path. (1962), 15, 328 Methods of testing combined antibiotic bactericidal action and the significance of the results L. P. GARROD' AND PAMELA M. WATERWORTH From the Department ofPathology, St. Bartholomew's Hcspital, London SYNOPSIS A description is given of two methods of measuring combined antibiotic bactericidal action: a test in liquid medium with subculture and the cellophane transfer method. It is emphasized that information so obtained is necessary in order to predict the effect ofcombined treatment, particularly in bacterial endocarditis due to organisms not fully sensitive to penicillin. Eight case histories are given, in all of which such a prediction was fulfilled, one of failure and seven of success from the use of five different combinations. The cellophane transfer method was applied to the study of the nature of combined antibiotic action on multiple strains of several bacterial species. The results were rarely uniform for any given combination and species: the necessity for individual tests as a guide to treatment is thus confirmed. Modifications of the theory of combined action formulated by Jawetz are proposed. The advisability of prescribing antibiotics in com- that penicillin alone has been found not to exert binations rather than singly is a long-standing sub- this effect. It cannot be emphasized too strongly that ject of controversy. There are five usually accepted the object of treatment in this disease should be to indications for combined treatment (Garrod, 1953), sterilize the lesion: antibiotics which merely inhibit one of which is the attainment of a synergic effect. bacterial growth arrest its progress for as long as theyhttp://jcp.bmj.com/ When the indication is of a different nature, such as are given but cannot eradicate the infection. For an attempt to prevent acquired bacterial resistance, this reason tests of combined action depending it is advisable to remember that a given combination merely on inhibition of growth are inapplicable and may be either synergic, merely additive, or actually will not be considered here. Tests of bactericidal antagonistic, in accordance with the law formulated action are of the following two kinds. by Jawetz and Gunnison (1952). Although there are many individual exceptions to this, it is generally TESTS IN LIQUID MEDIA WITH SUBCULTURE on September 26, 2021 by guest. Protected copyright. true that synergy is rarely seen except in the action of a pair of bactericidal drugs, and that antagonism If this test is to embrace a number of antibiotics, and only results from combining a bactericidal with a unless it is to be inordinately complicated, each can bacteristatic. The reality of these effects, not only be used in only a single concentration, which should in vitro but in the experimental animal and in the be one attainable in the blood by full doses. In the clinical field itself, is indisputable. method devised by Martin, Sureau, and Chabbert Jawetz and others have emphasized that the (1952), who adduce abundant clinical evidence of action of a combination on a given organism cannot the significance of their results, antibiotics are added be certainly predicted on theoretical grounds. It to broth in a fixed concentration, singly and in requires verification, because not all strains behave every possible combination, these tubes being alike. It is with methods for doing this that this inoculated with the organism and plated after over- paper is concerned. They are called for only in night incubation. The most promising combination connexion with the treatment ofseriously ill patients, is that giving the fewest survivors. The technique of notably those with bacterial endocarditis. The object the test is described in more detail by Chabbert is then to identify a combination which is completely (1953a and b) who describes both an elaboration of bactericidal for the infecting organism, assuming it using multiple antibiotic concentrations, and two means of simplifying it: these are adding the anti- "Now retired. Any correspondence to 2 Cross Path, Radlett, Herts. biotics on discs which are prepared in quantity and Received for publication 15 March 1962. stored, and pre-inoculating the medium used in 328 J Clin Pathol: first published as 10.1136/jcp.15.4.328 on 1 July 1962. Downloaded from Methods of testing combined antibiotic bactericidal action and the significance of the results 329 bulk. A somewhat more elaborate form of this test more remote from the angle colonies are few is described by Jawetz, Gunnison, Coleman, and (antagonism). Between these extremes are other Kempe (1955), which begins by testing the inhibitory appearances indicating lesser degrees of both types action of two concentrations of each antibiotic, the of combined action. A notable advantage of the lower of these being used in combined tests if it method is that in the critical area of the culture the proves capable of inhibiting growth. two separate antibiotic diffusion gradients produce a range of relative concentrations, and it may be TRANSFER METHODS evident from the result that a certain effect is obtained only, or is more marked, when the concentration of REPLICA PLATE In this method, derived from that of one antibiotic exceeds that of the other. According Lederberg and Lederberg (1952), as described by to Chabbert and Patte (1960), such findings can Elek, Hilson, and Jewell (1953), Elek and Hilson successfully be translated in terms of therapeutic (1954), and Manten (1954, 1956), antibiotic- dosage. containing discs are placed on a heavily inoculated We have employed each of these principal plate, producing a zone of inhibition. A replica methods, the first almost exclusively as a guide to plate, inoculated by transfer on a velvet pad, the treatment of individual patients, the second indicates whether this zone contains survivors. Each (cellophane transfer) also for this purpose but with antibiotic should also be tested singly: combined another object in addition. This was to obtain a action is determined either by placing the two dif- general picture of the nature of combined action ferent discs near together or by including both among the more commonly used antibiotics. For antibiotics in one disc. From the results obtained it this purpose it was necessary to test multiple strains is evident that this method gives significant results: of some of the 'problem' bacteria. It was hoped also the objection to it is that velvet transfer carries over to determine whether the cellophane transfer method only about 1 % of the bacteria on the primary plate. gives more consistent results than others. What appears to be complete bactericidal action may therefore not be: indeed, it is not possible by METHODS this method to determine that an antibiotic or combination has sterilized the inoculum, and the LIQUID MEDIUM W1TH SUBCULTURE Antibiotics are dis- object of such a test is to identify a combination solved in broth in a concentrationof l00 pg./ml.(penicillin which will do this and no less. and bacitracin respectively 100 and 2-5 units/ml.) and 0.5 ml. volumes are pipetted into tubes, singly and in http://jcp.bmj.com/ CELLOPHANE TRANSFER The foregoing objection every possible combination. These tubes may conveniently does not to this the be accommodated in a square rack, arranged as shown apply method, since whole in Fig. 1, an arrangement which we are accustomed to operation is conducted on one surface, and every refer to as the 'half chess-board'. The number of tubes survivor from the period during which the anti- required varies from 15 for a test employing five anti- biotics were acting can form a colony. Details of this biotics to 55 for one employing 10. To each of these elegant and revealing method devised by Chabbert tubes is then added 4-5 or 4 ml. (according to whether (1957, 1960) are given in the succeeding section. In they contain one or two antibiotics) of broth preinoculated on September 26, 2021 by guest. Protected copyright. outline, it consists of applying blotting paper strips with the organism to be tested. The medium requires no containing different antibiotics as a rectangle on a addition if the organism is a staphylococcus or Str. plate of medium, and allowing time for their diffu- faecalis: for other streptococci the addition of 5% of sion before removal. A cellophane 'tambour' is then blood is advisable. The inoculum should be a reasonably heavy one: we have added 2 drops of undiluted broth applied, the interior surface of which has been culture to 100 ml. of broth, which results in a viable heavily inoculated with the organism. Both nutrients content of > 105/ml. One loopful from a control tube and antibiotics diffuse through the tambour. After containing no antibiotic is plated to give a standard with preliminary incubation on the original plate, the which the bacterial content of the other tubes can later be tambour is transferred to a plate of normal medium, compared. and bacteria surviving in the zone of inhibition will After overnight incubation a full 2 mm. loopful from then form colonies. each tube showing no growth is plated on blood agar. The final picture shows at a glance to what extent Plates of ordinary size may conveniently be divided into each antibiotic is bactericidal when acting alone, quarters for this purpose, but care must be taken to distribute the inoculum as widely and uniformly as and, from what happens in the area surrounding the possible: if it is concentrated in a small area, carried-over angle where the strips meet, how they act in com- antibiotic may inhibit growth. If the antibiotic is purely bination. This area may be the only part of the plate bacteristatic, the number of colonies is of the same order free of colonies (synergy) or may be occupied by a as in the control culture (denoted by '+' in Fig.