ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1979, p. 580-586 Vol. 15, No. 4 0066-4804/79/04-0580/07$02.00/0 Bactericidal Effect of Combinations of Antimicrobial Drugs and Antineoplastic Antibiotics Against Staphylococcus aureus JOHN Y. JACOBS,`* JACQUES MICHEL,2 AND THEODORE SACKS2 Departments ofPharmacy Services' and Clinical Microbiology,2 Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem, Israel Received for publication 29 January 1979 Six antineoplastic antibiotics were tested against ten strains of Staphylococcus aureus. Four showed bacteriostatic and/or bactericidal activity against each of the ten strains, and two were only bacteriostatic for seven and nine strains, respectively. Using the cellophane transfer technique, combinations of these antineoplastic antibiotics with 16 antibacterial drugs were screened for combined bactericidal activity. Synergism or antagonism was demonstrated in about one- third of the combinations. Checkerboard titrations and killing curves confirmed these findings and indicated that the effective concentrations of the antibacterial agents were similar to those attainable in the serum after therapeutic doses of these drugs. Although the pharmacokinetics of the six antineoplastic antibiotics in humans are not fully known, at least one of them has a peak serum level corresponding to those values at which a bactericidal effect was produced in vitro. Synergism and antagonism between combi- fulfillment of the requirements for a Ph.D. de- nations of antibacterial antibiotics has been well gree.) documented. Little attention, however, has been paid to possible synergism and antagonism be- MATERIALS AND METHODS tween antibacterial drugs and antineoplastic Ten strains of S. aureus were examined. Of these, antibiotics. Most antineoplastic antibiotics have nine were strains isolated from pus, blood, or urine measurable antibacterial activity but are not cultures in the Department of Clinical Microbiology used to treat bacterial infections because of a of the Hadassah-Hebrew University Hospital. (The low therapeutic index. minimal bactericidal concentrations [MBC] ofpenicil- In 1967, Manten and Terra described lin G for two of these nine strains were 16 and 64 ug/ (13) ml, respectively, and for seven strains MBCs were 256 antagonism and indifference, but no synergism, iLg/ml or higher. Strain 10 was S. aureus [Oxford] between combinations of two antineoplastic NCTC 6571, a non-penicillinas producer. The MBC antibiotics, dactinomycin and mitomycin C, and of penicillin G was 1.0 pug/mL) Minimum inhibitory ampicillin and penicillin G when tested on Strep- concentrations/MBCs were determined by using serial tococcus pyogenes, Staphylococcus aureus, dilutions of both the antibacterial and the antineo- Escherichia coli, and Candida albicans. Since plastic drugs inoculated with an overnight culture of then, many new antimicrobial agents and anti- the organism to give a final concentration of 106 bac- neoplastics have been introduced into clinical teria per ml. use, but there has been no systematic study of Each of 16 antibacterial drugs was combined with or each of 6 antineoplastic antibiotics, all of which are in possible synergism antagonism between these routine clinical use, and the 96 combinations were two groups of agents. Such a study might be of screened against 10 strains of S. aureus, using the clinical importance and possible theoretical in- cellophane transfer technique (3) as modified by Cluzel terest. et al. (6). Most patients receiving antineoplastic drugs Full details of the technique have been reported are potential recipients of antibacterial therapy (15). Commercially available cellophane (PO 300,0.019 because of their increased susceptibility to infec- mm thick) was used for the tambours. The impreg- tions, and they frequently receive both types of nated strips were prepared by immersing strips of agents simultaneously. We therefore decided to Whatman no. 3 chromatographic paper in solutions of investigate a variety of combinations of antibac- the drugs shown in Table 1 and drying at 37°C. These concentrations of the antibacterial drugs have been terial and antineoplastic agents against a series previously described as giving suitable test diffusion of different bacteria. This paper reports the re- levels (5), but these authors did not examine the sults with strains of S. aureus. antineoplastic antibiotics. To select the concentrations (This work has been submitted to the Hebrew of antineoplastic antibiotics shown in Table 1, we University of Jerusalem by J. Y. J. in partial initially prepared several strips for each antineoplastic 580 VOL. 15, 1979 ANTIMICROBIAL AND ANTINEOPLASTIC COMBINATIONS 581 TABLE 1. Concentrations used to impregnate paper drug in combination by the MBC of the drug alone. strips for the cellophane transfer technique The total of the FBCs of the two drugs tested is the Drug Symbol Concn (pg/ml) FBC index (ZFBC). By analogy with criteria defined by the Study Group (17) for the index of fractional Antibacterial inhibitory concentrations, a ZFBC of <0.6, which is Ampicillin AM 500 equivalent to reducing the concentration of each agent Carbenicilin CN 2,000 in the mixture to one-fourth of its MBC, was classified Cephaloridine CD 500 as significant synergy. An FBC index of >1.3 was Chloramphenicol C 1,000 regarded as significant antagonism. Clindamycin CL 500 Seven examples of synergism or antagonism were Colistin CO 650 examined to follow the kinetic action of the drugs Erythromycin E 200 individually and in combination during 24 h, using Gentamicin GE 500 concentrations of each drug selected from checker- Kanamycin K 500 board titrations. One-tenth milliliter of a suitably di- Methicillin ME 1,000 luted culture (prepared as above) was added to 2 ml of Nalidixic acid NA 500 Trypticase soy broth containing the selected concen- Penicillin G P 50 trations of the drugs alone or in combination. The Rifampin R 10 tubes were incubated at 37°C, and samples were taken Streptomycin S 50q for viable counts at time intervals from 0 to 24 h. Tetracycline T 200 Vancomycin V 400 RESULTS Antineoplastic Bleomycin B 100 Table 2 shows the antibacterial activity de- Dactinomycin AD 50 tected by the cellophane transfer technique of Daunorubicin DN 200 the antineoplastic agents acting alone. On re- Doxorubicin DX 100 moval of the tambour from the antibiotic- Mithramycin MT 50 treated agar medium and its transfer to antibi- Mitomycin C MC 100 otic-free medium, growth occurred on the tam- bour where the drug had been bacteriostatic and not where it had been bactericidal. drug, using varying concentrations of the drug in the Dactinomycin was bacteriostatic and mito- impregnating solutions. The strip chosen for the tam- mycin C was bactericidal for all 10 strains. Dau- bour test was one that gave a zone of inhibition of 4 to norubicin was usually bactericidal, whereas its 5 mm with strains of S. aureus seeded on agar plates. 14-hydroxy derivative, doxorubicin, was more Care was taken to avoid "carryover" (5). Oxoid DST bacteriostatic. and mith- agar was used for the diffusion and transfer plates. frequently Bleomycin The inoculum contained approximately 106 organisms ramycin were either bacteriostatic or inactive. per ml and was obtained by dilution of an overnight Minimum inhibitory concentrations/MBCs of culture in Trypticase soy broth (Difco). All the results the antimicrobial drugs and the antineoplastic were classified as synergism, indifference, or antago- antibiotics, determined by the serial dilution nism by two of the authors independently, using pre- method, are shown in Table 3. viously published criteria (4, 10). Doubtful results were Ninety-six combinations were tested by the duplicated or even triplicated. On the basis of the cellophane transfer technique on each strain, cellophane transfer results, 11 combinations were cho- and the synergistic and antagonistic results are sen from groups of drugs that frequently showed syn- shown in Table 4. Although indifference was the ergism or antagonism, and these combinations were subjected to quantitative evaluation on several strains, most common outcome, synergism and antago- using the checkerboard technique. Combinations nism occurred in 16.9 and 15.1% of the combi- which rarely showed synergism or antagonism were nations, respectively. not chosen for quantitative analysis. Box titrations The highest frequency of synergistic combi- were performed, with doubling dilutions in Trypticase nations was seen with mitomycin C (39%) and soy broth of the antimicrobial drug, the antineoplastic dactinomycin (35%). Antagonism was most fre- antibiotic, and every combination of each dilution of each agent. An inoculum of 0.05 ml of a 1:100 dilution of an overnight culture was added to each tube to give TABLE 2. Qualitative anti-staphylococcal activity of a final concentration of 10' to 10' organisms per ml. the antineoplastic antibiotics as determined by the Minimum inhibitory concentrations were read after cellophane transfer technique incubation at 37°C ovemight. Quantitative subcul- No. of strains tures were made of those tubes not showing growth Activity on no. of strainLs indicated ADa MC DN DX B MT by plating 0.01 ml on DST agar supplemented with 2% whole blood. Colonies were counted, and MBCs Inactive 0 0 0 0 3 1 (99.9% kill) were recorded after overnight incubation Bacteriostatic 10 0 2 7 7 9 of the plates at 37°C. Bactericidal 0 10 8 3 0 0 Fractional bactericidal concentrations (FBC) of each drug were calculated