Combinations - Antimicrobial Agents and Chemotherapy

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1980, p. 732-735 0066-4804/80/04-0732/04$02.00/0

Vol. 17, No. 4

In Vitro Susceptibility of Pseudomonas aeruginosa to Carbenicillin, Glycine, and Ethylenediaminetetraacetic Acid Combinations G. FRANKLIN GERBERICKt* AND PETER A. CASTRIC Department of Biological Sciences, Duquesne University, Pittsburgh, 15219

Striking bacterial activity against Pseudomonas aeruginosa 9D-2 was achieved by glycine-carbenicillin, ethylenediaminetetraacetic acid-carbenicilhin, and glycine-ethylenediaminetetraacetic acid combinations, whereas none of the agents used alone was capable of the same degree of bactericidal activity. Studies using a microtiter modification of the checkerboard technique were performed to evaluate the comparative activity of these antimicrobial combinations. Isobolograms showed synergistic effects with carbenicillin-glycine, carbenicillin-ethylenediaminetetraacetic acid, and glycine-ethylenediaminetetraacetic acid combinations. Bacterial growth inhibitory curves with subinhibitory concentrations of these agents in combination confirmed these findings. Pseudomonas aeruginosa is frequently involved in nosocomial disease of debilitated patients such as those with burns and those receiving antibiotic, cytotoxic, or immunosuppressive therapy (9-11). Controlling invasion of P. aeruginosa is difficult; such microbial invasion often arises because of microbial resistance to antibiotics (12). A variety of topical agents are currently used to control proliferation of P. aeruginosa and other organisms associated with hospital-acquired infections (8). The ability of P. aeruginosa to exist as a resistant pathogen and the disadvantages of the topical creams now employed have led us to investigate other control methods. In this investigation the comparative activity of ethylenediaminetetraacetic acid (EDTA), glycine, and carbenicilhin against a P. aeruginosa isolate was evaluated. The inhibitory effect of glycine (4, 5), EDTA (13), and carbenicillin (7) on bacterial growth has been known for a long time. In addition, combinations of these antimicrobial agents were investigated for synergistic effects. P. aeruginosa strain 9D-2 was obtained from S. D. Kominos (Mercy Hospital, Pittsburgh, Pa.). This organism was an isolate from a septic human burn. Stocks were prepared by growing the organism to approximately 1.0 x 109 cells per ml in minimal media. Samples (1.0 ml each) were placed into sterile tubes containing 5% (vol/vol) dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) and then stored at -70°C. The minimal growth medium (GM III) contained the following: L-glutamic acid, 20 mM; t Present address: Department of Microbiology, West Virginia University Medical Center, Morgantown, 26505

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DL-methionine, 5.0 mM; MgSO4. 7H20, 2.0 mM; tris (hydroxymethyl) aminomethane, 50 mM; NaH2PO4, 5.0 mM; K2HPO4, 5.0 mM. This medium was adjusted to pH 7.5 with concentrated HCl and sterilized by autoclaving. Ferric citrate (20 mM) was filter sterilized and added to the sterilized medium to give a final concentration of 0.02 mM. All reagents were obtained from Fisher Scientific Co., Pittsburgh, Pa. Carbenicillin (disodium salt) was supplied by Sigma Chemical Co. Carbenicillin stock solutions were prepared at a concentration of 2,000 ,ug/ml (diluted with distilled water) and stored in 1.5-ml samples at -70°C. Glycine stock solutions were prepared in concentrations of 1,600, 1,200, 800, 600, and 200 mM and were stored at 8°C. EDTA stock solutions (500 mM) were stored at 8°C. Both EDTA and glycine were diluted with distilled water. Solutions of EDTA were filter sterilized and added to the sterile GM III medium to the desired concentration, whereas glycine was added with the other constituents of the GM III minimal medium and then autoclaved. All stock solutions were stored for no longer than 14 days. Carbenicillin disks (100 ,ug) were supplied by BBL Microbiology Systems, Cockeysville, Md. The procedure for susceptibility testing by a standardized single disk has been described by Bauer et al. (1). However, GM III miniimal medium with 1.5% agar was used in place of the standard Mueller-Hinton medium. The minimal medium GM III was employed so that the exact concentration of glycine could be controlled. A reference organism, P. aeruginosa ATCC 27853, was used to standardize the GM III media.

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VOL. 17, 1980

The tests for synergism with various drug combinations were carried out by the microtiter broth dilution method as described by Sabath (14). In this method, two-dimensional miniimum inhibitory concentrations were measured in microtiter disposable plates containing 96 wells. All wells contained a final inoculum of 1.0 x 105 cells per ml. After 30 h of incubation at 370C, the minimum inhibitory concentrations were calculated for all of the possible combinations of concentrations of each drug pair, and the results are presented in the form of isobolograms. To further determine the synergistic effect of two antimicrobial agents, we carried out bacterial inhibition studies. GM III broth containing subinhibitory concentrations of glycine, carbenicillin, EDTA, and combinations of these agents was inoculated with 1.0 x 107 cells of P. aeruginosa 9D-2 per ml. The cultures were incubated at 37°C on a rotary shaker, and samples were removed at appropriate intervals for optical density readings. Bacterial inhibitory studies are comparable to bacterial killing studies as described by Sabath (14). On GM III plates without glycine, the zone of inhibition around a carbenicillin disk was 25.2 mm, whereas on plates containing 62.5, 125, and 175 mM glycine, the zone of inhibition increased to 30.3, 36.0, and 39.0 mm, respectively. The results of testing combinations of carbenicillin and glycine against P. aeruginosa 9D-2 are presented in an isobologram (Fig. 1A). Each plotted point represents the minimum amount of carbenicillin and glycine for growth inhibition. The points intersecting the ordinate and abscissa represent the mimimum inhibitory concentra-

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tions of carbenicillin (128 ,Lg/ml) and glycine (300 mM). The isobole of this carbenicillin-glycine combination is bowed inward, suggesting a synergistic effect. Figure 1A also shows that subinhibitory concentrations of carbenicillin as low as 24 ,ug/ml and 75 mM glycine in combination produced an antibacterial effect against P. aeruginosa 9D-2. The results of combining carbenicillin and EDTA are shown in Fig. 1B. The minimum inhibitory concentrations for carbenicillin and EDTA were 128 ,ug/ml and 12.5 mM, respectively. The isobole is bowed inward, suggesting a synergistic effect. Subinhibitory concentrations of carbenicillin as low as 16 ,ug/ml and 1.56 mM EDTA in combination produced an inhibitory effect against P. aeruginosa 9D-2. Figure 1C illustrates the results of combining glycine and EDTA. The minimum inhibitory concentrations for glycine and EDTA were 300 and 12.5 mM, respectively. With this combination, the glycine-EDTA isobole is bowed slightly inward, suggesting a moderate synergistic effect. With low glycine concentrations (50 mM) and high EDTA concentrations (6.25 mM) a synergistic effect existed. However, as the concentrations of glycine increased and those of EDTA decreased, the synergistic effect lessened. Growth curves of P. aeruginosa 9D-2 in carbenicillin (32 jig/ml), glycine (150 mM), and the combination of carbenicillin (32 ,ug/ml) and glycine (150 mM) are shown in Fig. 2. Figure 2A shows that subinhibitory concentrations of carbenicillin and glycine in combination produced a greater inhibitory effect. A synergistic effect (Fig. 2B) was also indicated with a combination

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.5 3. ~~~~ 6.' .5 .' 100 200 300 0 100 200 EDTA (mbM) GLYCINE (mM) GLYCINE (mM)

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FIG. 1. Isobolograms illustrating the combined activity of carbenicillin, EDTA, and glycine against P. aeruginosa strain 9D-2 as determined in a checkerboard broth dilution assay. With both the carbenicillinglycine combination (A) and the carbenicillin-EDTA combination (B), the isobole is bowed inward, suggesting a synergistic effect. With glycine-EDTA (C), the isobole is bowed slightly inward, suggesting a moderate synergistic effect.

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2 4 6 8 10 HOURS OF INCUBATION

ANTIMICROB. AGENTS CHEMOTHER.

HOURS OF INCUBATION

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FIG. 2. Growth curves of P. aeruginosa strain 9D-2 with subinhibitory concentration of carbenicillinglycine, carbenicillin-EDTA, and glycine-EDTA combinations. (A) Symbols: 0, carbenicillin, 32 pg/ml; U, glycine, 150 mM; m combination of carbenicillin (32 pg/ml) and glycine (150 mM). (B) Symbols: 0, carbenicillin, 32 pg/ml; E EDTA, 2.0 mM; mg combination of carbenicillin (32 pg/ml) and EDTA (2.0 mM). (C) Symbols: U, EDTA, 2.0 mM; 0, glycine, 150 mM; _, combination of EDTA (2.0 mM) and glycine (150 mM). The controls are represented by open circles on all graphs.

of carbenicillin (32 ,ug/ml) and EDTA (2.0 mM). However, with 150 mM glycine and 2.0 mM EDTA, the inhibitory effect was only slightly enhanced, suggesting moderate synergy (Fig. 2C). We have demonstrated that growth of P. aeruginosa 9D-2 was inhibited by glycine, EDTA, and carbenicillin and that combinations of carbenicillin-glycine, EDTA-carbenicillin, and to some degree glycine-EDTA produced synergistic effects on P. aeruginosa 9D-2. The mechanism for the synergy demonstrated with these agents is unknown. EDTA is known to cause release of a protein-polysaccharidephospholipid complex from the outer membrane, thereby decreasing stability and increasing permeability of the cell wall. The role of EDTA in the synergism it demonstrated with carbenicillin may be twofold. First, it may facilitate the penetration of carbenicillin into its target site. Second, because EDTA alters the cell envelope, it may contribute to the instability of the cell wall which results from the action of carbenicillin. Carbenicillin, which decreases the stability of the cell wall by inhibiting both the transpeptidase and the D-alanine carboxypeptidase enzymes, also acts synergistically with glycine. Glycine inhibits bacterial growth by replacing both D- and L-alanine residues of the peptidoglycan (5, 6). Thus, carbenicillin and glycine, both of which inhibit cross-linking of peptidoglycan strands by different modes of action, would be expected to have a synergistic mode of action. The mechanism of synergism demonstrated with EDTA and glycine is probably similar to that of EDTA and carbenicillin in that EDTA

may contribute to the instability of the cell wall caused by the action of glycine on transpeptidation, or it may improve delivery of glycine to its site of action. The utilization of EDTA and glycine as antimicrobial agents is not new. Failla et al. (3) demonstrated that total parenteral nutrition solutions can be rendered antibacterial by decreasing the content of alanine and increasing that of glycine. They proposed that high concentrations of glycine are antibacterial either by inhibiting such enzymes as D-Ala-D-Ala ligase, alanine racemase, or L-alanine "adding" enzymes or by replacing both D- and L-alanine residues in peptidoglycan subunits, thereby impairing transpeptidization within the cell wall (3). Tomoeda et al. (15) have shown that glycine is effective in eliminating drug resistance of Escherichia coli K-12 JE2100 strain harboring the R100-1 factor, although at lower levels than that of sodium dodecyl sulfate. However, the mechanism by which glycine acts as a curing agent is still unknown. EDTA has been shown to substantially increase the antibacterial activity of polymyxin B sulfate, benzalkonium chloride, and chlorohexidine diacetate against P. aeruginosa (2), probably by increasing the permeability of the cells to the antimicrobial agents. Although high concentrations of glycine and EDTA are required in comparison to that of carbenicillin, we feel that further research should be done with other strains of P. aeruginosa. Also, in vivo research could be designed with laboratory animals to investigate the efficiency of EDTA and glycine as topical agents when used in conjunction with antibiotic treatment. However, it is important to note that

VOL. 17, 1980

activities of aminoglycosides, such as gentamicin and kanamycin, were not enhanced by the presence of glycine (unpublished data), whereas the activity of carbenicillin was greatly enhanced. In conclusion, this investigation suggests that glycine, EDTA, and carbenicillin in various combinations might be used in the treatment of P. aeruginosa infections and that this approach to the control of P. aeruginosa colonization (especially as it occurs in burn wounds) warrants further study. This investigation was supported by United Way Health Research and Services Foundation grant T-53. We thank Spyros Kominos, Mercy Hospital, Pittsburgh, Pa., for his expert assistance and guidance in this project. We also express our sincere appreciation to Irvin Snyder for reviewing the manuscript.

LITERATURE CITED 1. Bauer, A. W., W. M. Kirby, J. C. Sherris, and M. Truck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 36: 493-496. 2. Brown, M. R. W., and R. M. E. Richards. 1965. Effect of ethylenediamine tetraacetic acid on the resistance of Pseudomonas aeruginosa to antibacterial agents. Nature (London) 207:1391-1393. 3. Failla, M. L, C. D. Benedit, and E. D. Weinberg. 1975. Bacterial and fungal growth in total parenteral nutrition solutions. Antonie van Leeuwenhoek J. Microbiol. Serol. 41:319-328. 4. Hammes, W., K H. Schleifer, and 0. Kandler. 1973. Mode of action of glycine on the biosynthesis of peptidoglycan. J. Bacteriol. 116:1029-1053. 5. Hishinuma, F. 1970. Inhibition of incorporation of Lalanine into uridine-diphospho-N-acetylmuramic acid by glycine. Agric. Biol. Chem. 34:655-657.

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6. Hishinuma, F., K. Izaki, and H. Takashashi. 1969. Effects of glycine and D-amino acids on growth of various microorganisms. Agric. Biol. Chem. 33:15771586. 7. Izaki, K., M. Matsuhaski, and J. L. Strominger. 1968. Biosynthesis of the peptidoglycan of bacterial cell walls. VIII. Peptidoglycan transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reaction in strains of Escherichia coli. J. Biol. Chem. 243:31803192. 8. Jones, R. J. 1975. Topical chemoprophylaxis against infection of burns, p. 22. In R. Hermans (ed.), Proceedings of the Dutch Burns Association. Beueruijk, Holland. 9. Kominos, S. D., C. E. Copeland, and B. Grosiak. 1972. Mode of transmission of Pseudomonas aeruginosa in a burn unit and an intensive care unit in a general hospital. Appl. Microbiol. 23:309-312. 10. Lowbury, E. J. L. 1972. Infection associated with bums. Postgrad. Med. J. 48:338-346. 11. Lowbury, E. J. L. 1975. Ecological importance of Pseudomonas aeruginosa: medical aspects, p. 37-65. In P. H. Clarke and M. H. Richmond (ed.), Genetics and biochemistry of Pseudomonas. John Wiley & Sons, Inc., London. 12. Lowbury, E. J. L, and R. J. Jones. 1975. Treatment and prophylaxis for pseudomonas infections, p. 237-248. In M. R. W. Brown (ed.), Resistance of Pseudomonas aeruginosa. John Wiley & Sons, Inc., London. 13. Roberts, N. A., G. W. Gray, and S. G. Wilkinson. 1970. The bactericidal action of ethylenediaminetetra-acetic acid on Pseudomonas aeruginosa. Microbios 7-8:199208. 14. Sabath, L D. 1968. Synergy of antibacterial substances by apparently known mechanisms, p. 210-217. Antimicrob. Agents Chemother. 1967. 15. Tomoeda, M., M. Inuzuka, and M. Hayashi. 1976. Eliminatory action of glycine on drug resistance of Escherichia coli K12 harboring R factor. Jpn. J. Microbiol. 20:27-32.

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