Mechanism of the antibacterial action of spermine

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

36-54 (1959)

81,

Mechanism of the Antibacterial Action of Sperminel S. Razin and R. Rozansky From

the Department of Clinical Microbiology, Medical School and Hadassah University Received

July

The Hebrew University-Hadassah Hospital, Jerusalem, Israel 16, 1958

INTRODUCTION

Spermine, NH~(CHZ),NH(CH,)~NH(CH~)~NH~ , is a biological polyamine widely distributed in many animal tissues (1) and various microorganisms (2, 3). It has been shown to possess growth-inhibiting properties against various bacteria (4, 5). The antibacterial action of spermine was found to be antagonized by inorganic salts (6). This antagonism suggested a competition between spermine and cations for acidic cell components. To elucidate this point, the binding of spermine by the cell and the cell components responsible for the binding have been investigated in the present study. Since spermine did not kill washed staphylococci suspended in saline, it appeared that the bactericidal effect of the drug was dependent on some metabolic activity of the cell (6). The effect of spermine on cells in various stages of metabolic activity was thus further investigated, and attempts were made to elucidate the metabolic processes involved. MATERIALS

AND METHODS

Chemicals Spermine tetrahydrochloride and yeast ribonucleic acid were kindly supplied by Hoffmann-LaRoche & Co. Ltd., Basle, Switzerland. Deoxyribonucleic acid from sperm was the product of Nutritional Biochemicals Corporation, Cleveland, Ohio. Poly-n-lysine (n-34) was obtained by the courtesy of Professor E. Katchalski of the Weizmann Institue of Science, Rehovoth, Israel.

Test Organisms The following bacteria were examined for their capacity ~OCOCCUS aureus NO. 23 (sensitive to 15 pg./ml. spermine)

to bind spermine: Staphyand its spermine-resistant

1 This investigation was supported by a grant from the Hadassah Medical Organization Research Fund and taken in part from a dissertation presented by one of us (S. R.) in partial fulfillment of the requirments for the Ph.D. degree at the Hebrew University, Jerusalem.

36

ANTIBACTERIAL

ACTION

OF

37

SPERMINE

mutant No. 23R* (sensitive to 4000 pg./ml. spermine) ; Staphylococcus aureus No. 46 (sensitive to 30pg./ml. spermine) ; Escherichia coli (sensitive to 5OOpg./ml. spermine); Proteus vulgaris (not inhibited by 4000 pg./ml. spermine). The sensitivity of the strains mentioned was determined by the serial dilution method in Difco nutrient broth, pH 7.6 (6).

Adsorption

Technique

Test organisms, grown on Difco brain-heart infusion agar for 18 hr. at 37”C., were harvested and washed three times with distilled water; 1.5 ml. of cell suspension (containing 50 mg. dry weight of cells/ml.) was added to 1.5 ml. of various concentrations of spermine in 0.05 M boric acid-borax buffer (pH 7.6). The tubes were incubated in a shaking bath at 37°C. for 1 hr. Thereafter the cells were sedimented by centrifugation at 9000 X g for 15 min. The amount of spermine in the supernatant was determined chromatographically (7) and biologically (see below). From the difference between the initial concentration of spermine and the concentration of spermine in the supernatant, the amount of the drug bound by the cells could be determined.

Elution of Spermine The cells, after adsorbing spermine, were washed once with boric acid-borax buffer (pH 7.6) and agitated for 1 hr. in 0.1 N HCl at room temperature. The amount of spermine eluted was determined chromatographically or biologically.

Biological Determination

of Spermine

Serial dilutions of the supernatant fluid were arranged in Difco nutrient broth, pH 7.6 and inoculated with Staph. aureus No. 23, sensitive to 15 pg./ml. spermine. The highest dilution inhibiting growth multiplied by 15 gave the approximate concentration (pg./ml.) of spermine in the supernatant fluid.

Agglutination

Technique

Serial double dilutions of spermine from 14,000 to 1.5 pg./ml. were set up. The dilutions were made in distilled water, each tube receiving 2 ml. of solution and 0.2 ml. of distilled water containing 2 X lo9 washed cells. The tubes were incubated for 2 hr. at 37°C. and inspected for agglutination; they were then left overnight at 6°C. and reinspected.

Preparation of Cell-Free Extract Washed cells of E. coli were subjected to the action of the Mickle tissue disintegrator (8)) using ballotini beads size 14. After agitation for 1 hr., the unbroken cells and debris were sedimented by centrifugation at 9000 X g for 10 min. The clear viscous supernatant was used.

Bactericidal

Tests

The bactericidal effect of spermine was examined in the following media: 1. Difco nutrient broth, pH 7.6. 2. 0.3yo casein hydrolyzate (Nutritional Biochemicals Corporation, Cleveland, Ohio) pH 7.6. * Obtained of spermine.

by subculturing

the original

strain

through

increasing

concentrations

38

RAZIN

AND ROZANSKY

free” casein hydrolyzate, acid (Nutritional Biochemicals Cor3. 0.37, “vitamin poration), pH 7.6. 4. 0.05 M tris(hydroxymethyl)aminomethane buffer, pH 7.6. This buffer will be referred to as Tris buffer. 5. Tris buffer enriched with 0.04% and 0.47c glucose, respectively, or 0.4% sodium lactate, pH 7.6. 6. Triple-distilled water. Staph. aureus No. 23 served as test organism. Cells from a 24-hr.-old broth culture were thrice washed with distilled water, inoculated in the above-mentioned media, and incubated either at 37°C. or at 4°C. At various time intervals samples were taken and the number of viable cells was determined by the plate count technique. Difco Proteose No. 3 agar was used for plating, and the number of colonies was counted after incubation at 37°C. for 48 hr. All experiments were done in triplicate.

Measurements of Anaerobic and Aerobic Oxidation Anaerobic oxidation was tested by a modification Aerobic oxidation was measured manometrically method as described by Umbreit et al. (IO).

Adaptation

of the Thunberg method (9). by the conventional Warburg

Technique

Formation of &galactosidase by washed cells of Staph. aureus No. 23 was examined by a modification of the method described by Creaser (11). The test organism was grown on the following medium: Marmite O.l%, arginine 0.2%, glucose O.l%, KHzPOl O.l%, MgS04.7Hz0 0.07%, NaCl O.l%, (NHd)tHPOd 0.4’%, FeSOn.7H20 0.003’$&. After 15 hr. incubation at 37”C., cells were harvested, washed once in distilled water, and suspended in an induction medium containing o-galactose as an inducer. The 1’ hr. at 37”C., air being bubbled through continususpension was incubated for 2,s ously. Cells were sedimented by centrifugation at 9000 X g for 15 min., washed once with distilled water, and resuspended in distilled water. The fl-galactosidase activity of the cells was determined manometrically; oxygen uptake was measured in a Warburg apparatus, lactose serving as substrate. RESULTS

The Amounts of Spermine Adsorbed by Various Bacteria The amount of spermine adsorbed by cells of Staph. aureus No. 23 is shown in Fig. 1. It may be seen that up to 600 pg./ml. spermine the amount of the drug bound by the cells rose with its concentration in the buffer. Thereafter there was no increase in the amount adsorbed. Twenty micrograms spermine was the maximum bound by 1 mg. dry weight of cells. The same amount of spermine was bound by the spermine-resistant P. vulgaris and E. coli. However, a markedly smaller amount of spermine, namely 8 pg./mg. dry weight of cells, was bound by the trained resistant Staph. aureus No. 23R. The same amount of spermine was adsorbed by the cells when they were incubated with the drug at 37°C. or at 4°C. At 37°C. the adsorption of

ANTIBACTERIAL

0

ACTION

200 400 SPERMINE

OF

SPERMINE

3’3

800 800 1000 CONCENTRATlON(/‘f~l)

FIG. 1. The amount of spermine adsorbed by cells of Staph. aureus No. 23 at various concentrations of the drug in the medium. Washed cells were incubated with buffer, pH 7.6 various concentrations of spermine solution in 0.05 M boric acid-borax at 37°C. After incubation for 1 hr. the cells were sedimented and the amount of spermine in the supernatant was determined chromatographically.

spermine by cells of S. aureus No. 23 and P. vulgaris was quite complete within 5 min. Cells killed by heating at a temperature of 100°C. for 10 min. showed the same capacity as living cells to adsorb spermine. Approximately one half of the spermine adsorbed was eluted by 0.1 N HCl; when 0.01 N HCl was used, only one fourth of the adsorbed spermine could be eluted. These findings were observed with all bacteria examined irrespective of their sensitivity to spermine. The E$ect of pH and Inorganic Salts on the Binding of Spermine bg the Bacterial Cell In order to test the effect of the pH on spermine adsorption, cells of Stzph. aureus No. 23 were incubated with spermine at 37°C. in acetate buffer, pH’s 3.6 and 5.6 and in boric acid-borax buffer, pH’s 7.6 and 8.6. The determination of spermine in the supernatant could not be accomplished chromatographically because at the extreme pH values the chromatogram became unclear. The amount of spermine adsorbed was determined therefore by its elution from the cells with 0.1 N HCl, having in mind that approximately 50% of spermine adsorbed could be eluted by this method. It is seen from Fig. 2 that the amount of spermine adsorbed rose with increasing pH. The influence of inorganic salts is shown in Table I; 0.25 M of NaCl, CaClz , MgCl, , and NapHP04 reduced the adsorption of spermine by approximately 50%, while Na2S04 reduced the adsorption of the drug even more.

40

RAZIN

o-

3.6

AND

ROZANSKY

5.6

7.6

0.6

PH 2. The amount of spermine adsorbed by cells of Staph. aureus No. 23 at various pH values. Washed cells were incubated in a solution of spermine (concentration 600 pg./ml.) in 0.1 M acetate buffer, pH 3.6 or 5.6 or in 0.05 M boric acid-borax buffer, pH 7.6 or 8.6. After incubation for 1 hr. at 37”C., the cells were sedimented, washed once with distilled water, and resuspended in 0.1 N HCl. The amount of spermine eluted by the acid was determined chromatographically. FIG.

Agglutination

of Bacterial Cells by Spermine

Like other basic substances (12-14), spermine has been found to agglutinate bacterial cells. Aggregates were formed after 2 hr. incubation at 37°C. These aggregates sedimented during the night and adhered firmly to the bottom of the tube, the supernatant becoming completely clear. Gram-positive and some of the gram-negative bacteria tested were agglutinated within a certain range of spermine concentrations, the range varying with the organism concerned; staphylococci were agglutinated by spermine concentrations of from 27 pg./ml. to 3500 pg./ml. Escherichia coli or Klebsiella were agglutinated by 54-1750 pg./ml. spermine, whilst Serratia marcescens and Proteus vulgaris were not agglutinated at all. The gram-negative bacteria which were agglutinated by spermine were found to be in the rough form, whereas those not agglutinated were in the smooth form (12). Inorganic salts interfered with the agglutination of bacteria by spermine; they were even able to disperse an already existing aggregate. Thus 0.03 M NaCl, 0.003 M NazSOl, 0.003 M MgS04, and 0.007 M CaClz

ANTIBACTERIAL

BCTION

TABLE

OF

41

SPERMINE

I

Amount

of Spermine Adsorbed by Cells of Staph. aureus No. RS in Presence of Various Inorganic Salts Washed cells were incubated in a solution of spermine (concentration 600 pg./ml.) in 0.05 M boric acid-borax buffer containing 0.25 M of one of the salts indicated. After incubation for 1 hr. at 37”C., the cells were sedimented and the amount of spermine remaining in the supernatant was determined chromatographically. The cells were washed once with distilled water and resuspended in 0.1 N HCl. The amount of spermine eluted by the acid was determined chromatographically. Amount of spermine adsorbed, pg./mg. dry wt. of

Salt

Determined on the basis of the amount &ted from the cells

Determined on the basis of the amount remaining in the supernatant

12.0 10.0 10.0 10.6 7.6

11.0 11.0 10.0 n n

22.0

21.0

NaCl CaC12.2HzO MgC12.6HzO Na2HP04.12Hz0 NazSOd Control, a In the presence as the chromatogram

without

cells

salt

of these salts spermine could not be quantitatively of the supernatant was unclear.

were sufficient to prevent the agglutination spermine at a concentration of 700 pg./ml.

of Staph.

aweus

determined

No. 23 by

Formation of Insoluble Complexes between Sperm&e and Nucleic Acids The ability of spermine and other amines to form complexes with nucleic acids was examined in the following manner: Increasing amounts of diamines and polyamines were added to test tubes containing 3 ml. of 0.25% sodium ribonucleate (Na RNA) or sodium deoxyribonucleate (Na DNA). When insoluble complexes were formed, an immediate turbidity was observed. Overnight the complexes sedimented and the supernatant cleared. The minimal concentrations of amines forming insoluble complexes and the concentrations required to inhibit growth of Staph. aUreUS No. 23 are given in Table II. The diamines putrescine and cadaverine did not form insoluble complexes with nucleic acids and were devoid of antibacterial action. The larger the number of amino groups in the compound, the greater was its ability to form insoluble complexes with nucleic acids and the stronger was its antibacterial action. Apart from nucleic acids, phospholipides are known also to be components of the bacterial cell. As a representative phospholipide, lecithin was tested for its ability to form insoluble complexes with spermine and

42

RAZIN

AND

ROZANSKY

TABLE Growth

Inhibitory

Test Growth

organism: inhibition:

II

Concentrations of Amines in Comparison with Their Ability to Form Insoluble Complexes with Nucleic Acids Staph. aureus No. 23. Medium: Difco nutrient broth, pH 7.6. no visible growth after incubation for 24 hr. at 37°C.

-

Minimal linimal tration

Amine

molar concen inhibiting growth

molar insoluble

concentration complexes

O.‘ZSy, Na RNA

Putrescine Cadaverine Diethylene triamine Triethylene tetramine Tetraethylene pentamine Spermidine Spermine Dihydrostreptomycin Poly-L-lysine a Maximal

concentration

-

No inhibition of growtha No inhibition of growtha 1.2 x 10-Z 3.8 x 10-d 9.0 x lo-6 5.6 X 1OW 2.2 x 10-6 3.0 x 10-T 5.0 x IO-7

tested:

-

of Insoluble

Complexes

tested:

L

Na DNA

No complexes forme@ No complexes forme& 5.0 x 10-Z 1.4 x 10-Z 3.0 x 10-S 4.6 X lo+ 1.3 x 10-a 1.0 x 10-a 7.0 x 10-S

III between Amines

and Lecithin

No complexes 2.5 X 3.0 x 4.0 x 7.0 x 2.0 x 4.0 x

Putrescine Propane diamine Diethylene triamine Triethylene tetramine Spermidine Spermine Dihydrostreptomycin concentration

No complexes formed” No complexes formed0 5.0 x 10-S 7.0 x 10-s 3.0 x 10-Z 4.6 X 10-a 2.2 x 10-Z 2.0 x 10-s 1.5 x 10-J

Minimal molar concentration insoluble complexes with

Amine

0 Maximal

0.25%

5.0 X 1OW’1M. TABLE

Formation

forming with

5.0 X lo+

of amine forming 0.25% lecithin

formeda 1OW 10-d 10-s lo-5 10-S 10-h

M.

other amines. Heavy floccules appearing almost immediately after the setting up of the test, indicated the formation of insoluble complexes. The ability of the amines to form insoluble complexes with lecithin increased with the number of amino groups in the molecule (Table III). Extracts of gram-negative bacteria are rich in nucleic acids and phospholipides (15). The ability of a cell-free extract of E. coli to form insoluble complexes with spermine was therefore examined. Ascending concentrations of spermine were added to a series of tubes containing 0.5 ml. of the cell-

ANTIBACTERIAL

ACTION

TABLE Dissociation

of Spermine-Nucleic

OF

43

SPERMINE

IV

Acid Complexes by Inorganic Salts

Amounts of 1.5 ml. of 0.574 Na RNA or of 0.5% Na DNA were added to 1.5 ml. of a series of double dilutions of each of the salts indicated. Each tube received also 0.5 ml. of a 2 X 10-Z M spermine solution.

Salt

NaCl Na acetate NatSO NaHC03 Na K tartrate Na citrate KC1 LiCl NH&l MgS04.7HzO

Minimal molar concentration of salt inhibiting formation of complexes between spermine and: 0.22% Na RNA

0.22% Na DNA

0.12 0.12 0.03 0.015 0.015 0.003 0.12 0.12 0.12 0.03

0.25 0.25 0.03 0.06 0.06 0.015 0.12 0.12 0.12 0.06

free extract. A heavy precipitate, appearing immediately, spermine at 3 X 1O-4 M or higher.

was formed by

Dissociation of the Insoluble Complexes between Nucleic Acids and, Spermine by Inorganic Salts Table IV shows that all the salts tested inhibited the formation of the insoluble complexes between spermine and nucleic acids. The inhibitory effect of the anions decreased in the following order: citrate, sulfate, tartrate, bicarbonate, and chlorine. There was no difference in the effect of various univalent cations tested (Na, K, Li, and NH,). Of the bivalent cations only Mg++ could be tested, as Ca++, Mn++, Zn++, Fe++, and Fe+++ themselves caused precipitation of the nucleic acids. The concentration of each salt required to prevent the precipitation of nucleic acid by spermine was sufficient to dissolve a precipitate already formed. The formation of insoluble complexes between spermine and cell-free extracts of E. coli, as well as between spermine and lecithin, was also prevented by inorganic salts, e.g., NaCl and MgS04 . Interference of Nucleic Acids, Lecithin, Inorganic Salts and Amines with the Antibacterial Activity of Spermine

Antagonism between spermine and nucleic acids has been reported by Bichowsky-Slomnitzki (16). In order to see whether mononucleotides and nucleosides exhibit a similar antagonistic effect, adenylic acid, guanylic

44

RAZIN

AND

ROZANSKY

acid, adenosine, and guanosine were tested. RNA decreased the antibacterial action of spermine in proportion to its concentration in the medium. Thus addition of 0.5 % (w/v) of Na RNA to Difco nutrient broth increased the concentration of spermine required to inhibit the growth of Staph. aureus No. 23 from 8 pg./ml. to 120 pg./ml. Adenylic and guanylic acids showed a slightly lower antagonistic effect; adenosine and guanosine had no antagonistic effect at all. It would seem that the phosphoric radical of the nucleotide is responsible for the antagonistic effect. Na RNA exhibited the antagonism even when added after incubation of the bacteria with 60 pg./ml. spermine for 24 hr. in nutrient broth at 37°C. It was found by plate counting that at a concentration of 60 pg./ml. spermine several hundreds of cells survived after 24 hr. at 37°C. when the initial inoculum was approximately 10,000 cells/ml. Apparently the nucleic acids enabled the survivors to multiply by reducing the effective concentration of spermine in the medium. Evidence that there was no “revival” of damaged cells by addition of RNA was adduced by the following experiment. Staph. aureus No. 23 was incubated in Difco nutrient broth, pH 7.6 containing 60 pg./ml. spermine at 37°C. After 24 hr. the number of survivors was estimated by plate count. Parallel counting was done on plates of Difco nutrient agar and plates of the same medium enriched with 1% Na RNA. The number of colonies appearing on both media was nearly the same. Lecithin acted similarly as a spermine antagonist, its effect being proportional to its concentration. Thus 120 pg./ml spermine was required to inhibit growth of Staph. aureus No. 23 when 0.10% (w/v) lecithin was added to the Difco nutrient broth, as compared with 240 pg./ml. of spermine when 0.25 % (w/v) was added. The effect of a series of inorganic salts on the antibacterial action of spermine is seen in Fig. 3. Calcium and magnesium exhibited a stronger antagonistic effect than sodium, potassium, or ammonium. Raising the concentration of calcium and magnesium over a certain limit was not accompanied by a higher antagonistic effect because high concentrations of calcium and magnesium are themselves toxic to the bacterial cell. Among the anions the most effective antagonists were phosphate and sulfate. The effect of basic amino acids and amines on the antibacterial action of spermine is seen in Table V. All substances examined showed some antagonistic effect. The highest effect was exhibited by putrescine; the antagonistic effect was directly proportional to its concentration. The Bactericidal E$ect of Spermine in Nutrient Broth and in Casein Hydrolyzate Media As shown in Fig. 4, in nutrient broth in which Xtaph. aureus No. 23 multiplied readily, the lethal effect of spermine was pronounced, while

ANTIBACTERIdL

t

0

ACTION

0.03 0.06 CONCENTRATION

5

OF

45

SPERMINE

(XI2 OF SALT IN MEDIUM

a25 (M)

FIG. 3. The antagonistic effect of inorganic salts on the antibacterial action of spermine. Test organism: Staph. ~UWUS No. 23. Medium: 0.759” Bacto-Peptone. Growth inhibition: no visible growth after incubation for 48 hr. at 37°C. TABLE The Antagonistic Test Growth

organism: inhibition:

V

E$ect of Amines and Basic Amino Antibacterial Action of Spermine

Acids on the

Staph. aureus No. 23. Medium: Difco nutrient broth, No visible growth after incubation for 48 hr. at 37°C. Amine

or basic

amino

acid

tested”

Minimal spermine,

pH 7.6.

concentration of inhibiting growth

pg./ml.

Putrescine Cadaverine L-Histidine Agmatineh on-Ornithine L-Arginine n-Lysine Control (broth a Concentration b A concentration

only)

240 60 60 30 15 8 8 4

of amine or amino acid in the medium: 4 mg./ml. of 2 mg./ml. was used as 4 mg./ml. inhibited growth.

in casein hydrolyzate multiplication was restricted and the killing effect less. In “vitamin-free” casein hydrolyzate medium no multiplication of cells took place and the bactericidal effect of spermine was minimal. Thus, sensitivity to spermine was correlated with the rate of multiplication of the cells. The bactericidal effect of spermine on Staph. aureu.s No. 23 in nutrient broth :at 4°C. was much lower than at 37”C., and cells were affected mainly

46

RAZIN

0

AND

ROZANSKY

4

24 POURS

FIG. 4. The bactericidal effect of spermine on Staph. aureus No. 23 in nutrient broth and in casein hydrolyzate media. pH of media, 7.6. Temperature of incubation 37°C. O--C Difco nutrient broth, 0-0 Difco nutrient broth containing lOO/ 0.3y0 casein hydrolyzate; A-A 0.3y0 casein hydrolypg./ml. spermine; A--A zate containing 100 pg./ml. spermine, O---O 0.3y0 “vitamin-free” casein hydrolcasein hydrolyzate containing 100 pg./ml. yzate; w 0.3yn “vitamin-free” spermine .

during the first hours of incubation. After 6 hr., 46% survived; after 24 hr., 16% survived. The number of surviving bacteria remained unchanged during a further 48 hr. incubation. The E$ect of Sperm&e on Washed Cells of Staph. aureus No. 23 in Tris Bu$er and in Distilled Water Fifty per cent of washed cells of Staph. aureus No. 23 suspended in Tris buffer died after 5 hr., and nearly all were dead after 24 hr. incubation at 37°C. (Table VI). Addition of spermine did not speed up the mortality. When glucose or lactate was added to the buffer, more cells survived: the higher the concentration of glucose the slower the death rate. Addition of spermine together with glucose or lactate resulted in a pronounced bactericidal effect . Washed cells of Staph. aureus No. 23 suspended in distilled water died rather rapidly. Addition of spermine to the distilled water slowed down the rate of death. This paradoxical “protective” effect was exerted by a concentration as low as 40 pg./ml. spermine. When an inoculum of 10’ cells/ml. was used, all bacteria died out in distilled water after 22 hr. incubation at 37”C., whereas 1.5 X lo4 cells/ml. survived in the presence of 40 ,ug./ml. spermine and 4 X lo4 cells/ml. when the concentration of spermine was 400 pg./ml. Spermidine, cadaverine, and putrescine exhibited a similar effect.

ANTIBACTERIAL

ACTION

TABLE

The Effect

47

SPERMINE

VI

of Glucose or Lactate

on Staph. Inoculum 0.05 Y Tris

on the Bactericidal Action aureus No. 83 in Tris Buffer

size 3 X 107 cells/ml. buffer.

pH 7.6, plus:

GlUCOSe

Lactate

Spermine

PK./ml.

PK./ml.

LG./ml.

0 0 400 400 4000 4000 0 0

OF

0 0 0 0 0 0 4000 4000

Temperature

of incubation

Per cent of survivors after 5 hr. incubation

0 100 0 100 0 100 0 100

of Spermine

37°C.

Per cent of survivors after 24 hr. incubation

50.0 50.0 66.6 8.2 91.6 4.1 46.0 1.8

1.2 6.2 2.5 0 5.5 0 12.5 0.03

The finding that spermine did not injure the washed cells in distilled water or in Tris buffer indicates that the drug did not impair the osmotic barrier of the cells. The amount of inorganic phosphorus released from the cells in the presence of spermine was taken as an indicator of injury to the cell permeability (17) and was determined as follows. Cells of Staph. aUreU.s No. 23 grown on Difco brain heart infusion agar for 18 hr. at 37°C. were harvested and washed twice with saline. The cells were suspended in distilled water or in Tris buffer enriched with 0.4% glucose, and spermme was added to a final concentration of 100 pg./ml. Controls without spermine were run simultaneously. The suspensions were incubated at 37”C., and aliquots of 3 ml., withdrawn at time intervals indicated in Table VII, were centrifuged at 9000 X g for 15 min. The amount of inorganic phosTABLE Release of Inorganic

VII

Phosphorus from Cells of Staph. Presence or Absence of Spermine

aureus No. 23 in

Number of cells 3 X log/ml. Temperature of incubation 37°C. At times indicated cells were removed by centrifugation and the amount of inorganic phosphorus in the supernatant was determined [Ref. (lo)]. Amount Medium

Distilled

water

0.05 M Tris buffer, pH 7.6, enriched with 0.4yo glucase

Spa-mine

&-./ml. 0 100 0 100

of inorganic phosphorus, released after:

cg./ml.,

10 min.

2 hr.

4 hr.

24 hr.

1.0 1.0 1.0 1.0

5.0 6.5 1.0 1.0

7.0 8.5 1.0 1.0

18.5 19.0 12.0 11.0

48

RAZIN

AND

ROZANSKY

phorus was determined in 2 ml. of the supernatant by the method of Fiske and SubbaRow (10). As seen in Table VII, there was no significant difference in the amount of inorganic phosphorus released from the cells in the presence or absence of spermine. Thus, spermine did not behave as a surface-active substance; its bactericidal effect was not connected with physical injury to the cell envelope. The Injluence of Spermine on Some Metabolic Processes of Staph. aureus No. $3 1. The E$ect of Spermine on the Oxidation of Various Substrates. Staph. aureus No. 23 was grown on Difco brain heart infusion agar for 18 hr. at 37°C. Cells were harvested and washed twice in saline, and suspensions were made to contain 2 mg. dry weight of cells/ml. The anaerobic and aerobic oxidation of glucose, succinate, pyruvate, lactate, acetate, and formate were examined. Table VIII shows that spermine did not affect the dehydrogenation of glucose, lactate, and formate. The dehydrogenation of succinate, pyruvate, and acetate, however, was enhanced markedly even by the lower concentrations of spermine. Spermine had no significant effect on the aerobic oxidation of all the above-mentioned substrates. 2. The Efect of Spermine on Oxygen Uptake in Broth. The oxygen uptake of cells of Staph. aureus No. 23 suspended in nutrient broth was markedly inhibited by spermine (Fig. 5). Viable cell counts at the end of the experiment (3 hr.) showed no increase in the number of bacteria in the control. In the presence of 28 pg./ml. spermine, 98% of bacteria were killed and the oxygen uptake was 40% of the control. In the presence TABLE

VIII

The E$ect of Sperm&e on the Dehydrogenation of Various Substrates by Staph. aureus No. 25 Test tubes contained 1.0 ml. of 0.05 M Tris buffer, pH 7.6, 1.0 ml. of washed cells suspension (2 mg. dry wt.), 0.5 ml. of a spermine solution, 1.0 ml. of 0.1 M substrate and 0.5 ml. of 1: 10,000 methylene blue solution. Cells were incubated with spermine for 1 hr. before addition of substrate. Temperature of incubation 37°C. Methylene

blue reduction

time

(nzin.)

with:

Spermine Glucose

Succinate

Pyruvate

280 210 153 154

90 64 63 60

Lactate

Acetate

Formate

50 38 36 35

22 21 22 22

w/ml.

0 7 175 700 a No reduction

24 24 24 26

was noted within

7 hr.

7 7 8 9

NO substrate

ANTIBACTERIAL

ACTION

49

OP SPERMINE

3y 300 F 2 200 0" 100 0 3

2

I

0

4

HOURS FIG. 5. The effect of spermine on the oxygen uptake of cells of Staph. aureUs No. 23 in nutrient broth. Warburg flasks contained 2.5 ml. IXfco nutrient broth, pH. 7.6, inoculated with 5 X lo* organisms/ml. and 0.5 ml. of a spermine solution or distilled water; 0.2 ml. of 15y0 KOH was in the center well. Gas phase, air. Temperature 30°C. A-A control, no spermine; O--O final concentration of spermine 28 pg./ml.; O--C final concentration of spermine 561 pg./ml.

r

0 0

IS

30

45

60

MINUTES FIG. 6. The effect of spermine on the formation of /3-galactosidase by washed cells of Stccph. aUreU.s No. 23. Warburg Aasks contained 1.0 ml. of 0.067 M phosphate buffer, pH 7.2,1.5 ml. of cell suspension (3 mg. dry wt.), and 0.5 ml. of 0.02 M lactose; 0.2 ml. of 15yn KOH was in the center well. Gas phase, air. Temperature 30°C. Values corrected for endogenous activity. O--O cells taken from induction medium without spermine, 0-0 cells taken from induction medium with spermine.

50

RAZIN

AND

ROZAMKY

of 569 Bg./ml. spermine, 99.5% of bacteria were killed and the oxygen uptake was reduced to 30% of the control. The reduction of oxygen uptake due to spermine was thus not directly proportional to the killing effect of the drug. 3. The Efect of Sperm&e on the Formation of fl-Galactosidase. Spermine was added to the induction medium to a final concentration of 3 mg./ml.” A control without spermine was run simultaneously. As seen in Fig. 6, spermine markedly inhibited the formation of p-galactosidase. Oxygen uptake by cells taken from the induction medium which contained spermine was about one-third of the amount consumed by the control cells. DISCUSSION

Binding of spermine by the cells followed an adsorption isotherm. It is evident from our results that an active biological process was not involved in the adsorption of spermine. An amount of 8-20 pg. spermine (0.020-0.057 frmole) was adsorbed/mg. dry weight of cells. Spermine was thus bound to bacteria to a lesser extent than the cationic detergent cetyltrimethylammonium bromide or polymyxin. Three hundred micrograms cetyltrimethylammonium bromide (0.82 pmole) (18) or polymyxin (0.25 pmole) (19) was adsorbed by 1 mg. dry weight of cells. According to McCalla (20), a single bacterial cell is able to bind lo* ions of hydrogen. Katchalski et al. (13), working with polylysine, found that each cell adsorbed approximately lo8 lysine residues. Calculations based on the adsorption tests showed that each bacterial cell binds about 10’ molecules of spermine. Since a molecule of spermine contains four ionizable groups, the number found by us is in agreement with the calculations of McCalla and Katchalski et al. Neumark and Pasynskii (21) found that the adsorption of streptomycin by resistant and sensitive varieties of the same strain of Staph. aureus was about equal. In our experiments the trained spermine-resistant Staph. aureus No. 23R was found to adsorb about one third of the amount of spermine adsorbed by the sensitive parent strain. The resistance of the trained strain could therefore be due to the reduced adsorption of the drug. However, spermine resistance of gram-negative bacteria could not be explained by the lower adsorption of the drug as these organisms adsorbed the same amount as the sensitive gram-positive bacteria. The quantity of spermine adsorbed was greater at an alkaline pH. This finding was to be expected as hydrogen ions are known to interfere with the adsorption of cations by bacterial cells (20). A high concentration of hydrogen ions, i.e., 0.1 N HCI, was able to set free 50% of the spermine 3 A comparatively medium contained spermine-phosphate

high concentration of spermine was necessary as the induction 1.5oj, phosphates, and part of the spermine formed an insoluble complex.

ANTIBACTERIAL

ACTION

OF

SPERMINE

51

adsorbed by the cell. The adsorption of spermine, although reduced, still remained considerable at pH 3.6. The interference by hydrogen ions with the adsorption of spermine cannot therefore by itself explain t,he small antibacterial effect of the drug at lower pH values (4). The agglutination of bacteria by spermine is apparently due to the neutralization of their negative surface charge. A similar agglutination phenomenon has been described for various basic substances, e.g., trypaflavine (12), polylysine (13)) and streptomycin (14). The inagglubinability of gram-negative bacilli in the “S” phase by spermine is explained by their behavior as a hydrophilic colloid; such colloids are not agglutinated by neutralization of the electric charge only. Gram-positive and gram-negative bacteria in the “R” phase behave like hydrophobic colloids; the latter agglutinate upon abolition of their surface charge (22). Spermine, like polylysine, agglutinated bacteria within a limited range of concentrations. Katchalski et al. (13) have shown that low concent,rations of polylysine were unable to nullify the negative charge of bacterial cells, whereas high concentrations conveyed to the cell a positive electric charge. Only concentrations by which t,he electric charge is fully or nearly neutralized caused agglutination. Nucleic acids and phospholipides seem to be the cell constituents mainly responsible for the binding of spermine to the bacterium. A parallelism was observed between the antibacterial effect of the amines examined and their abilit,y to form insoluble complexes with nucleic acids and lecithin. These findings are in accordance with the view of Dubos (23) and Tolstoouhov (24) t,hat the formation of undissociable compounds between the drug and cell components is a prerequisite for antibacterial action. Inorganic salts enhanced the dissociation of the “drug-bacterium complex” and decreased the antibact,erial a&ion of spermine. The nontoxic cation of t,he salt competes with t,he basic drug for t,he anionic sites of the cell, cations of higher vnlency being more effective (25, 26): The anions also played a part in the ant,agonistic effect, sulfate and phosphate ions being most effective. The antagonistic effect of amines and basic amino acids may also be explained by the ca,tion-exchange t,heory (26). The configuration of the organic antagonist seems to be of importance; putrescine, an amine more closely related to spermine structurally than cadaverine, was a more effective ant,agonist than the latt,er. Spermine had no bactericidal effect on washed cells of Staph. aureus suspended in dist.illed water or buffer, but exhibited a remarkable bactericidal action when glucose or lactate was added to t,he buffer. It was therefore evident t,hat spermine affected cells in a state of metabolic activit,y only. The higher the metabolic activity, the more pronounced was

52

RAZIN AND ROZdNSKY

the bactericidal effect of spermine. Cells were killed more rapidly in nutrient broth than in casein hydrolyzate, a medium in which growth was restricted. At low temperature, when the metabolic activity of the cells was minimal, the bactericidal effect of spermine was very small. This dependence of bactericidal activity on the metabolic state of the cell has been observed with various antibacterial substances, e.g., penicillin, streptomycin, and polylysine (27, 28, 13). The fact that spermine prolonged the viability of cells of Staph. aureus suspended in distilled water leads to the suggestion that the drug might act as an osmotic regulator in certain conditions. ,4n osmotic regulatory effect of spermine on Neisseria perflava was suggested by Mager (29). The dehydrogenation of glucose, lactate, and formate by washed cells of Staph. aureus was not impaired by spermine, and that of succinate, pyruvate, and acetate was even enhanced. The dehydrogenation of these latter substrates by Staph. aureus is known to be slow and to be affected by dilution of the cell suspension or by its storage (9). Jeffree (30) and Bernfeld et al. (31) have shown that the activity of acid phosphatase and p-glucoronidase which had been weakened by dilution or long storage was enhanced by spermine. The aerobic oxidation of all these substrates was not affected by spermine, but the oxygen uptake of cells suspended in nutrient broth was diminished markedly. A similar effect on washed cells suspended in buffer and in nutrient broth has been described for penicillin and streptomycin (28, 32, 33). Some authors have assumed that anabolic processes are injured primarily by penicillin (34) and streptomycin (28) and suggested that the inhibition of respiration is a secondary effect. It seems plausible that interference with anabolic processes is responsible also for the antibacterial action of spermine. The inhibition of synthesis of /3-galactosidase by cells of Staph. aureu.s under the influence of spermine supports this assumption. ACKNOWLEDGMENTS The authors wish to thank Professor J. Gurevitch ment, and Dr. U. Bachrach for helpful advice.

for his interest and encourage-

SUMMARY

1. Spermine was adsorbed by various bacterial cells, following an adsorption isotherm. No correlation was observed between the quantity of spermine adsorbed by the bacteria and t,heir sensitivity to the drug. The adsorption of spermine decreased in t,he presence of high concentrations of hydrogen ions or other cations. 2. Various bacteria were agglutinated by spermine. Inorganic salts interfered with t,he agglutination.

aNTIBACTERIAL

ACTION

OF SPERMINE

53

3. Spermine formed insoluble complexes with nucleic acids, lecithin, and also with cell-free extracts of E. coli. Inorganic salts interfered with the formation of these complexes. 4. Nucleic acids, lecithin, basic organic compounds, and inorganic salts antagonized the antibacterial action of spermine. 5. Spermine exhibited a higher bactericidal effect on cells of Staph.. aurews in nutrient broth than in casein hydrolyzate media. Spermine did not kill washed cells suspended in Tris buffer. However, a marked bactericidal action was observed when glucose or lactate was added to the buffer. 6. Spermine inhibited the oxygen uptake of Staph. aweus in nutrient broth. The aerobic oxidation of glucose, succinat,e, pyruvabe, lactat,e, acetate, and format,e by washed cells of Staph. aweus was not affected by spermine. The anaerobic oxidation of glucose, lactate, and formate was not impaired; dehydrogenation of succinate, pyruvate, and acetate was even enhanced. Spermine interfered with the formation of /3-galactosidase by washed cells of Staph. uweus. REFERENCES 1. ROSENTHAL, S. RI., .~ND T~BOR, C. W., J. Pharnmcol. Exptl. Therap. 116, 131 (1956). 2. DIJDLEY, H. W., AND ROSENHEIM, O., Biochem. J. 19, 1031 (1925). 3. AMES, B. N., DUBIN, D.T., AND ROSENTHAL, S. N., Science 127, 814 (1958). 4. RozhNs~Y, R., BACHRACH, U., AND GROSSOWICZ, N., J. Gen. IlIicrobiol. 10, 11 (1954). 5. HIRSCH, J. G., AND DUBOS, R. J., J. Exptl. Med. 96, 191 (1952). 6. G~ossowrcz, N., R~ZIN, S., AND ROZANBKY, R., J. Gen.. Iliicrobiol. 13, 436 (1955). 7. RAZIN, S., AND ROZANSKY, R., J. Lab. Clin. Med. 49, 877 (1957). 8. MICKLE, H., J. Bou. Microscop. Sot. 68, 10 (19iSj. 9. BICHOWSKY-SLOMNITZKI, L., Arch. Biochem. 27. 294 (1950). 10. UMBREIT, W. W., BURRIS,R. H., AND STAUFFER, J.F., “Msnometric Techniques and Tissue Met.abolism.” Burgess Publ. Co., lklinneapolis, 1949. 11. CREASER, E. H., J. Gen. Microbial. 12, 288 (1955). 12. PAMPANA, E. J., J. Hyg. 33,502 (1933). 13. &TCHALSKI, E., BICHOWSKY-SLOMNITZKI, L., ANLI VOL~ANI, B. E., Biochem. J. 66. 671 (1953). 14. ~VCQUILLEN, K., Biochirn. et Biophys. Acta 7, 54 (1951). Met.abolism,” p. 1-l-l. Longmans, Green and Co., 15. STEPHENSON, PII., “Bacterial London, 1919. 16. BICHOWSKY-SLOMNITZKI, L., J. Bacterial. 66,33 (1948). 17. HOTCHKISS, R. D., Ann. N. I’. Acad. Sci. 46,179 (1946). 18. ~JCQUILLEN, K., Biochim. et Rio&s. Acta 6, 463 (1950). 19. FEW, A. V., AND S~HUL~~AN, J. H., J. Gen. Microbial. 9, 15-l (1953). 20. hfcCA~~.4, T. PII., J. Bacterial. 40, 23 (19-10). 21. NEUM.4RK, -4. hI., END P~~YNSKII, .4. c:., Doklady. Dad. ivauk S.S.S.R. 96, 3% (195ij. Its Biological and 22. LAMaNNa, C., .4ND RIALLETTE, hf. F., “Basic Bacteriology:

23.

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Chemical Background,” p. 195. The Williams and Wilkins Co., Baltimore, hid., 1953. Cell In Its Relation to Problems of Virulence, DUBUS, R. J., “The Bacterial Immunity and Chemotherapy,” p. 288, Harvard Univ. Press, Cambridge, Mass., 1945. of Drug Action in Chemotherapeutic TOLSTOOUHOV, A.V., " Ionic Interpretation p. 119. Chemical Publ. Co., New York, 1955. Research,” VALKO, E. I., AND DUBOI~, -4. S., J. Bacterial. 47, 15 (1944). MASSART, L., AND v.4~ DER STOCK, J., Natloe166.852 (1950). HOBBY, G. L., MEYER, K., AND CHAFFEE, E., Proc. Sot. Erptl. Biol. Afed. 60, 281 (1912). ROSENBLUM, E. D., .~NU BRYSON, V., dntibiotics & C’hemofherapy 3, 957 (1953). MAGER, J., Nature 176, 933 (1955). JEFFREE, G. M., Nature 176, 509 (1955). BERNFELU, P., BERNFELD, H. C., NIS~ELB.~UM, J. S., AND FISHMAN, W.,J. drrc. Chem. Sot. 76, 4872 (1954). HIRSCH, J., AND DOSDOGRU, S., rlrch. Biochem. 14, 213 (1947). & Chemoth.erapy 4, 262 (1954). PAINE, T. F., JR., AND CLARK, L. S., Antibiotics P.~RK, J. T., AND STROMINGER, J. L., Science 136, 99 (1957).