Of Significant in Vitro Antagonism Between Penicillin, Cephalothin, and Rolitetracycline FRANZ D

ANTIMICROBIAL AGENTS AND CHEmoTHzRAPY, Nov. 1976, p. 802-808 Vol. 10, No. 5 Copyright © 1976 American Society for Microbiology Printed in U.S.A. Combination of Bacteriostatic and Bactericidal Drugs: Lack of Significant In Vitro Antagonism Between Penicillin, Cephalothin, and Rolitetracycline FRANZ D. DASCHNERI Children Hospital, Division ofAntimicrobial Therapy, University ofMunich, 8 Munich 2, Germany Received for publication 16 April 1976 Although it is generally believed that bactericidal and bacteriostatic drugs should not be combined in vivo, in vitro experiments using the checkerboard dilution technique revealed no antagonism between penicillin/cephalothin and rolitetracycline but rather additive or synergistic activity of either drug combination in 40 to 50% of20 Escherichia coli and 14 Staphylococcus aureus strains. Slight antagonism occurred only between 3 and 8 h after combining penicillin/ cephalothin and rolitetracycline in either bacteriostatic or bactericidal concentrations, but not after 24 h of incubation, nor was antagonism found with combinations of these drugs in bacteriostatic concentrations. Neither bacteriostatic nor bactericidal activity of penicillin/cephalothin and rolitetracycline was inhibited by pretreatment of one E. coli strain with bacteriostatic rolitetracycline or bacteriostatic penicillin/cephalothin concentrations. Penicillin and cephalothin could exert a bactericidal effect after 2-h exposure of theE. coli strain to bacteriostatic rolitetracycline concentrations. Combined action of subinhibitory penicillin and rolitetracycline concentrations resulted in more pronounced inhi- bition of growth than either drug alone. The higher activity of penicillin/ cephalothin in combination with rolitetracycline on some E. coli and S. aureus strains might be due to a better access of rolitetracycline into bacterial cells whose cell walls have been weakened by cell wall-active, bactericidal drugs. Thus, growth ofpenicillin-induced spheroplasts ofE. coli and stable staphylococcal L-forms was inhibited by much lower concentrations of rolitetracycline than were the corresponding parent cells with intact cell walls. According to Jawetz and Gunnison, the anti- streptomycin is not needed for effective treat- biotic and chemotherapeutic drugs can be di- ment of patients with S. viridans endocarditis vided into substances the effect of which is (2). Furthermore, it has long been known from mainly bactericidal (e.g., penicillin, cephalo- in vitro studies that decreasing or increasing sporins) and substances that generally have the concentration of primarily bactericidal and only bacteriostatic effects (e.g., tetracyclines) bacteriostatic drugs results in either bacterio- (11). They noted that whereas two bactericidal static or bactericidal activity (19). None ofthese drugs were often synergistic, a combination of studies, however, considered the original find- bactericidal and bacteriostatic antibiotics often ing of Jawetz that even within a given test resulted in antagonism in vitro. Several excep- system, the results of drug combinations may tions, however, to this chemotherapeutic rule depend on drug concentrations, time of interac- have been found. The bacteriostatic sulfona- tion, inoculum size, and other laboratory varia- mides may act synergistically with the bacteri- bles (10). I therefore investigated some of these cidal polymyxins against Proteus species and variables, especially subinhibitory, bacterio- Pseudomonas aeruginosa (20, 22). Antagonism static, and bactericidal drug concentrations and has been described between two penicillins, time of interaction, combining bactericidal ampicillin and carbenicillin, when tested (penicillin G, cephalothin) and bacteriostatic against Enterobacter cloacae (2). Although in- (rolitetracycline [RTCI) drugs in vitro. In fur- creased killing ofpenicillin-susceptible Strepto- ther experiments with cell wall-defective bacte- coccus viridans by penicillin plus streptomycin ria (L-forms, spheroplasts), I examined why the can be demonstrated in vitro, the addition of combination of bactericidal and bacteriostatic to instead of antago- I Present address: Wissenschaftlicher Rat und Professor, drugs may lead synergism Leiter der Klinkhygiene in der Universitets-Fraunklinik, nism (16). RTC was used because with this drug 7800 Freiburg, West Germany. very high serum levels can be obtained in pa- 802 VOL. 10, 1976 COMBINATION OF BACTERIOSTATIC/CIDAL DRUGS 803 tients after intravenous application, levels that the usual manner. The isobole defining additive exceed the concentrations found to be bacteri- effects of the two antibiotics was obtained by draw- cidal in this study (7, 15). ing a straight line between the minimal inhibitory concentrations (MICs) of each drug acting alone. MATERIAL AND METHODS Synergism would be evident if the line representing combined action of both drugs at various concentra- Test organisms. All Escherichia coli and Staphy- tions fell below the additive isobole (21). Since one- lococcus aureus strains used in this study were iso- tube variations in MICs are commonly observed lated from children with nosocomial diseases, with serial dilution tests, which have a marked mostly urinary tract infections and septicemia. One effect on the shape of the isobole, synergism was E. coli strain isolated from urine was used through- considered present only when there was, with the out the study for the growth and killing curves. The combination, at least a fourfold reduction in the four strains of stable staphylococcal L-forms (L-36, MICs of both antibiotics (e.g., a twofold reduction L-65, L-68, L-101) and the corresponding bacterial with one antibiotic and a fourfold with the other forms were obtained from B. M. Kagan, Cedars one, causing only a slight concave bowing of the Sinai Medical Center, Los Angeles, Calif., and sub- isobole was not accepted as evidence of synergistic cultured weekly as described previously (5). activity). A twofold reduction of the MICs of both or Antibiotics. Potassium penicillin G was supplied either was considered additive, and no lowering of by Bayer (Leverkusen), cephalothin by Eli Lilly & the end points was regarded as indifference. Co. (Bad Homburg), and RTC by Hoechst AG Growth curves. Growth curves (Fig. 4) of one E. (Frankfurt), each as standard powder. Laboratory coli strain in Mueller-Hinton broth or osmotically standard solutions were prepared by dissolving the stabilized brain heart infusion broth were obtained antibiotic in Mueller-Hinton broth; they were stored by measuring optical density, as described previ- at -22°C and thawed immediately before use. Fur- ously (5, 6). Subinhibitory penicillin (150 ,ug/ml) or ther dilution was done in Mueller-Hinton broth RTC concentrations (0.9 ,ug/ml) were added alone, (Merck AG, Darmstadt). in combination, or following pretreatment with one Media. Mueller-Hinton broth adjusted to pH 7.4 of these antibiotics after 3 h of incubation. or brain heart infusion broth (Difco) supplemented Killing curves. Serial colony counts were per- with 10% sucrose for osmotic stabilization was used formed with broth cultures of one E. coli strain to for the growth and killing curves. All synergy stud- determine the time-related bactericidal or bacterio- ies using the checkerboard dilution technique were static effects of penicillin or cephalothin alone or in done in freshly prepared Mueller-Hinton broth, ad- combination with RTC. Broth with one or both anti- justed to pH 7.4 and supplemented with phenol red as biotics was inoculated with bacteria from an over- indicator to facilitate reading the end poiints, as night culture and adjusted photometrically to con- described previously (4). Salt serum agar was pre- tain an average final inoculum of 0.4 x 106 orga- pared according to Kagan (12) using gamma globu- nisms/ml. At 0, 1, 2, 3, 4, 5, 6, and 24 h, 0.1 ml from lin-free horse serum (Grand Island Lab., N.J.) in- each tube was transferred undiluted or in 10-fold stead of human serum. dilutions in broth to petri dishes with Endo agar Synergy studies. The effect of RTC on the inhibi- (Difco). Colony-forming units per milliliter were tory activity of penicillin G or cephalothin was ob- counted after overnight incubation. Typical experi- served using a microtiter variation of the checker- ments are shown in Fig. 1 through 3. board dilution technique, as described previously Antibiotic susceptibility testing of L-forms. Anti- (4). Briefly, twofold dilutions of RTC in 0.025 ml biotic susceptibility testing was done by using the were made in one direction with a microtiter dilutor. agar dilution technique. From salt serum agar with Twofold dilutions of penicillin or cephalothin were equal growth of single L-form colonies, 0.5- by 0.5- added in the same volume manually in the perpen- cm agar blocks were cut out and inversely moved to dicular direction, using a disposable pipette deliver- one-half of the salt serum agar plates containing ing 0.025 ml/drop. Where each ofthe antibiotics was twofold dilutions of RTC (range, 0.45 to 1,000 ,ug/ used alone, 0.025 ml ofbroth was added to achieve a ml). The other halves of the plates were inoculated volume of 0.05 ml. The inoculum of 0.05 ml from an with 0.005 ml of a standardized inoculum (approxi- overnight culture, adjusted to contain approxi- mately 5 x 105 organisms) of the corresponding par- mately 106 organisms/rnl with a Coleman Junior ent forms, which resulted in growth of single colo- spectrophotometer, was added by delivering 1 drop nies.

Load more