Synergism of Fosfomycin-Ampicillin and Fosfomycin-

ANTIMICROBiAL AGENTS AND CHEMoTHERAPY, May 1978, p. 75-79 66-484/78/13-75$2./ Copyright 1978 American Society for Microbiology Vol. 13, No. 5 Printed in U.S.A. Synergism of Fosfomycin-Ampicillin and Fosfomycin- Chloramphenicol Against Salmonella and Shigella E. J. PEREA,* M. A. TORRES, AND M. V. BOROBIO Department ofmicrobiology, School ofmedicine, University of Seville, Seville 9, Spain Received for publication 14 November 1977 Ninety strains of Salmonellaand 5 strains of Shigella were tested for susceptibility to fosfomycin, chloramphenicol, and ampicillin by the agar dilution method. Drug interaction between fosfomycin-ampicillin and fosfomycin-chloramphenicol was studied by the agar dilution method. The fractional inhibitory concentration was calculated. The combination of fosfomycin-ampicillin was synergistic against Salmonella in 74 cases, additive in 7, indifferent in 7, antagonistic in none, and nonevaluable in 2; against Shigella it was synergistic in 27 cases, additive in 9, indifferent in 14, and antagonistic in none. The combination of fosfomycin-chloramphenicol was synergistic against Salmonella in 56 cases, additive in 9, indifferent in 13, nonevaluable in 12, and antagonistic in none; against Shigella it was synergistic in 29 cases, additive in 1, indifferent in 9, nonevaluable in 2, and antagonistic in none. Killing curves with combinations of each antimicrobial agent showed that the cultures that had proven to be indifferent by the agar dilution method showed a bactericidal effect until h 4, with posterior regrowth of the culture after this time period. For the strains in which synergism was demonstrated, total bactericidal activity was reached at 24 h. Fosfomycin (FO) is a broad-spectrum bactericidal antibiotic of simple chemical structure and low molecular weight (8). Its action is due to inhibition of the first step in peptidoglycan synthesis among processes of cell wall biosynthesis in bacteria, incorporating itself into the organism by an active transport system (8). It is active against both gram-positive and gram-negative organisms, including Pseudomonas aeruginosa (6, 1); however, resistance to this agent emerges rapidly, suggesting that FO is likely to be useful only if combined with other antibiotics (6). Previous studies from our laboratory (1) and one other (2) have shown that FO acts synergistically with penicillin on Staphylococcus aureus and Escherichia coli. The aim of the present study was to investigate the effect on Salmonella and Shigella of the combination of FO with antibiotics that act on bacteria either by inhibiting protein synthesis (chloramphenicol [CAF]) or by inhibiting cell wall synthesis (ampicillin [AMP]). MATERIALS AND METHODS Bacteria. Bacterial strains were isolated and identified in the Bacteriology Laboratory of the University Hospital of Seville. A total of 14 clinical isolates recovered from fecal specimens were studied: 5 Shigella (2 S. flexneri 1, 12 S. flexneri 2, 1 S. flexneri 4, 12 S. flexneri 6, and 23 S. sonnei) and 9 Salmonella (5 S. typhi, 3 S. ariona, and 82 S. enteriditis [serotypes: 5paratyphi B, 1 chester, 1 kingston, 1 budapest, 23 typhimurium, 22 thompson, 15 blockley, 12 enteriditis, 1 goverham, and newlands]). Standard inocula were prepared by diluting an 18-h Mueller-Hinton broth culture of the test organisms with saline so that the turbidity matched a.5 Mc- Farland standard. The final inoculum was a 1:1 dilution of the standard. Saline was the diluent used in the agar plate dilution method, and Mueller-Hinton broth was used for the killing curves. Media. Mueller-Hinton agar or broth (Difco Laboratories) adjusted to ph 7.4 was used for cultures and serial dilutions. MIC of individual antimicrobial agents. The individual minimal inhibitory concentrations (MICs) of FO, AMP, and CAF for the 5 Shigella and 9 Salmonella were determined by the agar plate dilution method with a Steers replicator (13). The plates were incubated at 37C for 18 h. The MIC was recorded as the lowest concentration of antimicrobial agent that inhibited growth. Single colonies and a fine barely visiblq growth were ignored (13). Studies of combined antimicrobial activity. Checkerboard MIC studies with two drug combinations were performed with FO-AMP and FO-CAF by an agar plate dilution method with a Steers replicator (12). The checkerboard was achieved by combining in separate plates each of the 11 concentrations of one antibiotic (.25 to 256 jg/ml) with each of the 11 concentrations of the second antibiotic. MICs were determined for each antibiotic of the combination, and isobolograms were plotted (1, 12). Fractional inhibitory concentration (FIC) was the minimum concentration of each of the two antibiotics that had an inhibi- 75 Downloaded from http://aac.asm.org/ on June 19, 218 by guest

76 PEREA, TORRES, AND BOROBIO tory effect when acting together (1). The sum of the FICs of both antibiotics was the FIC index. The results were expressed as synergism, addition, indifference, and antagonism when the values of the FIC index were c.75, 1, higher than 1, and higher than 2, respectively (5, 11). Killing curves. Killing curve assays of combined antimicrobial activity were performed against eight bacterial isolates following a slight modification of the method of Rosenblatt and Stewart (12), using 25-ml flasks. In the FO-AMP combination, two Salmonella and two Shigella were tested to establish the killing curves of the different concentrations of the two drugs used singly or in combination. The combinations had previously proved synergistic in one of the two salmonella and one of the two Shigella; in the other two, they were indifferent. The same procedure was followed with the FO-CAF combination. For the isolates considered to be indifferent to the combination, the concentrations used in the killing curves were subinhibitory both when the agents were used alone and when the agents were used in combination. For the strains affected synergistically, the agents were used in subinhibitory concentrations when used singly and in three- or fourfold dilutions when used in combination. All concentrations of antibiotics selected for testing were clinically achievable in blood after normal dosage. Colony counts were performed at the start of incubation and after 1, 2, 4, 8, 18, and 24 h (12). With this method, synergism was defined as a decrease of 1 log or more in the colony count at the end of h 4 when the combination was used as compared with the count obtained with the most active single agent (7, 9). The results were also interpreted in bactericidal terms (14) as follows: (i) indifference, in which the action of the two drugs together was no greater than that of the more active agent alone; (ii) antagonism, in which the bactericidal effect of one of the drugs was reduced by its combination with the other; (iii) synergism, in which neither drug alone was completely bactericidal, but the two together sterilied the inoculum. RESULTS MICs of individual antimicrobial agents. Susceptibilities of the 9 isolates of Salnonella and 5 isolates of Shigella are given in Table 1. At a concentration of 8 ug/ml, FO inhibited 4% of the Salmonella isolates versus 73% for CAF ANTimICROB. AGFNTs CHEMOTHER. and 87.7% for AMP. At 32,ug/ml, a readily achievable serum concentration, FO inhibited 81% of the isolates, five strains being resistant to more than 256,ug/ml. Seven isolates were resistant to more than 256 pg of AMP per ml, and eight were resistant to the same concentration of CAF. No correlation was observed between susceptibility or resistance and species or serotypes, except for the strains belonging to serolog,cal group B, which showed the highest level of antibiotic resistance, two strains of Salmonella serotype paratyphi B and two strains of Salmonella serotype typhimurium being resistant to more than 256,ug of all three antibiotics per ml. Two strains of Salmonella serotype typhimurium and two isolates of Salmonella serotype thompson were resistant to more than 256 pg of AMP per ml. On Shigella, AMP and CAF were more active than FO, but this difference was observed only at low concentrations. At 32 pg/ml, the percentage of strains inhibited was similar for the three antibiotics. There was a relationship between susceptibilities and species but not between susceptibilities and serotypes. S. flexneri strains were uniformly resistant to AMP and CAF, whereas S. sonnei strains were susceptible to these antimicrobial agents. Five Shigella isolates were found that were simultaneously resistant to all three antibiotics as well as three strains of S. flexneri that were resistant to CAF and FO. Two isolates of S. flexneri and one of S. sonnei were resistant to AMP and FO. Synergism as determined by isobolograms. The results of the different interactions are shown in Table 2. FO and AMP on Salmonella showed synergism in 74 cases (82%), of which 39 (43%) had FICs below.5, indicating a high degree of synergism. In seven (7.7%) there was addition. Therefore, there was a favorable interaction in a total of 81 isolates (9%). In seven cases there was no effect, and no instances of antagonism were encountered. The same combination (FO and AMP) upon Shigella was synergistic in 27 cases (54%), 22 of which had FICs below.5. Addition was observed in nine cases. Downloaded from http://aac.asm.org/ on June 19, 218 by guest TABLE 1. FO, AMP, and CAF MIC results for Salmonella and Shigella isolates Organism (no. of Varying % of inhibited test strains at MIC (jg/m1) of: strains) Test agent 1 2 4 8 16 32 64 128 256 >256 Salmonella (9) FO 4.4 4 71.1 81.1 92.2 94.4 1 AMP 7.7 66.6 81 87.7 89.8 92 1 CAF 23.3 73.3 9 91.2 1 Shigella (5) FO 4 8 22 46 6 86 1 AMP 2 36 4 42 46 48 1 CAF 2 42 44 46 74 1

VoiL. 13, 1978 IN VITRO SYNERGISM OF FOSFOMYCIN 77 TABLE 2. FO-AMP and FO-CAF combinations against Salmonellae and Shigella No. of strains (%) exhibiting: Organism (no. of Test agent combination strains) Synergism Addition Indifference (so.75)a (.75-1.)a (>1.)a Not evaluable Salmonella (9) FO + AMP 74 (82.2) 7 (7.7) 7 (7.7) 2 (2.2) FO + CAF 56 (62.2) 9 (1.) 13 (14.4) 12 (13.3) Shigella (5) FO + AMP 27 (54.) 9 (18.) 14 (28.) FO + CAF 29 (58.) 1 (2.) 9 (18.) 2 (4.) a FIC index. 7 SALMONELLA THOMPSON 9762 *- uwoo posmins :) 4 Z - *'~~~ACONINL -Nmgftd ACILLIN 3 o&-4eeoas m1amp 21 > I 2 3 * 5 6 7 8 18 24 u w a SIOELLA SONNEI 9766 SALMONELLA TYV4 SHUGELLA SONNEI 9767 TIME OF INCUBATION (hrl FIG. 1. Killing curves constructed from colony counts after incubation in FO and AMP individually and in c,ombination. The FIC indexes in the FO-AMP combination were as follows: Salmonella serotye thompson 9762,.9; S. sonnei 9766,.13; Salmonella serotype typhimurium 9761, 2; and S. sonnei 9767, 2. 97U Downloaded from http://aac.asm.org/ on June 19, 218 by guest A favorable interaction was thus found in 36 cases (72%); there was no effect on the other 14 (28%). These belonged to S. flexneri, and all of them had MICs to AMP above 256 jag/ml. The interaction of FO and CAF on Salnonella was synergistic in 56 cases (62%), with an FIC below.5 in 17 isolates (18.8%). Addition was observed in nine isolates (1%). A total of 65 (72%) showed favorable interaction, and there were no cases of antagonism. With Shigella, there were 29 cases (58%) of synergism, with FICs below.5 in 11 (22%) of them. There was addition in 1 cases (2%). Favorable interactions totaled 39 (78%), and there were no cases of antagonism. Killing curves. FO and AMP (synergistic by

78 PEREA, TORRES, AND BOROBIO SALMONELLA PARAlYPI B 9764 ANTIMICROB. AGENTS CHEMOTHER. SALMONELLA TYPHI 9763 : u SHIGELLA SONNEI 9768 SHIGELLA SONNEI 9765 Downloaded from http://aac.asm.org/ 2 3 4 ' ' 7 a w 24 * 2 3 4 5 5 7 8 8 24 TIME OF INCUBATION (hr) FIG. 2. Killing curves constructed from colony counts after incubation in FO and CAF individually and in combination. The FIC indexes in the FO-CAF combination were as follows: Salmonella serotype paratyphi B 9764,.13; S. sonnei 9768,.25; S. typhi 9763, 2; and S. sonnei 9765, 2. the checkerboard method) showed bactericidal activity in the killing curves of S. enteriditis serotype thompson 9762 and S. sonnei 9766, which is complete at 24 h (Fig. 1). On the other hand, FO or AMP alone showed only a bacteriostatic effect that persisted for 4 h, with progressive regrowth after this time period. The same combination (indifferent by the checkerboard method) was bactericidal on Salmonella serotype typhimurium 9761 until h 4, with slow growth thereafter. In the case of S. sonnei 9767, the effect was similar but of a lesser degree. The effect of the combination FO and CAF (Fig. 2) on Salmonella serotype paratyphi B 9764 and S. sonnei 9768 (which had previously proven to be synergistic by the checkerboard method) shows that either antibiotic alone had only bacteriostatic activity, whereas the combination was bactericidal, with total killing at 18 h. In cases in which this combination had previously proven to be indifferent by the checkerboard method, i.e., in strains 9763 and 9765, FO and CAF showed a synergistic effect, since their activity was more than 1 log greater than either antibiotic alone, but their effect was only bactericidal in the first few hours, with regrowth of the cultures after 4 to 6 h. DISCUSSION Two methods for evaluating synergism between FO and AMP or FO and CAF were evaluated in this study. Although the checkerboard agar dilution method yields results that indicate whether there is synergism, antagonism, or indifference of antibiotic combinations, it provides quantitative data for inhibitory activity only; however, it is efficient for studies of a great number of strains. On the other hand, killing curves are cumbersome, time-consuming, and expensive techniques. Nevertheless, the results on June 19, 218 by guest

VOL. 13, 1978 of this study showed good agreement between these two techniques. We found that the first combination (FO- AMP) always achieved more favorable results, as would be expected from a combination of two bactericidal agents acting by inhibition of cell wall synthesis in two different steps (2, 1). The association FO-CAF, a bactericidal and a bacteriostatic agent, proved to be synergistic in more than half of the cases, and there were no cases of antagonism. It is, however, generally believed that two drugs, a bactericidal and a bacteriostatic antibiotic, should not be combined because of their in vitro and in vivo antagonism. Some recent experimental studies question the concept of general antagonism between bacteriostatic and bactericidal drugs (4). Synergism by the killing curves method, was demonstrated by all eight strains tested. All but two of the strains exhibited a decrease of 1 log or more with the combination at 4 h. One strain (S. sonnei 9766) failed to maintain a 1-log difference throughout the entire 24-h test period, but, as suggested by Jawet (7), this is probably not significant as long as an increased rate of early killing is present. If we use a bactericidal criterion (14), we find that the antibiotic combinations were only synergistic (bactericidal) against those strains that showed FICs below.75 by the checkerboard method. Thus, we feel there is complete agreement between both methods or criteria of interpretation. The difference between synergistic or additive effects of the FO-AMP and FO-CAF combinations is clearly shown by the checkerboard studies and by the killing curves at 18 and 24 h, but not at 4 h, since at this point there are no significant differences between the various curves (3). Our data suggest that the combinations of a bactericidal drug (FO) with a bacteriostatic (CAF) or bactericidal (AMP) antibiotic may be useful in treating infections due to Salmonella and Shigella. IN VITRO SYNERGISM OF FOSFOMYCIN 79 LITERATURE CITED 1. Barry, A. L 1976. The antimicrobic susceptibility test: principles and practices, p. 11-112. Lea & Febiger, Philadelphia. 2. Borowski, J., and H. Linda. 1977. Combined action of fosfomycin with B-lactam and aminoglycoside antibiotics. Chemotherapy 23(Suppl. 1):8245. 3. Bulger, R. J., and W. M. Kirby. 1963. Gentamicin and ampicillin: synergism with other antibiotics. Am. J. Med. Sci. 246:717-726. 4. Daschner, F. D. 1976. Combination of bacteriostatic and bactericidal drugs: lack of significant in vitro antagonism between penicillin, cephalotin, and rolitetracycline. Antimicrob. Agents Chemother. 1:82-88. 5. Elion, G. B., S. Singer, and G. H. Hitchings. 1954. Antagonists of nucleic acid derivatives. J. Biol. Chem. 28:477-488. 6. Hendlin, D., B. M. Frost, E. Thiele, H. Kropp, M. E. Valiant, B. Pelak, B. Weissberger, C. Corni, and A. K. Miller. 197. Phosphonomycin. III. Evaluation in vitro, p. 297-32. Antimicrob. Agents Chemother. 1969. 7. Jawet, E. 1968. Combined antibiotic action: some definitions and correlations between laboratory and clinical results, p. 23-29. Antimicrob. Agents Chemother. 1967. 8. Kahan, F. M., J. S. Kahan, P. J. Cassidy, and H. Kropp. 1974. The mechanism of action of fosfomycin (phosphonomycin). Ann. N.Y. Acad. Sci. 235:364-386. 9. Libke, R. D., C. Regamey, J. T. Clarke, and W. M. Kirby. 1973. Synergism of carbenicillin and gentamicin against enterococci. Antimicrob. Agents Chemother. 4:564-568. 1. Moreno-Lope, M., D. Damaso, E. J. Perea, A. Sanche-Sousa, M. L Martine, and M. L Marco. 1971. Phosphonomycin. Quantitative sensitivity spectra of various hospital strains to this new antibiotic. Microbiol. Esp. 24:79-85. 11. Phillips, I., and C. Warren. 1976. Activity of sulfamet4oxaole and trimethoprim against Bacteroides fragilis. Antimicrob. Agents Chemother. 9:736-74. 12. Rosenblatt, J. E., and P. R. Stewart 1974. Combined activity of sulfamethoxaole, trimethoprim, and polymixin B against gram-negative bacilli. Antimicrob. Agents Chemother. 6:84-92. 13. Washington, J. A., H, and A. L Barry. 1974. Dilution test procedures, p. 41-417. In E. H. Lennette, E. H. Spauling, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 14. Waterworth, P. M. 1973. Laboratory control, p. 49-531. In L. P. Garrod, H. P. Lambert, and F. O'Grady (ed.), Antibiotic & chemotherapy, 4th ed. Churchill Livingstone, London. Downloaded from http://aac.asm.org/ on June 19, 218 by guest