Hotchkiss and Dubos(1) 1940 polymyxin peptides; Investigations … · The peptides, circulin, isolated from trials, have shown that the polymyxins are culturesofBacilluscirculans(12),andpolypeptin,

THE PROPERTIES AND MODE OF ACTION OF THE POLYMYXINS

B. A. NEWTONMedical Re8earch Council Unit for Chemical Microbiology, Biochemical Laboratory, University of

Cambridge, England

CONTENTSIntroduction................................................................................... 14Chemical and Physical Properties of the Polymyxins ........................................... 14Biological Properties and Bactericidal Activity................................................. 15The Mode of Action of Polymyxin.............................................................. 15

Effects on Cell Permeability ............................................................... 15Morphological Changes .................................................................... 16Effects on Cellular Metabolism............................................................. 17

The Site of Action of Polymyxin............................................................... 18Absorption of Polymyxin by Isolated Cell Wall Preparations................................ 18Effect of Polymyxin on the Electrophoretic Mobility of Bacteria............................ 19The Use of a Fluorescent Derivative of Polymyxin in Studying the Site of Action of the Anti-

biotic.............................................................................. 19Chemical Nature of the Polymyxin-Binding Components of Bacterial Cells................. 21

The Selective Activity of Polymyxin ........................................................... 23Conclusions.................................................................................... 24References..................................................................................... 25

INTRODUCTION tensive use of a 1 per cent polymyxin cream.The success of these clinical trials initiated a

The polymyxins belong to the group of peptide dealdsuyoth mdefacin fteantibiotics, of which more than 20 have beenisolated*since Hotchkiss and Dubos(1)\ 1940 polymyxin group of peptides; the results of theseisolated since Hotchkiss and Dubos (1) in 1940udescribed the separation of gramicidin and Investigations will be the subject of this review.

tyrocidin from cultures of Bacillus brevis. These CHEMICAL AND PHYSICAL PROPERTIES OF

have not all been characterized, but from the THE POLYMYXINSdata available it seems that the majority of them Antibiotic substances produced by Bacillus(including gramicidin, tyrocidin, subtilin, baci- polymyxa were discovered independently by twotracin, polypeptin, licheniformin and polymyxin) groups of workers in America in 1947 (6, 7).are peptides of a predominantly basic character. In the same year workers in England (8) isolatedBricas and Fromageot (2) have recently reviewed an antibiotic material ("aerosporin") producedour knowledge of the properties, composition and by Bacillus aerosporus. These two organisms werestructure of these and other naturally occurring subsequently shown to be similar and thepeptides. antibiotic materials to be closely related. FiveThe peptide antibiotics have not found as wide chemically distinct peptides produced by different

a clinical application as was originally hoped, strains of B. polymyxa have now been recognizedbecause they have all proved to be, to some (9); they all contain threonine, a,,y-diamino-extent, nephrotoxic when administered paren- butyric acid and a C9 saturated fatty acid whichterally. The polymyxins, however, have been has been identified as (+)6-methyl-octan-1-oicused successfully for the treatment of alimentary acid (10). A cyclic structure has been postulatedtract infections (3) and meningitis (4); they are for the molecule (11). Table 1 summarizes thealso particularly suitable for local application. amino acid composition of the various poly-Jackson et al. (5), in a report of extensive clinical myxins. The peptides, circulin, isolated fromtrials, have shown that the polymyxins are cultures of Bacillus circulans (12), and polypeptin,effective prophylactically and therapeutically isolated from a mutant of B. circulans (13), arein Pseudomonas aeruginosa infections of bums; closely related to the polymyxins in chemicalno toxic symptoms were observed after an ex- composition.

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1956] PROPERTIES AND MODE OF ACTION OF POLYMYXINS 15

TABLE 1 tionship between intramolecular structure andComposition of polymyxins (2) toxicity.

In vitro tests have shown that the five poly-Peptide - - - myxns possess similar antibacterial spectra and

A B C D E are active in about the same concentrations (16);D-Leuine_................ +

- - - - in general, they are more active against gramL-Lhreucine.............. + + + negative than gram positive organisms. RhodesD-Serine ......... 0 0 0 1 + et al. (19) found that magnesium ribonucleateL-Phenylalanine......... O + + O o antagonized the action of polymyxin and sug-L-a ,y-Diaminobutyric gested that the magnesium ribonucleate content

acid.+ + + 5 + of gram positive organisms may be a factor con-(+)6-Methyloctanoic tributing to the selective action of polymyxin.acid.+ + + 1 + The polymyxins are bactericidal rather than.______________-bacteriostatic (15, 20). Bliss et al. (21), studyingPresence (+) or number of residues in one the effect of polymyxin on the growth of Bac-peptide molecule. terium colt (Escherichta coli), found that the

The molecular weights of the polymyxins are minimal concentration required to inhibit growthall about 1200; Bell et al. (11) obtained a value of depended on the size of the inoculum used in the1150 for polymyxin D by an osmometric method; growth test, suggesting that there was a con-

more recently, Few and Schulman (14) have siderable absorption of the antibiotic by thestudied the properties of unimolecular films of bacteria. These workers also found that thefouriof the prolymyins whenispread atth air,' nature of the growth medium did not affect thewater interface on concentrated salt solutions limiting inhibitory concentration of polymyxinand have determined their molecular weights by for B. coli; however, more recently Haas andapplication of a two-dimensional gas-law equa- Sevag (22) have reported that the sensitivity oftion to measurements carried out at low surface tr Aerobacter an dP yuoha aeinosaconcentrations. Their results show that there is to polymyxin is iluenced by the amno acidno significant difference between the molecular content of the growth medium.weights of polymyxins A, B, D and E, the mean THE MODE OF ACTION OF POLYMYXINvalue being 1250.

Effects on Cell PermeabilityBIOLOGICAL PROPERTIES AND BACTERICIDAL Aqueous solutions of polymyxin are markedlyACTIVITY OF THE POLYMYXIN5 surface active, and the antibiotic resembles theThe biological properties of polymyxins D and cationic detergents in that its bactericidal activity

A were compared by White et al. (15); their is decreased by soap, lipositol and certain phos-paper has an excellent bibliography on the early phatides (21, 23). Although polymyxin, tyrocidininvestigations of the antibiotic activities of the and cationic detergent molecules are similarlypolymyxins. More recently the pharmacological charged at neutral pH values, the antibacterialand chemotherapeutic properties of these anti- spectra of these substances differ, the cationicbiotics have been studied by Brownlee et al. (16); detergents and tyrocidin being more activethey showed that polymyxins B and E were against gram positive organisms while, in general,considerably less nephrotoxic than polymyxins polymyxin is more active against the gram nega-A, C and D when administered parenterally. tive group of bacteria.It has been suggested (17, 18) that the toxicity Many surface active agents are bactericidal,of these peptides may be partly due to their and various explanations have been put forwardD-amino acid content, since substances which to account for this action. Baker et al. (23) sug-afford protection against the toxicity of D-serine, gested that detergents disorganize the cell mem-such as glutathione and methionine, also protect brane and denature certain proteins. Hotchkissanimals against the nephrotoxicity of the poly- (24) reported the release of nitrogenous andmyxins. From a study of the properties of mono- phosphorus-containing compounds from cellslayers of the peptides Few and Schulman (14) which had been treated with detergents in bac-have suggested that there may be some rela- tericidal concentrations. Gale and Taylor (25)


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16 B. A. NEWTON [VOL. 20

Log IO/I at between the amount of 260 mjs absorbing ma-260 mp/mg terial released and the percentage of cells killeddry wt cells (28). For the majority of organisms studied the2.0 release of 260 m;& absorbing materials was rapid

Sensitive- 5 hr and correspond in amount to the free purine,pyrimidine and nucleotide content of the cells,autolytic breakdown contributing little. Resultsobtained with strains of Pseudomonas aeroginosa

\.0 differed from those obtained with other poly-myxin-sensitive organisms; in the presence ofbactericidal concentrations of polymyxin the

Sensitive- 0.5 hr release of 260 m;L absorbing materials continuesover a period of hours, is paralleled by a release

Resistan-5 hr -of equimolecular amounts of pentose and phos-L a a, , ophate, and has been correlated with a breakdown

100 200 300 400 500 of the ribonucleic acid of the cells (30). ThisPolymyxin Concentration breakdown does not occur at 2 C (29), in the

0'g/ml presence of concentrations of magnesium whichFigure 1. Effect of polymyxin concentration inhibit ribonuclease (31, 32) or in the presence of

on the release of 260 my absorbing materials from high concentrations of polymyxin (29). Figure 1a polymyxin-sensitive and a polymyxin-resistant records the effect of polymyxin concentration onstrain of Pseudomonas aeruginosa. Cells were the leakage of 260 m; absorbing materials from asuspended in 1.0 per cent w/v saline + polymyxin strain of P. aeruginosa. Tai and van Heyningenat 30 C and the absorption of the supernatant (33), studying mechanisms of bacteriolysis, havesolution measured at 260 my after the indicated also observed that high concentrations of poly-various periods of incubation. myxin inhibit autolytic mechanisms.

The use of a dye-forming fluorescent conjugatestonivmedorg ismandstheiogenousa posi- with protein in studying the change in permeabilityleased in fe aitronouac.Santon (26 of polymyxin-treated cells. A more direct demon-founed puincldes pyrimidnes p.entosa.n stration of the immediate change m cell perme-forgand phos.p tobemrel,.eatose fro bin ability which occurs in the presence of polymyxin

.phosphorus to .releasedfromboth has been made possible by studying the penetra-gram positive and gram negative organisms when tion of Ntolyl-a-naphthylamine-8-sulphonic acidthese were treated with the cationic detergent (TNS) into polymyxin-treated cells (34). Aqueouscetyltrimethylammonium bromide (OTAB), and solutions of this dye do not fluoresce when excitedMcQuillen (27) found that this action was ac- by ultraviolet light; in the presence of protein thecompamed by a marked alteration of surface y g pcharge...dye combines with negatively charged groups,charge....and the conjugate fluoresces strongly whenConsiderable evidence now exists to support.

, . ., ,.......... .] .excited by light of wavelength 436my(35). Whenthe view that the bactericidal activity of poly- ecdblhowvnt4m.3. Wmyxin is due to its ability to combine with and washed cells of P. aerugino8a were suspended in

disorganize structures of the bacterial cell which dilute solutions of this dye, no fluorescence was

are responsible for the maintenance of the observed, indicating that there were no groupsosmotic equilibrium of the cell. on the surface of the cell with which the dye

Leakage of cell constituents. The addition of a could combine. The addition of polymyxin to

bactericidal concentration of polymyxin to such cell suspensions resulted in an immediate

washed cell suspensions of sensitive organisms fluorescence because of an alteration in cells* *ss r j 1 a ..........permeability that allowed the dye to penetrateresults in a rapid release of materials having an permeabilit t a t

absorption maxima at 260 mru from the cells; to protein-containing portions of the cell.no such leakage occurs from polymyxin-resistant Morphological Changesorganisms (28, 29). In the presence of concen-trations of polymyxin less than those required Detergent-treated cells do not undergo com-for 99 per cent killing there is a linear relationship plete lysis, but electron micrographs have shown


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19561 PROPERTIES AND MODE OF ACTION OF POLYMIYXINS 17

an alteration in the surface structures of the cells nonviable, but no release of protein from the cells(36, 37). Electron-micrographs of polymyxin- occurs, and certain enzyme systems may remaintreated cells have shown that the leakage of active (38, 39). Sykes (40) attempted to evaluatecell constituents is accompanied by marked the relationship between the effect of surfacemorphological changes (29); cells treated with active agents on bacterial metabolism and the25 1.g polymyxin/mg dry weight cells for 1 hr effect on the viability of the cells. He concludedhad lost the majority of their electron-dense that, in general, the viability is affected by con-material and appeared as flattened empty centrations lower than those which inhibit metab-"ghosts" (figure 2, part 2). In contrast cells olism. Baker et al. (23) found that cationictreated with higher concentrations of the anti- detergents inhibit the respiration and acid pro-biotic (figure 2, part 3) were as electron-dense duction of gram positive and gram negative or-as the control cells (figure 2, part 1) and had ganisms to the same degree; manometric studiesmaterial adhering to their surface. showed that this action was rapid, complete

inhibition generally occurring in less than 15Emm.n They suggested that the most reasonable

The disorganization of cell permeability bar- working hypothesis to explain the rapid actionriers that results when bacteria are treated with of synthetic detergents on bacterial metabo-many surface active compounds renders the cell lism and viability would be based on a two-

Figure 2. Part 1, Pseudornonas aeruiginosa. Washed susp~ension of cells from a 15-hr cultutre, (X 16,800).Part 2, Washed cells treated with 25 ,sg p~olymnyxin/mng dry wAeight cells for 1 hr. This concentration ofpolymyxin causes maximum release of 260 mA absorbing materials from the cells (X 15,600). Part 3,Washed cells treated with 500 ,sg polymyxin/mg (Iry weight cells for 1 hr (X 16,800). Originally publishedin the Journal of General Microbiology (29). We thank the copyright owner for permission to reproduce.


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fold action: (a) a disorganization of a cell mem- and Bacterium coli Few (44) has shown thebrane by virtue of the surface activity of the presence of a secondary layer or membranecompounds; and (b) denaturation of certain pro- within the outer cell wall; he found no similarteins essential to growth and metabolism. structure within cell walls prepared from Staphy-Few reports have been made on the effect of lococcus aureus (Micrococcus pyogenes var.

polymyxin on bacterial metabolism. Cohen et al. aureus). In discussing these results, Few states,(41) have shown that polymyxin B inhibits the "It is tempting to suggest that the primary siteesterase activity of several species of myco- of attack of this antibiotic is at the inner layerbacteria. Newton (30, 42) found that polymyxinB and E, at a concentration three times the TAB3LE 2bactericidal level, inhibited the endogenous Maxinmim amiounts of polymnyxin absorbed by bac-respiration of washed cells of Pseudomonas teria in 20 minutes at 25 C and its relationshipaeruginosa and their ability to oxidize 2-keto- to their gramt reaction and polymyxingluconate, acetate, pyruvate, oxalacetate and sensitivity (28)succinate. They still were able however, to Poly-oxidize glucose with an oxygen uptake of two myxin

Gram Ab-atoms per glucose molecule, 2-ketogluconate Organism reac- Polymyxin sorbedaccumulating in the medium. Bactericidal dirnensitivity wt

concentrations of polymyxin doubled the en- teria)dogenous respiration of P. aeruginosa and had nosignificant inhibitory effect on the oxidation of Pseudomonas denitrifi-glucose, acetate, pyruvate, oxalacetate or suc- cans. sensitive 350cinate. These findings fit in with the general Micrococcus lysode-observations of Sykes (40) for other surface ikticus. + sensitive 375active compounds that viability is affected by Bacteriunm coli . - sensitive 220.ilBacillus subtilis.........+ sensitive 310

concentrations lower than those which inhibit Streptococcus faecalis. + resistant 71metabolism. rStaphylococcus aureus... + resistant 84

Proteus vulgaris . - resistant 82THE SITE OF ACTION OF POLYMYXIN

The Absorption of Polymyxin by Isolated Cell Suspensions in 0.01 Am phosphate buffer, pH 6.3.

Wall Preparations . I I I I

Few and Schulman (28), studying the absorp- Poly/myxin Eabsodrbedtion of polymyxin E by bacteria, found that (Jig-/mg dpolymyxin-sensitive organisms absorbed about P itriansfour times as much antibiotic as resistant or- Iiganisms (table 2). They also obtained isotherms E co ( s

for the absorption of polymyxin E by suspensions | aeruainosa (sensitive)of cell walls; walls prepared from polymyxin- 0sensitive bacteria had a much higher affinity for .the antibiotic than walls of polymyxin-resistant aorganisms (figure 3). Similar results were ob-tained wvith cell walls prepared from polymyxin- I 00 P.aeruc~o a(eIstant)sensitive and a polymyxin-resistant strain of * _ _ x xP. aeruginosa (43).

Studies with the electron microscope (43, 44) aS.fecallshave shown that the absorption of polymyxin by 2 0 40 60cell walls prepared from sensitive organisms is Polymyxin E in supernLtant solutionaccoml)anie(l l)y a marked increase in their (p g/ml)electromI density; no similar change was observed Figure 3. Absorption of polymyxin E by bac-when cell walls of polymyxin-resistant organisms terial cell walls (28, 43). Cell walls were incubatedwere treated with the antibiotic. By careful at 25 C for 20 min in 0.01 a I phosphate bufferrupture of cells of Pseudomonas denitrificans (pH 6.3) containing polymyxin.


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19561 PROPERTIES AND MODE OF ACTION OF POLYMYXINS 19

of these duplex structures, and that this inner were agglutinated by polymyxin, and its elec-layer or membrane is closely associated with trophoretic mobility, which was more thanosmotic pressure regulation in the intact cell. double that of the other strains, was markedlyThe apparent absence of this membrane from reduced in the presence of the antibiotic. Oncell wall preparations of the polymyxin resistant plating, this organism was found to be a "roughStaph. aureus, and the lack of any pronounced strain"; the high electrophoretic mobility ofmorphological changes when these preparations cells of this strain fits in with the observations ofare exposed to the antibiotic, would perhaps Moyer (48) and of Stearns and Roepke (49)support this view." More recent work (45), to be who found that rough strains of Bacterium colidescribed, has provided a direct demonstration of and Brucella abortus have high mobilities, whilethe combination of polymyxin with a membrane the mobilities of smooth forms are relatively lowunderlying the bacterial cell wall. when measurements are made in the presence of

high concentrations of salts. Latterrade andThe Effect of Polymyxin on the Electrophoretic Macheboeuf (50) also observed that polymyxin

Mobility of Bacteria only agglutinates bacteria which lack the 0

Many workers have suggested that the primary antigen.site of action of surface active agents is at thecell surface; this hypothesis is supported by T heUeoaF oresctivaof the Antibioticstudies on the effect of these substances on the ioelectrophoretic mobility of bacteria. Dyar and From a consideration of isotherms obtained forOrdal (46), studying the effect of the cationic the absorption of polymyxin by intact bacterialetergent cetylpyridinium chloride on a number and bacterial cell wall preparations Few andof bacteria, found that, in the absence of the Schulman (28) suggested that absorption takesdetergent, the cells were negatively charged place mainly at sites in the cell wall with butwhile, as the concentration of the detergent was little penetration inside the cell. However, ex-

raised, the charge decreased, became in turn periments with isolated cell walls are difficult toneutralized, reversed and finally stabilized at a interpret and give no indication of the amount ofpositive value. McQuillen (27) obtained similar antibiotic which would be absorbed by the cellresults when he studied the effect of CTAB on walls when other cellular components are

B. coli and he suggested that, ". . . an adequate present. A more direct approach to this problemexplanation of these results is to be found has been made possible by the use of a fluorescentin the supposition that increasing quantities ofthe detergent combine with the negatively TABLE 3charged organism resulting in reduction and Effect of polymyxin on the electrophoretic mobilityeventual reversal of the net negative charge." of a number of strains of PseudomonasUnpublished observations by McQuillen on the aeruginosa (McQuillen, unpublishedeffect of polymyxin on the electrophoretic observations)mobility of a number of strains of Pseudomonas Electrophoretic Mobility (j/sec/v/cm)aeruginosa are recorded in table 3. These results Pseudomonas Polymyxin concentration (g/ml)may be taken to indicate that, with the excep- Strain*tion of P. aeruginosa Molyneux, polymyxin is not _ 1 10 100

combining at the cell surface of these organisms P. 15 2.10 2.13 2.12 2.06in spite of the fact that they absorb as much as 5526 2.03 - 1.92300 Ag polymyxin/mg dry weight of cells. It is 6371 2.00 1.86possible to calculate an approximate value for P. 36 2.18 - 1.97the minimum depth below the surface at which P. 50 2.18 - 1.98polymyxin could combine with components of the McAlleese 1 .98 1.96 1.75 1.54cell without affecting the surface change (47); in Molyneux 4.45 3.90 1.85 0.98a buffer of ionic strength 0.001, as used in these * Washed cells suspended in M/300 phosphateexperiments, this would be c. 100 A. P. aeruginosa buffer, pH 7.0. Cell density = 50 ,g dry wt cells/Molyneux differed from the other six strains ml. Measurements were made at 25 C in a micro-examined in that suspensions of this organism electrophoresis cell (27).


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.er~entage'.il , by centrifugation at 100,000 X G; and only 10Percentage killing per cent with the cell walls. This distribution wasand fluorescence constant for a range of concentrations of DANSP,100 -____,Percentage killin the highest concentration tested being more than

___________ * four times the bactericidal level (figure 4).

Small,-rtice fraction-When cell walls alone were treated with DANSP-80 Smallparticle fraction they absorbed up to 270 ,tg DANSP/mg dry

/ weight walls. This was not removed by repeated6q washing in distilled water, but incubation with

the small particle fraction from the cells resultedin more than 90 per cent being removed from thewalls and taken up by the small particles.The polymyxin-sensitive, gram positive or-

°_0 Cell wall fraction ganisms studied were all lysozyme-sensitive.0 o 0~20 6,0, Weibull (51) has shown that controlled lysozyme

treatment of B. megaterium in the presence ofjug DANSP/mg dry wt cells 0.2 M sucrose results in the "depolymerization"

Figyue 4. Distribution of a fluorescent deriva- of the cell wvall, the rest of the cell remaining as antive of polymyxin (DANSP) in the cell fractions of intact structural unit-a spherical protoplast.Bacillus megaterium. Washed cells of B. negateriumn Such protoplasts can be lysed by dilution of thetreated with varying concentrations of DANSP sucrose and the resulting protoplast ghostsfor 20 min at 30 C; the intensity of fluorescence ofthe intact cells and cell fractions obtained after seimen aa dark yellow fractionbycen-mechanical disintegration and differential centrifugation at 10000 X G electronmicroscopytrifugation were measured in a modified Pulfrich shows this fraction to be composed of flatphotometer (34). Number of surviving cells esti- membrane-like bodies (52). Weibull found thatmated by plate counts. Fluorescence expressed as treatment of protoplast ghosts for a short periodpercentage of that of intact cells. in a sonic oscillator destroyed the membrane-like

structure, but the yellow material could be

derivative of polymyxin prepared by coupling resedimented as a small particle fraction by1-dimethyl-aminonaphthalene-5-sulphonyl chlo- centrifugation at 100,000 X G. This techniqueride with the y-amino groups of a,-y-diamino- has been used to study the distribution ofbutyric acid in the polymyxin molecule (45); DANSP in Bacillus megaterium (45); fluorescentlimiting amounts of the sulphonyl compound microscopy of DANSP-treated B. megateriumwere used so that the majority of polymyxin before and after lysozyme treatment has shownmolecules carried only one naphthalene group. that the fluorescent compound is associated withFluorescent microscopy showed that 1-dimethyl- the protoplast membrane (figure 5, parts 1 and 2).amino - naphthalene - sulphonamido - polymyxin After treatment of fluorescent protoplasts in a(DANSP) was readily absorbed by polymyxin- sonic oscillator all the DANSP was found to besensitive organisms and in growth tests it was associated with the small particle fraction sedi-found to have approximately the same bac- mented by centrifugation at 100,000 X G.tericidal activity as the untreated antibiotic. Fractionation of DANSP-treated P. aeruginosaThe distribution of DANSP in a number of did not yield such clear-cut results as those ob-polymyxin-sensitive organisms has been studied tained with B. megaterium. The fluorescent com-(45); cells treated with a bactericidal concentra- pound was equally distributed between the celltion of the derivative were disrupted by me- wall fraction and the small particle fraction;chanical agitation, fractionated by differential this may be due to a closer association betweencentrifugation and the intensity of fluorescence the cell wall and the underlying membrane-ifof the various fractions measured. Examination one exists-in gram negative organisms. Such anof Bacillus megaterium, Sarcina lutea and Micro- explanation would fit in with the observations ofcoccus lysodeikticus in this way showed that 90 Few (44) that cell wall preparations from gramper cent of the fluorescent conjugate was as- negative organisms retain a secondary innersociated with a small particle fraction sedimented layer, while cell walls prepared in a similar manner


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Figure 6. Part 1, Fluorescence photomicrograph of Bacillus megaterium treated with 25 ijsg I1-dimethyl -

aminon'itphthalene-5-sulphonamido-polymyxin (DANSP)/mg dry weight cells (X 750). Part 2, Fluores-cence photomicrograph of DANSP-treated B. meqaterium after "depolymerization" of the cell wallby treatment with lysozyme shoin.gthe association of DANSP with the protoplast membrane (X 750).Originally published in the Journal of General Microbiology (45). We thank the copyright owners forp~ermission to reproduce.

from gram positive organisms contained no point has been obtained from a study of thesuch structure. It will be difficult to make further competition between polymyxin and certainprogress with gram negative organisms until a cations for these groups. Bacteria can be pro-cell wall depolymerizing enzyme can be found or tected against the bactericidal activity ofsome other technique is developed for the com- polymyxin by pretreatment with certain non-plete separation of the cell wall from underlying toxic cations whose presence prevents the ab-structures. However, it seems possible that the sorption of the antibiotic by the cells (31).small particles obtained by fractionation of Certain antibiotics appear to have an avidity fordisrupted organisms may be derived from a cations (55), the annulment of aureomycin in-membrane underlying the cell wall. Mitchell hibition of a cell-free nitroreductase system byand Moyle (53) isolated a similar fraction from manganese being attributed to the formation of aStaphylococcus aureus. They found that these chelate (56). There is no evidence for the forma-particles contained a high proportion of phos- tion of a chelate between polymyxin and cations,pholipid and suggested that they may be derived and it has been shown that the antagonism in thisfrom the lipid layer described by Burdon (54) case is due to a competition between polymyxinwhich lies inside the cell wall and is readily and the cations for sites on the cells (34). Bungen-stained with Sudan Black. burg de Jong (57), studying the reversal of

charge on colloids by cations, found that certainThe Chemical Nature of the Polymyxin-Binding groups of colloids had characteristic "reversal of

Components of Bacterial Cells charge spectra." With "phosphate colloids" (i.e.,Polyyxirectswit man acdicsubtanes,"colloids" wvith ester-phosphate as ionogenic

includingnuceiacidswtmand phshoiidicsubsandesa groups, e.g., soya-bean phosphatides and egg leci-number of workers (21, 50) have observed thatthnicesgteiorausftrvet(C+these substances antagonize the antibacterial - Lga++) increased the reversal of chargeactivity of the antibiotic. However, such ob- concentration. This did not apply to bivalentservations do not give any indication of the cations in which the order of efficacy of a numberchemical nature of the polymyxin-combining of cations differed irregularly with the particulargroups of bacterial cells; some information on this phosphate colloid studied. However, the uranyl


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ion (UO2i), though bivalent, showed reversal boxyl colloids" (i.e., "colloids" with carboxylof charge at extraordinarily low concentrations, groups as ionogenic groups, e.g. sodium arabinateless than that of tervalent ions. and sodium pectinate) and for "sulphate col-These results, together with the "reversal of loids" (i.e. "colloids" with ester-sulphate as

charge spectra" obtained by de Jong for "car- ionogenic groups, e.g. potassium chondroitin

Ca MgBa Sr4+ ++ + 4++

++ +44 4+4 CMSrBaSoU02 Ce La Ca Ba Sr Mg

B Ca Sr Mg

I. Phosphate Colloids

U02 4As4+ +4 ++Ba Sr Ca Mg

-14+ +++Ce La

2. Corboxyl Colloids

Bo Sr Ca Mg

La Cep I I I

3. Sulfate Colloids

U ~i2 Ce Ld Ba Co Sr Mg

dIncreasing affinity for polymyxin-combining aroups_| 4. . Polymnxin. Combining, GrouDs p

-5 -4 -3 -2 -I 0

Log ConcentrationFigure 6. Comparison of specific cation sequences for reversal of charge on certain types of colloids

with the affinities of these cations for the polymyxin combining groups on washed cells of Pseudomonasaeruginosa (de Jong, 57). Cation sequences 1, 2, and 3 summarize the results of de Jong (57) and showthe concentrations at which cations reverse the charge on certain types of colloids. Cation sequence 4summarizes the results of Newton (34) and shows concentrations at which cations protect washed cellsof P. aeruginosa against the bactericidal activity of polymyxin. There is a close relationship betweenthe cation sequence of the reversal of charge on "phosphate colloids," and the affinity of these ions forthe polymyxin-combining groups of washed cells of P. aeruginosa.


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sulphate and sodium agar) are summarized in Pseudomonas aeruginosa are compared with thefigure 6. cation sequences obtained by de Jong (57)

In view of these results it seemed possible that for the reversal of charge on various groups ofa study of the relative affinities of uni-, bi-, and colloids. Clearly, a close relationship exists be-tervalent cations for the polymyxin-combining tween the cation sequence for the reversal ofgroups of bacteria might give some indication charge on phosphate colloids and the affinities ofof the chemical nature of these groups. The use these ions for the polymyxin-combining groups ofof the dye N-tolyl a-naphthylamine-8-sulphonic P. aeruginosa, suggesting that the polymyxin-acid (TNS) to detect the change in cell permea- binding loci of the cell surface may be poly-bility which results on treating sensitive bacteria phosphates. The observations of Rothstein andwith polymyxin provided an ideal system for associates (58, 59) add further support to thisstudying the competition between cations and hypothesis; they found that uranyl ions formedpolymyxin for sites on the cells. The addition of a highly undissociated complex at the surface ofpolymyxin to cells suspended in a dilute solution yeast cells and presented evidence that theof TNS results in an immediate increase in uranium-complexing groups of the cell surfacefluorescence of the suspension; if certain cations were polymers of phosphate.are added to the cell suspension before the 1\ore recently Few (60) has studied the inter-addition of polymyxin, the fluorescence of the action of polymyxin with bacterial and othersuspension increases gradually at a rate de- lipids using the monolayer technique and haspendent on the cation concentration. obtained further evidence of electrostatic bond-

It was found that univalent ions (Na+, K+, ing between polymyxin and ionized phosphateLi+ and NH4+) did not compete with polymyxin groups. He found that cephalin and lipid ex-for sites on the cells, with the result that the tracted from disrupted Pseudomonas denilrifiansaddition of polymyxin to cells suspended in the formed strong complexes with polymyxin;presence of these cations and TNS produced moderate complex formation also occurred withimmediate maximum fluorescence. Four bivalent cardiolipin and lipid extracted from 'staphy-ions were tested at a concentration of 10 ,umole/ lococcal fractions, but lecithin did not react withmug dry weight cells; it was found that the rate of the antibiotic. Discussing these results, Fewincrease in fluorescence varied with the different suggests that the positively charged cholinecations in the order Mg++ > Sr+ > Ca+ > group of lecithin effectively shields the ionizedBa+. The rate for each was taken as a measure of phosphate group, thus preventing electrostaticthe affinity of a particular cation for the poly- bonding between the phosphate groups andmyxin-combining groups of the cells, i.e., the amino groups of the polymyxin molecule.greater the degree of the dissociation of the Analysis of the bacterial lipids showed thatcation-cell complex, the greater the rate of in- choline was not present in the nitrogenous con-crease in fluorescence on the addition of poly- stituents; ethanolamine and serine were de-myxin. Tervalent cations were effective at lower tected.concentrations (0.2 ,umole/mg dry weight cells)than bivalent cations; cerium was found to have THE SELECTIVE ACTIVITY OF POLYMYXINa greater affinity than lanthanum for the poly- The reasons for the selective activity of poly-myxin-combining groups of the cells. In contrast myxin and the nature of the resistance shown byto the results obtained with other cations the some organisms to this antibiotic have not yetdivalent uranyl ion (UO2+) formed a relatively been adequately explained. Studies of the ab-undissociated complex with the cells so that sorption of polymyxin by cell walls prepared frompolymyxin did not combine with uranyl-treated polymyxin-sensitive or polymyxin-resistant or-cells even after long exposures to the antibiotic. ganisms (28, 43) suggest that this resistance de-Uranyl ions could be removed from the cells by pends to some extent on the chemical composi-washing with sodium hexametaphosphate, so- tion and structure of the cell wall; the cell walldiun) pyrophosphate or adenosine triphosphate of resistant organisms has a low affinity for thebut not with hexose diphosphate or inorganic antibiotic, and there is little penetration ofphosphate. polymyxin to the underlying membrane or

In figure 6 the affinities of bi- and tervalent osmotic barrier.cations for the polymyxin-combining groups of From the results described in the preceding


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TABLE 4Chemical composition of cell walls preparedfrom some polymyxirn-sensitive or polymyxin-resistant organisms

Pseudomonas Pseuomonas Pseudomonas Staphylococcusaeruginosa, denitrificanst aeruginosa, aureus

P. 15* McAlleese* Fractionst

Polymyxin sensitivity.............. ..Sensitive Sensitive Resistant ResistantTotal nitrogen...................... 9.9 - 10.1Total phosphorus................... 1.7 - 0.8Reducing substances§ ........ ........ 16.9 - 16.8Hexosamine§........................ 2.1 - 2.7Total lipid: ......................... 11.9 12.8 10.7 22.3Lipid phosphorus¶ ................... 3.8 4.3 2.7 1.8Lipid nitrogen..................... 1.0 1.1 1.8 0.8Lipid P/N ........................... 3.8 3.9 1.5 2.2

* Data from Newton (30).t Data from Few (60).t Expressed as percentage dry weight of cell walls.§ Determined after 2 N HCl hydrolysis for 2 hr at 100 C, expressed as percentage dry weight cell

walls.T Expressed as percentage dry weight of lipid material extractedl.

section a difference in the phospholipid content twice the amount of lipid phosphorus found inof the cell walls of polymyxin-sensitive and gram positive organisms. In general, polymyxin ispolymyxin-resistant organisms might be ex- more effective against gram negative organisms,pected; however, there is little information avail- and from the data available it is tempting toable on this point at the present time. Newton suggest that the difference in the lipid phos-(30) analyzed cell walls prepared from a poly- phorus of these two groups of organisms is amyxin-resistant and a polymyxin-sensitive strain factor contributing to the selective action ofof Pseudomonas aeruginosa; the results sum- polymyxin.marized in table 4 show that the only significantdifferences are in the total phosphorus content of CONCLUSIONSthe cell walls and in the phosphorus/nitrogen Umbreit (63) pointed out that biochemicalratio of the phospholipid fraction isolated from knowledge of the mode of action of antibioticsthe cell walls. The values obtained by Few (60) involves three aspects of their action: (a) thefor the phosphorus and nitrogen contents of vulnerable point in the biochemistry of the celllipids extracted from P. denitrificans and staphy- which the antibiotic attacks; (b) the reasons forlococcal fractions are also recorded in table 4. the specificity of antibiotics; and (c) the phe-The lipids extracted from cell walls of the poly- nomena of resistance and dependence. At themyxin-sensitive P. aeruginosa and P. dentrificans present time our knowledge of any one of thesecontained a high percentage of phosphorus aspects for most of the known antibiotics is stillcorresponding to that of many phosphatides, fragmentary; however, from the data reviewed inwhereas the lipids from cell walls of the poly- this article it is clear that we can now paint amyxin-resistant strain of P. aeruginosa and from fairly complete picture of the mode of action ofstaphylococcal fractions contained nitrogen and the polymyxins. The bactericidal activity ofphosphorus in approximately equimolecular the polymyxin peptides can be adequately ex-amounts, the quantities suggesting that less than plained in terms of a disorganization of a cell50 per cent of these lipids were phosphatides. membrane or barrier which controls the osmoticWith these results in mind it is interesting that equilibrium of the cell. This disorganization maySalton (61) found that the walls of a number be the result of a decrease in interfacial tensionof gram positive organisms contained con- at the membrane produced by the combinationsiderably less lipid than the walls of gram nega- of the antibiotic with ionized phosphate groupstive organisms, while Mitchell and Moyle (62) of phospholipid components of the membrane.found that gram negative organisms possess The resistance to polymyxin shown by the


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majority of gram positive organisms and some the fatty acid component. Nature, 164,of the gram negative group may be attributed 622.to the low affinity of the cell walls of these bacteria 11. BELL, P. H., BONE, J. F., ENGLISH, J. P.,for the antibiotic, there being little penetration FELLOWS, C. E., HOWARD, K. S., ROGERS,for~~~~~~~th.niitchr eigltl eerto M. M., SHEPHERD, R. G., WINTEHEOTTOMof polymyxin through the wall to the underlying R.,D HRNHUSH,A. G.,KSNEROTANR., DORNBUSH, A. C., KUrSHNER, S., ANDmembrane in these caseS. SUBBAROW, Y. 1949 Chemistry of poly-Although it was suggested more than 15 years myxin: comparison with 'aerosporin'. Ann.

ago (235 that the bactericidal activity of surface N. Y. Acad. Sci., 51, 897-908.active substances may be due to their ability to 12. MURRAY, F. J., TETRAULT, P. A., KAUFMANN,disorganize a cell membrane, the recent use of a 0. W., KOFFLER, H., PETERSON, D. H., ANDfluorescent derivative of polymyxin has provided COLINGSWORTH, D. R. 1949 Circulin, anthe first direct demonstration that such sub- antibiotic from an organism resemblingstances do combine specifically with a membrane Bacillus circulans. J. Bacteriol., 57, 305-underlying the bacterial cell wall. It seems likely 312.from the few detailed studies that have been 13. HAUSMANN, W., AND CRAIG, L. C. 1952faromeduth few dtalemod of action of other peptide Polypeptin: purification, molecular weightarnied out on the mode of action of other peptide determination, and amino acid composi-

antibiotics that tyrocidin (24), subtilin (64) tion. J. Biol. Chem., 198, 405-419.and circulin (65) act at a similar point. 14. FEw, A. V., AND SCHULMAN, J. H. 1953

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3. KAGAN, B. M., KREVSKY, D., MILZER, A., AND E. I. 1952 The chemotherapy and pharma-LOCKE, M. 1951 Polymyxin B and poly- cology of the polymyxins. Brit. J. Pharma-myxin E, clinical and laboratory studies. col., 7, 170-188.J. Lab. Clin. Med., 87, 402-414. 17. BROWNLEE, G., AND SHORT, E. I. 1948

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9. BROWNLEE, G., AND JONES, T. S. G. 1948 A 22. HAAs, G. J., AND SEVAG, M. G. 1953 Criticalrelated series of antibiotics derived from role of amino acids on the sensitivity andBacillus polymyza. Biochem. J., 43, xxv- development of resistance to polymyxin B.xxvi. Arch. Biochem. and Biophys., 43, 11-24.

10. WILKINSON, S. 1949 Crystalline derivatives 23. BAKER, Z., HARRISON, R. W., AND MILLER,of the polymyxins and the identification of B. F. 1941 Action of synthetic detergents


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24. HOTCHMSS, R. D. 1944 Gramicidin, tyro- 39. KREBS, H. A. 1948 Quantitative deter-cidin and tyrothricin. Advances in Enzy- mination of glutamine and glutamic acid.mol., 4, 153-199. Biochem. J., 43, 51-57.

25. GALE, E. F., AND TAYLOR, E. S. 1947 The 40. SYKES, G. 1939 The influence of germicidesaction of tyrocidin and some detergent on the dehydrogenases of Bacterium coli.substances in releasing amino-acids from J. Hyg., 39, 463-469.the internal environment of Streptococcus 41. COHEN, S., PURDY, C. V., AND KUSHNICK,faecalis. J. Gen. Microbiol., 1, 77-84. J. B. 1952 Inhibition of bacterial esterase

26. SALTON, M. R. J. 1951 The adsorption of activity by polymyxin B. Federationcetyltrimethylammonium bromide by bac- Proc., 11, 463-464.teria, its action in releasing cellular con- 42. NEWTON, B. A. 1953 The action of poly-stituents and its bactericidal effects. J. myxin on Pseudomonas pyocyanea. J. Gen.Gen. Microbiol., 5, 391-404. Microbiol., 8, vi-vii.

27. MCQUILLEN, K. 1950 Effect of cetyltri- 43. NEWTON, B. A. 1954 The absorption ofmethylammonium bromide on the electro- polymyxin by cell-wall preparations fromphoretic mobility of certain Gram positive Pseudomonas aeruginosa. J. Gen. Micro-bacteria. Biochim. et Biophys. Acta, 5, biol., 10, iii-iv.463-471. 44. FEW, A. V. 1954 Electron microscopy of

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29. NEWTON, B. A. 1953 The release of soluble in studying the site of action of the anti-constituents from washed cells of Pseudo- biotic. J. Gen. Microbiol., 12, 226-236.monas aeruginosa by the action of poly- 46. DYAR, M. T., AND ORDAL, E. J. 1946 Themyxin. J. Gen. Microbiol., 9, 54-64. effects of surface-active agents on the

30. NEWTON, B. A. 1954 Thesis, University of electrophoretic mobilities of bacteria. J.Cambridge, England. Bacteriol., 51, 149-167.

31. NEWTON, B. A. 1953 Reversal of the anti- 47. MITCHELL, P. 1949 The osmotic barrier inbacterial activity of polymyxin by divalent bacteria. In The nature of the bacterialcations. Nature, 172, 160-161. surface, pp. 55-75. Edited by A. A. Miles

32. LAMANNA, C., AND MALETTE, M. F. 1949 and N. Pirie. Blackwell Scientific Publica-Magnesium ion, an inhibitor of ribonuclease tions, Ltd., Oxford, England.activity. Arch. Biochem., 24, 451-458. 48. MOYER, L. S. 1936 Changes in the electro-

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54. BURDON, K. L. 1946 Fatty material in 60. FEW, A. V. 1955 The interaction of poly-bacteria and fungi revealed by staining myxin E with bacterial and other lipids.dried fixed slide preparations. J. Bac- Biochim. et Biophys. Acta., 16, 137-145.teriol., 62, 665-678. 61. SALTON, M. R. J. 1953 The composition of

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Documents

Hotchkiss and Dubos(1) 1940 polymyxin peptides; Investigations … · The peptides, circulin, isolated from trials, have shown that the polymyxins are culturesofBacilluscirculans(12),andpolypeptin,