Sulfanilamide Therapy in Bovine Mastitis
295
TABLE 4
Antisulfanilamide activity of p-aminobenzoic acid for Streptococcus agalactiae in tryptose broth
Sulfanilamide-mg. = .
P-aminobenzoic acid-gm.
F-12a-4 Culture—1 : 100,000
Growth in original tabes
Tube number
1
1,000
0.001
0.1 cc.
—
2
1,000
0.002
0.1
—
3
1,000
0.003
0.1
—
4
1,000
0.004
0.1
—
5
1,000
0.005
0.1
+
6
1,000
0.006
0.1
+
7
1,000
0.007
0.1
+
8
1,000
0.008
0.1
+
0
1,000
0.009
0.1
+
10
1,000
001
01
+
11
1,000
0
01
—
12
0
0.01
01
+
13
0
0
0.1
+
TABLE 4A
Results of subcultures made from above tubes into tryptose broth
Hours incubated before
11 . . . .
24 . . . .
48 . . . .
72 . . . .
06 . . . .
123 . . . .
Tube number
1
—
—
—
—
—
—
2
—
—
—
—
—
—
3
—
—
+
—
—
—
4
—
+
+
—
—
—
5
—
+
+
+
+
+
6
—
+
+
+
+ +
7
+
+
+
+
+
+
8
—
+
+
+
+
+
9
+
+
+
+ +
+
10
—
+
+
+
+
+
11
—
—
—
—
—
—
12
+
+
+
+
+
+
13
+
+
+
+
+
+
of the bactericidal action of sulfanilamide has been
made much clearer by Woods and Fildes 5 who
discovered that p-aminobenzoic acid* possesses
antisulfanilamide activity. It subsequently was
shown by Woods 6 that yeast extracts contain a
sulfanilamide inhibiting substance, and he presented
evidence to indicate that the yeast factor may be
p-aminobenzoic acid. Fildes 7 considered that p-
aminobenzoic acid was an essential metabolite for
bacteria. Rubbo and Gillespie 8 have claimed the
recovery of p-aminobenzoic acid from Clostridium
acetobutylicum and showed that the acid acts as a
growth factor for this organism. Since it may be
possible that Lancefield group B streptococci may
also produce this acid as an essential metabolite, the
following experiment was conducted to determine
whether p-aminobenzoic acid exerts the same inhibi-
tory effect on sulfanilamide for group B streptococci
as it does for other bacterial species.
A series of 13 tubes were set up, each containing
10 c.c. of tryptose broth. Into each of the 11 tubes,
100 mg. of sulfanilamide was added. P-aminobenzoic
acid in increasing amounts was also added, the first
tube receiving 0-001 gm., and the tenth, 0.01 gm.
Tubes 11, 12, and 13 served as controls for sulfa-
nilamide, p-aminobenzoic acid and tryptose broth,
respectively. Strain F-12a-4 was used as the test
organism. Approximately 240 bacteria were added
in each tube. The tubes were then incubated at
37. 5° C., and at 24-hour intervals subcultures were
made into tryptose broth, using a 4 mm. loop.
RESULTS
The data presented in Tables 4 and 4A show that
0.005 gm. of p-aminobenzoic acid was sufficient to
nullify the bactericidal effect of 1,000 mg. per cent
sulfanilamide. In the sulfanilamide control tube
(No. 11), which contained 1,000 mg. per cent of the
drug, the organisms were killed within a period of
eleven hours or less. The maximum concentration of
p-aminobenzoic acid (0.01 gm.) used in this experi-
ment had no inhibitory effect on the organisms. In
Table 4A, in which the results of the subcultures are
recorded, it will be noted that 0.003 gm. and 0.004
gm. of p-aminobenzoic acid exerted partial inhibi-
tion on sulfanilamide activity since the organisms
were not killed until after forty-eight hours' incuba-
tion. When p-acetylaminobenzoic acid was sub-
stituted for the p-aminobenzoic acid, the sulfanila-
mide activity was not inhibited. These results are
shown in Table 5. It is of interest to note that the
loss of activity of the acetylated form of the acid
corresponds to the loss of the bactericidal activity of
the conjugated sulfanilamide. These findings lend
further support to the theory that sulfanilamide
exerts its bacteriostatic action by competing (on
account of similarity in molecular structure) with
p-aminobenzoic acid in some enzymatic reaction
essential to growth. 7 Further studies in the field of
the antisulfanilamide factors and the possible dis-
covery of substances which will neutralize their
activity should lead to valuable and far-reaching
results.
* The p-aminobenzoic acid used in this work was supplied
by the E. I. duPont' de Nemours and Company.