ANTcROBIAL AGzNTs AD CHzMOTHAPY, May 1975, p. 500-506 Copyright 0 1975 American Society for Microbiology
Vol. 7, No. 5 Printed in U.SA.
Antibacterial Activity of Tropolone T. J. TRUST Department of Bacteriology and Biochemistry, University of Victoria, Victoria, British Colwmbia, Canada
Received for publication 4 November 1974
Tropolone was shown to be bacteriostatic and bactericidal for a wide range of bacterial species. This antibacterial activity was quantitated using standard methods. Tropolone treatment of whole cells resulted in cell lysis characterized by bleb formation and subsequent loss of cell contents after rupture of the bleb. Although bleb formation and lysis did not occur in the presence of 20% sucrose, cell viability was lost. Spheroplasts and protoplasts also lysed in the presence of tropolone. These results indicate that tropolone acts on the cell wall or envelope and on the plasma membrane.
The extreme durability of wood from the genera Chamaecypris and Thuja in the family Cupressaceae is attributed in part to the presence of nonbenzenoid aromatic compounds with the trivial name tropolones (3). These nonstructural components can be extracted from the wood without impairing its structure or strength. The compounds, which include ,B-dolabrin, nootkatin, a-, s-, and y-thujaplicin, and ,8-thujaplicinol contribute to the smell, taste, and color of the wood (3). The thujaplicins (isopropyltropolones) are the simplest of these extractives and are present in the largest amounts (9). These compounds have been reported to possess antifungal and antibacterial activity (1, 14-16). ,8-Thujaplicin is the best studied of the thujaplicins, and has been demonstrated to be bacteriostatic for gram-positive and gramnegative species and bactericidal for a number of gram-negative species (16). The study by Trust and Coombs (16) also showed that the factor which appears to limit the bactericidal activity of 6-thujaplicin is the poor solubility of this compound in water. This low solubility also hinders studies designed to elucidate the mode of antibacterial action of this novel seven-membered ring compound. There is one chemical relative of ,B-thujaplicin that is readily soluble in water. This compound is 2-hydroxy-2,4,6-cycloheptatriene1-one, which has the trivial name tropolone. The configuration of this compound, which has been recognized since 1945 (7), is shown in Fig. 1. Tropolone does not seem to occur naturally, but can be readily synthesized. Its solubility in water makes it a useful compound to use as a model for studies on the antibacterial action of the troponoids. Since little
quantitative information is available concerning its antibacterial activity, it would appear that few, if any, workers have,availed themselves of this model. The purpose of the current study was then to quantitate the antibacterial activity of tropolone using accepted and readily duplicated assay procedures (2, 4). Furthermore, the study aLso examined the activity of tropolone against a wide range of bacterial species, as well as protoplasts and spheroplasts, to provide information on the mode of antibacterial action of seven-membered troponoid compounds. MATERIALS AND METHODS Organisms. The organisms used in this study were obtained from the American Type Culture Collection (ATCC) and are listed in Table 1. Cultures were grown aerobically on Trypticase soy agar (Baltimore Biological Laboratories) at 30 or 37 C. Tropolone. Two samples of tropolone were used. Sample 1, from Aldrich Chemicals Co., was used routinely throughout the study. Sample 2, from K and K Chemical Co., was used to confirm results obtained with sample 1. Melting point determination, nuclear magnetic resonance, and mass, infrared, and ultraviolet spectral analysis confirmed that sample 1 had a purity of 98% and sample 2 had a purity of 99% (5, 11). Stock solutions were prepared by dissolving 250 mg of tropolone in 25 ml of the appropriate solvent. These solutions were stored at 10 C in the dark for no longer than 24 h before use. All procedures involving tropolone were carried out in low actinic glassware in the absence of fluorescent light and sunlight to protect the compound from photodecomposition (6). Plate assays of antibacterial activity. Antibacterial activity was assayed quantitatively by the procaine penicillin (52)-Official Final Action method of the Association of Official Analytical Chemists (2). The organism employed for these assays was Chromobacter violaceum. This organism was chosen
500
VOL. 7,1975
ANTIBACTERIAL ACTIVITY OF TROPOLONE
solution to the cylinders, the agar plates were allowed to stand for a 30-min diffusion period and then incubated on a leveled 2-cm plywood tray at 30 0.5 C for 18 to 24 h. Sizes of the inhibition zones were then measured to the nearest 0.5 mm, and corrected values
0 OH
FIG. 1. Configuration heptatriene-l-one.
of 2-hydroxy-2,4,6-cyclo-
TABLE 1. Bacteriostaticactivityoftropolone Species
Inoculum (no. of colonyforming units/ml)
MIC (M)
Gram positive
Bacillus subtilis
1.1 x 10'
5.7 x 10-'6
(ATCC 6633) (spores) Micrococcus lysodeihticus (ATCC 4698) Mycobacterium marinum (ATCC 927) Staphylococcus aureus (ATCC 6538)
1.1 x 107 1.9 x 103 1.9 x 10' 7.0 x 10' 7.0 x 105 9.6 x 10' 9.6 x 10'
4.1 2.0 4.1 2.0 4.1 1.2 4.1
x 10-4 x 10-4 x 10-4 x 10-' x 10-' x 10-'f x 10-4
x 10' x 107
1.2 1.0 4.9 1.0 4.1
x 10-'f x 10-4 x 10- 5 x 10- 4 x 10-4 x 10- 4 x 10-4 x 10-4 x 10- 4
Gram negative Aeromonas hydrophila (ATCC 9071) Chromobacter violaceum (ATCC 12472) Escherichia coli
(ATCC 9661) Pseudomonas aenrginosa
(ATCC 15442) Pseudomonas fluorescens
(ATCC 13525)
Salmonella typhi
(ATCC 167-T) Serratia marcescens (ATCC 8701)
6.0 6.0 1.1 1.1 8.0
x
10'
x 10'
x 10' 8.0 x 10' 9.9 x 10' 9.9 x 10' 1.2 x 104
1.2 x 10'
3.5 x 10'
3.5 x 10'
1.6 x 104
1.6
501
x
101
4.1 8.2 8.2 4.1
8.2 x 10-4 4.1 x 4.1 x 2.0 x 2.1 x
10-4
10-4 10-4
10-4
for these plate assays after preliminary experiments had shown that this species displayed very distinct zones of inhibition with tropolone and appeared to be the most suitable test species of those used in this study. Control assays were also performed using sodium penicillin G and Micrococcus lysodeikticus to allow variations due to technique to be accounted for and the reproducibility and validity of assays to be established (15). The results obtained with the control assays were consistent with those obtained previously (15) and confirmed the validity of the test assays. Inoculum cultures were grown in antibiotic assay broth (Difco) to the specifications of the Association of Offlcial Analytical Chemists (2), and 0.5 ml of test organism was added to each seed layer. The optimum cell concentration for assays with C. violaceum was about 5 x 10' viable cells/ml. Fisher stainless-steel cylinders (8 mm outer diameter, 6 mm inner diameter, 10 mm in height) were used. The assay solutions for the tropolone standard curve contained 156.3 usg (1.3 x 10-8 M), 312.5 sg (2.6 x 10-' M), 625 jig (5.1 x 10-' M, reference solution), 1,250 jg (10.2 x 10-' M), and 2,500 ,ug (20.5 x 10- ' M) of tropolone per ml. After addition of antibacterial
recorded (2). Broth assay of bacteriostatic activity. The minimal inhibitory concentration (MIC) was determined in antibiotic assay broth (pH 7.0) by the method of Kavanagh (11) and was defined as the lowest concentration showing absence of growth after 72-h incubation. Duplicate twofold dilutions (5 ml) were inoculated with 0.03 ml of an inoculum culture and incubated under appropriate conditions. The MIC for each species was determined using two inocula levels. Quantitative bactericidal assays. The number of viable cells present after exposure of cell suspensions to tropolone was determined in distilled water by the method of the British Standards Institution (4). Inoculum cultures were prepared by washing growth from a 24-h heart infusion agar (Difco) slant with 5 ml of l/4-strength Ringer solution (Oxoid). This suspension was shaken with glass beads for 30 s, the volume was made to 20 ml with ¼/4-strength Ringer solution, and 5.0 ml was added to 20 ml of distilled water. A 4.0-ml aliquot of this bacterial suspension was used in assays. The Bacillus subtilis spore suspension used in assays was prepared by the method of the Association of Official Analytical Chemists (2). After the appropriate contact time, 0.5 ml was removed to 4.5 ml of ¼/4-strength Ringer solution containing 0.5 ml of fetal calf serum to neutralize the antibacterial activity of tropolone, and duplicate serial dilutions were preapred in ¼/4strength Ringer solution. Tropolone did not produce clumping of bacterial suspensions, and therefore the number of viable cells present was determined on heart infusion agar by the drop pipet method of Miles and Misra (13). Effect on celi growth and morphology. The effect of tropolone on the rate of growth and on the viability and turbidity of exponential and stationary phase cells was determined. Cultures were incubated at 30 or 37 C in antibiotic assay broth without shaking, and changes in turbidity before and after addition of tropolone were followed at 560 nm with a Bausch and Lomb Spectronic 20 spectrophotometer. In several experiments, antibiotic assay broth and tropolone solutions containing 20% (wt/vol) sucrose were used. Viable cell counts were followed by the method of Miles and Misra (13) using the same dilution and plating procedures as in the quantitative bactericidal assays. In assays performed in the presence of 20% sucrose, dilution blanks also contained 20% sucrose. Effect on spheroplasts and protoplasts. Spheroplasts of Aeromonas hydrophila and Escherichia coli were prepared by the method of Lederberg (12). A 3-ml sample of an overnight broth culture was added to 10 ml of antibiotic assay broth containing 1,000 U of penicillin per ml, 20% (wt/vol) sucrose, and 0.2% (wt/vol) MgSO. and incubated for 2 to 3 h. The spheroplasts were then centrifuged, washed, and resuspended in the sucrose-MgSO4-antibiotic were
502
TRUST
assay broth. Protoplasts of M. Iysodeikticus were prepared by the method of Furr and Russell (8). Cells from an overnight broth culture were washed with distilled water, and protoplasts were formed in a medium containing 0.5 M sucrose, 0.013 M phosphate buffer (pH 7.2), and 10 ug of lysozyme (Eggwhite, grade 1, Sigma Chemical Co., St. Louis, Mo.) per ml. Protoplast formation was complete after 15 min at 20 C. The protoplasts were then centrifuged and resuspended in the sucrose-phosphate buffer. The effect of tropolone on the spheroplasts and protoplasts was determined by following changes in turbidity at 560 nm in a Bausch and Lomb Spectronic 20 spectrophotometer and microscopically with the aid of a Petroff-Hauser counting chamber.
RESULTS Dose versus response relationship. A standard curve was established fox tropolone with C. violaceum. A plot of dose (molar) versus loge of the size of the zone of growth inhibition (millimeter) between 2.5 x 10- M and 20.5 x 10- 8 M was linear. The following regressed equation was obtained: ln R = 21.43 D + 2.41 where R is the size of the zone of inhibition in millimeters and D is the molar concen-' tration of tropolone. For this assay the standard error of estimation of R on D was 0.12 mm, and the coefficient of correlation was 0.989. No quantitative difference in antibacterial activity was detected in samples of tropolone from the two suppliers. With this organism the zone of complete growth inhibition was surrounded by a zone of reduced bacterial growth and lack of pigmentation. This was surrounded by normal growth and pigmentation. MIC. The results obtained in broth-dilution assays to determine the bacteriostatic activity of tropolone are presented in Table 1. The lowest concentration of tropolone preventing bacterial growth was 5.4 x 10-6 M, whereas inhibition of Pseudomonas aeruginosa required tropolone at 8.2 x 10-4 M. Incubation of assay tubes for longer than 72 h did not affect the titer. The results in Table 1 also show that whereas the inoculum size did not greatly alter the MIC of tropolone for most of the assay species, the bacteriostatic activity of tropolone for A. hydrophila, B. subtilis, and Staphylococcus aureus was greatly altered by different inoculum sizes. Pigmentation of chromogenic species was also affected by tropolone. In assays of C. violaceum where the inoculum size was 108 cells/ml, pigmentation was prevented at 2.5 x 10-5 M, even though good growth was obtained at this concentration. Similarly, in
ANTIMICROB. AGENTS CHEMOTHER.
assays of Serratia marcescens with an inoculum of 108 cells/ml, pigmentation was inhibited by tropolone at 4.9 x 10 5 M. Solutions of tropolone were stable to autoclaving, and no loss of bacteriostatic activity was shown after storage of solutions in the dark at 10 C for 5 days. However, the bacteriostatic activity of tropolone was affected by pH. The MIC for A. hydrophila at pH 5.7 and pH 6.2 was 5.7 x 10-5 M, at pH 6.5 the MIC was 1.2 x 10- 4M, and at pH 6.8 and pH 7.0 the MIC was 2.5 x 10-4M. Quantitative bactericidal activity. Solutions of tropolone at 0.041 or 0.02 M were rapidly bactericidal for A. hydrophila and C. violaceum (Table 2). The bactericidal activity of tropolone at 0.041 was reduced in the presence of 10% fetal calf serum, and the serum completely neutralized the bactericidal activity of 0.004 M tropolone. This result confirms that sufficient serum was present in the first dilution blank to neutralize any residual tropolone carried over from the assay tube. The maximum concentration of tropolone in the first dilution blank in this study was 0.004 M. The results presented in Table 3 show that tropolone was also bactericidal for vegetative cells of the other assay species. With these species the bactericidal action of tropolone was decidedly slower than in the case of A. hydrophila or C. violaceum. The results in Table 3 also show that tropolone has little sporicidal activity. Effect on cell growth and morphology. Cells of the various test species failed to form filamentous forms when grown in the presence of tropolone at 0.5 or 0.1 times the MIC for that particular species. At the higher concentration of 0.041 M, however, tropolone caused the lysis and death of both exponential and stationary phase cells of A. hydrophila, B. subtilis, E. coli, P. aeruginosa, Salmonella typhi, and S. marcescens. Typical results of the lysis of A. hydrophila, B. subtilis, E. coli and P. aeruginosa by tropolone are shown in Fig. 2. Phase contrast microscopy revealed that the decrease in absorbance (560 nm) of suspensions of gram-negative species was preceded by the appearance of "blebs" on the cell envelope followed by rapid breakage of the bleb and loss of the cell contents. In the case of exponential phase cells, these blebs characteristically appeared. at points of cell division and at the ends of cells. The blebbing of stationary phase cells was not as pronounced. This was also true for B. subtilis. In these cases, the refractivity of the cells was lost gradually until only an empty cell wall remained.
VOL. 7, 1975
ANTIBACTERIAL ACTIVITY OF TROPOLONE
503
TABLE 2. Bactericidal activity of tropolone for Aeromonas hydrophila and Chromobacter violaceum No. of colony-forming units/ml of medication tnixture at (min):
Species
Tropolone (M)
0
Aeromonas hydrophila
(ATCC 9071)
Chromobacter violaceum (ATCC 12472)
0.041 0.02 0.008 0.004 DWb 0.041 + 10% fetal calf serum 0.004 + 10% fetal calf serum DW + 10% fetal calf serum 0.041 0.02 DW
aNCFU, No countable colony-forming units. b DW, Distilled water. Unlike the other test species, cells of S. aureus were not lysed by tropolone. Although tropolone halted the growth of this species, no decrease in absorbance (560 nm) was demon-
strated (Fig. 3). The presence of 20% sucrose protected the cells of sensitive species from lysis. Microscopic examination revealed that blebbing did not occur when cells in 20% sucrose were treated with tropolone. No decrease in absorbance (560 nm) occurred after the addition of tropolone to cells of A. hydrophila and B. subtilis in 20% sucrose (Fig. 4). However, Fig. 4 also shows that although no decrease in absorbance (560 nm) was demonstrated, cell viability was lost in the presence of 20% sucrose. Effect on spheroplasts and protoplasts. Spheroplasts of A. hydrophila and E. coli and protoplasts of M. lysodeikticus were lysed on addition of tropolone (Fig. 5). DISCUSSION The results presented here indicate that tropolone is a broad-spectrum antibacterial agent. It is bacteriostatic for both gram-positive and gram-negative species, and has bactericidal activity against both types of cells when used at concentrations higher than 0.02 M. This ability to kill both gram-positive and gram-negative cell types distinguishes it from ,3-thujaplicin, which has been shown to be bactericidal only for a number of gram-negative species (14). It should be noted, however, that the concentrations achievable with ,8-thujaplicin are markedly lower than those achievable with
5
30
60
107 107 107 107 107 107
1.6 x 101 1.9 X 107 4.7 x 107
7.7 x 107 7.9 x 107 3.0 x 107
NCFUa NCFU 6.0 x 106 1.6 x 107 7.9 x 107 4.9 x 103
NCFU NCFU 6.9 x 105 1.3 x 107 7.9 x 107 NCFU
7.0 x 107
7.0 x 107
7.0 x 107
7.0 x 107
7.0 x 107
7.0 x 107
7.0 x 107
7.0 x 107
1.1 x 108 1.1 x 108 1.1 x 108
1.0 x 106 1.0 X 107 1.1 X 106
NCFU
NCFU
2.0 x 106
8.0 x 104
1.1 x 108
8.0 x 107
7.9 x 7.9 x 7.9 x 7.9 x 7.9 x 7.0 x
tropolone, since ,B-thujaplicin is relatively insoluble in water. Although vegetative cells were killed by 0.02- M solutions of tropolone, the compound was not sporicidal at 0.04 M. The solubility of tropolone in water also accounts for its markedly different behavior from ,Bthujaplicin in agar diffusion assays, tropoloneexhibiting much greater diffusibility than ,3thujaplicin. Comparison of the bacteriostatic activity of tropolone with /3-thujaplicin on a molar basis shows that tropolone exhibits 12% of the bacteriostatic activity of ,B-thujaplicin for Mycobacterium marinum and 95% the activity of /3-thujaplicin for A. hydrophila. The values obtained for the other assay organisms were between these limits except in the case of P. aeruginosa and S. marcescens. Although tropolone inhibited the growth of these species, they were not inhibited by ,B-thujaplicin. These findings show that the isopropyl side chain on the seven carbon ring structure of /3-thujaplicin is not essential for antibacterial activity. Although this side chain appears to increase the bacteriostatic activity of the ring structure against some species, the increased activity was not found with all species. Moreover, the side chain markedly reduces the water solubility of the seven carbon ring, and so reduces the range of bactericidal activity. This can be seen when the spectrum of bactericidal activity of the seven carbon ring with and without its isopropyl group is compared. However, this side chain markedly enhanced the bactericidal activity of the ring compound for both A. hydrophila and C. violaceum. In the case of
504
TRUST
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. Bactericidal activity of tropolone for other species No. of colony-forming units/ml of medication mixture at (h):
Species
6
Tropolone (M) |
0.041 DWa
8.3 x 107 8.3 x 107
8.3 x 107 8.3 X 107
7.7 x 107 7.2 x 107
6.1 x 107 7.6 x 107
Escherichia coli (ATCC 9661)
0.041 0.02 DW
1.2 x 108 1.2 x 108 1.2 x 108
8.3 x 107 1.4 x 108 1.8 x 108
4.0 x 107 9.0 X 107
5.6 x 104 3.2 x 107
1.0 x 10'
1.3 x 108
Micrococcus lysodeikticus
0.041 0.02 DW
1.8 x 108
8.8 x 10'
NCFUb
1.8 x 10 1.8 x 108
4.2 x 107
1.1 x 108
1.3 x 106 1.1 x 10'
NCFU NCFU 1.1 x 10'
0.041 0.02 DW
1.2 x 106 1.2 x 10' 1.2 x 106
9.0 X 105 4.0 x 105 1.2 x 106
2.9 x 105 2.0 x 105 1.2 x 106
9.6 x 103 2.7 x 104 1.2 x 106
0.041 0.02 DW
1.8 x 10'
4.8 x 103 2.4 x 103 1.8 x 108
NCFU 2.4 x 103 1.3 x 10'
NCFU NCFU 1.3 x 10'
Pseudomonas fluorescens (ATCC 13525)
0.041 0.02 0.008 DW
2.0 x 2.0 x 2.0 x 2.0 x
NCFU 2.6 x 104 6.5 x 107 2.0 x 10'
NCFU NCFU 4.5 x 107 2.0 x 10'
NCFU NCFU 1.5 x 106 2.0 x 108
Salmonella typhi
0.041 0.02 DW
9.6 x 107 9.6 x 107 9.6 x 107
3.0 x 102 NCFU 1.6 x 10 | 1.2 x 103 9.6 x 107 9.6 x 107
NCFU NCFU 9.6 x 107
Serratia marcescens
0.041 0.02 DW
2.7 x 10'
5.5 x 105 4.0 x 107 2.7 x 108
1.2 x 103 2.0 x 10' 2.7 x 108
NCFU 1.6 x 10'
0.041 0.02 DW
9.0 X 107 9.0 X 107 9.0 X 107
4.0 x 106
7.2 x 10'
5.9 x 107 9.0 X 107
4.1 x 107 9.0 x 107
Bacillus subtilis (spores)
(ATCC 6633)
(ATCC 4698) Mycobacterium marinum
(ATCC 927) Pseudomonas aeruginosa
(ATCC 15442)
(ATCC 167-T) (ATCC 8701)
Staphylococcus aureus
(ATCC 6538)
Distilled water control. b NCFU, No countable colony-forming units.
a DW,
1.8 x 108
1.8 x 10'
10' 10'
10' 10'
2.7 x 108
2.7 x 10'
2.7 x 108 NCFU 4.6 x 10. 1.0 x 10.
505
ANTIBACTERIAL ACTIVITY OF TROPOLONE
Voi- 7, 1975
lysis of whole cells of both gram-positive and gram-negative species. The rapid death of the whole cells after blebbing itself presumably reflects a weakening of the cell wall or envelope material in some manner. In growing cells, the blebbing in the region of cell division and cell elongation could also result by inhibition of the enzymes in that area which are involved in the synthesis of new cell wall. However, if tropolone were to inhibit these enzymes, fila-
05 OA
42
Eoi tOS
9'
10.5
a 4C
A
03
10.2 E
a .011 2
2
4
4
2
0
I-
0
4
0
6
2
0
6
4
u
B
B
9
OA
6
Time, h
-4
0.3
FIG. 2. The effect of tropolone at a final concentration of 0.041 M on the growth of Aeromonas hydrophila ATCC 9071 (A), Escherichia coli ATCC 9661 (B), Pseudomonas aeruginosa ATCC 15442 (C), and Bacillus subtilis ATCC 6633 (D). Control growth in antibiotic assay broth (0); addition of tropolone (>); tropolone treatment in antibiotic assay broth (a).
E
8
4R° 4D °I
0.2
I o
7
Q.1
0
0 4 2 Time after tropolone addition, h
2
6
4
FIG. 4. The effect of tropolone at a final concentration of 0.041 M on the turbidity and viability of 18-h cultures of Aeromonas hydrophila ATCC 9071 (A) and Bacillus subtilis ATCC 6633 (B) in 20% (wt/vot) sucrose-antibiotic assay broth. Control growth (O3); addition of tropolone (>); tropolonetreated cells (U). Tropolone was prepared in the 20% sucrose-antibiotic assay broth. A
I
C
B
OA
E C
0.3
2
4
6
8
3
4
5
7
Time, h
FIG. 3. Effect of tropolone at a final concentration of 0.041 M on the growth of Staphylococcus aureus ATCC 6538. Control growth in antibiotic assay broth (0); addition of tropolone (>); tropolone treatment in antibiotic assay broth (-).
40.1
.
L
1
2
.--
.
1
2
.
2.
1
2
Time after tropolone added, h
FIG. 5. Effect of tropolone at a final concentration of 0.041 M on suspensions of spheroplasts and proto(A) Suspension of 2 x 100 spheroplasts of these organisms, comparison of the bactericidal plasts. Aeromonas hydrophila ATCC 9071 per ml; (B) that reveals two of the compounds activity suspension of 2 x 108 spheroplasts of Escherichia tropolone exhibits only 4% of the bactericidal coli ATCC 9661; (C) suspension of 1 X10x protoplasts activity of ,B-thujaplicin. of Micrococcus lysodeikticus ATCC 4698. Addition of The results indicate that at high concentra- tropolone (>); tropolone treatment in 20% sucrose tions, tropolone acts at cell surfaces causing (T); control (0).
506
TRUST
mentous forms would be expected at subinhibitory concentrations of tropolone. Such filamentous forms were not observed. The loss of viability of cells treated with tropolone in the presence of sucrose suggests a second mechanism of action for tropolone. The lysis of spheroplasts and protoplasts is evidence that this second action of tropolone is at the level of the plasma membrane. The lysis could occur in one of two ways: either the tropolone causes direct dissolution of the physical structure, or it could disrupt the membrane permeability causing rapid uptake of water and lysis of the spheroplasts and protoplasts due to mechanical pressure. The lack of lysis of whole cells in the presence of sucrose suggests that the cell wall prevents access of sufficient tropolone to bring about lysis of the cell membrane. When the wall is preweakened, as in the case of penicillin-treated cells, or removed, as in the case of lysozyme-treated cells, then access of sufficient tropolone to the membrane is allowed and lysis occurs. The loss of viability of whole cells in the presence of sucrose does, however, suggest that sufficient tropolone reaches the membrane of whole cells to cause damage which leads to cell death. The exact mechanics by which tropolone damages the cell membrane remains obscure and will require further experimentation. Similarly, the inability of tropolone to lyse cells of Staphylococcus aureus is difficult to explain at this time, except that this species is particularly refractory to many compounds and enzymes, including lysozyme. The observation that this organism is also eventually killed by tropolone could, however, indicate a third mechanism for tropolone activity, one not associated with the cell surface. ACKNOWLEDGMENTS This work was supported by a grant from the National Research Council of Canada. I am indebted to Karen Bartlett, Diane Chipman, and Vida Money for skilled technical assistance. J. T. Buckley is also thanked for supplying lysozyme.
ANTIMICROB. AGENTS CHEMOTHER. LITRATURE CITED 1. Anderson, A. B., and J. Gripenberg. 1948. Antibiotic substances from the heartwood of Thuja plicata D. Don. IV. The constitution of ,-thujaplicin. Acta Chem. Scand. 2:644-650. 2. Association of Official Analytical Chemists. 1970. Official methods of analysis of the Association of Official Analytical Chemists, 11th ed. Association of Official Analytical Chemists, Washington, D.C. 3. Barton, G. M., and B. F. MacDonald. 1971. The chemistry and utilization of western red cedar. Dept. Fish. For. Can. For. Serv., publication no. 1023, Ottawa. 4. British Standards Institution. 1960. Method for laboratory evaluation of disinfectant activity of quaternary ammonium compounds by suspension test procedure publication no. B.S.3286: 1960. British Standards House, London. 5. Cook, J. W., A. R. Gibb, R. A. Raphael, and A. R. Somerville. 1951. Tropolones. 1. The preparation and general characteristics of tropolone. J. Chem. Soc. 1951:503. 6. Coombs, R. W., and T. J. Trust. 1973. The effect of light on the antibacterial activity of ,B-thujaplicin. Can. J. Microbiol. 9:1177-1180. 7. Dewar, M. J. S. 1945. The structure of stipitatic acid. Nature (London) 155:50-51. 8. Furr, J. R., and H. D. Russell. 1972. Effect of phydroxybenzoic acid on spheroplasts of Serratia marcescens and protoplasts of Bacillus megaterium. Microbios 6:47-54. 9. Gardner, J. A. F., and G. M. Barbor. 1958. The extraneous components of western red cedar. For. Prod. J. 8:189-192. 10. Greenwood, D., and F. O'Grady. 1973. The two sites of penicillin action in Escherichia coli. J. Infect. Dis. 128:791-794. 11. Kavanagh, F. 1963. Dilution methods of antibiotic assay, p. 125-140. In F. Kavanagh (ed.), Analytical microbiology_Academic Press, New York. 12. Lederberg, J. 1956. Bacterial protoplasts induced by penicillin. Proc. Natl. Acad. Sci. U.S.A. 42:574577. 13. Miles, A. A., and S. S. Misra. 1938. The estimation of the bactericidal power of the blood. J. Hyg. 38:732-747. 14. Pauson, P. L. 1955. Tropones and tropolones. Chem. Rev. 55:9-136. 15. Rennerfelt, E. 1948. Investigations of thujaplicin, and fungicidal substance in the heartwood of Thuja plicata D. Don. Physiol. Plant. 1:245-254. 16. Trust, T. J., and R. W. Coombs. 1973. Antibacterial activity of ,8-thujaplicin. Can. J. Microbiol. 19:13411346. 17. Von Doering, W., and F. L. Detert. 1951. Synthesis of tropolone. J. Am. Chem. Soc. 73:876.
Vol. 7, No. 5 Printed in U.SA.
Antibacterial Activity of Tropolone T. J. TRUST Department of Bacteriology and Biochemistry, University of Victoria, Victoria, British Colwmbia, Canada
Received for publication 4 November 1974
Tropolone was shown to be bacteriostatic and bactericidal for a wide range of bacterial species. This antibacterial activity was quantitated using standard methods. Tropolone treatment of whole cells resulted in cell lysis characterized by bleb formation and subsequent loss of cell contents after rupture of the bleb. Although bleb formation and lysis did not occur in the presence of 20% sucrose, cell viability was lost. Spheroplasts and protoplasts also lysed in the presence of tropolone. These results indicate that tropolone acts on the cell wall or envelope and on the plasma membrane.
The extreme durability of wood from the genera Chamaecypris and Thuja in the family Cupressaceae is attributed in part to the presence of nonbenzenoid aromatic compounds with the trivial name tropolones (3). These nonstructural components can be extracted from the wood without impairing its structure or strength. The compounds, which include ,B-dolabrin, nootkatin, a-, s-, and y-thujaplicin, and ,8-thujaplicinol contribute to the smell, taste, and color of the wood (3). The thujaplicins (isopropyltropolones) are the simplest of these extractives and are present in the largest amounts (9). These compounds have been reported to possess antifungal and antibacterial activity (1, 14-16). ,8-Thujaplicin is the best studied of the thujaplicins, and has been demonstrated to be bacteriostatic for gram-positive and gramnegative species and bactericidal for a number of gram-negative species (16). The study by Trust and Coombs (16) also showed that the factor which appears to limit the bactericidal activity of 6-thujaplicin is the poor solubility of this compound in water. This low solubility also hinders studies designed to elucidate the mode of antibacterial action of this novel seven-membered ring compound. There is one chemical relative of ,B-thujaplicin that is readily soluble in water. This compound is 2-hydroxy-2,4,6-cycloheptatriene1-one, which has the trivial name tropolone. The configuration of this compound, which has been recognized since 1945 (7), is shown in Fig. 1. Tropolone does not seem to occur naturally, but can be readily synthesized. Its solubility in water makes it a useful compound to use as a model for studies on the antibacterial action of the troponoids. Since little
quantitative information is available concerning its antibacterial activity, it would appear that few, if any, workers have,availed themselves of this model. The purpose of the current study was then to quantitate the antibacterial activity of tropolone using accepted and readily duplicated assay procedures (2, 4). Furthermore, the study aLso examined the activity of tropolone against a wide range of bacterial species, as well as protoplasts and spheroplasts, to provide information on the mode of antibacterial action of seven-membered troponoid compounds. MATERIALS AND METHODS Organisms. The organisms used in this study were obtained from the American Type Culture Collection (ATCC) and are listed in Table 1. Cultures were grown aerobically on Trypticase soy agar (Baltimore Biological Laboratories) at 30 or 37 C. Tropolone. Two samples of tropolone were used. Sample 1, from Aldrich Chemicals Co., was used routinely throughout the study. Sample 2, from K and K Chemical Co., was used to confirm results obtained with sample 1. Melting point determination, nuclear magnetic resonance, and mass, infrared, and ultraviolet spectral analysis confirmed that sample 1 had a purity of 98% and sample 2 had a purity of 99% (5, 11). Stock solutions were prepared by dissolving 250 mg of tropolone in 25 ml of the appropriate solvent. These solutions were stored at 10 C in the dark for no longer than 24 h before use. All procedures involving tropolone were carried out in low actinic glassware in the absence of fluorescent light and sunlight to protect the compound from photodecomposition (6). Plate assays of antibacterial activity. Antibacterial activity was assayed quantitatively by the procaine penicillin (52)-Official Final Action method of the Association of Official Analytical Chemists (2). The organism employed for these assays was Chromobacter violaceum. This organism was chosen
500
VOL. 7,1975
ANTIBACTERIAL ACTIVITY OF TROPOLONE
solution to the cylinders, the agar plates were allowed to stand for a 30-min diffusion period and then incubated on a leveled 2-cm plywood tray at 30 0.5 C for 18 to 24 h. Sizes of the inhibition zones were then measured to the nearest 0.5 mm, and corrected values
0 OH
FIG. 1. Configuration heptatriene-l-one.
of 2-hydroxy-2,4,6-cyclo-
TABLE 1. Bacteriostaticactivityoftropolone Species
Inoculum (no. of colonyforming units/ml)
MIC (M)
Gram positive
Bacillus subtilis
1.1 x 10'
5.7 x 10-'6
(ATCC 6633) (spores) Micrococcus lysodeihticus (ATCC 4698) Mycobacterium marinum (ATCC 927) Staphylococcus aureus (ATCC 6538)
1.1 x 107 1.9 x 103 1.9 x 10' 7.0 x 10' 7.0 x 105 9.6 x 10' 9.6 x 10'
4.1 2.0 4.1 2.0 4.1 1.2 4.1
x 10-4 x 10-4 x 10-4 x 10-' x 10-' x 10-'f x 10-4
x 10' x 107
1.2 1.0 4.9 1.0 4.1
x 10-'f x 10-4 x 10- 5 x 10- 4 x 10-4 x 10- 4 x 10-4 x 10-4 x 10- 4
Gram negative Aeromonas hydrophila (ATCC 9071) Chromobacter violaceum (ATCC 12472) Escherichia coli
(ATCC 9661) Pseudomonas aenrginosa
(ATCC 15442) Pseudomonas fluorescens
(ATCC 13525)
Salmonella typhi
(ATCC 167-T) Serratia marcescens (ATCC 8701)
6.0 6.0 1.1 1.1 8.0
x
10'
x 10'
x 10' 8.0 x 10' 9.9 x 10' 9.9 x 10' 1.2 x 104
1.2 x 10'
3.5 x 10'
3.5 x 10'
1.6 x 104
1.6
501
x
101
4.1 8.2 8.2 4.1
8.2 x 10-4 4.1 x 4.1 x 2.0 x 2.1 x
10-4
10-4 10-4
10-4
for these plate assays after preliminary experiments had shown that this species displayed very distinct zones of inhibition with tropolone and appeared to be the most suitable test species of those used in this study. Control assays were also performed using sodium penicillin G and Micrococcus lysodeikticus to allow variations due to technique to be accounted for and the reproducibility and validity of assays to be established (15). The results obtained with the control assays were consistent with those obtained previously (15) and confirmed the validity of the test assays. Inoculum cultures were grown in antibiotic assay broth (Difco) to the specifications of the Association of Offlcial Analytical Chemists (2), and 0.5 ml of test organism was added to each seed layer. The optimum cell concentration for assays with C. violaceum was about 5 x 10' viable cells/ml. Fisher stainless-steel cylinders (8 mm outer diameter, 6 mm inner diameter, 10 mm in height) were used. The assay solutions for the tropolone standard curve contained 156.3 usg (1.3 x 10-8 M), 312.5 sg (2.6 x 10-' M), 625 jig (5.1 x 10-' M, reference solution), 1,250 jg (10.2 x 10-' M), and 2,500 ,ug (20.5 x 10- ' M) of tropolone per ml. After addition of antibacterial
recorded (2). Broth assay of bacteriostatic activity. The minimal inhibitory concentration (MIC) was determined in antibiotic assay broth (pH 7.0) by the method of Kavanagh (11) and was defined as the lowest concentration showing absence of growth after 72-h incubation. Duplicate twofold dilutions (5 ml) were inoculated with 0.03 ml of an inoculum culture and incubated under appropriate conditions. The MIC for each species was determined using two inocula levels. Quantitative bactericidal assays. The number of viable cells present after exposure of cell suspensions to tropolone was determined in distilled water by the method of the British Standards Institution (4). Inoculum cultures were prepared by washing growth from a 24-h heart infusion agar (Difco) slant with 5 ml of l/4-strength Ringer solution (Oxoid). This suspension was shaken with glass beads for 30 s, the volume was made to 20 ml with ¼/4-strength Ringer solution, and 5.0 ml was added to 20 ml of distilled water. A 4.0-ml aliquot of this bacterial suspension was used in assays. The Bacillus subtilis spore suspension used in assays was prepared by the method of the Association of Official Analytical Chemists (2). After the appropriate contact time, 0.5 ml was removed to 4.5 ml of ¼/4-strength Ringer solution containing 0.5 ml of fetal calf serum to neutralize the antibacterial activity of tropolone, and duplicate serial dilutions were preapred in ¼/4strength Ringer solution. Tropolone did not produce clumping of bacterial suspensions, and therefore the number of viable cells present was determined on heart infusion agar by the drop pipet method of Miles and Misra (13). Effect on celi growth and morphology. The effect of tropolone on the rate of growth and on the viability and turbidity of exponential and stationary phase cells was determined. Cultures were incubated at 30 or 37 C in antibiotic assay broth without shaking, and changes in turbidity before and after addition of tropolone were followed at 560 nm with a Bausch and Lomb Spectronic 20 spectrophotometer. In several experiments, antibiotic assay broth and tropolone solutions containing 20% (wt/vol) sucrose were used. Viable cell counts were followed by the method of Miles and Misra (13) using the same dilution and plating procedures as in the quantitative bactericidal assays. In assays performed in the presence of 20% sucrose, dilution blanks also contained 20% sucrose. Effect on spheroplasts and protoplasts. Spheroplasts of Aeromonas hydrophila and Escherichia coli were prepared by the method of Lederberg (12). A 3-ml sample of an overnight broth culture was added to 10 ml of antibiotic assay broth containing 1,000 U of penicillin per ml, 20% (wt/vol) sucrose, and 0.2% (wt/vol) MgSO. and incubated for 2 to 3 h. The spheroplasts were then centrifuged, washed, and resuspended in the sucrose-MgSO4-antibiotic were
502
TRUST
assay broth. Protoplasts of M. Iysodeikticus were prepared by the method of Furr and Russell (8). Cells from an overnight broth culture were washed with distilled water, and protoplasts were formed in a medium containing 0.5 M sucrose, 0.013 M phosphate buffer (pH 7.2), and 10 ug of lysozyme (Eggwhite, grade 1, Sigma Chemical Co., St. Louis, Mo.) per ml. Protoplast formation was complete after 15 min at 20 C. The protoplasts were then centrifuged and resuspended in the sucrose-phosphate buffer. The effect of tropolone on the spheroplasts and protoplasts was determined by following changes in turbidity at 560 nm in a Bausch and Lomb Spectronic 20 spectrophotometer and microscopically with the aid of a Petroff-Hauser counting chamber.
RESULTS Dose versus response relationship. A standard curve was established fox tropolone with C. violaceum. A plot of dose (molar) versus loge of the size of the zone of growth inhibition (millimeter) between 2.5 x 10- M and 20.5 x 10- 8 M was linear. The following regressed equation was obtained: ln R = 21.43 D + 2.41 where R is the size of the zone of inhibition in millimeters and D is the molar concen-' tration of tropolone. For this assay the standard error of estimation of R on D was 0.12 mm, and the coefficient of correlation was 0.989. No quantitative difference in antibacterial activity was detected in samples of tropolone from the two suppliers. With this organism the zone of complete growth inhibition was surrounded by a zone of reduced bacterial growth and lack of pigmentation. This was surrounded by normal growth and pigmentation. MIC. The results obtained in broth-dilution assays to determine the bacteriostatic activity of tropolone are presented in Table 1. The lowest concentration of tropolone preventing bacterial growth was 5.4 x 10-6 M, whereas inhibition of Pseudomonas aeruginosa required tropolone at 8.2 x 10-4 M. Incubation of assay tubes for longer than 72 h did not affect the titer. The results in Table 1 also show that whereas the inoculum size did not greatly alter the MIC of tropolone for most of the assay species, the bacteriostatic activity of tropolone for A. hydrophila, B. subtilis, and Staphylococcus aureus was greatly altered by different inoculum sizes. Pigmentation of chromogenic species was also affected by tropolone. In assays of C. violaceum where the inoculum size was 108 cells/ml, pigmentation was prevented at 2.5 x 10-5 M, even though good growth was obtained at this concentration. Similarly, in
ANTIMICROB. AGENTS CHEMOTHER.
assays of Serratia marcescens with an inoculum of 108 cells/ml, pigmentation was inhibited by tropolone at 4.9 x 10 5 M. Solutions of tropolone were stable to autoclaving, and no loss of bacteriostatic activity was shown after storage of solutions in the dark at 10 C for 5 days. However, the bacteriostatic activity of tropolone was affected by pH. The MIC for A. hydrophila at pH 5.7 and pH 6.2 was 5.7 x 10-5 M, at pH 6.5 the MIC was 1.2 x 10- 4M, and at pH 6.8 and pH 7.0 the MIC was 2.5 x 10-4M. Quantitative bactericidal activity. Solutions of tropolone at 0.041 or 0.02 M were rapidly bactericidal for A. hydrophila and C. violaceum (Table 2). The bactericidal activity of tropolone at 0.041 was reduced in the presence of 10% fetal calf serum, and the serum completely neutralized the bactericidal activity of 0.004 M tropolone. This result confirms that sufficient serum was present in the first dilution blank to neutralize any residual tropolone carried over from the assay tube. The maximum concentration of tropolone in the first dilution blank in this study was 0.004 M. The results presented in Table 3 show that tropolone was also bactericidal for vegetative cells of the other assay species. With these species the bactericidal action of tropolone was decidedly slower than in the case of A. hydrophila or C. violaceum. The results in Table 3 also show that tropolone has little sporicidal activity. Effect on cell growth and morphology. Cells of the various test species failed to form filamentous forms when grown in the presence of tropolone at 0.5 or 0.1 times the MIC for that particular species. At the higher concentration of 0.041 M, however, tropolone caused the lysis and death of both exponential and stationary phase cells of A. hydrophila, B. subtilis, E. coli, P. aeruginosa, Salmonella typhi, and S. marcescens. Typical results of the lysis of A. hydrophila, B. subtilis, E. coli and P. aeruginosa by tropolone are shown in Fig. 2. Phase contrast microscopy revealed that the decrease in absorbance (560 nm) of suspensions of gram-negative species was preceded by the appearance of "blebs" on the cell envelope followed by rapid breakage of the bleb and loss of the cell contents. In the case of exponential phase cells, these blebs characteristically appeared. at points of cell division and at the ends of cells. The blebbing of stationary phase cells was not as pronounced. This was also true for B. subtilis. In these cases, the refractivity of the cells was lost gradually until only an empty cell wall remained.
VOL. 7, 1975
ANTIBACTERIAL ACTIVITY OF TROPOLONE
503
TABLE 2. Bactericidal activity of tropolone for Aeromonas hydrophila and Chromobacter violaceum No. of colony-forming units/ml of medication tnixture at (min):
Species
Tropolone (M)
0
Aeromonas hydrophila
(ATCC 9071)
Chromobacter violaceum (ATCC 12472)
0.041 0.02 0.008 0.004 DWb 0.041 + 10% fetal calf serum 0.004 + 10% fetal calf serum DW + 10% fetal calf serum 0.041 0.02 DW
aNCFU, No countable colony-forming units. b DW, Distilled water. Unlike the other test species, cells of S. aureus were not lysed by tropolone. Although tropolone halted the growth of this species, no decrease in absorbance (560 nm) was demon-
strated (Fig. 3). The presence of 20% sucrose protected the cells of sensitive species from lysis. Microscopic examination revealed that blebbing did not occur when cells in 20% sucrose were treated with tropolone. No decrease in absorbance (560 nm) occurred after the addition of tropolone to cells of A. hydrophila and B. subtilis in 20% sucrose (Fig. 4). However, Fig. 4 also shows that although no decrease in absorbance (560 nm) was demonstrated, cell viability was lost in the presence of 20% sucrose. Effect on spheroplasts and protoplasts. Spheroplasts of A. hydrophila and E. coli and protoplasts of M. lysodeikticus were lysed on addition of tropolone (Fig. 5). DISCUSSION The results presented here indicate that tropolone is a broad-spectrum antibacterial agent. It is bacteriostatic for both gram-positive and gram-negative species, and has bactericidal activity against both types of cells when used at concentrations higher than 0.02 M. This ability to kill both gram-positive and gram-negative cell types distinguishes it from ,3-thujaplicin, which has been shown to be bactericidal only for a number of gram-negative species (14). It should be noted, however, that the concentrations achievable with ,8-thujaplicin are markedly lower than those achievable with
5
30
60
107 107 107 107 107 107
1.6 x 101 1.9 X 107 4.7 x 107
7.7 x 107 7.9 x 107 3.0 x 107
NCFUa NCFU 6.0 x 106 1.6 x 107 7.9 x 107 4.9 x 103
NCFU NCFU 6.9 x 105 1.3 x 107 7.9 x 107 NCFU
7.0 x 107
7.0 x 107
7.0 x 107
7.0 x 107
7.0 x 107
7.0 x 107
7.0 x 107
7.0 x 107
1.1 x 108 1.1 x 108 1.1 x 108
1.0 x 106 1.0 X 107 1.1 X 106
NCFU
NCFU
2.0 x 106
8.0 x 104
1.1 x 108
8.0 x 107
7.9 x 7.9 x 7.9 x 7.9 x 7.9 x 7.0 x
tropolone, since ,B-thujaplicin is relatively insoluble in water. Although vegetative cells were killed by 0.02- M solutions of tropolone, the compound was not sporicidal at 0.04 M. The solubility of tropolone in water also accounts for its markedly different behavior from ,Bthujaplicin in agar diffusion assays, tropoloneexhibiting much greater diffusibility than ,3thujaplicin. Comparison of the bacteriostatic activity of tropolone with /3-thujaplicin on a molar basis shows that tropolone exhibits 12% of the bacteriostatic activity of ,B-thujaplicin for Mycobacterium marinum and 95% the activity of /3-thujaplicin for A. hydrophila. The values obtained for the other assay organisms were between these limits except in the case of P. aeruginosa and S. marcescens. Although tropolone inhibited the growth of these species, they were not inhibited by ,B-thujaplicin. These findings show that the isopropyl side chain on the seven carbon ring structure of /3-thujaplicin is not essential for antibacterial activity. Although this side chain appears to increase the bacteriostatic activity of the ring structure against some species, the increased activity was not found with all species. Moreover, the side chain markedly reduces the water solubility of the seven carbon ring, and so reduces the range of bactericidal activity. This can be seen when the spectrum of bactericidal activity of the seven carbon ring with and without its isopropyl group is compared. However, this side chain markedly enhanced the bactericidal activity of the ring compound for both A. hydrophila and C. violaceum. In the case of
504
TRUST
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. Bactericidal activity of tropolone for other species No. of colony-forming units/ml of medication mixture at (h):
Species
6
Tropolone (M) |
0.041 DWa
8.3 x 107 8.3 x 107
8.3 x 107 8.3 X 107
7.7 x 107 7.2 x 107
6.1 x 107 7.6 x 107
Escherichia coli (ATCC 9661)
0.041 0.02 DW
1.2 x 108 1.2 x 108 1.2 x 108
8.3 x 107 1.4 x 108 1.8 x 108
4.0 x 107 9.0 X 107
5.6 x 104 3.2 x 107
1.0 x 10'
1.3 x 108
Micrococcus lysodeikticus
0.041 0.02 DW
1.8 x 108
8.8 x 10'
NCFUb
1.8 x 10 1.8 x 108
4.2 x 107
1.1 x 108
1.3 x 106 1.1 x 10'
NCFU NCFU 1.1 x 10'
0.041 0.02 DW
1.2 x 106 1.2 x 10' 1.2 x 106
9.0 X 105 4.0 x 105 1.2 x 106
2.9 x 105 2.0 x 105 1.2 x 106
9.6 x 103 2.7 x 104 1.2 x 106
0.041 0.02 DW
1.8 x 10'
4.8 x 103 2.4 x 103 1.8 x 108
NCFU 2.4 x 103 1.3 x 10'
NCFU NCFU 1.3 x 10'
Pseudomonas fluorescens (ATCC 13525)
0.041 0.02 0.008 DW
2.0 x 2.0 x 2.0 x 2.0 x
NCFU 2.6 x 104 6.5 x 107 2.0 x 10'
NCFU NCFU 4.5 x 107 2.0 x 10'
NCFU NCFU 1.5 x 106 2.0 x 108
Salmonella typhi
0.041 0.02 DW
9.6 x 107 9.6 x 107 9.6 x 107
3.0 x 102 NCFU 1.6 x 10 | 1.2 x 103 9.6 x 107 9.6 x 107
NCFU NCFU 9.6 x 107
Serratia marcescens
0.041 0.02 DW
2.7 x 10'
5.5 x 105 4.0 x 107 2.7 x 108
1.2 x 103 2.0 x 10' 2.7 x 108
NCFU 1.6 x 10'
0.041 0.02 DW
9.0 X 107 9.0 X 107 9.0 X 107
4.0 x 106
7.2 x 10'
5.9 x 107 9.0 X 107
4.1 x 107 9.0 x 107
Bacillus subtilis (spores)
(ATCC 6633)
(ATCC 4698) Mycobacterium marinum
(ATCC 927) Pseudomonas aeruginosa
(ATCC 15442)
(ATCC 167-T) (ATCC 8701)
Staphylococcus aureus
(ATCC 6538)
Distilled water control. b NCFU, No countable colony-forming units.
a DW,
1.8 x 108
1.8 x 10'
10' 10'
10' 10'
2.7 x 108
2.7 x 10'
2.7 x 108 NCFU 4.6 x 10. 1.0 x 10.
505
ANTIBACTERIAL ACTIVITY OF TROPOLONE
Voi- 7, 1975
lysis of whole cells of both gram-positive and gram-negative species. The rapid death of the whole cells after blebbing itself presumably reflects a weakening of the cell wall or envelope material in some manner. In growing cells, the blebbing in the region of cell division and cell elongation could also result by inhibition of the enzymes in that area which are involved in the synthesis of new cell wall. However, if tropolone were to inhibit these enzymes, fila-
05 OA
42
Eoi tOS
9'
10.5
a 4C
A
03
10.2 E
a .011 2
2
4
4
2
0
I-
0
4
0
6
2
0
6
4
u
B
B
9
OA
6
Time, h
-4
0.3
FIG. 2. The effect of tropolone at a final concentration of 0.041 M on the growth of Aeromonas hydrophila ATCC 9071 (A), Escherichia coli ATCC 9661 (B), Pseudomonas aeruginosa ATCC 15442 (C), and Bacillus subtilis ATCC 6633 (D). Control growth in antibiotic assay broth (0); addition of tropolone (>); tropolone treatment in antibiotic assay broth (a).
E
8
4R° 4D °I
0.2
I o
7
Q.1
0
0 4 2 Time after tropolone addition, h
2
6
4
FIG. 4. The effect of tropolone at a final concentration of 0.041 M on the turbidity and viability of 18-h cultures of Aeromonas hydrophila ATCC 9071 (A) and Bacillus subtilis ATCC 6633 (B) in 20% (wt/vot) sucrose-antibiotic assay broth. Control growth (O3); addition of tropolone (>); tropolonetreated cells (U). Tropolone was prepared in the 20% sucrose-antibiotic assay broth. A
I
C
B
OA
E C
0.3
2
4
6
8
3
4
5
7
Time, h
FIG. 3. Effect of tropolone at a final concentration of 0.041 M on the growth of Staphylococcus aureus ATCC 6538. Control growth in antibiotic assay broth (0); addition of tropolone (>); tropolone treatment in antibiotic assay broth (-).
40.1
.
L
1
2
.--
.
1
2
.
2.
1
2
Time after tropolone added, h
FIG. 5. Effect of tropolone at a final concentration of 0.041 M on suspensions of spheroplasts and proto(A) Suspension of 2 x 100 spheroplasts of these organisms, comparison of the bactericidal plasts. Aeromonas hydrophila ATCC 9071 per ml; (B) that reveals two of the compounds activity suspension of 2 x 108 spheroplasts of Escherichia tropolone exhibits only 4% of the bactericidal coli ATCC 9661; (C) suspension of 1 X10x protoplasts activity of ,B-thujaplicin. of Micrococcus lysodeikticus ATCC 4698. Addition of The results indicate that at high concentra- tropolone (>); tropolone treatment in 20% sucrose tions, tropolone acts at cell surfaces causing (T); control (0).
506
TRUST
mentous forms would be expected at subinhibitory concentrations of tropolone. Such filamentous forms were not observed. The loss of viability of cells treated with tropolone in the presence of sucrose suggests a second mechanism of action for tropolone. The lysis of spheroplasts and protoplasts is evidence that this second action of tropolone is at the level of the plasma membrane. The lysis could occur in one of two ways: either the tropolone causes direct dissolution of the physical structure, or it could disrupt the membrane permeability causing rapid uptake of water and lysis of the spheroplasts and protoplasts due to mechanical pressure. The lack of lysis of whole cells in the presence of sucrose suggests that the cell wall prevents access of sufficient tropolone to bring about lysis of the cell membrane. When the wall is preweakened, as in the case of penicillin-treated cells, or removed, as in the case of lysozyme-treated cells, then access of sufficient tropolone to the membrane is allowed and lysis occurs. The loss of viability of whole cells in the presence of sucrose does, however, suggest that sufficient tropolone reaches the membrane of whole cells to cause damage which leads to cell death. The exact mechanics by which tropolone damages the cell membrane remains obscure and will require further experimentation. Similarly, the inability of tropolone to lyse cells of Staphylococcus aureus is difficult to explain at this time, except that this species is particularly refractory to many compounds and enzymes, including lysozyme. The observation that this organism is also eventually killed by tropolone could, however, indicate a third mechanism for tropolone activity, one not associated with the cell surface. ACKNOWLEDGMENTS This work was supported by a grant from the National Research Council of Canada. I am indebted to Karen Bartlett, Diane Chipman, and Vida Money for skilled technical assistance. J. T. Buckley is also thanked for supplying lysozyme.
ANTIMICROB. AGENTS CHEMOTHER. LITRATURE CITED 1. Anderson, A. B., and J. Gripenberg. 1948. Antibiotic substances from the heartwood of Thuja plicata D. Don. IV. The constitution of ,-thujaplicin. Acta Chem. Scand. 2:644-650. 2. Association of Official Analytical Chemists. 1970. Official methods of analysis of the Association of Official Analytical Chemists, 11th ed. Association of Official Analytical Chemists, Washington, D.C. 3. Barton, G. M., and B. F. MacDonald. 1971. The chemistry and utilization of western red cedar. Dept. Fish. For. Can. For. Serv., publication no. 1023, Ottawa. 4. British Standards Institution. 1960. Method for laboratory evaluation of disinfectant activity of quaternary ammonium compounds by suspension test procedure publication no. B.S.3286: 1960. British Standards House, London. 5. Cook, J. W., A. R. Gibb, R. A. Raphael, and A. R. Somerville. 1951. Tropolones. 1. The preparation and general characteristics of tropolone. J. Chem. Soc. 1951:503. 6. Coombs, R. W., and T. J. Trust. 1973. The effect of light on the antibacterial activity of ,B-thujaplicin. Can. J. Microbiol. 9:1177-1180. 7. Dewar, M. J. S. 1945. The structure of stipitatic acid. Nature (London) 155:50-51. 8. Furr, J. R., and H. D. Russell. 1972. Effect of phydroxybenzoic acid on spheroplasts of Serratia marcescens and protoplasts of Bacillus megaterium. Microbios 6:47-54. 9. Gardner, J. A. F., and G. M. Barbor. 1958. The extraneous components of western red cedar. For. Prod. J. 8:189-192. 10. Greenwood, D., and F. O'Grady. 1973. The two sites of penicillin action in Escherichia coli. J. Infect. Dis. 128:791-794. 11. Kavanagh, F. 1963. Dilution methods of antibiotic assay, p. 125-140. In F. Kavanagh (ed.), Analytical microbiology_Academic Press, New York. 12. Lederberg, J. 1956. Bacterial protoplasts induced by penicillin. Proc. Natl. Acad. Sci. U.S.A. 42:574577. 13. Miles, A. A., and S. S. Misra. 1938. The estimation of the bactericidal power of the blood. J. Hyg. 38:732-747. 14. Pauson, P. L. 1955. Tropones and tropolones. Chem. Rev. 55:9-136. 15. Rennerfelt, E. 1948. Investigations of thujaplicin, and fungicidal substance in the heartwood of Thuja plicata D. Don. Physiol. Plant. 1:245-254. 16. Trust, T. J., and R. W. Coombs. 1973. Antibacterial activity of ,8-thujaplicin. Can. J. Microbiol. 19:13411346. 17. Von Doering, W., and F. L. Detert. 1951. Synthesis of tropolone. J. Am. Chem. Soc. 73:876.
