THE EFFECTIVE CONCENTRATIONS OF PENICILLIN IN VITRO AND

IN VIVO FOR STREPTOCOCCI, PNEUMOCOCCI, AND

TREPONEMA PALLIDUM
HARRY EAGLE, RALPH FLEISCHMAN, AND ARLYNE D. MUSSELMAN
Section on Experimental Therapeutics, National Institutes of Health, Bethesda 14, Maryland
Received for publication November 2, 1949
In the therapeutic use of penicillin, the question as to whether the minimum
effective concentration in vivo is the same, greater, or less than that necessary to
kill the same organ'sm in vitro is of obvious importance.
Data are here reported with respect to the minimal effective serum concentra-
tions of penicillin in a number of experimental infections in mice and rabbits. As
will be shown, these serum levels were usually 2 to 5 times higher than those
necessary to kill the same organism in vitro. However, in view of the known con-
centration differential between the serum and tissue fluids, this suggests that the
effective concentration of penicillin at the actual site of infection is in most in-
stances the same as that effective in vitro.
METHODS AND MATERIALS
The use of procaine penicillin to provide reasonably stable serum concentrations of
penicillin. In order to obtain a reliable measure of the effective serum concentra-
tions of penicillin, it was necessary to keep these levels reasonably constant for
the time that the drug was acting on the organisms. This was accomplished by
the use of procaine penicillin G suspended in peanut oil, gelled by the addition of
2 per cent aluminum monostearate (Buckwalter and Dickison, 1948). A series of
such suspensions were prepared, varying in penicillin content in approximately
2-fold steps from 320,000 down to 80 units per ml. The co-operation of the staff
of the Bristol Laboratories, by whom these penicillin suspensions were prepared,
is gratefully acknowledged. The various suspensions were injected intramuscu-
larly in a fixed volume of 0.1 ml in mice weighing 17.5 to 22.5 grams. In rabbits
(2.5 to 3.5 kg) the dosage volume in ml was one-fifth of the body weight in kilo-
grams. The serum concentrations of penicillin at varying intervals thereafter are
summarized in table 1. The relatively slow rate at which the penicillin concentra-
tion fell, reflecting its slow and continuing absorption from the intramuscular
depot, is evident from the table. For purposes of interpolation, lines were fitted
to the experimental data by the least squares method.
Penicillin assays. Serum penicillin assays were carried out by a serial dilution
technique previously described (Eagle and Newman, 1947). Ten-ml specimens
were obtained from rabbits by cardiac puncture, and mice were exsanguinated
from the exposed heart. Horse blood was used as the hemolytic indicator in
assaying the mouse sera, and rabbit blood for the rabbit sera. The results shown
in the tables have been corrected for the inhibitory effect of serum in the assay
(Eagle and Tucker, 1948). These corrective factors in the case of mouse serum
were found to be 1.9 for whole serum, 1.3 for 1:2 serum, and 1.15 for 1:4 serum.
625
626 H. EAGLE, R. FLEISCHMAN, AND A. D. MUSSELMAN [VOL. 59
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1950] CONCENTRATIONS OF PENICILLIN 627
In order to make the dosages and concentrations comparable to those reported in
a previous paper dealing with sodium penicillin, they have been expressed as mg
per ml or mg per kg of sodium penicillin G, assuming an activity of 1,667 units
per mg.
One hour after the injection of the penicillin, the animals were inoculated with
an actively growing culture of the several organisms, calibrated by microscopic
count (Magnuson, Eagle, and Fleischman, 1948). The colony count on a poured
plate averaged 1.1 times the microscopic count, with a range of 0.13 to 2.8. The
smaller values usually reflected the presence of bacterial clumps or chains in the
culture, the average number of organisms per chain varying from 2 to 7 in indi-
vidual experiments.
Determination of minimum dose of penicillin with bactericidal action in vivo and of
minimum effective serum concentrations. Two types of experiment were conducted.
In one, a relatively small inoculum was used (100 organisms in the mice and 20
organisms in the rabbits), and death or survival served as the criterion of penicil-
lin efficacy. The size of the inoculum was deliberately set at this low level so that
the effective concentration of penicillin would have to be provided for only a
short period of time in order to abort infection (Eagle, Fleischman, and Mussel-
man, 1949b). Under these circumstances, the effective concentration of penicillin
would be that present during the first few hours after its administration (see
below). The abortive doses of penicillin (ED5o) indicated in the tables were calcu-
lated by Karber's procedure (1931). The assistance of Mr. Nathan Mantel, of
the Office of the Statistical Coordinator, Division of Public Health Methods,
Public Health Service, in calculating the EDi dosages and fitting lines to the
experimental data is gratefully acknowledged.
In the second type of experiment the bactericidal effect of various serum con-
centrations of penicillin was determined directly. One hour after the injection of
varying doses of procaine penicillin 20,000 organisms were inoculated intra-
muscularly into mice. At intervals thereafter the muscle was removed and emul-
sified in a Waring blender with 50 ml of blood broth; the number of viable organ-
isms was determined by plating out in blood agar (3 ml of the tissue emulsion in a
total of 12 ml, and serial 40-fold dilutions from the first tube). All plate counts
were made in duplicate.
This technique was not feasible in syphilitic infection, since Treponema palli-
dum has not yet been cultivated in artificial media. Instead, rabbits were treated
with penicillin 10 to 12 days after the intratesticular inoculation of approximately
4( million organisms and after the development of an acute orchitis. The im-
mobilization and disappearance of organisms from the primary lesion after vary-
ing doses of procaine penicillin served as the criterion of the minimum trepone-
micidal concentration.
EXPERIMENTAL RESULTS
The Effective Serum Concentrations in a Number of Experimental Infections Judged
by the Dosages of Procaine Penicillin Necessary to Abort an Early Infection
Experiments to determine the dosages of procaine penicillin necessary to abort
the several infections here studied are summarized in tables 2 and 3. As there
628 H. EAGLE, R. FLEISCHMAN, AND A. D. MUSSELMAN [VOL. 59

shown, the effective dosage of penicillin and, thus, the effective serum concen-
tration were usually sharply defined. In the mice particularly a 2-fold difference
in dosage often made the difference between cure rates of less than 20 per cent
and more than 80 per cent.
TABLE 2
The dosage of procaine penicillin G (in peanut oil gelled with 2 per cent aluminum
monostearate) necessary to abort a number of experimental mouse infections
Suspensions containing varying concentrations of penicillin were injected intramuscu-
larly in a fixed volume of 0.1 ml into mice weighing 17.5 to 22.5 g. The dosages in mg per
kg have been calculated on the basis of the sodium rather than the procaine salt (1,667
units per mg). The animals were inoculated 1 hour later.
GROup A GROUP B DIPLOCOCCUS DIPLOCOCCUS PNEUMONIAE
DOSAGE p-EEMOLYTIC p-MEMOLYTIC PNEUMONIAE TYPEm m (100 ORGAMISS)
STREPTOCOCCUS STREPTOCOCCUS TYPE I (100
(100 ORGAMSMS (100 ORGANSMS ORGAMSMS
INTRMUS- INTRAMUS- INTRAPERI-
units/kg mg/kg CUJLALLY) CULARLY) TONEALLY) Intonperi Intramuscularly
25,000 15 0/20
12,500 7.5 1/20 0/30 2/30 2/24
8,700 5.2 6/30 3/30 0/25
6,250 3.8 0/20 2/18 1/29 4/30 5/25
4,400 2.6 9/30 3/30 18/25
3,200 1.9 0/20 17/20 40/60 16/30 22/25
2,200 1.3 25/30 24/30 25/25
1,600 0.96 9/20 14/17 18/20
800 0.48 18/20 13/19
400 0.24 19/20 15/15
Mortality in con- 100 39/43 17/20 54/55 34/35 23/25
trol animals re- 10 25/33 3/15 27/30 9/10 16/25
ceiving indicated 1 5/35 0/15 15/30 6/10
no. of organisms
Dosage that abor- mg/ 1.0 4 0.11* 3.0 =1: 0.46t 2.2 1 0.13 2.0 0.12 2.85 j 0.16$
ted infection in kg
half the animals
(ED5o)
-~~~~~
* Natural survivorship of 10 per cent assumed in calculating ED5o dosage.
t Natural survivorship of 15 per cent assumed in calculating ED50 dosage.
t Natural survivorship of 5 per cent assumed in calculating ED60 dosage.
The serum levels of penicillin after these EDso dosages may be taken as those
that provide effective concentration at the site of inoculation. From the data of
table 1 one could estimate that in the mice inoculated intraperitoneally with (a)
group A streptococci, (b) type I pneumococci, and (c) type III pneumococci and
intramuscularly with (d) group B streptococci and (e) type III pneumococci, the
serum concentrations provided by the EDw dosages of procaine penicillin at the
time of inoculation' were (a) 0.015, (b) 0.06, (c) 0.055, (d) 0.11, and (e) 0.11
1 One hour after the injection of penicillin.
 19]0 CONCENTRATIONS OF PENICILLIN 629
micrograms per ml, respectively (table 4, coluimn 2). Similarly, in rabbits inocu-
lated with the group B streptococcus, the ED50 dosage had provided initial serum
concentrations of 0.07 to 0.14 micrograms per ml, varying according to the site of
inoculation. In all these cases, dosages of penicillin that initially provided ap-
proximately half these concentrations were almost wholly ineffective (table 4,
columns 1 and 2). The minimal effective serum levels were thus only slightly less
than those provided by the ED50 dose at the time of inoculation.
TABLE 3
The dosage of procaine penicillin G (in peanut oil gelled with 2 per cent aluminum
monostearate) necessary to abort infection in rabbits inoculated with group B
5-hemolytic streptococci (20 organisms)
Suspensions containing varying concentrations of penicillin were injected intramuscu-
larly in rabbits at a dosage of 0.5 to 0.7 ml bod . They were inoculated 1

hour later as indicated in the table. Dosages in mg per kg have been calculated as sodium
penicillin G (1,667 units per mg).
MORLTALTY IN RABBITS INOCULATED WITH GROUP B
p-HEMAOLYTIC STREPTOCOCCI
PENICILLIN DOSAGE
Intramuscularly Subcutaneously Intratesticularly Intrapuly
monarily*
urnis/kg mg/kg
8,000 4.8 0/6
4,000 2.4 0/8 0/7
2,000 1.2 3/8 0/8 0/6 1/6
1,000 0.6 5/8 5/8 2/6 2/6
500 0.3 7/8 7/8 4/6 5/6
250 0.15 4/4 4/4 9/10 6/10
Mortality in untreated 12/12 9/10 7/7 5/7
control animals
Dosage which aborted units/kg 1,300 1,000 660 970
infection in half of mg/kg 0.78 :1: 0.16 0.60 :i 0.10 0.40 i 0.08 0.6*
animals
* Fifteen per cent natural survivors assumed in calculating EDs0 dosage of penicillin;
if one assumes 30 per cent natural survivors, the EDso value becomes 0.72 + 0.25.

There was reason to believe that, under the conditions of the present experi-
ments, the infections had been effectively aborted by the ED6o dose of penicillin
within less than 1 hour, and that the group A infection had been aborted some-
what faster than the other three. (1) In mice inoculated intraperitoneally or intra-
muscularly with 20 to 100 organisms but treated with aqueous sodium penicillin
instead of the suspension of procaine penicillin in oil, the abortive doses in the
case of group B streptococci, Diploous pneumoniae type I, and Diplococcus
pneumoniae type III had previously been found to range between 3 and 10 mg
per kg (Eagle, Fleischman, and Muselman, 1949b). These doses of aqueous
penicillin provided serum concentrations in excess of 0.05 ug per ml, for 1 to 1.6
630 H. EAGLE, R. FLEISCHMAN, AND A. D. MUSSELMAN [VOL. 59

hours (Eagle, Fleischman, and Musselman, 1949a, 1950). The abortive dose of
0.35 mg per kg in a similar group A streptococcal infection provided that concen-
tration for approximately 0.5 hours and a concentration of 0.012 ,gg per ml for
0.82 hours. (2) When these organisms were exposed to effective concentrations of
penicillin for a period of 15 to 45 minutes, the then surviving bacteria did not
resume multiplication for a number of hours. During this recovery period varying
TABLE 4
The minimal effective serum concentration of penicillin in a number of
experimental infections (based on the dosage of procaine penicillin
necessary to abort early infections)
SERUM CONCS. OF
(O1A)G
DOSAGE
PENICILLIN (JG/ML) (4)
NO. OF PROVIDED BY EDio ESTDIATED
ORGAN- THAT DOSAGE OF PENICILLIN MNINMUM
ORGANISM
ORGAMSM HOST
HOST ~ ROUTE OP
INOCULATION sm
INOCU
ABORTED
INFECTION SERETIV
FET
LNATE IN HALF (2) SERUM
LATD E
ANIMALS*
At time of
inocula-
(3)ou zcONC.NO
1lhourtPNCILN
tiont itr
mg/kg pg/mi
,-Hemolytic Mice Intramuscular 100 1.0 0. 015 0.0081: 0.012
streptococcus,
group A
p-Hemolytic Mice Intramuscular 100 3.0 0.11 0.06 0.08
streptococcus, Rabbits Intramuscular 20 0.8 0.14 0.11 0.12
group B Subcutaneous 20 0.6 0.1 0.09 0.1
Intratesticular 20 0.4 0.07 0.065 0.07
Intrapulmonary 20 0.6 0.1 0.09 0.1
Diplococcus Mice Intraperitoneal 100 2.2 0.06 0.035 0.05§
pneumoniae,
type I
Diplococcus Mice Intraperitoneal 100 2.0 0.055 0.03 0.05§
pneumoniae, Intramuscular 100 3.0 0.11 0.06 0.08
type III
* From data of tables 2 and 3. All dosages and concentrations are expressed in terms
of the sodium rather than the procaine salt.
t From data of table 1.
t Based on an extrapolation of the serum penicillin curves beyond the measurable
range.
§ Although the effective concentration for the type III organisms seemed to be some-
what less than for type I, the difference was not significant, and the effective concentra-
tion for both organisms has been rounded off to 0.05 Ag per ml.
proportions of the damaged organisms were disposed of by host mechanisms, in
the absence of drug (Eagle, 1949; Eagle and Musselman, 1949; Eagle, Fleisch-
man, and Musselman, 1950b).
It thus appeared that, under the conditions of the present experiments, the
EDi dose of procaine penicillin had probably completed its abortive action within
approximately 1 hour. In that case, the minimum effective serum level would be
slightly less than the concentration provided by the EDo dose at the moment of
inoculation and slightly greater than that present an hour after inoculation. The
1950] CONCENTRATIONS OF PENICILLIN 631
serum concentrations at those two times are given in columns 2 and 3 of table 4
for each of the infections here studied; and the last column of that table lists an
intermediate value, which is the estimated minimum effective concentration of
serum for each of those infections. Because of the slow rate of fall of the serum
penicillin levels, the error in this approximation is relatively small. In mice those
effective levels were estimated at 0.012 ug per ml for the group A streptococcus
inoculated intramuscularly, 0.05 ug per ml for the type I or III pneumococcus
inoculated intraperitoneally, 0.08 ,g per ml for the group B streptococcus inocu-
lated intramuscularly, and 0.08 ,g per ml for the type III pneumococcus inocu-
lated intramuscularly. In rabbits inoculated with the group B streptococcus the
minimum effective serum concentration of penicillin was approximately 0.12 Mg
per ml for an intramuscular inoculum, 0.1 Mug per ml for a subcutaneous or intra-
pulmonary inoculum, and 0.07 ug per ml for an intratesticular inoculum.
These minimal effective serum concentrations are approximately 2 to 4 times
those necessary to kill the same organisms in vitro and are estimated to have pro-
vided just the latter bactericidal concentration at the actual site of inoculation
(see below).
The Minimal Effective Serum Concentration of Penicillin as Determined by the
Direct Measurement of Its Bactericidal Action in Vivo
The data and conclusions of the preceding section were confirmed by experi-
ments in which the bactericidal action of penicillin was studied directly. A
relatively large number of organisms (20,000) were inoculated intramuscularly in
mice, and the minimum dosage of sodium penicillin that sufficed to cause an
initial reduction in the number of viable organisms was then determined. These
experiments will be reported in detail elsewhere (Eagle, Fleischman, and Mussel-
man, 1950b), but a single illustrative figure (figure 1) is reproduced here.
With all four organisms tested (Diplococcus pneumoniae, type I and type III,
and group A and group B ,B-hemolytic streptococci) a dosage of 3 mg per kg,
supplying a 30-minute serum level of 0.5 Mg per ml, was actively bactericidal. A
dosage of 0.6 mg per kg, supplying a 30-minute level of 0.11 Mug per ml and an
estimated 1-hour level of 0.01 Mg per ml, was actively bactericidal against the
group A streptococcus (for which the effective serum concentration is estimated
as 0.012 Mg per ml); it was, however, only slightly active against the group B
streptococcus and the pneumococci (requiring approximately 0.08 ,ug per ml). A
dosage of 0.15 mg per kg, providing a 30-minute serum concentration of 0.023
Mg per ml, was relatively ineffective against all four species. (A somewhat higher
activity was perhaps to have been expected against the group A streptococcus.)
With the latter organism the minimum dosage that had a bactericidal effect in
vivo was also determined by means of a suspension of the procaine salt in oil. As
shown in figure 3, a dosage of 0.24 mg per kg was wholly ineffective, and a dosage
of 0.48 mg per kg was definitely bactericidal. Although the serum concentration
provided by the latter dose is less than the measurable level, it may be estimated
by extrapolation of the data in table 1 to be on the order of 0.01 to 0.02 ,ug per
ml one-half hour after the injection. This agrees with the previous estimate of
632 H. EAGLE, R. FLEISCHMAN, AND A. D. MUSSELMAN [VOL. 59
0.012 ;g per ml as the minimum serum concentration effective against this
organism.
A special case was afforded by syphilitic infection. When syphilitic rabbits were
given a single injection of procaine penicillin, the smallest dosage causing the
temporary disappearance of the organisms from the primary lesion was 0.3 mg
per kg (500 units per kg). At this threshold dosage the organisms began to
decrease significantly in numbers only after 12 to 24 hours and disappeared al-
together from the lesion after 48 to 60 hours. The mean serum concentration pro-
vided by this dosage was 0.067 ug per ml after 1 hour, falling slowly to 0.026 ug

10.000~~~~~~~~~~~~~~~~~~~~~~~~.1
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wl
a: . IN
ON TYP PNEUSEMOSCLECCUS

HOURS AfTER INJECTION OF PENICILLIN

Figure 1. The bactericidal action of penicillin on type III pneumococci in vivo (after
Eagle, Fleischman, and Musselman, 1950b). The dotted portion of each curve indicates the
time for which the indicated dose provided serum concentrations in excess of the bacteri-
cidal level.

per ml in 24 hours. There was, however, reason to suspect that even smaller
doses of penicillin, and correspondingly lowver serum concentrations might be
effective if those levels were sustained for a sufficiently long period. This proved
to be the case. As shown in table 5, when the injections were repeated every 12
hours, the minimal effective dosage of procaine penicillin per injection fell to
0.038 mg per kg (64 units per kg), at which dosage the organisms began to dis-
appear from the primary lesion only after 48 to 60 hours and after 3 to 4 injec-
tions. At this dosage of procaine penicillin in oil the serum never contains peni-
cillin in concentrations demonstrable by the bioassay technique here used. An
unreliable extrapolation of the data in table 2 indicates that the effective dosage
of 0.038 mg per kg might provide a serum concentration in excess of 0.01 pg per
1950] CONCENTRATIONS OF PEMCILLIN 633

ml for approximately 1 to 2 hours, and in excess of 0.001 ,ug per ml for perhaps
3 to 4 hours. That this rough approximation may be of the correct order of mag-
nitude is indicated by the fact that, when syphilitic rabbits were treated with
aqueous penicillin at 4-hour intervals to a total of 50 injections, the CDw dosage
was 0.004 mg per kg per injection (Eagle and Magnuson, 1946). This dosage
also fails to provide a measurable level in the serum, but it may be estimated to
have provided a 30-minute serum concentration of approximately 0.005 ,g per
TABLE 5
The doeage of procaine penicillin G (in peanut oil with 2 per cent aluminum
mono8tearate) nece8sary to affect T. pallidum in a rabbit testicular chancre
Rabbits weighing 2.5 to 3.5 kg were inoculated intratesticularly with T. pallidum.
Ten to 14 days later, at the height of the inflammatory reaction, the animals were injected
intramuscularly with suspensions containing varying concentrations (80 to 320,000 units
per ml) of procaine penicillin G. The dosage in ml was one-fifth the body weight in kg.
The testes were aspirated for dark-field examination 3, 6, 9, 12, 24, 36, 48, 60, and 72 hours
after treatment, or until they became dark-field-negative. Two to three rabbits were
tested at each time period.
PENICILLIN DOSAGE PER AVERAGE SERUM CONC. OF PENICILIN, HOURtS RlEQUIRED
INJECTION oG/L FOR SIGNICANT
__EDUCTION IN NO.
units/lcg mg/kg 1 hr after injec- 4 hr after injec-. ORT. OTILTY OF
units/kg
~~tion tion PALLMDUM
Single injection 64,000 38 1 1 6-9
16,000 9.6 1 1 6-9
4,000 2.4 0.36 0.18 6-9
2,000 1.2 0.18 0.11 6-9
1,000 0.6 0.096 0.06 12
500 0.3 0.067 0.044 24-36
Injections repeated 500 0.3 0.067 0.044 24
at 12-hour inter- 250 0.15 <0.05 24
vals 128 0.078 60-72
64 0.038 72-96
32 0.11 cot
16 0.1 00

* Concentrations and dosages are expressed in terms of sodium penicillin G rather than
the procaine salt (cf. table 1).
t No apparent effect on number or motility of organisms after 8 injections, 96 hours
after beginning of treatment.

ml; and the treponemicidal concentration provided by this dosage at the focus of
infection may be estimated to be on the order of 0.002 Ag per ml (see below).
Treponema pallidum is thus at the same time one of the most sensitive as
well as one of the most resistant of all bacteria to the action of penicillin. It is
one of the most sensitive in terms of the smallest concentration which is bacteri-
cidal; it is one of the most resistant in terms of the time for which it must be ex-
posed to that concentration in order to be killed. Syphilitic infection may there-
fore be cured by a relatively minute concentration acting over a relatively long
period of time. Treponema pallidum differs from other bacteria in another im-
6346H. EAGLE, R. FLEISCHMAN, AND A. D. MUSSELMAN [VOL. 59
portant respect. With all other species studied so far the maximally effective con-
centration of penicillin is usually no more than 2 to 4 times as great as that which
barely suffices to kill a significant proportion of the organisms (Eagle and Mussel-
man, 1948), and only occasionally, as with the C-203 strain of group A strepto-
cocci, is the ratio an 8-fold one. In the case of Treponema pallidum, however, there
was a more than 50-fold ratio between the concentration that was most rapidly
effective and that which was demonstrably, if slowly, treponemicidal. The maxi-
mal effect was produced by a dose of 2.4 mg per kg, which provided an initial
serum concentration of 0.36 Ag per ml (0.6 units per ml). At this dosage the
organisms disappeared from the primary lesion in 6 to 9 hours. At the other
extreme, at a dosage of 0.038 mg per kg, which provided an initial serum concen-
tration below the measurable level, but estimated to be on the order of 0.005
to 0.01 jig per ml (0.008 to 0.016 units per ml), the organisms disappeared in 48
to 96 hours. This means that the rate of its treponemicidal action in vivo increases
progressively over a 50-fold range of serum concentration. Although low doses
providing concentrations below the measurable level suffice to effect cure, larger
doses would be more rapidly effective.
The Correlation between the Effective Conceration of Penicllin in Vitro and in Vivo
When bacteria are exposed to penicillin in vitro, one may define four "effective"
concentrations of the drug. (1) At the lowest level the drug serves only to re-
duce the rate of multiplication but does not kill a measurable proportion of
the organisms. (2) A slightly higher concentration is bactericidal for the suscep-
tible organisms but does not kill the more resistant variants in the culture
and therefore fails to sterilize the suspension. (3) At a somewhat higher con-
centration the drug kills all the organisms in a given suspension. (The "sensi-
tivity" of a bacterial strain as ordinarily defined, i.e., the concentration at
which a given inoculum fails to grow out visibly in culture, lies between con-
centration levels 2 and 3 as here defined. It is not a fixed value, but will vary
in magnitude according to the size of the inoculum and the conditions of the
test). (4) Beyond the level that suffices to kill all the organisms still higher
concentrations serve to increase the rate of that bactericidal action. Eventually,
however, one attains a concentration at which organisms are killed at a maximal
rate and are not affected by further increase.
The latter three "effective" levels in vitro are given in table 6 for each of the
four bacterial species under consideration. With all four species the serum concen-
tration that sufficed to abort an early infection (table 6, column 1) was regularly
1.5 to 3 times the concentration that sufficed to sterilize suspensions containing
106 organisms per ml (column 3) and 2 to 5 times as great as the concentration
that killed the majority of the organisms in a culture suspension (column 2).2
The fact that approximately half the serum penicillin is bound to serum pro-
tein and thus not available for diffusion into the tissues (Tompsett, Shultz, and
2 Since only small inocula were used in the present experiments, the latter is the more
appropriate comparison. The minimal effective concentrations in vivo as here determined
are therefore not those that kill the organisms at the maximal rate but merely those that
have a definite bactericidal action.
1950] CONCENTRATIONS OF PENICILLIN 635

McDermott, 1947a,b) would account for most of the differences observed be-
tween the minimum effective serum concentrations and those barely effective in
vitro. In addition, there is probably a concentration gradient between the dif-
fusible penicillin in the serum and the concentration at the actual focus of
infection in the tissues.8 One must conclude that the effective concentration of peni-
cillin in vivo, i.e, the concentration necessary to kill an organism in the tissue fluids,
is essentially the same as that effective against that same organism in vitro.
TABLE 6
Correlations between the effective concentrations of penicillin in vivo and in vitro
EFFECTVE CONCENTRATION IN VITRO,
/AG/ML
ESTIMATED (2)
MINIMAL
OF INOCULATION EFFECTIVE Lethal for (3
ORGANISM HOST ROUTE most Suffices to (4)
SERUM sterilize Kils organ-

PENICI[L.LrN*
CONC.

irgnsterilizetsms suspensin
but fails
to
Cotainin=
up to
106/m1* ratet
aia

B-Hemolytic strep- Mice Intramuscular 0.012 0.005 0.008 0.032

tococcus, group A
B6-Hemolytic strep- Mice Intramuscular 0.08
tococcus, group B Intramuscular 0.12
Rabbits Subcutaneous 0.1 0.024 0.04 0.064
Intratesticular 0.07
Intraperitoneal 0.1
Diplococcus pneu- Mice Intraperitoneal 0.05 0.020 0.028 0.064
moniae, type I
Diplococcus pneu- Mice Intraperitoneal 0.05 0.020 0.028 0.064
moniae, type III Intramuscular 0.08
* From data of table 4: serum concentrations afforded by dose of penicillin
that aborted infection in half of animals. All dosages and concentrations are expressed in
terms of the sodium (1,667 units per mg) rather than the procaine salt.
t From Eagle and Musselman, 1948; Eagle, Fleischman, and Musselman, 1950.

These results suggest that in the therapeutic use of penicillin, a dosage regimen
which provides at the focus of infection that concentration which kills the
organism at a maximal rate in vitro would be similarly effective in vivo. The
3The widely varying concentrations of penicillin that have been demonstrated in dif-
ferent organs (e.g., Cooke and Goldring, 1945; Cutting et al., 1945; Richardson et al., 1946;
Struble and Bellows, 1944) offer no direct evidence on the actual concentration of the drug in
the extracellular tissue fluids. The magnitude of that gradient between serum and tissue
fluids therefore remains to be determined. In the present experiment the ED50 dosage,
and thus the effective serum concentrations, were the same in rabbits inoculated subcu-
taneously and into the lung, somewhat higher in those inoculated intramuscularly, and
somewhat lower in those inoculated in the testis. In mice also an intramuscular inoculum
required a higher serum concentration than an intraperitoneal inoculum with the same
organism. This suggests corresponding minor differences in the concentration of penicillin
in the tissue fluids of these organs.
 H. EAGLE, R. FLEISCHMAN, AND A. D. MUSSELMAN [voL. 59
63600
serum concentration necessary to provide that effective level at the focus will
vary with the tissue involved, the rate of penicillin diffusion into the involved
area, possible local destruction of penicillin, and similar factors.
Early vs. established infections. The experiments of the preceding sections
relate only to freshly inoculated animals, and a different situation might conceiv-
ably obtain after theinfection has become established, with widely disseminated
foci of inflammation and much larger numbers of bacteria. Such differences have,
however, not been encountered. It is true that when mice or rabbits were treated a
number of hours after inoculation with either pneumococci or streptococci, or
when rabbits were treated 6 weeks after inoculation with T. pallidum, the single
curative dose (CDwo) of aqueous penicillin was as much as 1,000 times greater
than when they were treated immediately after inoculation;4 (Eagle, Fleischman,
and Musselman, 1949b). However, When treatment was then continued over a
sufficiently long period of time, the curative dose per injection was of the same
order of magnitude as that which sufficed to abort infection with a small number
of organisms (Eagle, Fleischman, and Musselman, 1950). Clearly, the established
infection did not require higher concentrations of penicillin but merely the
provision of the same effective level for a longer period of time.'
The 2- to 5-fold ratio here noted between the serum level of penicillin that
sufficed to provide an effective concentration at a tissue focus and the minimal
bactericidal concentration in vitro clearly indicates that, under the conditions
of the present experiments, the penetration of penicillin into the bacterial depot
offered no problem. A larger excess in the serum would, however, be necessary
in order to provide the effective concentration in an area in which the penicillin
was being inactivated. Further, if the bacteria were in a walled-off densely
fibrotic or necrotic area, only poorly supplied with blood, the diffusion of peni-
cillin into the bacterial focus from the surrounding capillary bed and tissue
fluids might be greatly retarded. Under the latter conditions, the effective
concentration would have to be maintained in the serum for a relatively long
period of time in order to assure eventual penetration into the focus of infection,
and a high concentration in the serum and tissue fluids would accelerate the
diffusion of the drug into that focus. These considerations, however, do not af-
fect the validity of the thesis that the concentration of penicillin at the bacterial
focus which kills the organisms in vivo is of the same order of magnitude as that
which is effective against the same organism in vitro.
Supposed peristence of peniciUin in the tissues. This conclusion is apparently at
variance with the repeated observation that patients can be effectively treated
with penicillin even if the serum concentration falls below effective or even
measurable levels for a number of hours between injections (cf. Tillett, McCor-
4 With the group A i3-hemolytic streptococcal infections in mice and the group B infec-
tions in rabbits, it was not possible to cure a 24-hour infection with any single dose of
aqueous penicillin short of the toxic level (Eagle, Fleischman, and Musselman, 1949b);
and divided doses (4 times at 3- or 24-hour intervals) were similarly ineffective. This failure
was not related to the magnitude of the penicillin levels, and its cause is under present
study.
5 See footnote 4.
1950] CONCENTRATIONS OF PENICILLIN 637

mack, and Cambier, 1945; Tompsett, Timpanelli, Goldstein, and McDermott,

1949). Further, in experimental infections bacteria may continue to die for
hours after the serum penicillin has fallen below the concentration effective in
vitro (Jawetz, 1946; Grunberg, Schnitzer, and Unger, 1948; Eagle, Fleischman,
and Musselman, 1950b). Of the four bacterial species under present study the
group B streptococcus in particular was characterized by the continuing death
I I I .I I I

100- SERUM CONCENTRATION OF

PENICILLIN G IN MICE AFTER
INJECTION OF 200 MG /KG
10

.01-

00

so MORTALITY IN MICE INOCULATED

INTRAMUSCULARLY WITH 100 TYPE
z 60 - m PNEUMOCOCCI AT INDICATED
v TIME AFTER PENICILLIN
I&J 407
20-

o 2 3 4 5 6
TIME IN HOURS
Figure 2. The time for which a single injection of aqueous penicillin serves to protect
mice against a following small inoculum. Mice weighing 17.5 to 22.5 g were injected intra-
muscularly with 0.1 ml of 4 per cent aqueous penicillin G (200 mg per kg). At varying inter-
vals thereafter they were inoculated into the opposite leg muscle with 0.1 ml of a bacterial
suspension diluted to contain 100 Diplococcus pneumoniae type III (by microscopic count).
The upper curve in the figure is the mean serum concentration of penicillin after this dosage,
as previously reported (Eagle, Fleischman, and Musselman, 1949a). The lower curve shows
the proportion of animals that died during the period of observation. Each experimental
group comprised 25 to 37 animals, and the mortality in untreated controls was 96 per cent.

of the organisms for as long as 24 to 48 hours after penicillin was no longer demon-
strable in the serum.
The simplest explanation for this paradox would be that penicillin persists
at effective levels in the tissues for hours after it has disappeared from the blood
(cf. Ercoli, Lewis, Schwartz, and Whitehead, 1948). However, this has not been
found to be the case under the conditions of the present experiments. When mice
were injected intramuscularly with massive doses of aqueous penicillin, which
provided initial levels of several hundred ug per ml, the animals became suscep-
638 H. EAGLE, R. FLEISCHMAN, AND A. D. MUSSELMAN [VOL. 59
tible to a minimal inoculum of a pathogenic organism even before the serum
concentration had fallen below the effective level (figure 2). Similar results have
been obtained in rabbits inoculated with a group B i3-hemolytic streptococcus
(table 7). As the table shows, 4 hours after the intramuscular injection of peni-
cillin G at 60 mg per kg, when the serum concentration still averaged 0.6 pg
per ml, an intramuscular inoculum of 20 organisms killed 62 per cent of the
animals, and after 6 hours, when the serum level averaged 0.06 ,ug per ml, the
inoculum was regularly fatal. Since as little as 0.25 mg per kg sufficed to abort
TABLE 7
The time for which sodium penicillin G persists at effective level8 in rabbit muscle
Rabbits were injected intramuscularly with sodium penicillin G at either 60 or 3 mg
per kg. At varying intervals thereafter they were inoculated into the opposite leg muscle
with 0.5 ml of a suspension of group B fi-hemolytic streptococci calibrated by microscopic
count to contain 20 organisms. The minimal protective dosage of penicillin when it was
injected simultaneously with the organisms was 0.25 mg per kg; and it is a reasonable
surmise that rabbits that died contained less than this amount of available penicillin in the
body tissues at the time of inoculation.
TINE OF INOCULATION
DOSAGE OF (HOURS AFTER INJECTION OF PENICILLIN)
PENICILIN
1 2 3 4 5 6
mg/kg
Average serum cone. of peni- 20 5.9 2t 0.6 0.2t 0. 06t
60 Mortality in rabbits inocu- - 0/16 0/7 11/16 7/8 13/14
lated at indicated time
Average serum conc. of peni- 1 0.1 0. 064ft 0.03 -

3 Mortality in rabbits inocu- 0/7 3/7 7/8 6/6

lated at indicated time
Conclusion: Penicillin did not persist at effective levels in rabbit muscle for a signifi-
cant period after it had fallen below those levels in the serum. A small number of organ-
isms caused a fatal infection when inoculated even before the serum penicillin had fallen
to ineffective levels.
* After Eagle, Fleischman, and Musselman, 1949a.
t Estimated by interpolation.
this inoculum if given at the time of the injection, it is apparent that only neg-
ligible amounts of penicillin were present in the tissues 4 to 6 hours after the
injection, and that it did not persist at effective concentrations in the muscle
for a significant period after it had fallen below these effective levels in the
serum.
The sensitivity to penicillin of bacteria damaged by previous exposure to the drug.
A second possible explanation for the continuing death of bacteria in vivo might
be that bacteria, once exposed to bactericidal concentrations, continue to die
in the presence of much smaller concentrations, which are ordinarily wholly
ineffective (cf. Grunberg, Unger, and Eldridge, 1948). Since the hourly decrease
1950] CONCENTRATIONS OF PENICILLIN 639

in the serum penicillin concentration after the intramuscular injection of aqueous

solutions in mice averages more than 90 per cent, one would have to assume at
least a 10-fold increase in susceptibility in order to explain the persistence of
the bactericidal action for just 1 hour after the serum penicillin had fallen below
the normally bactericidal level. This was not found to be the case. Although
some bacterial species previously exposed to maximally effective concentrations of
penicillin were thereby rendered somewhat more sensitive to low concentra-
tions of the drug (cf. table 8), the differences were slight. Thus, when group
A streptococci and type III pneumococci were exposed in vitro to 0.12 ,ug per ml
TABLE 8
The susceptibility to sodium penicillin G of bacteria exposed to bactericidal
concentrations of the drug in vitro and damaged but not yet killed
by that exposure
Bacterial cultures in the logarithmic phase of growth were diluted to contain 106 to
107 organisms per ml by microscopic count and exposed to 0.12 ,ug per ml of penicillin for
the times indicated in the table. The suspensions were then plated out in blood agar con-
taining varying concentrations of penicillin in order to determine the levels bactericidal
for the surviving and damaged organisms.
PRELrMINAILY
SUSCEPTIBILITY OF SURVIVnG ORGANSMS TO PENICILLIN
EXPOSURE TO
pENICILLIN Smallest conc. with definite Conc. necessary to kill
BACTERIAL SPECIES bactericidal action, pg/ml 99t999%t
Hours hklled Penicillin-
%
exposed Normal bacteria Penicillin-
exposed Normal bacteria
bacteianl bacteria

Streptococcus pyogenes (C- 1j 99.3 0.002 0.005 0.006 0.007

203)
Diplococcus pneumoniae, 2 99.4 0.006 0.014-0.017 0.012 0.024
type III
Staphylococcus aureus 4 89 0.016 0.017-0.02 0.032 0.028-0.034
(Smith)
Group B ,-hemolytic strep- 3 98 0.012 0.017-0.02 0.024 0.028
tococcus (F-20)
* Determined by plating out the reacting mixture in blood agar containing varying
concentrations of penicillin (in the case of the group A streptococcus, e.g., 0.002, 0.003,
0.004, 0.006, 0.008, and 0.012 micrograms per ml).
t I.e., 99.999 per cent of original suspension.
long enough to kill approximately 99 per cent of the organisms, the concentra-
tions then necessary to kill the surviving organisms were approximately half
those necessary to kill normal organisms of the strain. (There is no information
as to whether this difference represents an actual increase in sensitivity to
penicillin or whether the organisms had irreversibly bound some penicillin
during their initial exposure to the higher concentration.) With the strains of
group B streptococcus and Staphylococcus aureus, however, the surviving organ-
isms were not abnormally susceptible to penicillin, requiring just as high a con-
centration as normal untreated bacteria.
In vivo also, when group A streptococci were exposed to relatively high con-
640 H. EAGLE, R. FLEISCHMAN, AND A. D. MUSSELMAN [VOL. 59
10,000 z A 10,000
V. A B
oX
6
,w 0.24 mg/kg
Z 1,000 - 1,000

0
o.j
z<
;i-
(90 00 0 Z 100
.. 0
048 mo
z aNOTREATMENTg
ADDITIONAL, _ 0.1

o !i 10 0 24 mg/kg

o1- 0 1048
g/kg m/kg

I,
- g 0~~~~~~~~926Mg
/kg12mgk
0 ~ ~ BCE /k A7
0.1 12 0.1
PENICILLIN-DAMAGED NORMAL
BACTERIA BACTERIA
0.01 ic "lw
v I 2 4 0
I
2
I
4
0.01
PRELIMINARY
WITREATMENTS
WITH AQUEOUS
PENICILLIN
HOURS AFTER ADMINISTRATION OF PROCAINE PENICILLIN

Figure S. The susceptibility to penicillin of group A streptococci that have previously

been exposed in vivo to bactericidal concentrations of the drug. Mice were inoculated intra-
muscularly with 200,000 group A streptococci (by microscopic count) and immediately
treated with 1 mg per kg of aqueous penicillin G. One and one-half hours later, when ap-
proximately 83 per cent of the organisms had been killed and the serum penicillin had fallen
to wholly ineffective levels, the mice were retreated with varying doses of procaine penicil-
lin G (suspended in oil with 2 per cent aluminum monostearate), as indicated in section A
of the figure. At intervals thereafter the inoculated muscle was removed from groups of 3
mice and emulsified in a Waring blender, and the number of surviving organisms was de-
termined by plating out on blood agar containing 0.02 per cent "clarase" (Takamine) in
order to inactivate the tissue penicillin. The points were plotted in the figure as the geomet-
ric means of the organisms surviving at each time period, referred to the number recovered
from control animals immediately after inoculation as 100. The number so recovered ini-
tially was 1.8 times the number of colonies obtained by plating out the actual inoculum,
the paradoxical increase reflecting the fact that small bacterial chains and clumps were
partially broken up in the blender.
Section B of the figure shows the results in a parallel experiment, in which mice inocu-
lated with 20,000 organisms were immediately treated with varying doses of procaine peni-
cillin, without preliminary exposure to penicillin. As shown by a comparison of sections A
and B, there was no difference in the effective dose of procaine penicillin in the two experi-
ments and, thus, no difference in the penicillin-sensitivities of normal bacteria and bacteria
previously exposed to maximally effective concentrations of the drug. In both instances a
dosage of 0.24 mg per kg had no significant effect, 0.48 mg per kg was definitely bactericidal,
and dosages of 0.96 and 1.92 mg per kg were correspondingly more effective.
Despite the small numbers of animals used and the large individual variation, the stand-
ard error of the logarithms of the means plotted in the figure varied only between 0.02 and
0.3 (X 1.05-2.0) in the 1-hour groups, and (1 value excepted) 0.098 and 0.294 (X 1.26-1.97)
in the 2-hour groups. By the fourth hour, the differences between individual mice were much
greater, and the standard error was correspondingly larger, 0.114 to 0.82 (X. 1.3-6.6).
1950] CONCENTRATIONS OF PENICILLIN 641
centrations of the drug, there was no indication that the surviving and damaged
organisms had thereby been rendered more vulnerable to low concentrations. In
the experiment of figure 3 more than 80 per cent of the organisms had been killed
by a preliminary dose of sodium penicillin, and the survivors had been so damaged
that even in the absence of further treatment they did not resume multiplication
for a period of more than 4 hours. Nevertheless, as is shown in the figure, the
same dosage of penicillin was necessary in order to continue the bactericidal ac-
tion on the damaged organisms as was required to initiate it in normal, pre-
viously untreated bacteria.
The continuing death of bacteria in vivo after the serum penicillin has fallen
below the supposedly minimum effective level is therefore not due to the increased
susceptibility of the surviving organisms to low concentrations of penicillin.
The phenomenon is, however, adequately explained by the fact that, with some
bacterial species, organisms that have been damaged but not killed by penicillin
remain susceptible to the defense mechanisms of the host for a number of hours
thereafter (Eagle, 1949; Eagle and Musselman, 1949). In this indirect manner
the therapeutic activity of penicillin continues for a time after the drug itself
has disappeared, until the bacteria have recovered sufficiently from the toxic
effects of the drug to resume multiplication.
SUMMARY AND CONCLUgIONS
The abortive dose of penicillin was determined for a number of experimental
infections in mice and rabbits, using the procaine salt suspended in oil with
aluminum monostearate. Because of the relatively stable and reproducible
serum concentrations observed with this preparation, one could thus estimate
from the ED5o dose the minimum serum concentration that provided an effective
level at the focus of infection.
(a) In mice the minimum effective serum concentrations were approximately
0.012, 0.08, and 0.08 &g per ml for a group A streptococcus, a group B strepto-
coccus, and a type III Diplococcus pneumonia, respectively, all inoculated in-
tramuscularly. For both type I and type III Diplococcus pneumoniae inoculated in-
traperitoneally, the minimum effective serum concentration was 0.05 ,ug per ml.
(b) In rabbits inoculated with the group B streptococcus intramuscularly,
subcutaneously, intratesticularly, and directly into the lung, the minimal effec-
tive serum concentrations were approximately 0.12, 0.1, 0.07, and 0.1 yg per ml,
respectively.
(c) In the case of Treponema pallidum in rabbits the minim serum concen-
trations that supplied a treponemicidal level at a testicular lesion were estimated
to be on the order of 0.005 to 0.01 Mg per ml. These values were not significantly
affected by the size of the inoculum or the age of the infection.
With each of four organisms tested the smallest dose of aqueous sodium
penicillin that had a bactericidal effect at an intramuscular focus provided
serum concentrations of the same order as those provided by the minimum effec-
tive dose of procaine penicillin in oil.
These minimum effective serum concentrations were regularly 2 to 5 times
642 H. EAGLE, R. FLEISCHMAN, AND A. D. MUSSELMAN [VOL. 59
the concentrations necessary to kill the same organisms in vitro. However, since
approximately half of the serum penicillin is bound to the serum proteins and
not immediately available for diffusion into the tissues, and since in addition
there is probably a concentration gradient between the diffusible serum peni-
cillin and the tissue fluids, one may conclude that the effective concentration
of penicillin at the actual focus of infection is of the same order of magnitude as
that which is operative in vitro.
There was no evidence in these experiments that penicillin persists at ef-
fective levels in the tissues for a significant period after it has fallen below those
levels in the serum. Further, when bacteria were exposed to lethal concentra-
tions of penicillin in vivo or in vitro, the surviving and damaged organisms were
not thereby sensitized to the drug. The concentrations necessary to continue
the bactericidal action on the damaged bacteria were essentially the same as
those originally necessary to initiate it.
From the foregoing considerations one may conclude that for the foci of
infection here studied the duration of the direct bactericidal action of penicillin
in vivo is generally approximated by the time for which the serum concentrations
remain in excess of 2 to 5 times the minimal bactericidal level in vitro.
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