MUTATIONS TO SULFONAMIDE RESISTANCE IN
STAPHYLOCOCCUS AUREUS*

EUGENE F. OAKBERG? ann 5. E, LURIA*
Carnegie Institution of Washington, Department of Genetics,
Cold Spring Harbor, New York

Received December 24, 1946

WO general hypotheses have been postulated to explain the origin of

drug-resistant strains of bacteria. These hypotheses are: (x) Resistant
cells arise by mutation and are selected when grown in the presence of drugs.
(2) Resistance is the result of a specific adaptation of bacteria in response to
inhibiting agents.

Resistance of Escherichia coli to bacteriophage (Luri1A and DELBRUCK, 1943)
resistance of Staphylococcus aureus to penicillin (DEMEREC 1945), and resist-
ance of Escherichia coli to ultraviolet radiation (WITKIN 1946) have been
shown to be the result of mutations in individual cells. These mutant cells and
clones formed from them possessed selective advantages in the presence of the
destructive agent concerned. It is generally accepted that naturally occurring
strains of sulfonamide-sensitive species of bacteria exhibit low tolerance for
these drugs (Scumipr et al., 1940; FriscH 1942; SCHMIDT et al. 1943; LANDY
and GERSTUNG 1945; BERNHEIMER 1946). Resistance to sulfonamides is char-
acteristic of a large number of bacterial cultures isolated from patients who
have undergone sulfonamide therapy (FRISCH 1942; Scumipt ef al. 1942;
EprpemioLocy Unit No. 22, 19453 LANDY and GERSTUNG 1945; BERNHEIMER,
1946). However, the mechanism by which sulfonamide fastness is acquired is
not clearly understood. Strauss et al. (1941a and b), SESLER and ScHMIDT
(1942), Kirpy and Rantz (1943), and ScHMIDT and SESLER (1943) reported
that sulfonamide tolerance is the result of physiological adaptation of bacteria,
but their experiments do not give conclusive evidence for this mechanism.
Lanxrorp et al. (1943) and Spicer (1945) concluded that naturally occurring
bacterial populations are a mixture of cell types; sulfa drugs act merely as
selective agents in the development of resistant strains. No data were given
on the origin of these few resistant cells or on their rate of occurrence.

As the mode of origin of sulfonamide-resistant strains of bacteria is subject
to debate, it was felt that this phenomenon needed additional clarification.
Such information would be valuable in therapy, for if mutation and selection
would prove to be the mechanisms involved, doses of sulfonamides should be
high enough to inhibit either the few resistant cells that may be present in a
sensitive bacterial strain or resistant cells that may arise by mutation. Also,

2 This work was done in part under a contract recommended by the COMMITTEE ON MEDICAL
ResEarcu between the Orrice or Screntiric RESEARCH AND DEVELOPMENT and the CARNEGIE
INSTITUTION OF WASHINGTON.

2 Now at RecronaL Poutrry Researce Lasorarory, East Lansing, Michigan.

3 On leave of absence from Inpiawa Unrverstry, Bloomington, Indiana.

GENETICS 32: 249 May 1947
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

250 EUGENE F. OAKBERG AND S. E. LURIA
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Ficure 1.—Resistance of S. aureus during serial transfer in Sodium Sulfathiazole.
SULFONAMIDE RESISTANCE 251

as the action of sulfonamides has been associated with specific cell processes—
especially p-aminobenzoic acid metabolism—it was hoped that changes in
physiological characteristics could be correlated with the change from sensi-
tivity to resistance.

MATERIALS

The strain of Staphylococcus aureus used was obtained originally from the
NortHern REGIONAL RESEARCH LaBoratory, Peoria, Illinois. This same
strain, designated as NRRL-313 was used by DEMEREC (1945) in a study
of mutations to penicillin resistance.

Sodium sulfathiazole (NaST) was chosen because of its high antibacterial
activity and because of its high solubility. As strains resistant to one sul-
fonamide usually show cross-resistance with most sulfa derivatives, with the
exception of sulfamylon (ScHMITH 1943), the experiments were confined to
one drug. NaST was prepared in'sX10~ M solution in distilled water and
diluted to the desired concentrations.

A semisynthetic casein hydrolysate medium described by STRAUSS et al.
(1941b) was used in order to avoid sulfonamide antagonists present in or-
dinary bacteriological media. Twenty-five gms of powdered agar were added
per 1900 ml of medium for pouring plates.

DEVELOPMENT OF RESISTANT STRAINS BY SERIAL TRANSFER

Initial attempts to establish resistant strains by isolation of the colonies
that developed when a sensitive culture was plated in sulfonamide-agar were
precluded by the effects of inoculum size. Large inocula gave complete growth
even in agar saturated with NaST. No colonies developed when the number
of organisms plated was small enough for inhibition of bacterial growth.
Therefore, preliminary experiments were based upon observation of the
magnitude and distribution of increases in resistance that occurred during
serial transfer in liquid medium containing NaST.

“:X10-5 M was the maximum concentration of NaST in which an inoculum
of 10? or 10* cells per ml of strain NRRL-313 grew. At each transfer inocula of
10, 10°, 10%, 10%, and ro? bacteria per ml of culture were tested with a range of
1X 107? to 1X10-§ M NaST. Growth was estimated from turbidity after 48
and 72 hours incubation at 37°C. Bacteria for the next transfer were taken
from the highest concentration of NaST in which an inoculum of 105 cells per
ml had grown to almost full turbidity in 72 hours. This inoculum was selected
because it was inhibited by the same NaST molarities as inocula of 10* and
ro? cells per ml, yet it was large enough to allow detection of resistant cells.
As resistance of the strains increased the higher concentrations of NaST
were spaced at closer intervals, and some of the lower concentrations were
omitted.

Results of three transfer series, A, B, and C are given in figure 1. A total
increase in NaST tolerance of 1,000 times occurred in series A and C, and an
increase of ten times occurred in series B. The characteristic resistance of each
252 EUGENE F, OAKBERG AND §. E. LURTA

resultant strain was maintained through 15 transfers in the absence of NaST,
and therefore appeared to be a stable, heritable property.

Increases in resistance appeared as discrete steps that were of different mag-
nitude and were separated by flat plateaus. The number of transfers separating
the steps differed both between different series and between successive in-
creases within the same series. It is possible that the steps observed were some-
what forced by the NaST concentrations used. The data do not support this
hypothesis, for in nine instances intermediate NaST concentrations were by-
passed at a single transfer.

Stepwise increases in resistance occurring at random time during the course
of serial transfers, and stability of resistance in the absence of sulfonamides,
are better explained by mutation and selection than by physiological adapta-
tion. The resistant mutants that arise in a sensitive strain possess selective
advantages when grown in the presence of sulfonamides, and should constitute
a large proportion of the inoculum used for the next transfer. Consequently,
serial transfer in liquid medium is unsuitable either for estimation of mutation
rates or for isolation and study of individual mutants.

DERIVATION OF RESISTANT STRAINS BY COLONY ISOLATIONS

When Seitz filtrates from cultures in NaST were tested, it was observed
that a reduction of sulfonamide activity had occurred even when visible growth
was not reached. No loss of NaST could be demonstrated by colorimetric
titration, but this method may not have given a selective differentiation be-
tween biologically active and biologically inactive sulfonamide molecules.
Another hypothesis is suggested by the observation of Rose and Fox (1942)
that bacteria exposed to sulfonamides undergo a certain number of divisions
before growth is stopped. Production of antagonists during this noninhibited
phase of bacterial growth would result in reduction of effective sul-
fonamide concentrations. The relation of antagonist production to growth
also was indicated by the absence of inoculum-size effects when the bacteria
tested previously had been exposed to inhibitory concentrations of NaST.
CHANDLER and JANEWAY (1939) and LowELt ef al. (1941) made similar ob-
servations. Utilization of inhibited bacteria therefore should allow the plating
of inocula large enough for the detection of rare resistant cells.

The following technique was used for the isolation of resistant bacteria
from strain NRRL-313. Each of ten tubes containing ten ml of casein hydrol-
ysate broth was inoculated with approximately 10? sensitive bacteria from a
24-hour culture. These tubes were incubated for 48 hours at 37°C. From each
of these ten cultures grown from small inocula a 3.5-ml culture containing
1X10~ M NaST was inoculated with ro‘ cells per ml. These NaST cultures
had titers between 1 and 2X108 cells per ml after 18 hours of incubation at
37°C instead of the 8 to 10 X 108 cells per ml observed in control cultures. These
inhibited bacteria were tested in plates containing a final concentration of
3107 M NaST. One ml of each undiluted culture was poured into each of
three test plates. Three 1-ml portions of a 1o~* dilution were plated in agar
without NaST for assay of the bacterial titer.
SULFONAMIDE RESISTANCE 253

All colonies that developed in NaST agar were counted, and samples from
each culture were transferred to 5-ml tubes containing 1X10~§ M NaST.
Bacteria from strain NRRL-313 grew slowly in this concentration of NaST,
while resistant mutants were not inhibited. Thus overgrowth of the resistant
cells by more sensitive, faster-growing organisms was prevented. As 1X10§M
represented the minimum inhibitory concentrations of NaST, selection pres-
sure in favor of secondary mutants with still higher resistance should have

TABLE 1

Resistance of mutants from different strains of S. aureus.

 

 

 

MAXIMUM MOLAR PARENT SUBSTRAINS FROM SUBSTRAIN FROM
CONCENTRATION STRAIN NRRL-333 . STRAIN 38
or NaST
IN WHICH NRRL-313, STRAIN 33, STRAIN 38, STRAIN 67,
MUTANT RESISTANT TO RESISTANT TO RESISTANT TO RESISTANT TO

GREW* 1X10*M NaST 5X10°*M NaST 1X10°*M NaST 1X10? M NaST

 

5-6** X 1078 1
1-2X107+ 15
3-4X 107 I
5-6X 107+

g-10X 1074 18
2X10 6
3xX10%
4X1078 5

”

awe OO
oo

12

a

 

Total number of
independent mu-
tants isolated and
tested 17 go 34 17

 

* Test inoculum zo* cells per ml of final culture.
** Variation between different experiments necessitates expression of NaST tolerance as a
range for most strains.

been minimized. The resulting cultures were tested by adding approximately
10 bacteria per m! to 2-ml broth tubes containing varied NaST concentra-
tions. Resistant strains were established on nutrient agar slants from tubes
where complete growth had occurred in concentrations of NaST higher than
1X1075 M.

Similar techniques were used for isolation of highly resistant mutants from
strains obtained from NRRL-313. NaST concentrations were increased five
times for strain 33, ten times for strain 38, and 100 times for strain 67.

NaST resistance of the mutant sirains

Resistance of the substrains isolated is given in table 1. Variation of indi-
vidual cultures in different tests made it necessary to evaluate resistance in
terms of a range of NaST concentrations; the values given represent maximum
254 EUGENE F. OAKBERG AND S. E. LURIA

tolerance as measured by at least three different tests. Strain NRRL-313,
resistant to 1X 10-5 M NaST, gave mutants resistant to 5-6 X 10-8, 1-2 X 1074,
and 3-4X10+ M NaST. Strain 33, a substrain from NRRL-313 that was re-
sistant to 510-5 M NaST, gave mutants resistant to 1-2 X10-4, 5-6 X 1074,
and 9-10X1074 M NaST. Strain 38, a substrain from NRRL-313 that was
resistant to 1X10-4 M NaST, gave mutants resistant to 3-4 X 1074, 5-6 X 10-4,
and 9-10 10-4 M NaST. Strain 67, a substrain from strain 38 that was re-
sistant to 1X10-* M NaST, gave mutants resistant to 2X107*, 3X107*, and
41079 M NaST. Increases from strain 67 seem small, but these bacteria
already had undergone two mutations, each of which had resulted in an in-
crease of ten times in NaST tolerance. Also, the actual amount of sulfonamide

TABLE 2

Test for sulfonamide-resistant colonies in ten
“inhibited” cultures of NRRL-313.

 

 

 

 

 

TITER OF COLONIES IN TEST PLATES
7 TOTAL
CULTURE CULTURES
8 COLONIES
(X 108) PLATE I PLATE 2 PLATE 3
A 1.6 ° ° ° °
B I.E : 13 Ir Ir 35
Cc 1.5 32 40 39 Imt
D 5 ° ° ° °
E 0.9 ° ° ° °
F 2.3 ° ° ° °
G 2.4 ° ° ° °
H 1.5 2 8 10 20
I 2.8 6 4 3 13
J 2.1 2 2 1 5

 

* Mean of three assay plates.

involved in changing from 1X 10-* to 4 X 107? M concentrations is much greater
than for any previous step. These results indicate that the few colonies that
grow when a large inoculum is plated in NaST agar have increased resistance
to NaST. Variants with even higher resistance may be obtained from the
strains first isolated.

Origin of resistant bacteria

Whether the resistant variants arise by mutation or by physiological adap-
tation may be tested by the method proposed by Luria and DELBRicx (1943).
If cultures are started from an inoculum small enough to make the inclusion
of resistant cells highly improbable, random variation in the occurrence of
rare mutations should result in statistically significant fluctuations in the
number of resistant cells in different cultures. No significant differences be-
tween cultures would be expected if the process involved is one of adaptation.

Data for a typical experiment are given in table 2. The fact that some
SULFONAMIDE RESISTANCE 255

cultures contained no resistant cells while other cultures contained many is
conclusive evidence that resistance was not the result of physiological adapta-
tion.

The postulate that mutations arise at random time during the growth of
cultures was tested by variance analysis of those cultures that gave colonies in
each of three test plates (table 3). The omission of cultures with no colonies

TABLE 3

Variance analysis of the number of colonies per culture.*

 

 

 

DEGREES OF MEAN
SOURCE OF VARIATION
FREEDOM SQUARE
Total 14
Between cultures 4 614.05*%*
Between plates within cultures 10 8.07

 

* Data from Table 2, limited to cultures that showed colonies,
** Significant at one percent level.

TABLE 4

Significance of variation in number of resistant
cells in different cultures.

 

 

SIGNIFICANCE OF BETWEEN-CULTURE MEAN SQUARES

 

 

 

NUMBER OF
CULTURES
EXPERIMENTS
USED AS AVAILAELE NO SIGNIFICANT DIFFERENCES BE- DIFFERENCES BE-
PARENT FOR STATIS DIFFERENCES TWEEN CULTURES TWEEN CULTURES
STRAINS . BETWEEN SIGNIFICANT SIGNIFICANT
TICAL TEST
CULTURES AT 5% LEVEL AT 1% LEVEL
NRRL-313 2 ° ° 2
Strain 33 4 ° ° 4
Strain 38 2 ° I I
Strain 67 1 ° ° 1
Totals 9 ° 1 8

 

* Analysis limited to experiments with three or more cultures that gave resistant colonies on
each of three plates per culture.

minimizes differences between cultures. Nevertheless, variation in the number
of resistant cells per culture was highly significant. The results for nine inde-
pendent experiments are given in table 4. Variation between cultures was sig-
nificant at the one percent level in eight experiments and significant at the five
percent level in one experiment. This highly significant fluctuation in the
number of resistant cells in different cultures started from a common source is
strong evidence that resistant bacteria arise by mutation.
256 EUGENE F. OAKBERG AND S. E. LURIA
Mutation rates

The rate of mutation from sensitivity to resistance was estimated from the
percentage of cultures not giving resistant colonies (Luria and DELBRUCK
1943). Data for strain NRRL-313 and three substrains from NRRL-313 are
given in table 5. Different classes of resistant mutants were pooled to form the
total for each individual strain. The four strains tested all gave similar muta-
tion rates, with a range of 2X10~® to 4X107"° per bacterium per generation.
This close agreement between strains, even though some had undergone two
previous mutations, suggests a large number of different mutations to NaST
resistance.

The minimum number of mutations concerned probably is five; the maxi-
mum number may be considerably larger. The minimum figure is based on

TABLE 5

Mutation rates of different strains of S. aureus.

 

 

 

MEAN
NaST TOTAL NUMBEROF FRACTION OF NUMBER OF MUTATION
PARENT RESISTANCE NUMBER CULTURES CULTURES BACTERIA RATE PER
STRAIN OF PARENT OF WITHOUT WITHOUT TESTED PER BACTERIUM PER
STRAINS CULTURES MUTANTS MUTANTS CULTURE GENERATION
(X 108)
NRRL-313 1X10% 50 34 0.68 6.9 4X 10718
Strain 33 5X10% 40 12 0.30 4.3 2X1079
Strain 38 1X10 40 19 0.48 6.4 8X 10710
Strain 67 1X1073 40 21 0.53 5.0 gxX 10720

 

strain 67, which gave three classes of mutants after having undergone two pre-
vious mutations to NaST resistance. Except for increased p-aminobenzoic
acid production, no criterion was found for differentiation of mutants from
different strains. Equal magnitude of resistance changes cannot be used, for
it is impossible to determine how the same mutation may affect the phenotype
when it occurs in cells of different genetic constitution.

PRODUCTION OF p-AMINOBENZOIC ACID BY RESISTANT STRAINS

According to the Woops-FitpEs hypothesis (Woops 1940) that sulfonamides
act through competition for an essential metabolite—specifically, p-amino-
benzoic acid (PAB)—increased PAB synthesis could account for increased
bacterial resistance. Lanpy, et al. (1943) reported that sufficient PAB was
present in the filtrate of a sulfonamide-resistant culture of Staphylococcus
aureus to explain the level of resistance observed. However, LANDY and GERS-
TUNG (1945) found that while a high percentage of sulfonamide-fast strains of
gonococci showed increased PAB synthesis, some resistant strains produced
very little PAB. Conversely, some sensitive strains produced more PAB than
others. Therefore, while many resistant strains showed increased production
‘SULFONAMIDE RESISTANCE 257

of an antagonist identified as PAB, other mechanisms of resistance also were
involved.

Sulfonamide antagonists in filtrates of full-grown cultures of strains NRRL-
313, 33, 38, and 67, of strains A, B, and C from the serial transfers, and of a
sample of strains from each class of mutants isolated from plates were tested
by the following methods: (1) ability of filtrates to antagonize NaST, thereby
allowing growth of sensitive staphylococci; (2) ability of filtrates to replace
PAB as a growth factor for Acetobacter suboxydans (LANDY and DICKEN, 1942);
and (3) ability of filtrates to support growth of a p-aminobenzoicless strain of
Neurospora crassat (THompson et al. 1943). Presence of PAB in filtrates of

TABLE 6

 

p-aminobensoic acid production by sulf ide-resistant
mutants of Staphylococcus aureus.

 

 

 

 

 

MUTANTS ¥ROM STRAINS WITH Low PAB MUTANTS FROM STRAINS WITH INCREASED PAB
PRODUCTION PRODUCTION
Frou NRRL-313, FROM STRAIN 33,** FROM STRAIN 38,*** FROM STRAIN 67,°***
RESISTANT TO RESISTANT TO RESISTANT TO RESISTANT TO
1X107§ M NaST 5 X10~* M NaST 1X10 M NaST 1X10-3 M NaST
MUTANTS TESTED MUTANTS TESTED MUTANTS TESTED MUTANTS TESTED
INCREASE INCREASE . INCREASE INCREASE
T
prance my PAB peeienaNcE iN PAB pesierance In PAB eeserance mene
PRODUCTION PRODUCTION PRODUCTION PRODUCTION
5-6 X1075 -* 1-2 X107¢ - 3-4 X1074 - 2X10 -
I~2 X107# + 5-6 X10 + 5-6 X1074 - 3Xro8 -
3-4X1074 + 9-10 X1074 + g-10 X107¢ - 4xX1078 -

 

* —Indicates no increase over parent strain; -+indicates increase over parent strain. Increased PAB production
was by the same amount whenever it occurred.

** Colony isolation from NRRL-313 with same PAB production as strain NRRL-313.

*** Colony isolation from NRRL-313 with increased PAB production.

se** Colony isolation from strain 38 with same PAB production as strain 38.

S. aureus cultures is indicated by the fact that positive results were obtained
with all assay procedures used.

A summary of the data on PAB production by different strains is given in
table 6. Some resistant strains synthesized more PAB than the sensitive
NRRL-313 strain; others did not. Mutants from NRRL-313 that were re-
sistant to 5~6xX107~' M NaST showed no increase in antagonist production;
those resistant to NaST concentrations of 110-4 M and higher produced
more antagonists than the parent strain. When strain 33 was used as the
parent strain, mutants resistant to 1X10-* M NaST showed no increased
production of antagonist; mutants resistant to NaST molarities of 510+
or higher showed increased antagonist production. Strains 38 and 67 already
had undergone one mutation that resulted in formation of higher amounts of

‘4 This strain of Neurospora was kindly supplied by Dr. E. L. Tatum of YALE UNIVERSITY,
New Haven, Connecticut,
258 EUGENE F. OAKBERG AND §&. E. LURIA

PAB. None of the mutants from these strains showed further increases in
production of antagonist. Mutants with increased PAB production fall into
two classes when classified on the basis of NaST tolerance: those mutants with
resistance increased by 10-20 times and those with resistance increased by
30-40 times. Increase in PAB production always was by the same amount.
Therefore, while these mutations differed in effect on NaST tolerance, they
had a common effect on PAB production.

All increases in production of sulfonamide antagonists were by a factor of
ten times as measured by antagonism of NaST for sensitive Staphylococcus
aureus, by a factor of twenty times as measured by Acetobacter suboxydans, and
by a factor of 30 times as measured by g-aminobenzoicless Neurospora crassa.
An increase of 10-30 times in PAB production explained changes in sulfona-
mide tolerance of similar magnitudes when mutations affected both NaST re-
sistance and PAB synthesis simultaneously. While it was a factor, production
of antagonist did not account for the increases in resistance observed in all
mutant strains, because mutants with increased NaST resistance and with no
increase in PAB production were obtained from strain 38 and strain 67 (table
6). Also, strains with NaST resistance ten times that of NRRL-313 could be
obtained by two successive mutations that did not affect PAB production.

Conclusions based on filtrates may be misleading. The use of both filtrates
and culture extracts (MAcLEoD, 1940) offers another approach, but it is ques-
tionable whether or not extracts correctly represent conditions in living cells.
Also, it is difficult to determine if measurement of PAB in fully grown cultures
gives an accurate estimate of conditions existent during the early stages of
growth.

DISCUSSION

Discrete stepwise increases jn resistance that occur at random time during
serial transfer in NaST, and significant variation in the number of resistant
cells in independent cultures, indicate mutation and selection as the mechan-
isms by which sulfonamide-resistant strains of Staphylococcus aureus originate.
The same conclusion has been reached concerning resistance of Escherichia colt
to bacteriophage (LuRiA and DELBRicK (1943), resistance of Staphylococcus
aureus to penicillin (DEMEREC 1945), and resistance of Escherichia coli to
ultraviolet radiation (WITKIN 1946). Mutation and selection therefore appear
to be the mechanisms by which resistance to antibacterial agents originates in
many species of bacteria. In like manner, development of sulfonamide resist-
ance in most species of microorganisms probably occurs by the same mecha-
nisms as in Staphylococcus aureus. ,

SESLER and ScuMipt (1942) reported experiments on serial transfer of pneu-
mococci in sulfonamides. The data published showed stepwise increases in
resistance that occurred at random time during transfer, but they concluded
that the mechanism involved was one of adaptation, and that the steps were
forced by the concentrations of sulfonamides used. Nevertheless, in the nine
transfer series SESLER and ScuipT reported, there were seven instances where
increases in resistance bypassed one and often two intervening concentrations
of sulfonamide at one transfer. Also, adaptation does not explain the random-
SULFONAMIDE RESISTANCE 2509

ness in time of occurrence of the rises. Identical results obtained in the trans-
fer experiments reported here were interpreted as constituting evidence for
origin of resistance through mutation and selection.

Tests of the hypothesis that resistant bacteria arise by mutation should be
made with organisms that have been grown in absence of inhibiting agents.
This was impossible with sulfonamides because of inoculum-size effects. Never-
theless, validity of the data for determining the importance of mutations was
not affected by the techniques used, for many cultures contained no resistant
cells. This is conclusive evidence that physiological adaptation was not the
mechanism by which resistant bacteria were developed. Preliminary exposure
to sulfonamides could result in a serious error because of differential selection
if several discrete mutations were involved at each step, for statistical analyses
of the number of resistant cells in different cultures would be biased if the more
resistant mutants had higher selective values. Clone size would reflect different
resistance levels rather than random occurrence of mutations. The data gave
no indication that such a condition existed, for highly significant variation
between cultures was found in experiments where all mutants isolated showed
identical resistance. Also, in some cases the highest number of colonies per
culture was associated with low resistance.

Mutation rates were calculated from the percentage of cultures containing
no resistant cells. Computation of mutation rates from the actual number of
mutants present in the cultures could not be made from these data because of
selection pressures existent during preliminary exposure to NaST.

The majority of mutants from NRRL-313 had NaST tolerance increased by
about ten times, a few had NaST resistance increased by 30-40 times, and a
few had NaST resistance increased by five times (table 1). All mutants with
NaST tolerance increased by ten or by 30-40 times showed increased produc-
tion of a sulfonamide antagonist capable of replacing p-aminobenzoic acid as a
growth factor for Acetobacter suboxydans and for a p-aminobenzoicless strain of
Neurospora crassa. Strains already resistant to ten times as much NaST as the
parent strain NRRL-313 gave mutants with resistance increased by a second
factor of ten times. Antagonist production was not altered by the second muta-
tion. Therefore, some colony isolations from NRRL-313 might be expected to
show an increase in resistance of ten times and no increased antagonist produc-
tion. Such strains were not found. Either an insufficient number of colonies
was tested, or the action of the genes concerned is conditioned by the genotype
in which they occur. For example, mutations that increased sulfonamide toler-
ance by five times when they occurred in NRRL-313 may have given increases
of ten times in strain 38. Another possibility is that mutations in strain 38
affect metabolic systems that are of importance in sulfonamide resistance only
when PAB metabolism has been altered.

Several mechanisms of sulfonamide action also are suggested by the fact
that the antagonist titer of filtrates depends on the method of assay used. Tests
with Staphylococcus aureus, Acetobacter suboxydans, and Neurospora crassa
each gave different results. NaST resistance of the parent strain NRRL-313
probably is the summation of several effects. Therefore, variation in PAB pro-
260 EUGENE F. OAKBERG AND S. E. LURIA

duction by a factor of 30 times as measured by Neurospora could show an
increase of only ten times as measured by Staphylococci because some antag-
onists in the filtrate of NRRL-313 are effective in counteracting NaST but
do not replace PAB as a growth factor for p-aminobenzoicless N’. crassa. An-
tagonists other than PAB that may be present in the filtrate of NRRL-313
were not increased during the change from sensitivity to resistance, because
tests with S. aureus gave the same anti-sulfonamide value for filtrates from all
cultures with increased antagonist production.

Selection of resistant mutants probably is the primary factor responsible for
development of drug-resistant strains that frequently occurs when a low dosage
of sulfonamide is used either for prophylaxis or for long courses of treatment
(EprpEMIoLocy Unit No. 22, 1945; BERNHEIMER 1946). Single-step mutants
with high resistance occur at a low rate; for none were isolated either by
DEMEREC (1945) or during this investigation. Bacteria with low drug tolerance
were found to be relatively common, and if established as a strain, subse-
quently gave mutants with higher resistance. Therefore, if therapeutic doses
are large enough to inhibit the primary mutants that may occur, development
of highly resistant bacterial strains through selection of secondary mutants
will be prevented. Treatments should be as intensive as possible in order to
preclude development of resistant strains of bacteria,

SUMMARY

The mode of origin of sulfonamide-resistant strains of Staphylococcus aureus
was studied by means of serial transfer in the presence of sodium sulfathiazole ©
and by isolation of colonies from plates containing sodium sulfathiazole.

Stepwise rises in resistance that occurred at random time during serial trans-
fer, and significant variation between similar cultures in the number of resist-
ant cells they contained, indicated mutation and selection as the mechanisms
by which resistant strains of bacteria were developed.

Mutant strains with moderate resistance gave rise to variants with still
higher sulfonamide tolerance. Bacteria capable of growing in saturated solu-
tions of sodium sulfathiazole were obtained by successive mutations to resist-
ance.

Calculations based on the proportion of cultures that failed to show resist-
ant cells gave rates of mutation from sensitivity to resistance that ranged from
2 times 10-* to 4 times 107” per bacterium per generation.

At least five different mutations affect the sulfonamide resistance of S.
aureus. Only one or possibly two mutations are associated with increased pro-
duction of p-aminobenzoic acid. Therefore, it appears as if several cell mecha-
nisms are sensitive to sulfonamides and are altered by mutations.

LITERATURE CITED

BreRNHEMER, ALAN W., 1946 Sulfonamide susceptibility of stock strains of dysentery bacilli
and of strains from recent epidemics. Proc. Soc. Exp. Biol. 61: 325-330.

CHanpter, C. A., and C. A. JANEway, 1939 Observations on the mode of action of sulfanila~
mide in vitro. Proc. Soc. Exp. Biol. 40: 179-184.
SULFONAMIDE RESISTANCE 265

DeEMEREC, M., 1945 Production of staphylococcus strains resistant to various concentrations of
penicillin. Proc. Nat. Acad. Sci. 31: 16-24.

Eprpemrotocy Unir NumBer 22.,1945 Sulfadiazine resistant strains of Beta Hemolytic strep-
tococci. J. Amer. Med. Ass. 129: 921-927.

Fitpes, P., 1940 A rational approach to research in chemotherapy. Lancet 238: 955~957.

Friscu, A. W., 1942 The in vitro susceptibility of pneumococci to sulfapyridine and sulfathiazole.
J. Bact. 43: 72-73.

Kiesy, W. M. M., and L. A. Ranrz, 1943 Quantitative studies of sulfonamide resistance. J.
Exp. Med. 77: 29-39.

Lanpy, Maurice, and Dorotuy M. Dicken, 1942 A microbiological method for the determi-
nation of p-aminobenzoic acid. J. Biol. Chem. 146: 109-114.

Lanpy, Maurice, and Rutu B. GersTunG, 1945 p-aminobenzoic acid synthesis by Neisseria
gonorrhoeae in relation to clinical and cultural sulfonamide resistance. J. Immunol. 51: 269-
277.

Lanpy, Maurice, N. W, Larxum, E. J. Oswavp, and F, StREIGHTOFY, 1943 Increased synthesis
of p-aminobenzoic acid associated with the development of sulfonamide resistance in Staphy-
lococcus aureus. Science 97: 265-267.

Lanxkrorp, C. E., V. Scott, and W. R. Cooxe, 1943 Studies of sulfonamide resistance of the
gonococcus. J. Bact. 45: 201.

LoweLL, F. C., E. Srrauss, and M. Frntanp, 1941 Further studies on the action of sulfapyridine
on pneumococci. J. Immunol. 40: 311-323.

Louris, S. E., and M. Detsrtcx, 1943 Mutations of bacteria from virus sensitivity to virus
resistance. Genet, 28: 491-511.

MacLe£op, C. M., 1940 The inhibition of the bacteriostatic action of sulfonamide drugs by sub-
stances of anima) and bacterial origin. J. Exp. Med. 72: 217-232.

Ross, H. M., and C. L. Fox, Jr., 1942 A quantitative analysis of sulfonamide bacteriostasis.
Science 95: 412-413.

Scumipt, L. H., Carotyn Hires, H. A. Detrwier, and Errie Starxs, 1940 The response
of different types and strains of Pneumococcus to sulfapyridine. J. Infect. Dis. 67: 232-242.

Scuminr, L. H., C. Sester, and H. A. Derrwiter, 1942 Studies on sulfonamide-resistant organ-
isms. I, Development of sulfapyridine resistance by pneumococci. J. Pharmacol. 74: 175-
189.

Scumapt, L. H., C. L. Sester, and M. Hameurcer, JR., 1943 Clinical studies on sulfonamide-
resistant pneumococci. J. Introduction. Methods of study and occurrence of resistant pneumo-
cocci in patients before treatment. J. Bact. 45: 27.

Scusapt, L. H., and Ciara L. Sesier, 1943 Studies on sulfonamide-resistant organisms. ITI. On
the origin of sulfonamide-resistent pneumococci. J, Pharmacol. 77: 165-174.

ScumirH, K., 1943 Effect of »-(aminomethyl)-benzene-sulfonamide (Marfanil). A chemo-
therapeutic with a fundamentally new mode of action. Acta. Path. et Microbiol. Scand. 20:
563-572.

SEsLeR, Ciara L., and L. H. Scumipt, 1942 Studies on sulfonamide resistant organisms: II.
Comparative development of resistance to different sulfonamides by pneumococci. J.
Pharmacol. 75: 356-362.

SPICER, SOPHIE, 1945 A study of sulfapyridine resistance in pneumococci. J. Bact. 50: 23-29.

Strauss, E., J. H. DInGLe, and M. FrInuanp, 1941 (a) Studies on the mechamism of sulfonamide
bacteriostasis, inhibition and resistance experiments with E. colt in a synthetic medium. J.
Immunol. 42: 313-329.

1941 (b) Studies on the mechanism of sulfonamide bacteriostasis, inhibition and resistance
experiments with Staphylococcus aureus. J. Immunol. 42: 331-342.

Tuompson, Roy C., Evira R. Ispetz, and Herscnet K. MitcHet1, 1943 A microbiological
assay method for p-aminobenzoic acid. J. Biol. Chem. 148: 281-287.

Wrrkin, EveLtyn M., 1946 Inherited differences in sensitivity to radiation | in Escherichia coli
Proc. Nat. Acad. Sci. 32: 59-68.

Woops, D. D., 1940 The relation of p-aminobenzoic acid to the mechanism of the action of
sulphanilamide. Brit. J. Exp. Path. 21: 74-90. ©