Methods of Testing Combined Antibiotic Bactericidal Action and the Significance of the Results

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 antibiotics 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. 1). band of profuse growth while alongside the strips Bactericidal action may be partial, the numbers of J Clin Pathol: first published as 10.1136/jcp.15.4.328 on 1 July 1962. Downloaded from

330 L. P. Garrod and Pamela M. Waterworth colonies being diminished, sometimes to a very small whence the culture has been referred for examination. number, or complete, the subculture being sterile In some instances penicillin alone proved to be fully ('+' and '-' respectively). bactericidal, and treatment with it was successful. In others the disease was far advanced, and death from CELLOPHANE TRANSFER METHOD heart failure occurred at too early a stage for the results of treatment to be assessed. The patients whose CELLOPHANE TRANSFER METHOD Strips of sterilized Ford blotting paper 0 5 x 5 cm. are immersed completely in brief case histories follow were of more interest. The antibiotic solutions, briefly drained of surplus solution by first was a predicted failure in treatment: in the application to dry blotting paper, and placed at a right remainder several different combinations were recom- angle to one another with the angles in contact on the mended and treatment with them was successful. surface of a blood agar plate: this is incubated overnight Their combination antibiograms are given in Fig. 1. to permit diffusion. This use of freshly impregnated strips is a convenient modification, when large numbers of tests Case 1 A.W., a man aged 37, developed an endo- are not being done, of Chabbert's method of preparing carditis of the aortic valve due to Str. faecalis which was dried strips which are stored. The strengths of antibiotic diagnosed in a provincial hospital in February 1957. He solutions used are penicillin G 600 ,g./ml., methicillin, had had teeth extracted four months before, and 'cystitis' streptomycin, neomycin, kanamycin, polymyxin, and (with pain in the loin and 'brown' urine) two months vancomycin, 1,000 ,g./ml., tetracycline, chloramphenicol, before: in view of the nature of the infection the latter novobiocin, and erythromycin 200 ug./ml. These are the episode seems the more likely origin of the infection. He amounts used by Chabbert, but it is arguable that for was treated from February until the end of May, first some purposes some of them are too high. For testing a with penicillin + streptomycin + Benemid, then with highly sensitive organism the concentration of penicillin streptomycin + erythromycin, but the fever remained is better reduced: this was done in the test of a pneumo- uncontrolled. He was transferred to the National Heart coccus shown in Fig. 3F. Hospital, whence we received his culture, on May 29 The tambour is prepared by stretching a circle of cello- under the care of Dr. Paul Wood. This organism proved phane (those sold as jam pot covers are suitable) over a to be abnormally resistant to penicillin (M.I.C. 6,ug./ml. glass cylinder 7 5 cm. in diameter and 2-5 cm. deep, on by tube dilution), and, although it was normally sensitive which it is held in place by two rubber bands, the whole to streptomycin (10 pg./ml.), this combination did not being sterilized by autoclaving. The inner surface of this kill it nor did any other in a 55-tube test. It was not is inoculated by flooding with a I in 10 dilution of a broth possible to suggest any combination likely to succeed. culture of the organism, the surplus being removed from Seven different antibiotics, some in combinations, were the tilted tambour with a Pasteur pipette. The tambour is given without and effect, after progressive deterioration, http://jcp.bmj.com/ then placed on the surface of the plate from which the the patient died on July 7. At necropsy all three cusps of antibiotic strips have been removed, covered with a the aortic valve were found to be affected, and there was filter paper and transferred to the incubator. When the an aneurysm of the sinus of Valsalva, which had ruptured surface of the tambour is dry, the whole is inverted. After shortly before death. seven hours' incubation the tambour is removed from Case 2 W.S., a man aged 47, was admitted to St. the original plate and transferred to a second plate of Bartholomew's Hospital in February 1958 with an medium containing no antibiotics and incubated over- endocarditis due to Str. faecalis apparently precipitated night. Blood agar is the usual medium, but others may by dilatation of a urethral stricture. This organism was on September 26, 2021 by guest. Protected copyright. be used, e.g., 'chocolate' agar for H. influenzae or simple inhibited by 4 units (2-4 ug.) penicillin per ml., but only nutrient agar with no addition for hardier species. The by 50,000 jig./ml. of streptomycin, and this combination, surface of the second plate should be dry before the as might be expected from the high degree of resistance tambour is applied. to streptomycin, was not totally bactericidal, nor was any other in a 36-tube test found to have such an effect RESULTS (see Fig. 1). A course of penicillin alone was followed by relapse, and it was then found that the combination penicillin + neomycin was totally bactericidal: a course TESTS IN LIQUID MEDIUM WITH SUBCULTURE We of treatment with these two antibiotics purchased full re- employed some variant of this method for over 10 covery at the price oftotal loss ofhearing. This case is fully years: the results with penicillin-resistant streptococci described by Havard, Garrod, and Waterworth (1959). from cases of endocarditis (including three strains of Str. faecalis) are referred to by Cates, Christie, and This result may be compared with that in a patient Garrod (1951). The usual synergic action ofpenicillin described in the following section, treated for six and streptomycin on Str. faecalis has been confirmed weeks with penicillin + kanamycin and recovering repeatedly since that time. without any eighth nerve damage. More recently a fuller range of tests of combined action has been carried out, as described in the pre- Case 3 R.K.G., a man aged 27, with a history of ceding section, on bacteria from patients with rheumatic fever 12 years earlier, was admitted to Queen endocarditis not only in this hospital, but in others, Mary's Hospital, Roehampton, under the care of Dr. G. J Clin Pathol: first published as 10.1136/jcp.15.4.328 on 1 July 1962. 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Methods of testing combined antibiotic bactericidal action and the significance of the results 331 Penicillin Strepto- Tetra- Chloram- Erythro- Novo- Vanco- Bacitracin Oleando- E129 (ostreo- mycin cycline phenicol mycin biocin mycin mycin gricin) Penicillin +- + + + + + + + Streptomycin - + (+) + + () + (4+) Tetracycline + + - - + + - Chloramphenicol --T- + + Erythromycin - + + + + Novobiocin -- -4- - Vancomycin + ± (4 ) Bacitracin + -+ Oleandomycin + + E129 (ostreogricin) + Case 1

Penicillin Streptomycin Tetracycline Chloram- Erythromycin Novobiocin Vancomycin Bacitracin phenicol Penicillin + + (+) (+) + + Streptomycin + + + + ++ + +-+ Tetracycline + + + -- + + + Chloramphenicol + + + + Erythromycin + + Novobiocin r + + Vancomycin - Bacitracin + Case 2

Penicillin Streptomycin Tetracycline Chloram- Erythromycin Novobiocin Vancomycin Bacitracin phenicol Penicillin (+) - + (+) (4) _ (4) Streptomycin (-) + (-) (4) _ + Tetracycline + + ± + + + Chloramphenicol + +- (4+) + Erythromycin + + (4+) (+ Novobiocin + U+) + Vancomycin (4+) (4+) Bacitracin +±+ Case 3 http://jcp.bmj.com/ Penicillin Strepto- Tetra- Chloram- Erythro- Novo- Vanco- Ristocetin Neomycin E129 (ostreo- mycin cycline phenicol mycin biocin mycin gricin) Penicillin (- ) - ( ) () _ Streptomycin -- -)( Tetracycline (-) Chloramphenicol ( ) (-) (-) (4 Erythromycin

Novobiocin (-) (-) U) C . on September 26, 2021 by guest. Protected copyright. Vancomycin Ristocetin Neomycin E129 (ostreogricin) Case 4

Penicillin Strepto- Tetra- Chloram- Erythro- Novo- Vanco- Ristc- Neomycin Bacitracin mycin cycline phenicol mycin biocin mycin cetin

Penicillin + + ± + (4)I (1 - Streptomycin (4-) (4-) (+-) (+) ( ) Tetracycline (4l-) ) (4-) ( ) (U) (+) Chloramphenicol I- Ut) (4-) U+) Erythromycin (+) Ut) (-4) (j-4 - (4-) Novobiocin ( -) Vancomycin (+) - (4-) Ristocetin Neomycin Bacitracin Case 5

FIG. 1. Combination antibiograms for Cases I to 5. + + = growth + = bacteristasis (numbers as in control) (+) = partial bactericidal actioni - = complete bactericidal action J Clin Pathol: first published as 10.1136/jcp.15.4.328 on 1 July 1962. Downloaded from

332 L. P. Garrod and Pamela M. Waterworth Hosking, on 5 November 1957, having been febrile for 0-25 g. q.d.s. orally. The temperature fell to normal in several weeks. He had recently had teeth extracted. He 48 hours and remained so for about 10 days, when it had signs of both mitral and aortic lesions, and blood began to rise again (? cause). The ductus was ligated by culture yielded Str. viridans. This organism required Mr. G. Flavell on 15 September and the child made an 0-5 unit/ml. (0-3 ,ug.) of penicillin to inhibit its growth, uninterrupted recovery. and penicillin was not totally bactericidal, but penicillin Case 6 C.G., a girl aged 10 with a ventricular septal + streptomycin was (Fig. 1). He was treated with defect (Roger type), developed endocarditis in December penicillin alone from 6 November and from 9 November 1958. Str. faecalis was cultivated from her blood. She with penicillin + erythromycin, but in view of these find- was treated elsewhere for one month with penicillin and ings he was given from 22 November penicillin 1-5 mega tetracycline, for two months with tetracycline only, and unit four-hourly (later reinforced with benemid) and 0-5 g. for three months with erythromycin, after which she was streptomycin b.d. This treatment was continued with admitted to the Hospital for Sick Children, Great short intermissions for two months, despite some febrile Ormond Street, under the care of Dr. R. E. Bonham episodes, one of which was associated with a severe Carter on 24 June 1959. Our initial investigations in this Ps. pyocyanea urinary tract infection. He recovered, and case were confined to showing that the combination of is still well and active four years later. penicillin + streptomycin was totally bactericidal for Case 4 A full history ofD.D., a man aged 40, has been this strain of streptococcus, and she was treated with given by Garrod and Waterworth (1962). Briefly, he was these two antibiotics for three months, making a com- found to have an endocarditis due to a Str. viridans nor- plete recovery. She had 0 5 g. streptomycin b.d. through- mally sensitive to penicillin and other antibiotics, and had out: initially 5 mega units penicillin were given six-hourly, a full course oftreatmentwith penicillin and streptomycin. but after two weeks of this the hope was expressed that Fever recurred soon after this was completed, and blood a regime could be devised involving less frequent in- culture was again positive, the streptococcus being now jections. It was suggested that the two daily injections highly penicillin-resistant. The first inquiry made by one should contain penicillin 2 mega units with streptomycin of us (L.P.G.) was whether any teeth had been extracted 0-5 g. and that six hours after each, penicillin V 2 mega during the course: this proved to be so, multiple extrac- units and benemid 0 5 g. be given orally; blood assays tions having been done three weeks after the course showed that this treatment maintained adequate levels of started. The streptococcus was abnormally resistant only both antibiotics. to the two antibiotics administered (M.I.C. penicillin 8 pg./ml.: streptomycin >512 ug./ml.) and a selective effect of treatment on the oral flora was evident, with This case illustrates three points: the inefficacy of extractions resulting in re-infection with a streptococcus the 'static' antibiotics in this disease, treatment for no less than six months having failed, the efficacy of havingtheseunusualcharacters. Despite its high resistance http://jcp.bmj.com/ to these two antibiotics, the combination antibiogram the classical combination, and the possibility of (Fig. 1) was highly encouraging: even the com- making its administration more tolerable for a child bination penicillin+streptomycin was totally bactericidal. who had had a very rough time. That recommended was penicillin + erythromycin, both because of greater ease and safety of administration and because erythromycin was totally bactericidal alone and CELLOPHANE TRANSFER METHOD Typical results are in every combination. This treatment was given and was illustrated in Figs. 2 and 3. They are of three kinds.

successful. Indifference The zones of effect (Fig. 2A) form on September 26, 2021 by guest. Protected copyright. Case S J.S., a girl aged 3 years, and known to have a a right angle, scarcely blunted at all. Streptomycin patent ductus arteriosus, became ill with high fever while is completely bactericidal and vancomycin incom- resident in Ibadan in June 1958. After unsuccessful treat- pletely so, but there is no extension of the strepto- ment at home with penicillin, tetracycline, and chlortetra- mycin effect where this antibiotic is diffusing into the cycline she was admitted to University College Hospital, other zone. Ibadan, where a penicillin-resistant Staph. pyogenes was grown from her blood. Treatment successively with Synergy Fig. 2B shows synergic action by vanco- chlortetracycline and erythromycin, to both of which the mycin and kanamycin against Str. faecalis. (This organism was sensitive, had little effect, and she was formidable combination of two ototoxic drugs is not transferred to the London Hospital under the care of Dr. likely to commend itself therapeutically except as a Wallace Brigden. Her temperature, on erythromycin, was last resort.) There are many survivors in the remoter 99 to 1010F.: when treatment was stopped to obtain a part of the vancomycin zone, but the effect of kana- further culture it rose to 105°F. This culture (obtained on mycin extends deeply into this, converting it into 24 August) kindly referred to us by Professor C. F. total bactericidal effect where the kanamycin con- Barwell, was sterilized byseveralcombinations (seeFig. 1): centration must be quite low. Fig. 2C is an example those including vancomycin, neomycin, and bacitracin of the most observed synergic effect, that were naturally not favoured, and the most promising frequently appeared to be streptomycin and novobiocin, particularly of penicillin and streptomycin on Str. faecalis. The since the latter was bactericidal even when acting alone. penicillin (vertical) zone shows the paradoxical zone As from 29 August, the child was accordingly given phenomenon (Eagle and Musselman, 1948), bacteri- streptomycin 0-2 g. intramuscularly b.d. and novobiocin cidal action being more nearly complete in a band J Clin Pathol: first published as 10.1136/jcp.15.4.328 on 1 July 1962. Downloaded from

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FIG. 2C FJG. 2F FIG. 2. Results oftests by the cellophane transfer method.

Organism Antibiotric Organism Antibiotic Vertical Horizontal Vertical Horizontal A Staph. pyogenes Vancon.iycin Streptomycin E P. mirabilis1 Polymyxin BW 56-72 B Str. faecalis Vancomiycin Kanamycin F P. mirabilis Polymyxin BW 56-72 C Str. faecalis Penicilliin Streptomycin D Str. faecalis Penicilliin Streptomycin 'Test of simple bacteristatic action without transfer (for further Erythro:omycin Erythromycin description see text). J Clin Pathol: first published as 10.1136/jcp.15.4.328 on 1 July 1962. Downloaded from

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C Staph. pyogenes Methicillin Novobiocin (250 igg/mI.) D Staph. albus Novobiocin Penicillin 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 335 to the right of the position of the strip, where the of which must presumably mean that the relative concentration is optimum. Diffusion of streptomycin proportions for maximum antagonism vary some- into this zone converts this partial to total bacteri- what with absolute concentration. cidal action, again to such a distance that the con- Figure 3E illustrates antagonism between tetra- centration of streptomycin required for the effect is cycline and penicillin acting on Staph. pyogenes. evidently low. Figure 2D (see text, Case 7) is the Each is partially bactericidal, penicillin much more same test, but with erythromycin also included in so, but between them is a very broad zone of profuse both strips, showing that this antibiotic antagonizes growth, indicating antagonism over a wide range of both the individual and the combined action of the relative concentrations. Figure 3F shows antagonism other two. between chloramphenicol and penicillin acting on a Figures 2E and 2F illustrate the combined action pneumococcus. Chloramphenicol alone is totally of polymyxin and BW 56-72, which is 2,4-diamino- bactericidal, and penicillin appears to be so at an 5-(3,4,5-trimethoxybenzyl) pyrimidine, an anti- optimal (low) concentration, but where they meet bacterial substance now undergoing investigation there are many survivors. This strain of pneumo- and kindly furnished to us by Dr. D. A. Long of the coccus was cultivated from the cerebrospinal fluid Wellcome Foundation. Figure 2E is not a cellophane of a patient whose meningitis was initially treated transfer preparation, but a simple nutrient agar plate with both of these antibiotics to cover the possi- inoculated with Proteus mirabilis after the two sub- bilities of both Gram-positive and Gram-negative stances had diffused from strips, and therefore infection. As soon as the pneumococcus was identi- illustrates only bacteristatic action. Polymyxin fied (and before any such tests as this were done) (vertical strip) is inactive alone, but is potentiated by treatment was continued with intramuscular peni- BW 56-72, which is also itself bacteristatic. Figure 2F cillin alone, with satisfactory immediate results. is a cellophane transfer test of the same combination, The two following cases illustrate the practical and shows that a bactericidal effect is only produced value of such tests. where BW 56-72 is diffusing into the polymyxin area. Although this combination, by reason of including a Case 7 Mrs. A.K., aged 48, was admitted to the Royal synthetic drug, is strictly outside the present subject, West Sussex Hospital under the care of Dr. J. D. it is included because such a picture is unique in our Whiteside on 13 January 1961 with a history of fever for experience. six weeks, a mitral lesion, and a positive blood culture

Antagonism A minor degree of antagonism (de- (Str. faecalis). On the basis of sensitivity tests she was http://jcp.bmj.com/ noted 'a' in Tables I to III), which is interference treated with penicillin (for most of the time at the rate of 2 mega units four-hourly, reinforced with benemid), with the action of neomycin by tetracycline on Str. streptomycin 0 5 g. b.d., and at first also with erythro- faecalis, is shown in Fig. 3A. Instead of forming a mycin 500 mg. q.i.d. The culture was referred to us simply right angle, the zone of profuse growth forms a for an opinion on whether erythromycin should also be pointed peripheral extension. (The rash of plaques given. As the result of tests to be described it was stopped in this preparation is due to imperfect contact half way through the six weeks' course: the patient made between the cellophane and the medium.) In Fig. 3B a good recovery and remains well. on September 26, 2021 by guest. Protected copyright. penicillin is only partially bactericidal for a (penicillinase-forming) strain of Staph. pyogenes: erythro- Figure 2C shows the synergic action of penicillin mycin, where its concentration is high, is totally and streptomycin on the streptococcus. The effect bactericidal, but where its concentration is only of adding erythromycin to this combination was inhibitory and penicillin is also present, there is a studied in two ways. When a cup containing erythro- baned of profuse growth. This is antagonism at only mycin solution was placed near the meeting point of a certain level of concentration of one of the anti- the antibiotic strips, there were numerous survivors biotics. Figure 3C shows a higher degree of the in this area. When erythromycin was added to each same effect. Novobiocin is totally bactericidal for antibiotic in both strips (Fig. 2D) total bactericidal this strain of Staph. py,ogenes: methicillin at optimal action was inhibited over the whole area of the concentration (exemplifying well the paradoxical plate. It is clear, therefore, that erythromycin can zone phenomenon) is almost totally so, but where it be antagonistic to this combination. A possible meets a concentration of novobiocin below the previous example of this was reported by Dormer bactericidal level for that antibiotic, there is a broad (1960): a girl aged 19 with a Staph. pyogenes infection zone of profuse growth. In Fig. 3D (penicillin and of a patent ductus arteriosus shows no sign in the novobiocin vs. Staph. albus from a case of chronic chart reproduced of responding to the triple com- septicaemia arising in a Spitz-Holter valve) two bination, but responded promptly to penicillin and antibiotics independently bactericidal are shown to streptomycin, shown by test to act synergically on be antagonistic by a band of growth, the curvature this staphylococcus. J Clin Pathol: first published as 10.1136/jcp.15.4.328 on 1 July 1962. Downloaded from

336 L. P. Garrod and Pamela M. Waterworth Case 8 J.N., a woman aged 42, with a history of methods. No explanation can be offered for this: it rheumatic fever at the age of 10, was found in November has simply to be accepted that in any critical 1960 to have an endocarditis due to Str.faecalis involving situation an ad hoc test of combined action on the the aortic valve. After ineffectual treatment elsewhere patient's organism must be done. Results of the with penicillin and chloramphenicol she was admitted to the National Heart Hospital under the care of Dr. Paul present kind can only show which combinations Wood on 31 January 1961. The streptococcus, re-isolated are worth testing and which are not. The most there in four separate cultures, was inhibited by 4 and noteworthy feature of these results is the very strong 32 ug./ml. respectively of penicillin and streptomycin, antagonism between methicillin and the 'static' but this combination, tested by cellophane transfer, was antibiotics, much more consistent than the corre- not totally bactericidal. Of eight other combinations so sponding findings with penicillin. Indeed methicillin tested, two were totally bactericidal, penicillin + kana- + chloramphenicol is the only combination in the mycin and streptomycin + novobiocin. The use of the whole table which was invariably antagonistic. former was advised, and for six weeks the patient had Streptococcus faecalis (Table II). All of the 10 penicillin, 2 mega units four-hourly, accompanied by kanamycin, 0-25 g. t.d.s., for 13 days and thereafter strains tested were from cases of endocarditis. The 0-166 g. four-hourly. Blood assays were done to verify results differ from those obtained with staphylococci that this dose was producing adequate blood levels. She in several ways. The usual synergy between penicillin recovered completely, and suffered no loss of hearing. and streptomycin is shown: other combinations with penicillin are more often antagonistic, including TESTS OF MULTIPLE STRAINS BY CELLOPHANE TRANSFER erythromycin and vancomycin. The few tests with METHOD This laborious series of tests was done to methicillin again show antagonism not only with determine whether this method gives more consistent statlc antibiotics but also with vancomycin. Several results than others. All the organisms used were other combinations differ in their action from that clinical isolates from infections, mostly recent. The on staphylococci, usually in the direction of being largest number of tests was done with the two more often antagonistic. following 'problem' bacteria. Escherichia coli Tests were done with a total of Staphylococcus pyogenes All the 12 strains used seven strains, and naturally with a more limited were penicillinase formers, but with the exception of range of antibiotics. The noteworthy feature in these three also resistant to streptomycin and to tetra- results (Table III) is that, whereas combinations of cycline, they were sensitive to all other antibiotics. static antibiotics (tetracycline and chloramphenicol) The results are given in Table I. Where fewer than with two of the bactericidal antibiotics (strepto-http://jcp.bmj.com/ 12 strains were tested, it is because the combination, mycin and kanamycin) are often antagonistic, e.g., tetracycline + chloramphenicol, was expected tetracycline and chloramphenicol are usually synergic to be indifferent. It will be seen that the results are with polymyxin. not consistent; with few exceptions they are no Other bacteria Tests were done with six strains more so than others have reported using different of Haemophilus influenzae. They showed synergy

TABLE I on September 26, 2021 by guest. Protected copyright. RESULTS OBTAINED BY CELLOPHANE TRANSFER METHOD WITH MULTIPLE STRAINS OF STAPHYLOCOCCUS PYOGENES Methicillin Streptomycin Tetracycline Chloram- Erythromycin Novobiocin Vancomycin Kanamycin phenicol Penicillin 00000 000000 000000 000000 000000 000000 000000 000000 O0aaa OOOOOS AAAAA OAAAAA OOOaaA OOOaaA OO0SS OOOOOS Methicillin 000000 OAAAA AAAAAA O0aaaa 00aaa 000000 000000 OOOOOa AAAAA AAAAAA aaaaAA aaAAAA OOOOSS OOSSSS Streptomycin 00000 000000 000000 000000 000000 000000 Oaaaa O0aaaa OaaaaS 00aaaS OOOOOS 000000 Tetracycline 000000 000000 000000 OO00aa 000000 a aaaaA OaaaA Chloramphenicol 00000 00000 O0aaaa OOOOOa Oaaaa OOaa aaaaA aaaaA Erythromycin 00000 00000 000000 OOaa OOOaa O0aaaa Novobiocin 000000 000000 OOOOaa OOOSSS Vancomycin 000000 000000

0 indifference S synergy a antagonism A = more pronounced antagonism 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 337 TABLE II RESULTS OBTAINED BY CELLOPHANE TRANSFER METHOD WITH MULTIPLE STRAINS OF STREPTOCOCCUS FAECALIS Methicillin Streptomycin Tetracycline Chloram- Erythromycin Novobiocin Vancomycin Kanamycin phenicol Penicillin Not done OOOSS 00000 OOAAA OOaaA 00000 OOOOa OOOOS SssSS OaAAA AAAAA AAAAA 0000a AAAAA SSSsS Methicillin SS AA AAA AA Oa AAA sss Streptomycin 00000 OOOOA OOaaA 00000 OOOOS 00000 Oaaaa AAAAA AAAA SSSA SSSSS 00000 Tetracycline 000 0000 0000 0000 00000 0000 OaaAA Chloramphenicol 000 0000 00000 OAAAA O0aaa AAAAA Erythromycin 0000 00000 OAAAA OOOaA AAAAA Novobiocin 00000 00000 OOOOa OOOSS Vancomycin OOSSS SSSSS

0 = indifference S = synergy a = antagonism A = more pronounced antagonism

'Some of these tests were done with neomycin in place of kanamycin: these two antibiotics behave very similarly.

TABLE III new developments are perhaps worth noting. It is RESULTS OBTAINED BY CELLOPHANE TRANSFER METHOD admitted that no combination will invariably behave WITH MULTIPLE STRAINS OF ESCH. COLI in the same way, but in so far as its action is reason- Tetracycline Chloram- Kanamycin Polymyxin ably consistent, how far does the simple law formu- phenicol lated by Jawetz and Gunnison (1952) still apply? It has been evident for some time that there are Streptomycin OOOa aaaa 0000 000 aaS aaa 000 000 frequent exceptions to this, and an attempt has now

Tetracycline 00 Oaa 000 been made by Manten and Wisse (1961) to elaborate http://jcp.bmj.com/ aaa sss the law to cover them. They propose that each class Chloramphenicol Oaa OOS AA sss of antibiotic, bactericidal and bacteristatic, requires Kanamycin 000 subdivision. In one class of the cidal are those, 000 including penicillin and vancomycin, which kill only 0 = indifference S = synergy growing bacteria and are therefore antagonized by a antagonism A = more pronounced antagonism. certain of the static (tetracyclines, chloramphenicol,

etc.). In the other are those lethal to resting bacteria, on September 26, 2021 by guest. Protected copyright. between either penicillin or methicillin and either and therefore not impeded by stasis due to another streptomycin or kanamycin, antagonism between agent: this class is said to include polymyxins, penicillin or methicillin and tetracycline, and con- streptomycin, and neomycin. This distinction must sistent indifference between penicillin or methicillin be agreed for polymyxin. Manten and De Nooy and chloramphenicol. On the other hand chloram- (1956, 1959) have previously shown that polymyxin phenicol + tetracycline was twice synergic, the only and chloramphenicol act synergically on species of example seen of synergy between two static drugs. Salmonella, the opposite of what is to be expected Two strains each of Proteus mirabilis and Proteus according to the Jawetz law. This action is verified, vulgaris were tested with most combinations of and supported by the behaviour of polymyxin + nine antibiotics. The usual antagonism between static tetracycline, in the few tests with Esch. coli reported (tetracycline and chloramphenicol) and bactericidal here (Table III). On the other hand, the present (streptomycin and kanamycin) was found. There results do not support the idea that streptomycin were very few examples of synergy (but see Figs. 2E should be placed in this category, and the under- and 2F). lying assumption that it is bactericidal to resting cells is incorrect (Garrod, 1948). In so far as neomycin DISCUSSION and kanamycin can be equated, the same argument PRESENT-DAY APPLICABILITY OF THE JAWETZ LAW applies to neomycin. Manten and Wisse also sub- No attempt can be made adequately to review the divide static drugs, proposing a small category, now extensive literature on this subject, but some including cycloserine (and sulphonamides), which do J Clin Pathol: first published as 10.1136/jcp.15.4.328 on 1 July 1962. Downloaded from

338 L. P. Garrod and Pamela M. Waterworth not antagonize penicillin, etc., 'because a compara- can be interpreted in therapeutic terms further tively long space of time elapses before these drugs experience will be necessary to decide. cause bacteriostasis' and meanwhile 'irretrievable injury' can be done by the cidal component. We are indebted to the physicians named in the text for We believe that a further and perhaps more permission to refer to their patients. Much of the work important step can be taken towards reconciling described has only confirmed the findings of Dr. Y. practical findings with the Jawetz law by recognizing Chabbert, who originated both of the methods used, but one of us (P.M.W.) is further indebted to him for the that some antibiotics have a dual type of action. hospitality of his laboratory and for kind instruction in Erythromycin and novobiocin, in particular, can be the cellophane transfer technique. This visit was made static in lower concentrations and cidal in higher, possible by a study grant from the Institute of Medical and the nature of combined action will depend on Laboratory Technology, to which we also express our which of these is at work. The cellophane transfer thanks. method brings this out well: it is clear in Fig. 3B that erythromycin antagonizes penicillin only in a static REFERENCES concentration. Even chloramphenicol may be cidal Cates, J. E., Christie, R. V., and Garrod, L. P. (1951). Brit. med. J., in a high concentration (Fig. 3F), and we have other 1, 653. evidence that it is cidal at much lower levels for Chabbert, Y. (1953a). Ann. Inst. Pasteur. 84, 545. (1953b). Ibid., 85, 122. H. influenzae, a highly sensitive organism. If this (1957). Ibid., 93, 289. additional factor is taken into account, and a few , and Patte, J. C. (1960). Applied Microbiol., Baltimore, 8, 193. antibiotics, certainly including polymyxin and other Dormer, A. E. (1960). Brit. med. Bull., 16, 61. Eagle, H., and Musselman, A. D. (1948). J. exp. Med., 88, 99. polypeptides, are excepted altogether, the Jawetz Elek, S. D., and Hilson, G. R. F. (1954). J. clin. Path., 7, 37. law appears still to be generally applicable. , and Jewell, Pamela (1953). Brit. med. J., 2, 1298. Garrod, L. P. (1948). Cadernos Cientificos, 2, 23. (1953). Brit. med. J., 1, 953. CLINICAL UTILITY OF TESTS OF COMBINED ACTION The and Waterworth, Pamela M. (1962). Brit. Heart J., 24, 39. Havard, C. W. H., Garrod, L. P., and Waterworth, Pamela M. (1959). purpose of citing a series of case histories here is to Brit. med. J., 1, 688. show that tests of combined bactericidal action are Jawetz, E., and Gunnison, J. B. (1952). Antibiot. Chemother., 2, 243. a reliable guide to effective treatment. These , Coleman, Virginia, and Kempe, H. C. (1955). Amer. J. clin. Path., 25, 1016. histories are not exceptional: successful instances, Lederberg, J., and Lederberg, E. M. (1952). J. Bact., 63, 399. particularly of the penicillin-streptomycin com- Manten, A. (1954). Antibiot. Chemother., 4, 1228. (1956). Ibid., 6, 480. bination for Str. faecalis infections, could be and De Nooy, J. A. (1956). Antonie van Leeuwenhoek J. Micro-http://jcp.bmj.com/ multiplied. Failures have been due either to the biol. Serol., 22, 231. - (1959). Ibid., 25, 183. advanced stage ofthe disease when the recommended and Wisse, M. J. (1961). Nature (Lond.), 192, 671. treatment was adopted, with such gross valvular Martin, R., Sureau, B., and Chabbert, Y. (1952). Bull. Mem. Soc. Med. deformity as to render heart failure inevitable, or to H6p. de Paris, 68, 1192. an infection (as in Case 1) due to an organism for which no effective combination could be found. If a ADDENDUM

combination can be identified which is totally on September 26, 2021 by guest. Protected copyright. bactericidal for a heavy inoculum, sterilization of This paper was written before we had seen that by the lesion can confidently be expected if a full course R. Tompsett and M. Pizette (Arch. int. Med., 1962, of treatment is not interrupted by death or intoler- 109, 146) describing four cases of enterococcal endo- ance to the drug. carditis, all of which responded to penicillin + Of the two methods described, that employing a streptomycin, although this combination was totally liquid medium with subculture is easier to perform, bactericidal in vitro for only two of the strains of and if applied only to a few likely combinations, enterococcus. We do not agree with their conclusion should indeed be within the capacity of almost any that such tests 'are not of value in predicting the use- clinical laboratory. The necessary effort to carry it fulness' of this treatment, but in view of these out for the very few serious problem cases which therapeutic results this combination should evidently occur annually in any hospital should surely be be tried, despite an unfavourable result in vitro, if no made. The cellophane transfer method is more better one can be found. It is noteworthy in this difficult and requires special apparatus, experience, connexion that according to E. Jawetz et al. (J. gen. and more time. Its advantage is that it gives a Microbiol., 1954, 10, 191) streptomycin may act picture of both individual and combined action at synergically with penicillin on Str. faecalis in as little different absolute and (in combined action) relative as 1/1000th of the concentration required for its concentrations. How closely the details so revealed independent effect.