Effect of Polymyxin B on Antibiotic-Resistant Proteus Mirabilis

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Effect of Polymyxin B on Antibiotic-Resistant Proteus mirabilis

INDER JIT SUD AND DAVID S. FEINGOLD lifectious Disease Unit, Beth Israel Hospital-Children's Hospital Medical Center; Departments of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215 Received for publication 3 April 1972

The surface properties of polymyxin B-resistant Proteus mirabilis are markedly altered by the antibiotic. The effects include the development of susceptibility to surface-active agents such as deoxycholate or tris(hydroxymethyl)aminomethane and a marked increased osmotic fragility. However, cell wall impermeability to various agents such as erythromycin, actinomycin D, bacitracin, lysozyme, and triphenyltetrazolium chloride apparently remains intact. These results support the concept that the actions of the polymyxins on gram-negative bacteria are multiple; the action(s) ofthe antibiotic responsible for lethality in vitro, presumably at the level of the cytoplasmic membrane, may represent only a portion of the potential thera- peutic effects of the antibiotic in vivo.

The polymyxin antibiotics are a family of sodium chloride adjusted to pH 7.2 with sodium cationic cyclic peptides with a broad spectrum hydroxide. The cultures were aerated on a rotary of bactericidal activity among gram-negative shaker at 37 C. The above media were solidified when It has been suggested that the lethal necessary with 1.5% agar (Difco); MacConkey agar bacteria. (Baltimore Biological Laboratory) was used where action ofthe polymyxins results from electrostatic indicated. MacConkey medium contains 0.15 g of interaction with the anionic phosphate groups in bile salts per 100 g of medium. To prevent swarming, the cytoplasmic membrane with resultant damage the surface of agar was dried before plating; the plates to the permeability barriers of the cell (9). were incubated at room temperature for 24 hr before Among the Enterobacteriaceae, only Proteus enumeration of the colonies. species and Serratia marcescens are regularly Treatment of the cells. Polymyxin B (Aerosporin, resistant to the bactericidal action of the poly- Burroughs Wellcome & Co.) was used at a concentra- myxin antibiotics. Studies on Proteus mirabilis tion of 20 jg/ml in liquid media and 100 sg/ml in have provided strong evidence that polymyxin solid media. Early log-phase cells were exposed to the sites are present but unac- antibiotic for 30 min at 37 C. The cells were then used B-susceptible target as indicated in various experiments. Sodium deoxy- cessible in these organisms (13, 15). cholate was used at a concentration of 0.15% (0.15 In the course of our studies of the mechanism g/100 ml). Triphenyltetrazolium chloride (ITC) was of polymyxin B resistance in P. mirabilis, we added to sterilized, cooled LB agar before the plates observed that after exposure to the antibiotic the were poured. organisms, while growing and remaining viable Osmotic stability of cells. Cells were washed once under most conditions, were not viable when with 0.9% saline and then suspended in saline. The plated on agar medium containing bile salts suspensions were diluted 100-fold into saline or dis- (MacConkey agar), and they were killed by vari- tilled water. Washings were performed by centrifuga- Herein the marked, but tion of the saline suspensions and suspensions of cells ous surface-active agents. in the wash medium to the initial volume. All sus- nonlethal, effects of the antibiotic on P. mirabilis poflL.w.Xe incubated at 37 C for 30 -min before are reported and th& impiatioosdiscussed. vi}+' comnt wre c-'& MATERIALS AND METHODS RESULTS Organism and culture media. The P. mirabilis strain Effect of deoxycholate. The observation, that used was a clinical isolate from the Diagnostic Bac- Proteus growing in the presence of polymyxin B teriology Laboratory of the Beth Israel Hospital; it gave low viable counts when plated on Mac- was maintained by monthly transfers. The bacteria to LB is shown in were grown in Trypticase soy broth (TSB; Baltimore Conkey agar as compared agar, Biological Laboratory) or LB broth consisting of 1% Table 1. Polymyxin B, when added to a growing tryptone (Difco), 0.5% yeast extract, and 0.5% culture, had no effect on the viability or the rate of 417 418 SUD AND FEINGOLD ANTIMICROB. AG. CHEMOTHER. growth of the cells (see Table 2). When these were suspended in deoxycholate-containing broth organisms were plated on MacConkey agar, and the deoxycholate-exposed organisms in more than 95% of them failed to form colonies. broth containing polymyxin B. The incubation In retrospect, the loss of viability on Mac- was continued at 37 C; Table 3 shows the viable Conkey agar was proposed to be due to the pres- counts performed at intervals. The cells exposed ence of bile salts. To test this hypothesis, the effect first to the antibiotic and then to deoxycholate of deoxycholate on the growth of P. mirabilis in were rapidly killed, whereas with the reverse the presence or absence of polymyxin B was de- sequence the cells continued to grow in poly- termined. Table 2 shows the results of this ex- myxin B. periment. Polymyxin B or deoxycholate had no When polymyxin B-exposed cells were washed effect on viability, although deoxycholate slowed and resuspended in saline and then treated with the rate of growth from a doubling time of 30 min deoxycholate, rapid lysis occurred during the to approximately 45 min. In the culture contain- first 10 min (Fig. 1). The rate of lysis then de- ing both the antibiotic and deoxycholate, the creased gradually, with no further decrease oc- number of viable cells decreased by more than curring after 30 min. The viable count in deoxy- 3 log units after 90 min of incubation. To define further the combined effect of polymyxin B and deoxycholate, the organism was TABLE 3. Sequlelntial actionI of sodiumn1 deoxycholate one or other The (DOC) and polymyxin B (PxB) oni Protelus exposed to the agent. organisms mirabilis were centrifuged at 10,000 X g and washed twice with broth; the polymyxin B-exposed cells Viable cells/ml Time (min) TABLE 1. Viability of Proteuts mirabilis, wit/h anzd PxB-treated cells DOC-treated cells without exposuire to polymyxini B, oni different in DOC in PxB solid miiedia 0 7.2X107 2X107 No. of colonies/ 30 2.7 X 105 4.0 X 107 Cultural con(lition' 90 1.5X10' 1.6X108 MacConkey LlB agar agar Cells growing in Trypticase soy broth (TSB) were exposed to PxB (20,ug ml) or DOC (0.15%) Control 80 72 for 30 min. Cells were centrifuged and washed With polymyxin B 130 0 twice with TSB. The PxB-treated cells were suspended in TSB which contained 0.15%c DOC, and a Part of a culture growing in Trypticase soy the DOC-treated cells were suspended in PxB- broth was exposed to polymyxin B (20 ,ug/ml) containing medium. Incubation was continued, and for 30 min. The treated and untreated cells were viable counts were performed at invervals. then plated on solid media at various dilutions. ,'Number when 0.1 ml of 10-1 dilution was plated. 100 .o-o. -o Control

TABLE 2. Synzergistic actioi of polymyxin B aiid K.. sodium deoxycholate (DOC) on Proteuts 90 mnirabilist L _~ 80 Viable cells/mi : o- 70 Time (min) TSB ox + poly- TSB TSB + p)lY- TSB myxin B 60 DOC myxin B + DOC DO 0 20 0 3.3 X 107 3.3 X 107 3.3 x 107 3. 3 x 107 40 60 80 30 7.5 X 107 8.9 X 107 4.2 X 107 4.9 x 106 M/IU TE5 60 1.7 X 101 2.1 X 108 8.3 X 107 2.0 X 105 FIG. 1. Lysis by sodium deoxycholate (DOC), of 90 3.5 X 108 5.2 x 108 1.1 X 108 3.0 X 104 Proteus mirabilis growni in the presence of polymyxin B. Bacteria growing ini Trypticase soy broth were a At time 0, a culture growing in logarithmic exposed to Polymyxin B (20 ,4g/ml) for 30 mini. They phase in Trypticase soy broth (TSB) was sub- were washed twice with 0.9% saline antdsuspended in sa- divided into four equal parts. Polymyxin B at 20 line. DOC (0.15%r fi,ial conceiitrationi) was added to half ,ug/ml and DOC at 0.15% were added where indi- Of the suispension, uiid Klett readings were taken at in- cated, and incubation was continued. Viable tervals. Sali,ie suispensioni of unitreated P. mirabilis counts were performed at indicated times. showed no decrease in turbiditY the,i exposed to DOC. VOL. 1, 1972 EFFECT OF POLYMYXIN B ON P. MIRABILIS 419 cholate suspension went down by 95 % as com- with water yielded a dramatic decrease in viability pared to no change in the control which had not of the treated as compared with the untreated been treated with the antibiotic. There was no P. mirabilis. change in the optical density of the polymyxin Entry of agents usually excluded from the cell. B-exposed bacteria when incubated in saline The outer aspects of the cell wall of gram-negative alone. organisms exclude various enzymes, dyes, and Effect of Tris. Cells exposed to polymyxin B antibiotics. Hence, it was of interest to determine were killed by over 99% when suspended in the susceptibility of polymyxin B-modified P. 0.05 M tris(hydroxymethyl)aminomethane (Tris) mirabilis to some of these agents. pH 8.0 (Table 4); the control cells were resistant. Lysozyme had no effect on the optical density The experiment was done at 37 C; at no time or viability of cells which had been previously were the cells exposed to chilled Tris which is known to damage Enterobacteriaceae (7). The TABLE 5. Durationi of the effect of polymyxini B on bactericidal effect of Tris on polymyxin B-treated Proteuis inirabilisa cells was on there was dramatically dependent pH; V-iable cells/ml no killing at pH 7.0. Time Duration of effect of polymyxin B. To examine (hr) Before exposure to After exposure to Per cent the duration of the effect of polymyxin B, or- DOC DOC survival ganisms were exposed to the antibiotic, washed twice with TSB broth, and suspended in broth; 0 3.3 X 106 2.8 X 105 8 the susceptibility to deoxycholate (exposure for 1 1.3 X 107 1.1 X 106 8 1 hr before plating) was determined at intervals 2 7.3 X 107 2.0 X 106 3 up to 5 hr (Table 5). The data show that even 2.5 1.4 X 108 6.6 X 106 5 3 hr 7 generations) of 3b 2.6 X 106 2.7 X 10' 10 after (approximately 4 1.2 X 107 4.9 X 106 41 growth in polymyxin B-free medium, the anti- 5 2.6 X 107 3.3 X 107 130 biotic-exposed cells were still sensitive to deoxycholate. The cells started to recover by the 4th a Cells growing in Trypticase soy broth (TSB) hr and became insensitive to deoxycholate lysis were exposed to polymyxin B (20 jug/ml) for 30 after 5 hr (11 to 12 generations) of growth. min. They were washed with and resuspended in Osmotic stability. The modification of the cell TSB at a concentration of 3 X 106 cells/ml. The surface of P. mirabilis caused by polymyxin B culture was incubated, and, at indicated times, also had a profound effect on their susceptibility samples were withdrawn and viable counts were performed before and after exposure to deoxy- to osmotic shock (Table 6). A 100-fold dilution cholate (DOC) for 1 hr at 37 C. into distilled water resulted in more killing of bCulture was diluted 100-fold with TSB at 2.5 polymyxin B-exposed organisms; one washing hr.

TABLE 4. Effect of Tris on Proteus mirabilis grown TABLE 6. Effect of osmotic shock on the survival of in the presenice of polymyxint B, polymyxin B-exposed Proteus mirabilisa

Viable cells/ml Survivalb Suspending medium Treatment of saline suspension Control Polymyxin ControlCnrl PolymnyxinB-treated B-treated

Saline 6 X 107 1 X 108 None 1.0 1.0 Tris (0.05 M) 100-Fold dilution into: pH 8.0 4.4 X 107 3.3 X 105 saline 0.83 0.75 pH 7.0 5.8 X 107 7.2 X 107 water 0.50 0.18

P04 buffer, 0.05 M One wash with water and pH 8.0 4.9 X 107 6.2 X 107 resuspension in water 0.25 1 X 1(5

a Part of a growing culture was exposed to a Part of a growing culture in Trypticase soy polymyxin B (20 ,ug/ml) for 30 min. The cells were broth was exposed to polymyxin B (20 Ag/ml) for centrifuged and suspended in saline. Equal vol- 30 min. The control and treated cells were centri- umes of saline suspension were centrifuged, and fuged and suspended in normal saline. the cells were suspended in various solutions. b Ratio of viable counts after and before the Viable counts were done after incubation at 37 C treatment. Counts were performed after incuba- for 30 min. tion at 37 C for 30 min. 420 SUD AND FEINGOLD ANTIMICROB. AG. CHEMOTHER.

treated with polymyxin B. The lysozyme was Ag of polymyxin B per ml; the usual disc sus- added at a concentration of 50 ,ug/ml in 0.05 M ceptibility testing yielded no zone of inhibition by P04 buffer, pH 7.0. Although the peptidoglycan the polymyxins. of some Proteus species is quite resistant to We have made three types of observations on lysozyme (4), the P. mirabilis used in these studies the nonbactericidal effects of polymyxin B on forms spheroplasts after a few cycles of freezing Proteus. (i) The bacteria become markedly more and thawing in the presence of the enzyme. susceptible to "surface-active agents" such as TTC is a dye which is reduced to a red formazan deoxycholate and Tris after exposure to poly- by enzymes associated with the cytoplasmic myxin B (Tables 2 and 4). It is clear from the re- membrane. Entry of this agent is retarded by sults summarized in Tables 2 and 3 that the intact cell wall (10); organisms with altered synergy between polymyxin B and deoxycholate wall allowing entry of the dye form bright red results from alterations caused by the antibiotic colonies on plates. TTC was included in LB which sensitize to deoxycholate and Tris and not plates, with or without polymyxin B, at concen- vice versa. Furthermore (Table 5), the effect of trations of dye ranging from 0.001 to 0.5 mg/ml. polymyxin B is a prolonged one which lasts for No difference was observed between the control over 10 generations after removal of antibiotic and polymyxin-containing plates. At 0.001 mg from the medium. (ii) The physical strength ofthe of TTC per ml, colonies had no color; at high bacterial cell wall is compromised by polymyxin concentrations, more color began to appear in B; this is reflected in a markedly increased sus- colonies on the plates with and without poly- ceptibility of the organisms to osmotic shock. myxin B; but even at the maximum TTC con- (iii) The cell wall retains some of its differential centration of 0.5 mg/ml, there was no difference permeability properties, since antibiotics which between them. may only penetrate gram-negative bacteria hav- The evidence suggests that certain antibiotics ing certain cell wall defects continue to be ex- such as erythromycin, actinomycin D, and cluded from these organisms. Also, neither TCC bacitracin are not effective against gram-negative nor lysozyme appears to penetrate to the cyto- bacilli because they are excluded by the intact plasmic membrane or the peptidoglycan, re- cell wall. For example, P. mirabilis spheroplasts spectively. are susceptible to erythromycin, whereas whole The mechanism of the lethal action of the organisms are not (14); actinomycin D inhibits polymyxins is not known with certainty. The only Escherichia coli cells that have had the en- data of Newton (9) and Few (3) suggest that velope damaged by ethylenediaminetetraacetic interaction of the cationic antibiotic with the acid (6); bacitracin inhibits the dephosphorylat- anionic polyphosphate of the cytoplasmic mem- ing enzyme of the antigen carrier lipid of E. coli brane phospholipids results in disruption of the but has no effect on whole E. coli. When erythro- cellular permeability barriers. Our previous data mycin (10 /hg) and bacitracin discs (10 units) (13) and those of Teuber (15) suggest that were used, there was no zone, regardless of Proteus is resistant to the lethal action of poly- whether or not polymyxin B (100 ,ug/ml) was in- myxin B because the drug is excluded from the corporated into the plates. Similarly, with ac- cytoplasmic membrane by the outer aspects of the tinomycin D at 100 ,ug/ml, growth of P. mirabills bacterial envelope. One can then clearly identify was not inhibited, whether or not polymyxin B at least two actions of polymyxin B in vitro: was present in the broth at 20 ,ug/ml. Thus, al- (i) a lethal one at the cytoplasmic membrane; though some wall-defective gram-negative bac- (ii) an apparent nonlethal one involving interac- teria become sensitive to these antibiotics, the tion with the outer aspects of the cell wall as surface damage caused by polymyxin B does not determined physiologically in our studies and result in entry through the cell wall of these those of Rifkind using endotoxin (11, 12), and bacteria. morphologically in electron micrographic studies of polymyxin-susceptible E. coli (5) and of iso- DISCUSSION lated lipopolysaccharide (8). The locus of poly- Usage of the term antibiotic resistance is gen- myxin binding at the cell surface is not clear, erally interpreted to mean that an antimicrobic but phospholipids as well as other polyphos- does not kill or markedly inhibit the growth of an phates such as lipid A and heptose or ethanolo- organism in vitro. This does not mean that the amine phosphates are possibilities. agent has no effect on the bacteria in question, Our results raise important questions regarding as demonstrated in the studies reported in this the clinical use of polymyxin B in particular and paper. The P. mirabilis tested, an example of of antimicrobial agents in general. Do tests of many Proteus species that we examined in lesser antibiotic susceptibility in vitro at times offer detail, grew with unaltered growth rate in 1,000 unacceptably limited information on the poten- VOL. 1, 1972 EFFECT OF POLYMYXIN B ON P. MIRABILIS 421 tial in vitro efficacy? Should we be asking other search Career Development Award from the same institute questions on the action of antibiotics which won't (KO3-AI-35455). show up under the limited and protective conditions of in vitro testing? For example, although LITERATURE CITED Proteus species are regularly resistant to the 1. Corrigan, J. J., and B. M. Bell. 1971. Comparison between the polymyxins and gentamicin in preventing endotoxin in- polymyxins, will the reported surface effects of duced intravascular coagulation and leukopenia. Infect. polymyxin B alter the organisms in vitro so that Immunity 4:563-566. they will be more susceptible to host defenses 2. Feingold, D. S. 1969. The serum bactericidal reaction. J. such as phagocytosis or the immune bactericidal Infec. Dis. 120:437-444. 3. Few, A. V., and J. H. Schulman. 1953. The absorption of reaction? If endotoxin mediates any of the serious polymyxin E by bacterial cell walls and its bactericidal ac- sequellae of sepsis with gram-negative bacteria, tion. J. Gen. Microbiol. 9:454-466. will the polymyxins be able to affect a beneficial 4. Fleck, J., M. Mock, R. Minck, and J. M. Ghuysen. 1971. The neutralization of the endotoxin of the cell envelope in Proteus vulgaris P18. Isolation and regardless characterization of the peptidoglycan component. Biochim. antibiotic susceptibilities of the offending bac- Biophys. Acta 233:489-503. teria? A recent demonstration of an animal sys- 5. Koike, M., K. lida, and T. Matsuo. 1969. Electron microscopic tem in which polymyxin B could lessen the action studies on mode of action of polymyxin. J. Bacteriol. 97: of endotoxin in precipitating disseminated in- 448-452. 6. Leive, L. 1965. A nonspecific increase in permeability in travascular coagulation suggests a positive Escherichia coli produced by EDTA. Proc. Nat. Acad. Sci. answer (1). U.S.A. 53:745-750. One of us (2) previously raised the question 7. Leive, L., and V. Kollin. 1967. Controlling EDTA treatment of the existence of antimicrobial to produce permeable Escherichia coli with normal metabolic of novel classes processes. Biochem. Biophys. Res. Commun. 28:229-236. agents that may have been missed because in 8. Lopes, J., and W. E. Inniss. 1969. Electron microscopy of vitro growth inhibition may not result from the effect of polymyxin on Escherichia coli lipopolysaccharide. action of the drug whereas survival in vivo may J. Bacteriol. 100:1128-1130. be was 9. Newton, B. A. 1956. The properties and mode of action of affected critically. Diphenylamine used polymyxins. Bacteriol. Rev. 20:14-27. as the example in that case, but the action of 10. Nordstrom, K., L. G. Burman, and K. G. Eriksson-Grenn- polymyxin B on Proteus may be an even better berg. 1970. Resistance of Escherichia col to penicillins. example. Studies are underway in our laboratory VIII. Physiology of a class II ampicillin-resistant mutant. to determine whether the functional J. Bacteriol. 101:659-668. same aspects 11. Rifldnd, D. 1967. Studies on the interaction between endo- of the polymyxin B molecule are responsible for toxin and polymyxin B. J. Infect. Dis. 117:433-438. both the bactericidal action and the action on the 12. Rifkind, D., and J. D. Palmer. 1966. Neutralization of endo- cell wall. If different chemical structures are re- toxin toxicity in chick embryos by antibiotics. J. Bacteriol. of the 92:815-819. sponsible for certain actions antibiotic, 13. Sud, I. J., and D. S. Feingold. 1970. Mechanism of poly- possibly less toxic agents to perform one func- myxin B resistance in Proteus mirabilis. J. Bacteriol. 104: tion can be devised. 289-294. 14. Taubeneck, U. 1962. Susceptibility of Proteus mirabilis and its ACKNOWLEDGMENTS stable L-forms to erythromycin and other macrolides. Nature (London) 196:196-197. These investigations were supported by Public Health Service 15. Teuber, M. 1969. Susceptibility to polymyxin B of penicillin grants AI-06313 and TOI-AI-00350 from the National Institute G-induced Proteus mirabilis L forms and spheroplasts. J. of Allergy and Infectious Diseases. D. S. F. is recipient of a Re- Bacteriol. 98:347-350.