Tobramycin Alone and in Combination Against Pseudomonas

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1982, p /82/ $02.00/0 Copyright C 1982, American Society for Microbiology Vol. 22, No. 5 Effect of Calcium, Magnesium, and Zinc on Ticarcillin and Tobramycin Alone and in Combination Against Pseudomonas aeruginosa JEFFREY J. ZURAVLEFF,1'2 VICTOR L. YU,2,3* ROBERT B. YEE,1 M. K. ZAPHYR,1 WARREN DIVEN,3 AND FLOYD B. TAYLOR2 Graduate School ofpublic Health' and School of Medicine,3* University of Pittsburgh, and the Veterans Administration Medical Center,2 Pittsburgh, Pennsylvania Received 6 May 1982/Accepted 3 August 1982 Correlation between in vitro and in vivo test results for synergy between carboxypenicillins and aminoglycosides against Pseudomonas aeruginosa is poor. Although the divalent cation content of culture media is known to affect aminoglycoside susceptibility testing for P. aeruginosa, this effect of divalent cations has not been examined for synergy testing of carboxypenicillin-aminoglycoside interaction against P. aeruginosa. The minimal inhibitory concentrations (MICs) of tobramycin and ticarcillin and the interaction of these drugs in combination were studied by a microtitration method for 36 strains of P. aeruginosa in Mueller-Hinton broth with varying supplements of calcium, magnesium, and zinc. The supplementation of Mueller-Hinton broth to 50 or 100 mg of calcium per liter had a significant effect in increasing the tobramycin MIC (P < 0.01), as well as decreasing the degree of synergy between ticarcillin and tobramycin (P < 0.01). Supplementation to 20 mg of magnesium per liter, 1.0 mg of zinc per liter, or both did not significantly affect tobramycin MIC or the interaction of tobramycin and ticarcillin. Supplementation to 50 or 100 mg of calcium per liter rendered any additional effect of magnesium and zinc on aminoglycoside MIC and aminoglycoside-carboxypenicillin interaction negligible. If these results for ticarcillin and tobramycin are confirmed for other carboxypenicillins and aminoglycosides, then the Mueller-Hinton broth used for P. aeruginosa aminoglycoside susceptibility and synergy testing may need to be supplemented only with calcium at a concentration of 50 mg/liter. In vitro synergy between carboxypenicillin (carbenicillin or ticarcillin) and an aminoglycoside against Pseudomonas aeruginosa is well documented (1, 13, 19). However, in vivo models examining the synergistic interaction of carboxypenicillin and an aminoglycoside against P. aeruginosa have produced conflicting results. For example, Chusid et al. (M. J. Chusid, S. D. Davis, and L. D. Sarff, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 11th, Boston, Mass., abstr. no. 1071, 1979) studied the efficacy of ticarcillin and tobramycin, singly and in combination, in a guinea pig model for P. aeruginosa bacteremia; they were unable to show an improvement in survival with combinations demonstrated to be synergistic in vitro against strains of Pseudomonas, as compared to a single-drug therapy. The results of in vitro studies examining the interaction between carboxypenicillin and aminoglycosides against P. aeruginosa are not easily compared. This is due to several reasons. First, the definition of synergy varies from study to study. Second, the divalent cation content of the culture media, which will affect aminoglycoside susceptibility (18, 22, 23), can also vary from study to study; unfortunately, the cation content is not even reported in a number of synergy studies of P. aeruginosa. We wondered whether some of the discrepancies in the literature concerning penicillin and aminoglycoside synergy might be due to differences in divalent cation content of the test media. To our knowledge the effect of divalent cations on the synergistic interaction of carboxypenicillins and aminoglycosides has not been systematically evaluated. We sought to do so by testing the interaction between ticarcillin and tobramycin against 36 clinical isolates of P. aeruginosa in 12 cation-defined media. MATERIALS AND METHODS Bacteria. Thirty-five clinical isolates of P. aeruginosa were obtained from the diagnostic microbiology laboratories of Presbyterian-University, Mercy, and 839

2 840 ZURAVLEFF ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Total concentration of calcium, magnesium, and zinc in 12 test media used for susceptibility and synergy testing Total concn (mg/liter) of divalent cationa Medium Supplement (mg/liter) Calcium Magnesium Zinc A None B Mg C Zn D Mg + Zn E Ca (50) F Ca (50) + Mg G Ca (50) + Zn H Ca (50) + Mg + Zn I Ca (100) J Ca (100) + Mg K Ca (100) + Zn L Ca (100) + Mg + Zn Normal human serum a Total concentrations of 50 or 100 mg of calcium, 20 mg of magnesium, and 1.0 mg of zinc per liter are expected concentrations after supplementation. Observed concentrations did not differ by more than ±7%. Veterans Administration Hospitals, Pittsburgh, Pa. All isolates were identified by standard criteria as P. aeruginosa. P. aeruginosa ATCC served as a control. Isolates were stored in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) with 15% glycerol (vol/vol) at -70 C. Weekly subcultures were made on Mueller-Hinton agar (BBL). Several colonies from the subculture were inoculated into Trypticase soy broth the day before susceptibility testing and incubated at 37 C overnight. Antibiotics. Stock solutions were prepared weekly from reagent-grade powders of ticarcillin disodium (Beecham Pharmaceuticals, Bristol, Tenn.) and tobramycin sulfate (Eli Lilly and Co., Indianapolis, Ind.). Culture media. Calcium, magnesium, and zinc concentrations were determined for the commercial Mueller-Hinton broth (BBL, lot no. E2DJAY) used for testing. Concentrations of the divalent cations were determined by flame atomic absorption spectrophotometry. In addition, the ionized calcium concentrations were determined with a calcium-specific electrode. Supplemented media were prepared by addition of sterile, stock solutions of reagent-grade MgCl2 * 6H20, CaC12 * 2H20, and ZnSO4 to 500-mI volumes of sterile Mueller-Hinton broth (Table 1). Total concentrations of the divalent cations and ionized calcium in the supplemented media were then measured. Susceptibility testing. The susceptibility of the 36 strains of P. aeruginosa was determined for ticarcillin and tobramycin alone and in combination by a previously described microtiter broth dilution checkerboard method (8, 17). Each test was performed in duplicate, and all strains were tested in parallel in each of 12 media by this method. The minimal inhibitory concentration (MIC) was designated as the lowest concentration of antibiotic(s) in a column of the plate needed to inhibit visible turbidity. Each microtiter test provided the MICs for ticarcillin and tobramycin alone and for combinations of the two drugs in concentration ratios of 1:1, 2:1, and 1:2. In addition, fractional inhibitory concentration (FIC) indices were calculated for the combinations of ticarcillin-tobramycin as described previously (6). The mean of the three FIC indices for the 36 strains was calculated and is recorded in Table 2. An FIC index of 1.0 denotes an additive or indifferent interaction; an index of less than 1.0 implies a synergistic interaction. Gilbert et al. (11) and D'Amato et al. (3) have previously demonstrated that aminoglycoside MICs of P. aeruginosa increase with magnesium supplementation. However, the concentrations of magnesium used in those studies, 45 and 37.9 mg/liter, respectively, were approximately twofold higher than currently proposed standards (15). We sought, therefore, to compare the effect on tobramycin MIC of a physiological concentration of magnesium (20 mg/liter) with that of an approximate concentration of magnesium as used in the above studies. Tobramycin MICs for 10 P. aeruginosa isolates were determined in Mueller-Hinton broth with the following concentrations of magnesium: 4.4 mg/liter (unsupplemented), 20 mg/liter (free physiological, as proposed by the National Committee for Clinical Laboratory Standards [15]), and 40 mg/ liter (approximating the concentrations used by Gilbert et al. and D'Amato et al.). Statistical analysis. Tobramycin MICs in each of 11 supplemented media were compared with tobramycin MICs in the unsupplemented media by Friedman's test (9). The main effects of calcium, magnesium, and zinc and the interaction of these factors with the ticarcillintobramycin interaction were determined by multifactorial analysis of variance (Prophet Computer System, Division of Research Resources, National Institutes of Health). Statistical significance was set at the 99% confidence interval. RESULTS Table 1 depicts the total concentration of divalent cations that were present in the 12 media and used for susceptibility and synergy testing. Ionized calcium composed ca. 55 to 65% of the total calcium present in the media. Table 2

3 VOL. 22, 1982 TABLE 2. Effect of divalent cation supplementation on tobramycin MIC and ticarcillin-tobramycin synergy (FIC index) against P. aeruginosa Tobramycin MIC Mean FIC Medium Supplement (mg/liter) Median Geometric pa index mean A None C Zn B Mg D Mg + Zn E Ca(50) < G Ca (50) + Zn < F Ca (50) + Mg < H Ca (50) + Mg + Zn < I Ca (100) < K Ca (100) + Zn < J Ca (100) + Mg < L Ca (100) + Mg + Zn < a Tobramycin MIC in each supplemented medium compared to the unsupplemented medium by Friedman's test. An FIC index of <1.0 indicates synergy. The lower the FIC index, the greater the degree of synergy. shows that divalent cations increased the MIC of tobramycin against P. aeruginosa. Mueller-Hinton broth supplemented to 50 or 100 mg of total calcium per liter (i.e., media E through L in Table 1) had significantly higher tobramycin MIC than unsupplemented Mueller-Hinton broth (P < 0.01). Median MICs of tobramycin were identical and geometric mean MICs of tobramycin were not significantly different in Mueller-Hinton broth with 100 mg of total calcium per liter as compared with 50 mg of total calcium per liter. The addition of magnesium or zinc or both to calcium-supplemented Mueller- TABLE 3. Cation Hinton broth had little effect on the tobramycin MIC. The increase in tobramycin MIC by supplementation of Mueller-Hinton broth to physiological concentrations of magnesium, zinc, or both was insignificant (Table 2). Ticarcillin MIC was not significantly affected by any of the cations or cation combinations. Table 3 summarizes the effect of total calcium, magnesium, and zinc on the interaction of ticarcillin and tobramycin against P. aeruginosa. Supplementation of commercial Mueller-Hinton broth to physiological concentrations of magnesium and zinc had no significant effect on the Significant effect of calcium supplementation on the synergistic interaction of ticarcillintobramycin against P. aeruginosa Designationa Mean FIC indexb PC Calcium (100) I, J, K, L 0.96 <0.01 Calcium (6.0) A, B, C, D 0.86 Calcium (50) E, F, G, H 0.97 <0.01 Calcium (6.0) A, B, C, D 0.86 Calcium (100) I, J, K, L 0.96 Calcium (50) E, F, G, H 0.97 Magnesium (20) B, D, F, H, J, L 0.95 Magnesium (4.4) A, C, E, G, I, K 0.92 CATIO AND SYNERGY 841 Zinc (1.0) C, D, G, H, K, L 0.93 Zinc (0.5) A, B, E, F, I, J 0.92 a See Table 1. b The FIC index is used as the measure for synergy. A FIC index of <1.0 indicates synergy. c Multifactorial analysis of variance., Not significant.

4 842 ZURAVLEFF ET AL. action of ticarcillin-tobramycin against the bacteria. However, supplementing the medium to 100 or 50 mg of total calcium per liter had a significant effect on the interaction of the drugs (P < 0.01). The FIC indices increased (i.e., the degree of synergy decreased) in Mueller-Hinton broth with calcium supplementation (Table 3). However, no significant difference in the degree of synergy of ticarcillin-tobramycin was seen when Mueller-Hinton broth with 50 mg of total calcium per liter was compared with Mueller- Hinton broth with 100 mg of total calcium per liter. Mean tobramycin MICs for 10 P. aeruginosa increased threefold in Mueller-Hinton broth supplemented to 40 mg of magnesium per liter as compared to 4.4 mg of magnesium per liter (unsupplemented). In contrast, there was no significant difference in tobramycin MIC between broth supplemented to 20 mg of magnesium per liter versus unsupplemented broth (4.4 mg/liter). DISCUSSION Since the early work with streptomycin in the 1940s, the ionic composition of the culture medium has been shown to influence measurement of susceptibility to aminoglycoside antibiotics (5). Although the drug susceptibility of many species of bacteria has been shown to be affected by the ionic composition of the test medium, variability is greatest with P. aeruginosa (7). The cation composition of both broth and agar has been reported to influence the susceptibility of P. aeruginosa to aminoglycosides and colistin (4, 10, 11, 14, 23). Calcium and magnesium concentrations of the media were implicated as the major cause of variation in susceptibility testing of P. aeruginosa. The exact mechanism for the increase in resistance of P. aeruginosa to aminoglycosides when calcium and magnesium concentrations are increased is uncertain, although the interaction of the cations with P. aeruginosa appears to occur at a locus on the cell wall (16, 23). Reller and co-workers recommended that Mueller-Hinton medium be supplemented to 50 to 100 mg of calcium and 20 to 35 mg of magnesium per liter for the susceptibility testing of P. aeruginosa (18). Subsequently, Kenny and co-workers reported that the soluble and ionized fraction of the total calcium and magnesium in agar correlates with susceptibility of P. aeruginosa to gentamicin (2, 12). Washington and coworkers reported, using a regression analysis, that zinc is the cation most highly correlated with aminoglycoside MIC in Mueller-Hinton agar (20). One current recommendation for aminoglycoside susceptibility testing of P. aeruginosa is that Mueller-Hinton broth be supplement- ANTIMICROB. AGENTS CHEMOTHER. ed with 50 mg of calcium and 25 mg of magnesium per liter (21). This recommendation provides a concentration of magnesium in the normal physiological range of humans, but provides a calcium concentration that is approximately one-half of the total and ionized calcium found in human sera. Therefore, we compared synergy and tobramycin susceptibility results for P. aeruginosa in media supplemented to 100 mg of calcium per liter (in which the total and ionized concentration approximates that of human sera) with those in media supplemented to 50 mg of calcium per liter. We also evaluated the effects of physiological concentrations of magnesium (20 mg/liter) and zinc (1.0 mg/liter) on tobramycin susceptibility testing and synergy testing of P. aeruginosa. Our study confirmed that calcium supplementation of Mueller-Hinton broth dramatically affects the susceptibility of tobramycin against P. aeruginosa. Interestingly, concentrations of 100 and 50 mg of calcium per liter had essentially the same effect on tobramycin susceptibility (Table 2). Although physiological concentrations of magnesium, zinc, or both increased the tobramycin MIC slightly, this effect was negligible if calcium was also present in the medium at a total concentration of 50 or 100 mg/liter (Table 2). We also show that the degree of synergy between ticarcillin and tobramycin against P. aeruginosa is decreased by the divalent cation content of the culture medium (the interactions of three to six isolates characterized as "synergistic" in noncalcium-containing media [media A through D in Table 2] were reclassified as "indifferent" or "additive" in the calcium-supplemented media [media E through L in Table 2]). Total concentrations of 100 and 50 mg of calcium per liter had essentially the same effect on the synergistic interaction of ticarcillin and tobramycin, whereas physiological concentrations of magnesium, zinc, or both had no significant effect (Table 3). In fact, culture medium with physiological concentrations of magnesium, zinc, and total and ionized calcium (medium L in Table 2) gave essentially the same results for synergy testing of ticarcillin-tobramycin (as well as susceptibility testing of tobramycin) as culture medium supplemented only with 50 mg of calcium per liter (medium E in Table 2). If similar results can be demonstrated for other penicillin-aminoglycoside combinations in other commercial Mueller-Hinton broths, this may suggest that Mueller-Hinton broth for P. aeruginosa susceptibility testing need only be supplemented to 50 mg of calcium per liter. Finally, until problems with divalent cation shifts in Mueller-Hinton agar have been resolved (12), consideration should be given to using Mueller-Hinton broth in preference to agar for

5 VOL. 22, 1982 the antibiotic susceptibility testing of P. aeruginosa. LITERATURE CITED 1. Anderson, E. L., P. K. Gramling, P. R. Vestal, and W. E. Farrar, Jr Susceptibility of Pseudomonas aeruginosa to tobramycin or gentamicin alone and combined with carbenicillin. Antimicrob. Agents Chemother. 8: Casillas, E., M. A. Kenny, B. H. Minshew, and F. D. Schoenknecht Effect of ionized calcium and soluble magnesium on the predictability of the performance of Mueller-Hinton agar susceptibility testing of Pseudomonas aeruginosa with gentamicin. Antimicrob. Agents Chemother. 19: D'Amato, R. F., C. Thornberry, C. N. Baker, and L. A. Kirven Effect of calcium and magnesium ions on the susceptibility of Pseudomonas species to tetracycline, gentamicin, polymyxin B, and carbenicillin. Antimicrob. Agents Chemother. 7: Davis, S. D., A. Ianetta, and R. J. Wedgwood Activity of colistin against Pseudomonas aeruginosa: inhibition by calcium. J. Infect. Dis. 124: Donovick, R., A. P. Bayan, P. Canales, and F. Pansey The influence of certain substances on activity of streptomycin. III. Differential effects of various electrolytes on the action of streptomycin. J. Bacteriol. 56: Elion, G. B., S. Singers, and G. H. Hitchings Antagonists of nucleic acid derivatives. VIII. Synergism in combinations of biochemically related antimetabolites. J. Biol. Chem. 208: Fass, R. J., and J. Barnishan Effect of divalent cation concentrations on the antibiotic susceptibilities of nonfermenters other than Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 16: Felegle, T. P., V. L. Yu, L. W. Rumans, and R. B. Yee Susceptibility of Pseudomonas maltophilia to antimicrobial agents, singly and in combination. Antimicrob. Agents Chemother. 16: Friedman, M The use of ranks to avoid the assumption of normality implicit in the analysis of variance. J. Am. Stat. Assoc. 23: Garrod, L. P., and P. M. Waterworth Effect of medium composition on the apparent sensitivity of Pseudomonas aeruginosa to gentamicin. J. Clin. Pathol. 22: Gilbert, D. N., E. Kutscher, P. Ireland, J. A. Barrett, and J. P. Sanford Effect of the concentrations of magnesium and calcium on the in vitro susceptibility of Pseudomonas aeruginosa to gentamicin. J. Infect. Dis. 124: Kenny, M. A., H. M. Pollock, B. H. Minshew, E. Casillas, and F. D. Schoenknecht Cation components of Mueller-Hinton agar affecting testing of Pseudomonas CATIO AND SYNERGY 843 aeruginosa susceptibility to gentamicin. Antimicrob. Agents Chemother. 17: Kluge, R. M., H. C. Standiford, B. Tatem, V. M. Young, W. H. Greene, S. C. Schmnpff, F. M. Calia, and R. B. Hornick Comparative activity of tobramycin, amikacin, and gentamicin alone and with carbenicillin against Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 6: Medeiros, A. A., T. F. O'Brien, W. E. C. Wacker, and M. F. Yulung Effect of salt concentrations on the apparent in vitro susceptibility ofpseudomonas aeruginosa and other gram-negative bacilli to gentamicin. J. Bacteriol. 143: National Committee for Clinical Laboratory Standards Standard methods for dilution antimicrobial susceptibility tests for bacteria which grow aerobically. PSM-7. National Committee for Clinical Laboratory Standards, Villanova, Pa. 16. Nicas, T. I., and R. E. W. Hancock Outer membrane protein H1 of Pseudomonas aeruginosa: involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin. J. Bacteriol. 143: Ostenson, R. C., B. T. Fields, and C. M. Nolan Polymyxin B and rifampin: new regimen for multiresistant Serratia marcescens infections. Antimicrob. Agents Chemother. 12: Reiler, L. B., F. D. Schoenknecht, M. A. Kenny, and J. C. Sherris Antibiotic susceptibility testing of Pseudomonas aeruginosa: selection of a control strain and criteria for magnesium and calcium content in media. J. Infect. Dis. 130: Sonne, M., and E. Jawetz Combined action of carbenicillin and gentamicin on Pseudomonas aeruginosa in vitro. Appl. Microbiol. 17:893-8%. 20. Washington, J. A., R. J. Snyder, R. C. Kohner, G. G. Wiltse, M. D. Ilstup, and J. T. McCall Effect of cation content of agar on the activity of gentamicin, tobramycin and amikacin against Pseudomonas aeruginosa. J. Infect. Dis. 137: Washington, J. A., H, and V. L. Sutter Dilution susceptibility test: agar and macro-broth dilution procedures, p In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant (ed.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, D.C. 22. Yu, V. L., R. M. Vickers, and J. J. Zuravleff Comparative susceptibilities of Pseudomonas aeruginosa to 1- oxacephalosporin (LY127935) and eight other antipseudomonal antimicrobial agents (old and new). Antimicrob. Agents Chemother. 17: Zimelis, V. M., and G. G. Jackson Activity of aminoglysode antibiotics against Pseudomonas aeruginosa: specificity and site of calcium and magnesium antagonism. J. Infect. Dis. 127: