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Prevention of the emergence of antibiotic-resistant strains of bacteria by atabrine
ARCHIVES
OF
BIOCHEMISTRY
Prevention
AND
BIOPHYSICS
108,
85-88
of the Emergence Bacteria
(1964)
of Antibiotic-Resistant by Ata brine”
Strains
of
2
M. G. SEVAG From the Department
of Microbiology, School of Medicine, Philadelphia, Pennsylvania Received
April
University
of Pennsylvania,
16, 1964
In addition to spermine and spermidine, atabrine has been found to prevent effectively the emergence of strains resistant to streptomycin, sulfathiazole, novobiocin, erythromycin, tetracycline, penicillin, and chloramphenicol. Since atabrine already has been used widely in medical practice, we believe that it can now be incorporated into antibiotic therapy to prevent the emergence of resistant strains of bacteria. These findings are suggestive also of their possible application in early cancerous and viral systems.
role of streptomycin by a firmer combination with DNA than that between the antibiotic and DNA may be considered here. Relative to this question it may be noted that the incorporation of valine-Cl4 by the ribosomal system of E. coli B was inhibited 92 % by 100 pg of streptomycin. This inhibition was reduced to 44 % in the presence of 100 pg of spermine which was the degree of inhibition by spermine alone. The reduction by spermine of the inhibition by streptomycin occurred independently of the order of addition of the two inhibitors. These results indicate that spermine combines with the ribosomal system more tightly than with streptomycin. For further study of the problem a search for other substances which could substitute for spermine was made. To be of value for medical use such compounds should not be rapidly metabolized and must be relatively nontoxic in the dosages required. Of the several substances known to combine with nucleic acids, such as thionin, pyronine or the acridine class of compounds, diamino-acridines appeared most to meet the criteria stated above. Of these, the first one chosen for experimentation was atabrine, because its pharmacological properties have been determined and it has been
In previous communications (l-3) we reported that the emergence of antibiotic-resistant strains of Staphylococcus aureus, Aerobatter aerogenes, and Escherichia coli is prevented by the combined presence of a polyamine, such as spermine or spermidine, and an antibiotic. This combination, effective only on bacteria which had never before been exposed to an antibiotic, had no effect on the growth of bacteria which had already experienced an exposure to an antibiotic and thereby had become resistant to it. It is known that both spermine and streptomycin combine with nucleic acids. In recent experiments (4) we found that spermine dissolved with DNA forms a complex which is nondialyzable through a cellophane membrane. It has been reported that DNA is the bearer of the drug-resistance behavior of bacterial cells (5). The question of whether spermine prevents the resistance-inducing 1 This work was aided by a contract between the Office of Naval Research, U. S. Department of the Navy, and the University of Pennsylvania. Reproduction in whole or part is permitted for any purpose of the United States Government. a The following trade names for atabrine [3-chloro-7-methoxy-9-(l-methyl-4-diethylaminobutylamineacridine dihydrochloride] are known: Mepacrine, Chinacrine, Metoquine, and Italchin. 85
86
SEVAG
widely used medically in humans as an antimalarial chemotherapeutic agent and against certain other infectious systems such as canine giardiasis, coccidiosis in rabbits, dogs and cattle, avian malaria, canine taeniasis, human intestinal amebiosis, auricular fibrillation, etc. We have found that the presence of atabrine2 in a growth medium containing streptomycin prevents the emergence of streptomycin-resistant strains. In the presence of streptomycin alone, strains of S. aureus or E. coli resistant to this antibiotic emerge.
MATERIALS
RESULTS
In a previous study (1) it was shown that one subculture of a drug-sensitive strain in streptomycin alone renders it insensitive to the combined presence of streptomycin and spermine. The results of the present study also show that streptomycin and atabrine in combination prevent the emergence of a
TABLE
I CELLS FROM THE OF STREPTOMYCIN Growth turbidity
Growth system additions
40 rg/ml 40 pg/ml 40 rg/ml
METHODS
The details of the materials and methods are same as those used earlier (Sevag and Drabble, 1962).
PREVENTION OF THE EMERQENCE OF DRUG-RESISTANT ESCHERICHIA COLI (STRAIN B)” BY COMBINATIONS
None Streptomycin, 10 pg/ml Streptomycin, 20 pg/ml Streptomycin, 30 j-g/ml Streptomycin, 50 rg/ml Atabrine, 40 pg/ml Streptomycin, 10 pg + atabrine, Streptomycin, 20 pg + atabrine, Streptomycin, 30 rg + atabrine,
AND
NORMAL STRAXN AND ATABRINE
OF
readings at hours of:
0
21
45
117
7 13 13 8 8 11 9 8 15
167 143 100 23 8 98 6 7 14
163 162 8 156 6 5 16
8 150 4 5 15
a Inoculum: 2 X lo* cells/5 ml of salts-glucose casein hydrolyeate medium. Growth in glucose broth is one half of the above control values. The effects of the drugs on the growth are, however, of the same degree. TABLE PREVENTION OF THE EMERGENCE STAPHYLOCOCCUS AUREUS (STRAIN
II
OF DRUG-RESISTANT CELLS FROM THE NORMAL STRAIN OF 3A)a BY COMBINATIONS OF STREPTOMYCIN AND ATABRINE Growth turbidity
Growth system additions 0
None Streptomycin, 10 @g/ml Streptomycin, 50 fig/ml Streptomycin, 100 pg/ml Atabrine, 20 fig/ml Atabrine, 50 pg/ml Atabrine, 100 pg/ml Streptomycin, 100 rg + atabrine, Streptomycin, 100 pg + atabrine, Atabrine, 100 pg f streptomycin, Atabrine, 100 cLg f streptomycin,
8
20 rg/ml 50 @g/ml 10 &g/ml 50 pg/ml
8 15 8 13 3 16 4 7 3 17
a Inoculum: 1.0 X lo8 cells/5 ml of 0.5% glucose nutrient from an overnight culture grown in the same medium. b Reading 118 at 187 hours.
readings at hours of:
24
48
116 68 22 16 121 115 128 9 13 61 22
110 118 133 13 106 96 114 5 5 112 18
broth.
The washed
70
312
140 68
135*
62
90 14 26
inoculum
was harvested
PREVENTION
OF ANTIBIOTIC
drug-resistant strain from a normal bacterial population, but fail to do so after a drugsensitive strain had been subcultured in streptomycin. The results presented in Table I show that a culture of E. coli capable of yielding resistant strains in the presence of 10, 20, or 30 pg of streptomycin or 40 pg of atabrine per milliliter remained sensitive when 40 pg of atabrine and as little as 10 ccgof streptomycin were added simultaneously. Similarly, the results presented in Table II show that a combination of 100 pg of streptomycin and 50 pg of atabrine prevented the emergence of the staphylococcal strain resistant to streptomycin or atabrine. The results in Table III show that E. coli already resistant to high levels of streptomycin grew in 1000 pg of streptomycin and 250 pg of atabrine (the sensitive strain fails to grow or, in exceptional cases, grows after
a delay period of 120 hours in 250 pg of atabrine) . In contrast, S. aureus, resistant to 1000 pg of streptomycin, failed to grow in this concentration of streptomycin when combined with 80 pg of atabrine (Table IV). The results presented in Table V show that the drug-sensitive strain of E. co&isurvives or emerges as a resistant strain in 1, 5, or 10 pg of sulfathiazole per milliliter, or 50 or 100 pg of atabrine per milliliter. If, however, the medium contains 100 pg of atabrine plus the lowest level of sulfathiazole (1 pg) the emergence of a resistant strain is prevented. Similar results, not reported here, show that 50 pg of sulfathiazole with 100 pg of atabrine per milliliter prevented the emergence of sulfathiazole-resistant strains of S. auyeus. Also, the combination of 0.2 unit of penicillin and 100 pg of atabrine, or 10 pg of
TABLE FAILURE
III
OF COMBINATIONS OF STREPTOMYCIN AND ATABRINE TO PREVENT THE GROWTH OF DRUG-RESISTANT CELLS OF ESCHERICHIA COLI (STRAIN B)a Growth Growth
system
250 pg/ml
a Inoculum: grown in 0.5% glucose-nutrient coli Resistant to 1000 pg of streptomycin
at hours
of:
0
4
22
47
0 5 10 8
41 38 33 27
61 61 74 57
67 68 91 61
broth, 1.0 X lo* cells/5 per milliliter. TABLE
VARYING
turbidity
additions
None Streptomycin, 1000 pg/ml Atabrine, 250 pg/ml Streptomycin, 1000 pg + atabrine,
i&a
87
RESISTANCE
118 74 78 100 70
ml of the same medium.
Escher-
IV
SENSITIVITY AUREUS
OF THE STREPTOMYCIN-RESISTANT STRAIN OF STAPHYLOCOCCUP TO THE COMBINATION OF STREPTOMYCIN .~ND VARYING CONCENTRATIONS OF ATABRINE Growth
Growth
None Streptomycin, 1000 Atabrine, 20 pg/ml Atabrine, 40 rg/ml Atabrine, 80 pg/ml Streptomycin, 1000 Streptomycin, 1009 Streptomycin, 1000 a Staphylococcus medium.
system
fig/ml
fig + atabrine, pg + atabrine, kg + atabrine, aureus
turbidity
at hours
of:
additions
resistant
20 rg/ml 40 rg/ml SO rg/ml
0
5
22
14 14 14 13 20 13 20 17
120 17 33 15 22 17 24 18
179 180 150 163 19 163 2s 16
to 1000 pg of streptomycin/ml.
Inoculum:
27
60
118
142
209 194 155 160 190 180 168 15
15
1.5 X lo8 cells/5
ml
88
SEVAG TABLE
V
PREVENTION OF THE EMERGENCE OF
SULFATHIAZOLE-RESISTANT COLI BY ATABRINE”
EXXXERICHIA
Growth turbidity
readings at hours of:
0
1s
42
71
137
160
0
18
42
71
131
10 1 5 4 9 2 0
161 85 6 8 113 61 0
197 140 16 7 174 184 0
98 6 0
300 134 3
157 7
4 4 13 8 8 0 0
160 160 20 12 105 0 1
144 124 172 15 127 17 13
112 87 92
222 154 180
0
0
0
0
2
4
0
1
1
0
6
7
+ atabrine,
1
2
2
2
8
13
0
2
1
1
8
11
+ atabrine,
6
7
7
7
17
21
2
2
2
2
10
13
pg f
1
1
1
1
11
11
2
2
2
2
9
13
None Sulfathiazole, 1 pg/ml Sulfathiazole, 5 pg/ml Sulfathiszole, 10 rg/nd Atabrine, 50 pg/ml Atabrine, 100 rg/ml Sulfathiazole,5pg + atabrine, 50 rg Sulfathiazole, 10 brine, 50 pg Sulfathiazole, lpg 100 Pg Sulfathiazole,5pg 100 Pg Sulfathiazole, 10 brine, 100 pg
OF
Strain resistant to 1000 pg S&/ml
Sensitive strain (B) Growth system additions
STRAINS
pg +
ata-
ata-
a Inoculum: about 1 X 106 cells/5 * SM = Streptomycin.
ml of salts-glucose
novobiocin and 50 pg of atabrine, or 5 pg of erythromycin and 50 pg of atabrine, or 1 rg of tetracycline and 80 fig of atabrine, or 0.5 pg of tetracycline and 160 pg of atabrine or 7 pg of chloramphenicol and 160 kg of atabrine prevented the emergence of the resistant strains from the normal strain of S. aureus. It is to be noted that, unlike spermine, atabrine alone exercises a bacteriostatic action only during the earlier period of growth. It can also be noted that the sensitivity to atabrine of the antibiotic-sensitive and -resistant strains varies (compare Table I with III, and Table II with IV). Nevertheless, the phenomenon described in this report appears to be a common occurrence with all the drugs mentioned. The possibility that the active component might be a derivative of atabrine produced by photosensitization in the laboratory light was investigated, using S. aureus, streptomycin, and atabrine as outlined in Table II.
casein hydrolyzate
160
-
medium.
During a period of 90 hours of growth, no difference in results was observed between the cultures protected from light with aluminum foil and those maintained in the usual laboratory light. ACKNOWLEDGMENT The author wishes to express his thankful acknowledgment to Dr. T. Shiio for the preliminary tests and Mrs. Barbara Ashton for her extensive and valuable technical assistance in the pursuance of this study. REFERENCES W. T., Biochem. 1. SEVAG, M. G., AND DRABBLE, Biophys. Res. Commun. 8, 452 (1962). 2. DRABBLE, W. T., AND SEVAG, M. G., “Antibacterial Agents and Chemotherapy” pp. 649-653, American Society for Microbiology, Ann Arbor, Michigan, (1962). 3. SEVAG, M. G., Naval Res. Rev. XVI, 1 (1963). 4. DRABBLE, W. T., AND SEVAG, M. G., unpublished data (1963). 5. HOTCHKISS, R. D., Harvey Lectures 49, 124-144 (1953-1954).
OF
BIOCHEMISTRY
Prevention
AND
BIOPHYSICS
108,
85-88
of the Emergence Bacteria
(1964)
of Antibiotic-Resistant by Ata brine”
Strains
of
2
M. G. SEVAG From the Department
of Microbiology, School of Medicine, Philadelphia, Pennsylvania Received
April
University
of Pennsylvania,
16, 1964
In addition to spermine and spermidine, atabrine has been found to prevent effectively the emergence of strains resistant to streptomycin, sulfathiazole, novobiocin, erythromycin, tetracycline, penicillin, and chloramphenicol. Since atabrine already has been used widely in medical practice, we believe that it can now be incorporated into antibiotic therapy to prevent the emergence of resistant strains of bacteria. These findings are suggestive also of their possible application in early cancerous and viral systems.
role of streptomycin by a firmer combination with DNA than that between the antibiotic and DNA may be considered here. Relative to this question it may be noted that the incorporation of valine-Cl4 by the ribosomal system of E. coli B was inhibited 92 % by 100 pg of streptomycin. This inhibition was reduced to 44 % in the presence of 100 pg of spermine which was the degree of inhibition by spermine alone. The reduction by spermine of the inhibition by streptomycin occurred independently of the order of addition of the two inhibitors. These results indicate that spermine combines with the ribosomal system more tightly than with streptomycin. For further study of the problem a search for other substances which could substitute for spermine was made. To be of value for medical use such compounds should not be rapidly metabolized and must be relatively nontoxic in the dosages required. Of the several substances known to combine with nucleic acids, such as thionin, pyronine or the acridine class of compounds, diamino-acridines appeared most to meet the criteria stated above. Of these, the first one chosen for experimentation was atabrine, because its pharmacological properties have been determined and it has been
In previous communications (l-3) we reported that the emergence of antibiotic-resistant strains of Staphylococcus aureus, Aerobatter aerogenes, and Escherichia coli is prevented by the combined presence of a polyamine, such as spermine or spermidine, and an antibiotic. This combination, effective only on bacteria which had never before been exposed to an antibiotic, had no effect on the growth of bacteria which had already experienced an exposure to an antibiotic and thereby had become resistant to it. It is known that both spermine and streptomycin combine with nucleic acids. In recent experiments (4) we found that spermine dissolved with DNA forms a complex which is nondialyzable through a cellophane membrane. It has been reported that DNA is the bearer of the drug-resistance behavior of bacterial cells (5). The question of whether spermine prevents the resistance-inducing 1 This work was aided by a contract between the Office of Naval Research, U. S. Department of the Navy, and the University of Pennsylvania. Reproduction in whole or part is permitted for any purpose of the United States Government. a The following trade names for atabrine [3-chloro-7-methoxy-9-(l-methyl-4-diethylaminobutylamineacridine dihydrochloride] are known: Mepacrine, Chinacrine, Metoquine, and Italchin. 85
86
SEVAG
widely used medically in humans as an antimalarial chemotherapeutic agent and against certain other infectious systems such as canine giardiasis, coccidiosis in rabbits, dogs and cattle, avian malaria, canine taeniasis, human intestinal amebiosis, auricular fibrillation, etc. We have found that the presence of atabrine2 in a growth medium containing streptomycin prevents the emergence of streptomycin-resistant strains. In the presence of streptomycin alone, strains of S. aureus or E. coli resistant to this antibiotic emerge.
MATERIALS
RESULTS
In a previous study (1) it was shown that one subculture of a drug-sensitive strain in streptomycin alone renders it insensitive to the combined presence of streptomycin and spermine. The results of the present study also show that streptomycin and atabrine in combination prevent the emergence of a
TABLE
I CELLS FROM THE OF STREPTOMYCIN Growth turbidity
Growth system additions
40 rg/ml 40 pg/ml 40 rg/ml
METHODS
The details of the materials and methods are same as those used earlier (Sevag and Drabble, 1962).
PREVENTION OF THE EMERQENCE OF DRUG-RESISTANT ESCHERICHIA COLI (STRAIN B)” BY COMBINATIONS
None Streptomycin, 10 pg/ml Streptomycin, 20 pg/ml Streptomycin, 30 j-g/ml Streptomycin, 50 rg/ml Atabrine, 40 pg/ml Streptomycin, 10 pg + atabrine, Streptomycin, 20 pg + atabrine, Streptomycin, 30 rg + atabrine,
AND
NORMAL STRAXN AND ATABRINE
OF
readings at hours of:
0
21
45
117
7 13 13 8 8 11 9 8 15
167 143 100 23 8 98 6 7 14
163 162 8 156 6 5 16
8 150 4 5 15
a Inoculum: 2 X lo* cells/5 ml of salts-glucose casein hydrolyeate medium. Growth in glucose broth is one half of the above control values. The effects of the drugs on the growth are, however, of the same degree. TABLE PREVENTION OF THE EMERGENCE STAPHYLOCOCCUS AUREUS (STRAIN
II
OF DRUG-RESISTANT CELLS FROM THE NORMAL STRAIN OF 3A)a BY COMBINATIONS OF STREPTOMYCIN AND ATABRINE Growth turbidity
Growth system additions 0
None Streptomycin, 10 @g/ml Streptomycin, 50 fig/ml Streptomycin, 100 pg/ml Atabrine, 20 fig/ml Atabrine, 50 pg/ml Atabrine, 100 pg/ml Streptomycin, 100 rg + atabrine, Streptomycin, 100 pg + atabrine, Atabrine, 100 pg f streptomycin, Atabrine, 100 cLg f streptomycin,
8
20 rg/ml 50 @g/ml 10 &g/ml 50 pg/ml
8 15 8 13 3 16 4 7 3 17
a Inoculum: 1.0 X lo8 cells/5 ml of 0.5% glucose nutrient from an overnight culture grown in the same medium. b Reading 118 at 187 hours.
readings at hours of:
24
48
116 68 22 16 121 115 128 9 13 61 22
110 118 133 13 106 96 114 5 5 112 18
broth.
The washed
70
312
140 68
135*
62
90 14 26
inoculum
was harvested
PREVENTION
OF ANTIBIOTIC
drug-resistant strain from a normal bacterial population, but fail to do so after a drugsensitive strain had been subcultured in streptomycin. The results presented in Table I show that a culture of E. coli capable of yielding resistant strains in the presence of 10, 20, or 30 pg of streptomycin or 40 pg of atabrine per milliliter remained sensitive when 40 pg of atabrine and as little as 10 ccgof streptomycin were added simultaneously. Similarly, the results presented in Table II show that a combination of 100 pg of streptomycin and 50 pg of atabrine prevented the emergence of the staphylococcal strain resistant to streptomycin or atabrine. The results in Table III show that E. coli already resistant to high levels of streptomycin grew in 1000 pg of streptomycin and 250 pg of atabrine (the sensitive strain fails to grow or, in exceptional cases, grows after
a delay period of 120 hours in 250 pg of atabrine) . In contrast, S. aureus, resistant to 1000 pg of streptomycin, failed to grow in this concentration of streptomycin when combined with 80 pg of atabrine (Table IV). The results presented in Table V show that the drug-sensitive strain of E. co&isurvives or emerges as a resistant strain in 1, 5, or 10 pg of sulfathiazole per milliliter, or 50 or 100 pg of atabrine per milliliter. If, however, the medium contains 100 pg of atabrine plus the lowest level of sulfathiazole (1 pg) the emergence of a resistant strain is prevented. Similar results, not reported here, show that 50 pg of sulfathiazole with 100 pg of atabrine per milliliter prevented the emergence of sulfathiazole-resistant strains of S. auyeus. Also, the combination of 0.2 unit of penicillin and 100 pg of atabrine, or 10 pg of
TABLE FAILURE
III
OF COMBINATIONS OF STREPTOMYCIN AND ATABRINE TO PREVENT THE GROWTH OF DRUG-RESISTANT CELLS OF ESCHERICHIA COLI (STRAIN B)a Growth Growth
system
250 pg/ml
a Inoculum: grown in 0.5% glucose-nutrient coli Resistant to 1000 pg of streptomycin
at hours
of:
0
4
22
47
0 5 10 8
41 38 33 27
61 61 74 57
67 68 91 61
broth, 1.0 X lo* cells/5 per milliliter. TABLE
VARYING
turbidity
additions
None Streptomycin, 1000 pg/ml Atabrine, 250 pg/ml Streptomycin, 1000 pg + atabrine,
i&a
87
RESISTANCE
118 74 78 100 70
ml of the same medium.
Escher-
IV
SENSITIVITY AUREUS
OF THE STREPTOMYCIN-RESISTANT STRAIN OF STAPHYLOCOCCUP TO THE COMBINATION OF STREPTOMYCIN .~ND VARYING CONCENTRATIONS OF ATABRINE Growth
Growth
None Streptomycin, 1000 Atabrine, 20 pg/ml Atabrine, 40 rg/ml Atabrine, 80 pg/ml Streptomycin, 1000 Streptomycin, 1009 Streptomycin, 1000 a Staphylococcus medium.
system
fig/ml
fig + atabrine, pg + atabrine, kg + atabrine, aureus
turbidity
at hours
of:
additions
resistant
20 rg/ml 40 rg/ml SO rg/ml
0
5
22
14 14 14 13 20 13 20 17
120 17 33 15 22 17 24 18
179 180 150 163 19 163 2s 16
to 1000 pg of streptomycin/ml.
Inoculum:
27
60
118
142
209 194 155 160 190 180 168 15
15
1.5 X lo8 cells/5
ml
88
SEVAG TABLE
V
PREVENTION OF THE EMERGENCE OF
SULFATHIAZOLE-RESISTANT COLI BY ATABRINE”
EXXXERICHIA
Growth turbidity
readings at hours of:
0
1s
42
71
137
160
0
18
42
71
131
10 1 5 4 9 2 0
161 85 6 8 113 61 0
197 140 16 7 174 184 0
98 6 0
300 134 3
157 7
4 4 13 8 8 0 0
160 160 20 12 105 0 1
144 124 172 15 127 17 13
112 87 92
222 154 180
0
0
0
0
2
4
0
1
1
0
6
7
+ atabrine,
1
2
2
2
8
13
0
2
1
1
8
11
+ atabrine,
6
7
7
7
17
21
2
2
2
2
10
13
pg f
1
1
1
1
11
11
2
2
2
2
9
13
None Sulfathiazole, 1 pg/ml Sulfathiazole, 5 pg/ml Sulfathiszole, 10 rg/nd Atabrine, 50 pg/ml Atabrine, 100 rg/ml Sulfathiazole,5pg + atabrine, 50 rg Sulfathiazole, 10 brine, 50 pg Sulfathiazole, lpg 100 Pg Sulfathiazole,5pg 100 Pg Sulfathiazole, 10 brine, 100 pg
OF
Strain resistant to 1000 pg S&/ml
Sensitive strain (B) Growth system additions
STRAINS
pg +
ata-
ata-
a Inoculum: about 1 X 106 cells/5 * SM = Streptomycin.
ml of salts-glucose
novobiocin and 50 pg of atabrine, or 5 pg of erythromycin and 50 pg of atabrine, or 1 rg of tetracycline and 80 fig of atabrine, or 0.5 pg of tetracycline and 160 pg of atabrine or 7 pg of chloramphenicol and 160 kg of atabrine prevented the emergence of the resistant strains from the normal strain of S. aureus. It is to be noted that, unlike spermine, atabrine alone exercises a bacteriostatic action only during the earlier period of growth. It can also be noted that the sensitivity to atabrine of the antibiotic-sensitive and -resistant strains varies (compare Table I with III, and Table II with IV). Nevertheless, the phenomenon described in this report appears to be a common occurrence with all the drugs mentioned. The possibility that the active component might be a derivative of atabrine produced by photosensitization in the laboratory light was investigated, using S. aureus, streptomycin, and atabrine as outlined in Table II.
casein hydrolyzate
160
-
medium.
During a period of 90 hours of growth, no difference in results was observed between the cultures protected from light with aluminum foil and those maintained in the usual laboratory light. ACKNOWLEDGMENT The author wishes to express his thankful acknowledgment to Dr. T. Shiio for the preliminary tests and Mrs. Barbara Ashton for her extensive and valuable technical assistance in the pursuance of this study. REFERENCES W. T., Biochem. 1. SEVAG, M. G., AND DRABBLE, Biophys. Res. Commun. 8, 452 (1962). 2. DRABBLE, W. T., AND SEVAG, M. G., “Antibacterial Agents and Chemotherapy” pp. 649-653, American Society for Microbiology, Ann Arbor, Michigan, (1962). 3. SEVAG, M. G., Naval Res. Rev. XVI, 1 (1963). 4. DRABBLE, W. T., AND SEVAG, M. G., unpublished data (1963). 5. HOTCHKISS, R. D., Harvey Lectures 49, 124-144 (1953-1954).