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IHIIIS '

" This is to certify that the

' thesis entitled

The effects of subinhibitory concentration of

I' chloromycetin, aureomycin and dehydrostreptomycin
on Salmonella choleraesuis, Salmonella gyphimurium
and Pasteurella multocida in Vitro arr! 1.13 Vivo.

presented by

  
   
       
 

Charles H. Kupsky

has been accepted towards fulfillment
of the requirements for

M. 5. degree in BaCteriOlogy

WWW

Major professor

Date J uly 29, 1952

 

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THE EFFECT OF SUBINHIBITORY CONCENTRATIONS OF
CHLOROMYCETIN, AUREOMYCIN, AND DIHYDROSTREP-
TOMYCIN ON SALMONELLA CHOLERAESUIS,
SALMONELLA TYPHIMURIUM, AND
PASTEURELLA MULTOCIDA IN
VITRO AND IN VIVO

 

 

 

BY

CHARLES H. KUPSKY
‘u—v

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Bacte riology

1952

 

 

Thieve-

'll.l|l['|llll||l

 

 

/,’a —/9""§ 3* l

“7* a

To Beverly

and Kathleen

rig-‘5‘“ "J ‘
“-«i §<)Q(),;

ACKNOWLEDGMENTS

The writer wishes to extend his sincere appreciation to
Dr. H. W. Dunne for his guidance and advice throughout this
inve stigation.

Gratefulness is expressed to Dr. H. J. Stafseth, Dr. C.
A. Hoppert, and Dr. R. Czarnecki for their constructive criti-
cisms and valuable suggestions.

The author expresses his gratitude to Miss E. M. Smith
for her aid and cooperation during this study.

Thanks are extended to Dr. J. P. Newman and Dr. J. J.
Stockton for use of their animal room during the in vivo portion
of this investigation.

Gratitude is expressed for the special graduate assistant-
ship received from V. M. Division, Biological Laboratories,
Chemical Corps, at Camp Detrick, Frederick, Maryland.

The writer is grateful for the bacterial cultures received
from Miss Lisa Neu, Michigan State College, East Lansing, Mich-
igan; Miss Mary Mayer, Michigan State College, East Lansing,

Michigan; Dr. W. W. Ferguson, Michigan State Health Laboratories,

iv
Lansing, Michigan; Dr. D. W. Bruner, Cornell University, Ithaca,
New York; Dr. L. C. Ferguson, Ohio State College, Columbus,
Ohio; Dr. S. M. Morrison, Colorado Agricultural and Mechan-
ical College, Fort Collins, Colorado; and Dr. E. Nelson, Iowa

State College, Ames, Iowa.

INTRODUCTION . .

TABLE OF CONTENTS

REVIEW OF LITERATURE ...................

Dihydrostreptomycin ......................

Aureomycin . . .

Chloromycetin (chloramphenical) .............

METHODS AND MA

TE RIALS ..................

Sensitivity Determinations ..................

In Vitro Experiments .....................

Inoculation of

media ....................

Morphologic observations .............. . .

In Vivo Experiments ........... . ..........

RES UL TS AND DISCUSSION ...................

Sensitivity Determinations ..................

Morphology . .. .

Biochemical Reactions ....................

In Vivo Re sults

Chloromycetin

15

Z4

Z4

Z9

30

32

33

40

40

50

50

52

53

Page
Dihydro streptomycin .................... 60
Aureomycin .......................... 61
SUMMARY ............................... 67

BIBLIOGRAPHY ........................... 71

INTRODUCTION

The field of antibiotics, though relatively new, has pro-
gressed at an extremely rapid pace. Since the discovery of
penicillin 12 years ago, many new antibiotics have been uncov-
ered from microorganisms that include bacteria, fungi, actino-
mycetes, and protozoa. These antibiotics have been extensively
investigated in vitro and in vivo for their chemotherapeutic val-
ues in combating pathogenic organisms. Nevertheless, there
are phases which require more complete study to better under-
stand the effect of antibiotics on pathogens; for example: (1)
possible immunogenic stimulation by an organism treated with
a subminimal concentration of an antibiotic when subsequently
inoculated into an animal; (2) morphological and biological vari-
ation in these antibiotic-treated organisms.

With these thoughts in mind, this investigation was un-
dertaken to add or confirm some knowledge of the in vitro and
in vivo effects produced on pathogenic organisms, subjected to

subinhibitory concentrations of an antibiotic.

REVIEW OF LITERATURE

Dihydrostreptomycin

The actinomycetes were recorded possessing lytic ac-
tion upon certain bacteria as far back as the late part of the
nineteenth century. It was not until 1944 that Schatz, Bugie,
and Waksman (1944) obtained the antibiotic, streptomycin, from

Actinomycetes griseus that possesses clinical value as a chemo-

 

therapeutic agent. However, from the very start it was realized
that streptomycin was not a good chemotherapeutic agent be-
cause of its toxic effects and severe side reactions. Many at-
tempts were made to produce a more nearly pure streptomycin
in hopes of reducing its toxicity, but this was to no avail as
acute toxic symptoms still accompanied treatment. In 1946,
Peck g al_. (1946) produced a modified form of streptomycin

by catalytically hydrogenating it. This man-made modification
was called dihydrostreptomycin. The reduced form of strepto-
mycin differed chemically from streptomycin in that the carbonyl
group of the streptobiosamine moiety was reduced to an alcohol,

and also differed in that it was less toxic.

Waksman (1947) described streptomycin chemically as a
nitrogen-containing glycoside that showed basic properties. He
further stated that it could be crystallized by many methods
and that it was a thermostable compound, soluble in water and
insoluble in ether.

Oswald and Nielsen (1947) determined the stability of
streptomycin by holding solutions of 100 micrograms per milli-
liter and 1,000 micrograms per milliliter at pH 6, pH 7, and
pH 8 at a temperature of 10 degrees centigrade. He found that
all solutions remained stable for 3 months. Similar studies
were made by Erlanson (1951), with concentrations of strepto-
mycin of 100,000 micrograms per milliliter and at a tempera-
ture of 5 degrees centigrade. He found that this solution re-
mained stable for 3 months. Wolinsky and Stukin (1946) found
that the bacteriostatic effect of streptomycin diminished con-
siderably as the culture neared a pH of 5.5. Bartz gt 9.1. (1946)
determined from their experiments that dihydrostreptomycin was
more stable in an alkaline solution than streptomycin. To con—
firm this, Donavich and Rake (1947) found dihydrostreptomycin
to be more effective when the medium was at pH 9. Pratt and

Dufrenoy (1949) revealed that at room temperature, solutions

4

at pH 6.6 lost about one-third of their activity in 2 weeks. In
the dry state dihydrostreptomycin remained stable for 18 months
at room temperature.’

The studies of Schatz gt a}, (1944) showed the mode of
action of streptomycin to be bacteriostatic, but under certain
conditions it possessed strong bactericidal properties like strep-
tothricin. Studies by Garrod (1948) brought forth various fac-
tors that determined whether streptomycin was bactericidal.

The factors were as follows: (1) concentration of antibiotic;
(2) optimum temperature; (3) strain of organism; (4) nature of
medium; (5) reaction of medium; (6) size of inoculum. Hamre
e_t 11, (1946) found streptomycin to be bactericidal for multiply—
ing and nonmultiplying Klebsiella Hneumoniae. The bactericidal
action was also confirmed by Erlanson (1951). The postulated
theory of the bacteriostatic action of streptomycin by Henry

e_t 9.1. (1948) and Berkman e_t a_l_. (1948) was that streptomycin
had “an inhibitory effect on an enzyme or enzymes of a suscep-
tible organism, that were involved in the metabolism of carbo-
hydrates or that streptomycin inhibited their formation. Their
experiments revealed that catalase, carbonic anhydrase, urease,

carboxylase, cytochrome, succinoxidose, trypsin, or cytochrome

oxidase were not inhibited by high concentrations of streptomy-
cin. Brink and Folkers (1950) found that there was an involve-
ment of the pyruvate-oxalacetate condensation in the action of
streptomycin and that the terminal respiration process was in-
hibited.

Donavich and Rake (1947) studied the toxic effects of
dihydrostreptomycin in mice and found the toxic manifestations
to be similar to those of streptomycin in mice. Pratt and
Dufrenoy (1949) indicated that although streptomycin and dihy-
drostreptomycin are similar in many respects, they did differ
pharmaceutically. Indications were that dihydrostreptomycin
was approximately one-third as toxic as streptomycin. Toxic
symptoms of streptomycin were of both the chronic and acute
types. The work of Brink and Folkers (1950) further confirmed
the conclusions of Pratt and Dufrenoy that dihydrostreptomycin
was less toxic than streptomycin. The L.D.50 toxicity level of
the dihydrostreptomycin in mice was shown by Baron (1950) to
be 225 milligrams per kilogram weight of mouse.

The antimicrobial spectrum of streptomycin was first
studied by Schatz, Bugie, and Waksman (1944). Streptomycin

was found to be active in vitro against gram-negative pathogens

and gram-positive mycobacteria when compared to streptothricin,
which is more specific for gram—positive organisms.
Streptomycin studies on salmonellae were undertaken by
Seligmann and Wassermann (1947), using sixty different types
and 226 strains of salmonella. The antibacterial range was
shown to be between four and eight units of streptomycin. Sen-
sitivity of animal-originated bacteria was investigated by Cole
(1948), in which the author had included eleven strains of S.

choleraesuis, three strains of S. typhimurium, and seventy-eight

 

strains of E... multocida. The ranges of sensitivities were, re-
spectively, 5.0 to 10.0 micrograms per milliliter of streptomy-

cin for S. choleraesuis, 10.0 micrograms per milliliter of strep-

 

tomycin for S. typhimurium, and 1.0 to 10.0 micrograms per
milliliter of streptomycin for I_3_. multocida.
Mice to be used in a Japanese Bacillus encephalitis vac-

cine were found to be infected with Salmonella enteritidis by

 

Slanetz (1946). The treatment used consisted of adding strep-
tomycin to the drinking water in concentrations sufficient to
give 100 units per mouse per day. This was proved very satis-

factory, as only one mouse out of six colonies was S. enteritidis

positive after a short duration. Further work was done by

Slanetz on the susceptibility of S. enteritidis and S. typhimurium

 

to streptomycin. The results indicated that _S_. enteritidis was

 

more susceptible to the antibiotic than S. typhimurium. S. t —

 

phimurium and S. choleraesuis experimentally infected mice

 

 

were studied by Seligmann and Wassermann (1947). The mice
were treated with streptomycin by the, oral route and by simul-
taneous oral and subcutaneous routes, with the resulting supres-
sion of the organisms. However, after termination of the experi-
ment, the organisms reappeared and the death-rate curve re-
sembled that of the control animals. It was concluded that the
only effect of the streptomycin was to prolong the survival time
of the mice. Cole (1948) made a similar type investigation of
E. multocida, except that sufficiently high concentrations were
used to produce 100 percent protection against the organism.
Mice, inoculated with 0.1 cubic centimeter of undiluted culture
intraperitoneally, received between 0 and 400 micrograms of
streptomycin per milliliter of broth and received no protection,
whereas mice obtaining 2,000 to 4,000 micrograms per milli-

liter were protected 100 percent.

Peck e_t ai. (1946) produced the modified streptomycin,
dihydrostreptomycin, and compared the activities of both anti-
biotics. They accomplished this by using the same quantity of
each antibiotic and then comparing the activities in units per
milligram. It was found that dihydrostreptomycin had an activity
of 750 units per milligram, whereas streptomycin had an activity
of 800 units per milligram. The antibiotics were further com-
pared by attempting to obtain 50 percent protection of mice
against one lethal dose. This comparison was accomplished
by Dr. H. Robinson and Mr. O. Graessle of Merck Institute
for Therapeutic Research (Peck e_t a” 1946). The results
show that it required 85 units of streptomycin trichloride. A
comparison of antibacterial activity in vitro was made by Bartz
g _a_1_1_. (1946) in the same year; the activity was found to be al-
most the same for both antibiotics. For example, Bacillus sub-
ti_l_i_§ required 1.0 microgram per milliliter of streptomycin, and
0.3 microgram per milliliter of dihydrostreptomycin. In 1947,
Donavich and Rake (1947) studied some of the biological aspects
of dihydrostreptomycin and compared them with those of strep-
tomycin. With the ten organisms used in their antibacterial

activity determinations, they found dihydrostreptomycin to be

9

slightly less active against all the organisms except Mycobac-

 

terium tuberculosis. Pratt and Dufrenoy (1949) concluded that
streptomycin and dihydrostreptomycin have essentially the same
antibacterial activity, since the examples cited revealed that the
concentration of the two antibiotics differed by less than a micro—

gram.
Aureomycin

After the discovery of streptomycin and its modification,
dihydrostreptomycin, screening programs to discover new anti-
biotics were greatly intensified, with the result that Duggar
(1948) discovered aureomycin. While studying many strains
and species of microorganisms in his particular screening pro-
gram, he discovered an actinomycete, Streptorryces aurefaciens,
which synthesized aureomycin.

Aureomycin was described by Duggar (1948) and Baron
(1950) as a crystallizable antibiotic which contained lemon-yellow
rhomboid crystals. Chemically, aureomycin was a weak base
that contained nitrogen and nonionic chlorine, as reported by
Broschard gt a1. (1949), and readily formed the hydrochloride

salt. Baron (1950) further revealed that it had a molecular

10
weight of 508, decomposed at 210 degrees centigrade, and had
a solubility of 14 milligrams per milliliter of water at 25 de-
grees centigrade.

The stability of the antibiotic was investigated by Bliss
and Chandler (1948) and Chandler and Bliss (1948), and also re-
ported by Baron (1950). The results indicated that room tem-
perature caused rapid deterioration of the antibiotic, as there
was a 50-percent loss of activity in 24 hours, 60 percent in
18 hours at 37 degrees centigrade, and 75 percent in 4 hours
at 56 degrees centigrade. Price, Randall, and Welch (1948)
used various media at room temperature, but found that even
in distilled water there was rapid deterioration. Paine g 31;
(1948) found aureomycin to be stable in solution at high concen-
tration, 200 milligrams per milliliter at 4 degrees centigrade,
for 2 weeks. Erlanson (1951) reported aureomycin stable for
3 months at 5 degrees centigrade at a concentration of 1,000
micrograms per milliliter. Paine gt _a_l_. also found aureomycin
to be most active and stable in an acid pH range, and much
less active and stable in the alkaline range.

The mode of action of aureomycin was found to be bac—

teriostatic by Chandler and Bliss (1948), as the growth curves

ll
of their experiments demonstrated a steady decline of growth
of organisms for 24 hours, and then a steady increased growth
thereafter. During the same year, Bryer _e_t_ 3.1. (1948) found
aureomycin to be bactericidal in high concentrations. Lomis
(1950) studied the mechanism of this action and postulated that
aureomycin depressed phosphorylation without inhibiting respira-
tion of the organism. Van Meter and Oleson (1951) studied the
action of aureomycin on the respiration of normal rat-liver
homogenates, and through their work and data, suggested that
aureomycin could possibly block some part of the Krebs cycle.

The toxicity of aureomycin was investigated by the de-
termination of the L.D. in mice for various routes of inocu-

50

.lations. The L.D.50 was 50 to 100 milligrams per kilogram
for the intravenous route, and was 3,000 to 4,000 milligrams
per kilogram for the subcutaneous route. The work was done
by Harned g; _a_l_. (1948), Bryer _e_t_ a}: (1948), and Goldin and
Stern (1951).

The antibacterial spectrum of aureomycin was shown by
Duggar (1948) to include both gram-positive and gram-negative

organisms. This work was confirmed by the investigations of

Paine, Collins, and Finland (1948), Bliss and Chandler (1948),

12

Price, Randall, and Welch (1948), Broschard _e_t_ §_1_. (1949), and
Neter (1950). The activity of aureomycin against gram—positive
organisms was shown to be of a lower order than that of peni-
cillin by Bliss and Chandler (1948), and polymyxin was more
active against gram—negative organisms than aureomycin. How-
ever, Paine, Collins, and Finland (1948) studied the effect of
aureomycin on penicillin-resistant strains, streptomycin-resistant
strains, and streptomycin-dependent strains, and indicated that
the resistance developed by these strains to a particular anti-
biotic (penicillin or streptomycin) had no effect on the activity
of aureomycin.

Included among the organisms Duggar used in his initial

work were fifty species of Salmonella pullorum and Eberthella

 

 

typhosa, which he indicated were readily susceptible to aureo-
mycin. Paine, Collins, and Finland (1948) obtained sensitivities

of many organisms in vitro, including S. choleraesuis. Their

 

data showed S. choleraesuis to be susceptible to 6.3 micrograms

 

per milliliter of aureomycin, whereas 250 micrograms per mil-
liliter of streptomycin was required to obtain the end point.

-1
They also studied the effect of aureomycin on large 10 dilu-

tion and small 10“4 dilution of members of Staphylococcus

 

l3
pyogenes, and found greatest activity of aureomycin on the small
inoculum, which had displayed the more vigorous growth. Price
_e_t _a_l. (1948) ran sensitivity tests in vitro on various gram-
positive and gram-negative organisms, among which were twelve

strains of salmonellae. One strain of S. typhimurium was in-

 

hibited by 1.56 micrograms of aureomycin per milliliter in 24
hours. Clinical studies of aureomycin on five typhoid-fever
patients and three patients with salmonella infections were re-
ported by Collins, Paine, and Finland (1948). Success was in-
dicated in the cases of typhoid fever, as all patients recovered;
however, two of the three salmonella-infected patients died from
a severe bacteremia.

Neter and Gorzynski (1950) investigated the susceptibility

of eight different strains of _P_. multocida to aureomycin in vitro.

 

They used a large inoculum of bacteria containing one million
to one hundred million organisms per milliliter, and a small
inoculum containing one hundred to one thousand organisms per
milliliter. The sensitivity range of the small inoculum of 2.

multocida was 0.01 to 0.3 microgram per milliliter, whereas

 

the large inoculum required from 0.1 to 3 micrograms per mil-

liliter for inhibition of growth. Little (1948) experimentally

14

infected one-day-old chicks with E. multocida, and then gave

 

doses of aureomycin by the following routes: intra—abdominally,
intramuscularly, and orally. Only the intra-abdominal route
gave lOO-percent protection, and this required 10 milligrams
per kilogram weight of the chick. Neter, Gorzynski, and Cass
(1951) infected mice intraperitoneally with a 10..4 dilution of

E. multocida, and then gave aureomycin subcutaneously in doses

 

ranging from 0.2 milligram to 2 milligrams to different groups
of mice. They concluded from their data that aureomycin was
effective in delaying death and lowering the fatality rate over
a 15-day period. The clinical usefulness of aureomycin was
investigated by Neter, DeKleine, and Egan (1951).) The case
reported was that of a lS-year-old boy who had been kicked
in the face by a horse in 1944. Loss of the nasal bones re-
quired plastic repair with the implantation of preserved human
cartilage in the summer of 1949. Inflamation and a purulent
discharge from the nose instigated a bacteriological investiga-

tion, revealing a pure culture of _Ii. multocida. Ten months

 

later, treatment with aureomycin was begun and continued for

3 weeks, resulting in marked clinical improvement.

15

Chloromycetin (chloramphenical)

The soils in the vicinity of Caracas, Venezuela, contain
a genus of actinomycetes (streptomyces) that is of great signif-
icance to the field of antibiotics. This genus is found in many
other soils throughout the world; however, it was from a soil
sample from this particular area that Dr. Paul R. Burkholder
(Ehrlich _e_t_ 11;, 1947) first isolated Streptomyces venezuelae.
Agar streak cultures of this organism by Ehrlich g; 11:. demon-
strated its antibacterial activity upon Bacillus subtilis, Strepto-
coccus pyoggnes, Salmonella schottmuelleri, and various other
organisms. Growing Streptomyces venezuelae in submerged
aerated culture with various types of media, it was discovered
that the organism produced Chloromycetin, a new antibiotic.
Since this initial discovery, a similar, if not identical, culture
of this streptomyces species has been isolated from a compost
pile in Illinois (Pratt and Dufrenoy, 1949). Parke, Davis and
Company (Neter, 1950) has furthered the importance of chloro-
mycetin by synthesizing it in its research laboratories. Chlor-
amphenical, the laboratory-synthesized compound in question, is

the only one of four possible isomers that possessed the same

l6
biologic, chemical, and physical properties as the biosynthesized
Chloromycetin.

Crystallization of the antibiotic was accomplished by
Ehrlich e_t a1. (1947), while going through the procedures of
purification and concentration of Chloromycetin. The crystals
are colorless needles of elongated plates. The crystallized
antibiotic is a neutral compound that contains nitrogen and non-
ionic chlorine within its structure. The name "Chloromycetin”
was originated because of the large amount of chlorine in the
molecule. The solubility of the compound in water was found
to be about 2.5 milligrams per milliliter at 25 degrees centi-
grade, but was freely soluble in methanol, ethanol, buranol,
acetone, and propylene glycol.

The initial work on Chloromycetin by Ehrlich 91 _a_l_. (1947)
included studies performed to determine the stability of the anti-
biotic in solution. The results of the work indicated that chloro-
mycetin remains stable for more than 24 hours at room tem-
perature within the range of pH 2 to pH 9. When suspended
in distilled water, boiling for 5 hours did not affect the anti—
biotic. The reports of Bartz (1948) and Baron (1950) confirmed

this pH range at which Chloromycetin was stable at room

17
' temperature.- Further work by Bartz (1948) revealed that at
pH 10.82 the antibiotic was only 13-percent active. He also
found that the antibiotic in distilled water remained stable for
a month at 37 degrees centigrade. Erlanson (1951) stored
Chloromycetin at 5 degrees centigrade at a concentration of
1,000 micrograms per milliliter with no loss of activity. In
the dry state, Pratt and Dufrenoy (1949) found the antibiotic

to be thermostable for a longer period of time.

The type of action by Chloromycetin was investigated by
Seligmann and Wassermann (1949), both in vitro and in vivo.
Bacteriocidal action was found to prevail in the in vitro work.
Sensitivity tests were run on twenty-three salmonellae and the
concentration of antibiotic required for growth supression of
these organisms did not increase over a 24-hour incubation
period. However, the course of the disease in treated animals
following termination of treatment was almost identical with
the course in control animals. Their conclusion was that
Chloromycetin reaction in animals was purely bacteriostatic.
Erlanson's (1951) work confirmed the bacteriostatic action of
this antibiotic. Smith g 11; (1949) observed a possible clue

to the bacteriostatic action of chloramphenical in their

18

investigations upon the correlation between the esterase activity

and the growth of Escherichia coli. There was a close agree-

 

ment in the final results, since a low concentration of the an-
tibiotic has no effect on its growth or esterase activity. How—
ever, with increased concentration ,of the antibiotic, there was
a decrease in the growth of the organism, and also a decrease in
esterase activity. Finally, at a therapeutic concentration of
chloramphenicol, there was neither growth of the organism nor
esterase activity.

Toxic manifestations of Chloromycetin were first inves-
tigated by Ehrlich gt 2i- (1947); its L.D. in mice was deter-

50

mined. For a ZO-gram mouse, the intravenous L.D.50 was 3.0

milligrams. The following year, Smith _e_t_ SI. (1948) studied
specific toxic effects on small animals and found Chloromycetin

to be only slightly toxic. The L.D.50 in mice, for the intra-

venous route, was reported as 245 milligrams of antibiotic per
kilogram weight of the mouse. Mice tolerated 1 gram per kilo-
gram orally and 400 milligrams per kilogram subcutaneously.

Baron (1950) determined the mouse L.D.50 for all the different

routes of inoculation. Intravenously, the L.D.50 was 109.5 to

202.6 milligrams per kilogram; intraperitoneally it was 1,320

l9
milligrams per kilogram; orally, 2,640 milligrams per kilogram;
and subcutaneously, 100 milligrams per kilogram.

The antibacterial spectrum of Chloromycetin (chloram-
phenical) was first uncovered by Ehrlich gt _ag1_. (1947), who showed
that the antibiotic was more active against gram-negative organ-
isms than against acid—fast and gram—positive organisms. The
gram-negative Salmonella schottmuelleri required 0.33 micro-
gram of Chloromycetin per milliliter of broth, while the gram-

positive M. tuberculosis required 12.5 micrograms of antibiotic

 

per milliliter of broth. Co-workers of Ehrlich, Smith _e_t_ al.
(1948), confirmed the specific activity of Chloromycetin against
gram-negative organisms by determining the activity of the
antibiotic on many more organisms, both gram-negative and
gram-positive. The activity of Chloromycetin was also compared
to penicillin and streptomycin, with the final results indicating
that Chloromycetin was more active against gram-negative or—
ganisms than either penicillin or streptomycin. McLean _e_t a_I_.
(1949) did a very thorough study of the susceptibility of various
bacteria (including gram-negative and gram-positive) and viruses.
Although many salmonellae were assayed, only three strains of

S. typhimurium were included. The inoculum was a one to ten

 

20
million dilution, and was found to be inhibited between 2.5 micro-
grams per milliliter and 5.0 micrograms per milliliter. Spe-
cific studies on the effect of Chloromycetin on salmonellae were
accomplished by Seligmann and Wassermann (1949). Twenty-
three salmonellae were studied, including S. typhimurium and

S. choleraesuis. The sensitivities indicated that nineteen sal-

 

monellae were susceptible to 2 micrograms per milliliter,
whereas the other four were inhibited by 4 micrograms per
milliliter. In the in vivo experiments which followed, mice were
inoculated either orally or intraperitoneally with 0.1 cubic centi-

meter of an 18-hour broth culture of S. typhimurium, 1908, after

 

which they were treated either orally or subcutaneously with
2,000 to 2,500 micrograms of Chloromycetin each day. At the
end of the treatment, the eighth day, the mice began to die at

a more rapid rate, and at the end of 21 days only a very few
remained alive. Seligmann and Wassermann concluded that
death could be delayed, but not prevented. Ross gt .a_1. (1949)
reported the effects of Chloromycetin in five clinical cases of
salmonellosis. Two cases were initially treated with strepto-
mycin and aureomycin for 4 months with no appreciable results.

However, treatment with Chloromycetin rendered the stool free

21
of salmonella without a later reappearance of the organism.
The third case had a positive test for salmonella in the blood.
This was rapidly cleared with the treatment of Chloromycetin.
The fourth case showed persistently positive stool samples,
and, at the time of publication, had not been affected by the
antibiotic. The last patient had a severe bacteriemia and died
12 hours after the first treatment. Successful treatment of
clinical cases of typhoid fever with Chloromycetin was reported
by Woodward _e_t a__l_. (1948). For the first 24 hours, 40 to 100
gamma per cubic centimeter was given orally. The next 72
hours, 20 gamma per cubic centimeter was administered orally.
Relapses were noticed in only two out of the ten cases. These
two cases were further treated with the same concentration of
Chloromycetin as initially, since no increase in resistance of the
organism was indicated in the in vitro assay. The patients
showed steady progress following the second treatment, and
recovered in the same manner as the other eight. A later pub-
lication by Woodward g §_1_. (1950) included twenty-two cases of
typhoid fever, which received an average Chloromycetin total
of 23.4 grams over an average period of 9.2 days. Four re-

lapses were reported. However, with further treatment these

22
cases recovered rapidly. Similar studies by Neter (1950) brought
forth the spectacular effects of Chloromycetin in typhoid fever in
four cases at Buffalo Children's Hospital. Salmonella or para-
typhoid infections were found to require a much higher dosage
of antibiotic than typhoid-fever infection.

Neter and Gorzynski (1950) determined the minimal
growth inhibitory concentrations of eight different antibiotics,
among which Chloromycetin was included, on eight different

strains of _P_. multocida. The range of inhibitory concentrations

 

for all eight strains of the organism was 0.01 microgram per
milliliter to 10 micrograms per milliliter, although it must be
pointed out that only one strain required 10 micrograms per
milliliter, whereas the other seven strains were inhibited by

1.0 microgram per milliliter. Two assays were run on each
strain; one in which one million to one hundred million organ-
isms per milliliter were inoculated, and the other in which one
hundred to one thousand organisms were inoculated. The smaller
number of bacteria required much less Chloromycetin. The
sensitivities of three strains of Pasteurella avicida were de-
termined by McLean gt _a_l_. (1949) in their broad study of various

organisms. An inoculum of one to two thousand organisms was

23
found to be effectively inhibited by 0.5 microgram per milliliter
to 0.25 microgram per milliliter. These experiments were fur-
ther followed by Gorzynski and Neter (1951), in which the effi-
cacy of Chloromycetin in vivo was determined. The mice were
infected with 104 to 5 x 104 MLD of E. multocida (strain No.
200) and series of experiments were carried out in which the
antibiotic was administered in single and divided doses. The
data show a total concentration of 2.0 to 2.4 milligrams of
Chloromycetin to be more effective when given in multiple doses,
while a total concentration of 0.125 to 0.3 milligram was not

effective in either single or divided doses.

ME THODS AND MATERIALS

Sensitivity Determinations

The fourteen strains of _S_. typhimuriumJ thirteen strains

 

of S. choleraesug, and sixteen strains of E- multocida used

 

 

in this experiment were obtained from various sources. The
strains and numbers of the organisms are as follows:

S. typhimurium:

 

SF-366, SF-361, SF-360, SF-368, 13-4, 13-5,
13—6, 13—7, 13-8, 4-7d, 73-6, O.S.U.-la., N.Y.S.-
3a, and L-169.

. cholerae suis:

IU‘)

 

15-1, 15-2, 15-3, 15-6, 15-9, 15-10, 15-12,
14-8, 8-Al, 19-8, o.s.U.-1, N.Y.S.-3, and L-2.

E. multoc ida:

 

49-50-800, 50-51-939, 651, 49-50-812, 50-51-111,
49-50-638, 1184, 49-50-829, 49-50-618, 49-50-681,
49-50-557, 50-51-110, O.S.U.-1b, N.Y.S.-3b, and

L-169.

25
A stock culture of each organism was maintained in a
semisolid medium that required transferring the organism every
3 to 4 weeks. The salmonellae were kept viable in the follow-

ing medium at 4 degrees centigrade:

Peptone - 1 gram
Sodium chloride - 0.5 gram
Agar - 0.3 gram

Distilled water 100 milliliters
Pasteurellae required a different type stock medium at

25 degrees centigrade, which was made up as follows:

Brain heart

infusion - 3.7 grams
(dehydrated)

Agar - 0.3 gram
Distilled water - 100 milliliters

Sensitivities of the above-mentioned organisms were de-
termined by use of the tube-dilution method. Aureomycin hydro-

l 2 3
chloride, Chloromycetin, and dihydrostreptomycin sulfate were

 

l
Aureomycin hydrochloride was supplied by Lederle
Laboratories.

Chloromycetin was supplied by Merck Laboratories.

3
Dihydrostreptomycin sulfate was supplied by E. R.
Squibb and Sons.

26

the three antibiotics employed in both the in vitro and in vivo
experiments. The procedure followed was the serial-dilution
method, in which twofold dilutions were utilized. This method
was modified to resemble closely the method used by Dorbush
(1949) for the determination of sensitivities of organisms to
aureomycin. Joslyn gt al. (1949) described a similar method
for the determination of sensitivities of organisms to chloro-
mycetin. The method specified the use of 0.5 milliliter of di-
luted antibiotic added to 1.5 milliliters of a one to one hundred
dilution of an l8-hour-old organism in Wasserman tubes, and
incubation for 4 hours. The usual serial dilution was made
with 9 milliliters of a seeded broth, plus 1 milliliter of diluted
antibiotic and was incubated for 16 to 24 hours (Waksman, 1947).
Preparation of the stock solution of Chloromycetin was
accomplished by dissolving the exact amount of antibiotic in
sterile distilled water in concentrations of 200 milligrams per
100 milliliters of solution. The 'desired concentrations of aureo-
mycin hydrochloride and dihydrostreptomycin sulfate were ob-
tained by the addition of an exact amount of sterile distilled
water to the dry powder contained within a sterile vial. Sub-
sequent dilutions in distilled water yielded the stock solution

containing 200 milligrams per 100 milliliters of solution.

27

All stock solutions not used during the day of preparation
were put in sterile stoppered flasks and placed in the freezer,
which maintained a temperature below 10 degrees centigrade.
Stock solutions held for more than 1 month in the frozen state
were not used.

Desired concentrations of the antibiotics were obtained
by diluting the stock solutions in sterile distilled water to pro-
vide an initial concentration of 200 micrograms per milliliter
of solution. This solution was then serially diluted by halves
with broth, through an additional eight dilutions. This was so
calculated that 0.5 milliliter of the diluted solutions, when added
to 1.5 milliliters of the seeded broth, gave the required concen-
trations of antibiotic.

Brain heart infusion broth was employed in the sensitivity
determination of aureomycin hydrochloride and Chloromycetin.
Mycin assay broth was used solely for dihydrostreptomycin
sulfate.

The sensitivity of each organism to each antibiotic was
tested in duplicate in all cases. Three controls were employed
with every assay of antibiotic. The first control consisted of

adding 0.5 milliliter of each concentration of antibiotic to 1.5

28
milliliters of uninoculated broth. A second control consisted
of one tube, which contained 2.0 milliliters of inoculated broth.
A third control also consisted of one tube that contained only
unseeded broth.

A culture known to be sensitive was also included in

each test for comparison. Bacillus cereus No. 5, known to be

 

sensitive, was replaced by S. typhimurium strain 13-4, which

 

was found to be sensitive to all three antibiotics.

The tubes were incubated at 37 degrees centigrade, ex-
amined for visible turbidity at the end of a 4-hour period, and
again at the end of 18 hours. The lowest concentrations of each
antibiotic inhibiting growth at the end of 4 hours and 18 hours
were taken as the end-point readings. Any degree of turbidity
contained in a tube was considered positive, and the end point
was the last clear tube, which was usually easily distinguished.

The subinhibitory concentration of the antibiotic used in
all following experiments was that which did not totally supress

the growth of the organism.

29

In Vitro Experiments

M2912: The media utilized were produced by the Difco
Laboratories. Preparation and sterilization of each individual
medium was accomplished according to directions.

The media used were:

1. Liquids: glucose, maltose, xylose, sorbital,
lactose, arabinose, dextrose, mannitol, mo—
tility medium, indol medium, urea medium,
brain heart infusion broth, and mycin assay
broth.

2. Solids: MacConkey's agar, S. S. agar, blood
agar, gelatin, and Kligler's iron agar.

Ten milliliters of rabbit blood were added to 100 milliliters of
agar base.

All the liquid media, except brain heart infusion broth and
mycin assay broth, were dispensed in sterile Wasserman tubes
and then sterilized according to specification of the Difco Manual.
Sterile Petri plates were used for dispensing the sterilized
solid media, except Kligler's iron agar. Brain heart infusion
broth, mycin assay broth, and Kligler's iron agar were dispensed

in regular-sized sterile test tubes, and then sterilized.

30

Sample tubes and Petri dishes containing the various
media were incubated at 37 degrees centigrade for 24 hours to
check for sterility.

Storage of the media was not required in most instances,
since it was most desirable to use freshly prepared media.
However, when the media were not used immediately, they were
placed in a refrigerator, at a temperature of 4 degrees centi-
grade. Media stored at 4 degrees centigrade for 2 weeks or

longer we re discarded.

Inoculation of media. Each organism was transferred
from its stock culture medium to brain heart infusion broth and
mycin assay broth, and then incubated for 4 hours and 18 hours
at 37 degrees centigrade. At the end of each period, a loopful
of the inoculated broths was transferred to specific media,
depending upon the organism.

Salmonella cultures were inoculated into the following
media:

1. Glucose, maltose, xylose, sorbital, lactose,
arabinose, dextrose, mannitol, motility me-

dium, indol medium, and urea medium.

31

2. MacConkey's agar, S. S. agar, gelatin, and
Kligler's iron agar.

Pasteurellae were inoculated into the following media:

1. Glucose, maltose, xylose, sorbital, lactose,
arabinose, dextrose, mannitol, motility me-
dium, indol medium, and urea medium.

2. Gelatin tubes and blood-agar plates.

All inoculated media were incubated at 37 degrees cen-
tigrade for 24 hours. The various reactions of the organisms
on the media were recorded.

The next phase required the organisms to be treated
with a subinhibitory quantity of antibiotic before being inoculated
in the above media, and then run in parallel with one of the
sensitivity determinations. The sensitivity determinations were
run as previously discussed. Aureomycin hydrochloride, di-
hydrostreptomycin sulfate, and Chloromycetin were used. At
the end of 4 hours, a loopful of the last'tube containing growth
was inoculated in the various media, and then further incubated
at 37 degrees centigrade for 24 to 48 hours. This procedure

was also used at the end of 18 hours. After 24 to 48 hours of

 

 

 

32
further incubation, the reactions obtained upon the media were

tabulated.

Morphol_ogic observations. Slides were made of normal

 

or untreated organisms, and also of organisms treated with
subinhibitory quantities of aureomycin hydrochloride, dihydro-
streptomycin, and Chloromycetin.

The organisms were transferred from the stock cultures
into brain heart infusion broth and mycin assay broth, and incu-
bated at 37 degrees centigrade for 4 hours and 18 hours. At
the termination of these two periods, each tube was agitated,
and a loopful of each culture was placed on a clean slide. The
material was allowed to air dry, and was then warmed over the
flame of a Bunson burner. The slide was stained by the Gram
stain. For the pasteurellae, an additional slide was prepared
and then stained with methylene blue. This procedure was for
the untreated organisms.

At the time of a sensitivity determination, slides, stained
as described above, were made of the organisms which were
treated individually with the antibiotic. Microscopic examination

of each set of slides followed the staining procedure.

33

In Vivo Expe riments

Mice were selected for the in vivo experiments. The
animals were of mixed breeds and came from different colonies.
The weight of each animal used was approximately 20 grams.

Number 10 tin cans were used to house the mice used
in the experiment. Inasmuch as all infected mice were kept in
the same room, these cans provided a means of isolation. Each
can was cleaned every other day to reduce the residual bacteria
contained in the fecal materials Upon completion of an experi-
ment, the can and its accessories (watering bottle and screen
top) were autoclaved and then thoroughly washed in a detergent
before being used in further experiments.

The organisms used in the in vivo experiments were S.

typhimurium strain SF-368 and strain 13—5; S. choleraesuis

 

strain 15-9 and strain 15-6, and _P_. multocida strain 49-50-638.

 

These organisms were subjected to in vitro sensitivity tests,
described in detail in the section on ”Sensitivity Determinations."
These tests served a dual purpose in that they were a check on
the previous sensitivity determinations, and the organism that
was partially inhibited by the antibiotic served as the inoculum

for the mice,

34

S. typhimurium strain SF-368 was employed in the ex-

 

periment with aureomycin, and also with Chloromycetin. At 4
hours, 1.56 micrograms per milliliter of aureomycin were re-
quired, while 6.25 micrograms per milliliter were necessary
at 18 hours for partial inhibition. The subinhibitory concentra-

tion of Chloromycetin for S. typhimurium strain SF-368 was a

 

constant 1.56 micrograms per milliliter for 4 hours and 18

hours. S. typhimurium strain 13-5 was subjected to dihydro-

 

strptomycin. The subinhibitory concentrations for 4 hours and
18 hours were, respectively, 3.13 and 25.0 micrograms per
milliliter.

Chloromycetin and dihydrostreptomycin were used for

S. choleraesuis strain 15-6 experiments. The concentration

 

of Chloromycetin required for subinhibition was 1.56 micrograms
per milliliter for the 4-hour and 18-hour cultures. The 4-hour

incubation of S. choleraesuis strain 15-6 required 0.78 micro-

 

gram per milliliter of dihydrostreptomycin, whereas the organism
required 3.13 micrograms per milliliter at 18 hours for sub-

inhibition. S. choleraesuis strain 15-9 was treated with a sub-

 

inhibitory amount of aureomycin which was 1.56 micrograms per

35
milliliter for 4 hours, and 6.25 micrograms per milliliter for
18 hours.

The subinhibitory concentration of aureomycin required

for 1:. multocida strain 49—50-638 was 0.39 microgram per mil-

 

liliter for both the 4- and 18-hour cultures. 2. multocida strain

 

49-50-638 was also subjected to subinhibitory amounts of dihy-
drostreptomycin. These concentrations were 0.78 and 1.56
micrograms per milliliter, respectively, at 4 and 18 hours of

incubation. 21. multocida strain 49-50-638 required 0.78 micro-

 

gram of Chloromycetin per milliliter for subinhibition at both
4- and 18-hour incubations.

The subinhibitory concentration of each antibiotic for
each organism for the 4- and 18-hour periods was inoculated
into nine series of mice. No further antibiotic treatment of
the organisms followed the initial exposure to in vitro subinhib-
itory concentrations. There were three mice in each series in
which Chloromycetin and dihydrostreptomycin were employed,
whereas six mice were used in each series subjected to aureo-
mycin treatment.

Inoculations were made with sterile 1.0 cubic centimeter

Tuberculin syringes and 27—gauge needles. The inoculum was

36

thoroughly agitated before being drawn into the syringe. The
mice were injected with 0.1 cubic centimeter of the inoculum
intraperitoneally.

The inoculated animals were observed twice a day through-
out the entire time of the experiment.

Control experiments were run in conjunction with the
above-mentioned experiments. Two series of mice were inocu-
lated with brain heart infusion broth and mycin assay broth. A
control series of mice was run on a broth suspension of each
organism, diluted one to one hundred, and incubated for 4 hours
and 18 hours. A third control group of mice was inoculated
with 1.0 milligram per milliliter of each antibiotic, which served
as a toxicity control. All control mice were inoculated intra-
peritoneally with 0.1 cubic centimeter of specified solutions or
suspensions (see Tables IV and V).

All mice which survived, following an injection with an
organism subjected to a subinhibitory concentration of one of
the antibiotics, were challenged with a normal culture of the
same organism to determine if some degree of immunity had

developed.

37
One-tenth cubic centimeter of an untreated, diluted, 18-
hour broth suspension of a specified strain of the organism was
inoculated intraperitoneally with a Tuberculin syringe using a
27-gauge needle. All syringes and needles were sterilized
before use. The quantity used in the case of each organism
was found to be a lethal dose for normal mice.

A brain heart infusion broth suspension of S. choleraesuis

 

strain 15-6 was incubated for 18 hours at 37 degrees centigrade.
This suspension was diluted so that the inoculum used was 0.1
cubic centimeter of a 10"4 dilution of the original 18-hour cul-
ture. One-tenth cubic centimeter of this 10"4 dilution of S.

choleraesuis was found to kill normal mice within six days.

 

Mice that had recovered from an initial injection of S. choleraesuis
strain 15-6 which had been treated with a subinhibitory concen-
tration of Chloromycetin for 4 hours and 18 hours were inocu-
lated with 0.1 cubic centimeter of a 10.4 dilution. Also, mice
recovering from infection with the organism treated with dihy-
drostreptomycin for 4 hours and 18 hours were challenged.

A similar procedure was followed with S. typhimurium

 

strain 13-5. The organism was inoculated into brain heart in-

fusion broth and incubated at 37 degrees centigrade for 18 hours.

38
Mice recovering from injection with S. typhimurium strain 13-5
which had been subjected to minimal concentrations of dihydro-
streptomycin for 4 hours or 18 hours were inoculated with 0.1
. . -4 . .
cubic centimeter of a 10 dilution of the untreated 18-hour
broth suspension of the organism.
A brain heart infusion broth suspension was made of

B. multocida strain 49-50-638 and incubated for 18 hours at

 

37 degrees centigrade. This suspension was diluted so that the
-3
inoculum was a 10 dilution of the original. Mice recovering

from a 2. multocida strain 49-50-638 suspension which had been

 

treated with a subinhibitory concentration of aureomycin for 4
hours or 18 hours were inoculated with the 0.1 cubic centimeter
-3 . . . .
of a 10 dilution of the normal culture. M1ce recovering from

infection with _P_. multocida strain 49-50-638 which had been sub-

 

jected to a subinhibitory concentration of Chloromycetin for 4
. . -3 . .

hours or 18 hours were also inoculated With the 10 dilution

of the normal culture, as were the mice recovering from E.

multocida strain 49—50-638 which had been treated with the

 

minimal concentration of dihydrostreptomycin for 4 hours and

18 hours.

39
Care was exercised in removing dead mice from the cans
containing the challenged mice, and also in removing fecal ma-

te rial.

RESULTS AND DISCUSSION

Sen 5 itivity De te rmination s

The validity of determinations of sensitive microorgan-
isms to antibiotics is based upon the ability to duplicate the
initial results. This applies to the tube-dilution method, or
any other method by which the sensitivity of an organism to
an antibiotic is determined.

Certain physical factors must be kept constant through
several assays to eliminate the variables which could produce
results entirely different from those of the initial determina-
tion.

Most significant is the sensitivity of an organism to the
antibiotic. Trials with test organisms must be run simultane-
ously with tests on organisms of unknown sensitivities. The
end point of the test organism and organism to be tested is
compared, and in this way the degree of sensitivity of the or-
ganism tested is determined. Also, a test organism, whose

sensitivity is known, can be utilized in determining the

41
concentration of antibiotic solutions, the approximate concen-
trations of which are not known.

Secondly, the organism must be of the same strain and
growth phase in each and every assay. Pratt and Dufrenoy
(1949) indicated that different strains of an organism that pro-
duced pulmonary tuberculosis showed a thirtyfold difference in
their sensitivities to streptomycin. The growth phase of the
organism is important because some antibiotics are more ac-
tive on actively metabolizing organisms than on less active,
older organisms.

Thirdly, it is important that the incubation temperatures,
as well as the length of incubation time, be identical in every
assay. If the incubation temperatures are increased, the incu—
bation period should be decreased, as thermolable antibiotics
are significantly affected by the temperature increase over a
specific period of time. This was found to be true of aureo-
mycin. This results in a variable end point, as the activity
of the antibiotic is destroyed or decreased.

A constant number of organisms per assay is essential
for some antibiotics. Increased numbers of organisms result

in a greater concentration of the antibiotic to produce inhibition.

42

Lastly, the medium in which the assay is performed
should remain constant. Waksman (1947) indicated that strep—
tomycin could be inactivated or antagonized when certain sugars,
cysteine, or ketone reagents were added to medium used in as-
saying the antibiotic. This resulted in a deviation of the anti-
bacterial activity of streptomycin.

The consistency of the pH of the medium is also essen-
tial. Streptomycin is most effective in an acid medium. Chlor-
omycetin is effective in either an acid or alkaline medium, since
its effectiveness lies in the pH range from 2 to 9.

There are uncontrollable factors that cause variation in
sensitivity tests, though the factors mentioned have been elim-
inated. The human element is one uncontrollable factor, influ-
encing accuracy of laboratory procedures and interpretation of
results. Another uncontrollable factor is the lack of constant
homogeneity within a bacterial population. Different degrees
of resistance or possibly antibiotic dependence of individual
organisms within a bacterial population could cause a fluctua-
tion of the end point. The growth phase of the organism at
the time of the sensitivity determination would also cause a

fluctuation of the end point. It is logical to assume that all

43
bacteria within a population are not at exactly the same stage
of growth at any specific time. Some bacteria are in the log-
arithmic growth phase, while others are in a lag phase. Gen—
erally, the organisms in the lag phase are least susceptible,
whereas those in the logarithmic phase are most susceptible,
depending, of course, upon the antibiotic used.

Waksman (1948) was of the opinion that the antibacterial
action of each antibiotic was distinct from that of all other anti-
biotics. The difference was determined by the site and type of
action upon the organism. Importance was attached to the vari-
ability of reactions on various substrates, serum, glucose, or
salt.

Jackson gt _a_l_. (1950) concluded from their results that
the enteric organisms as a group vary greatly in their sensi-
tivity to different antibiotics. However, the authors indicated
that the results obtained were valid only for the conditions
under which those tests were maintained, since there were
many factors which could influence the sensitivity determina-

tions, and those factors varied from one antibiotic to another.

44

Maintaining all factors as constant as possible, the sen-
sitivities of all organisms were determined and the results were
tabulated (see Tables I, II, and III).

The susceptibility of the organisms generally varied
from antibiotic to antibiotic, and also among species of the
bacteria within the group. There was also a noticeable change
of susceptibility of the organisms to the antibiotics in the 4-hour
and 18-hour results, with one exception. Chloromycetin main-
tained steady end points for 18 hours' incubation, as compared
to the 4-hour sensitivities. A11 salmonellae and pasteurellae
tested were readily susceptible to Chloromycetin.

The ability of organisms to develop resistance to chloro-
mycetin was indicated by Coffey _e_t_ EE- (1950). After twenty
transfers, the organisms had gradually developed an increased
resistance to the antibiotic in vitro. Woodward g ;a_1_. (1948)

noted lack of development of resistance of Salmonella typhosa

 

in vivo. Patients treated with Chloromycetin had relapses, and
the organism was reisolated from the patients to check the sen-
sitivity of this organism. The results of the sensitivity test
were identical with the initial tests, indicating the lack of de-

veloped resistance.

45

TABLE I

RESULTS OF IN VITRO TESTS FOR SENSITIVITY OF FOUR-
TEEN STRAINS OF SALMONELLA TYPHIMURIUM TO
AUREOMYCIN, CHLOROMYCETIN, AND
DIHYDROSTREPTOMYCINI

 

 

 

 

 

 

 

 

Culture stiigbtgdin Aureomycin Chloromycetin
Number

4 hrs.2 18 hrs. 4 hrs. 18 hrs. 4 hrs. 18 hrs.
SF-360 6.25 50.0 6.25 25.0 6.25 6.25
SF-361 12.5 50.0 12.5 25.0 12.5 12.5
SF-366 6.25 50.0 6.25 25.0 6.25 6.25
SF-368 6.25 50.0 3.13 12.5 3.13, 3.13
13-5 6.25 25.0 12.5 25.0 6.25 6.25
13-6 6.25 50.0 3.13 12.5 6.25 6.25
13-7 8.13 50.0 3.13 25.0 6.25 6.25
13-8b 12.5 50.0 12.5 12.5 3.13 3.13
4-7d 6.25 50.0 1.56 6.25 3.13 3.13
73-6 6.25 50.0 6.25 12.5 6.25 6.25
L-88 12.5 50.0 1.56 12.5 6.25 6.25
OSU-la 6.25 50.0 12.5 25.0 12.5 12.5
13-43 1.56 3.13 1.56 3.13 1.56 1.56
NYS-3a 25.0 50.0 1.56 12.5 6.25 6.25

Micrograms _per milliliter inhibiting growth of or-
ganisms.

Hours of exposure to antibiotic.

Assay organism.

TABLE II

46

RESULTS OF IN VITRO TESTS FOR SENSITIVITY OF THIR-
TEEN STRAINS OF SALMONELLA CHOLERAESUIS TO
AUREOMYCIN, CHLOROMYCETIN, AND

DIHYDROSTREPTOMYCINI

 

 

 

 

 

 

 

 

Hours of exposure to antibiotic.

Culture 5 t:1:t::;;in Aureomyc in Chloromycetin
Number

4 hrs.2 18 hrs. 4 hrs. 18 hrs. 4 hrs. 18 hrs.
14-8 25.0 25.0 12.5 25.0 12.5 12.5
15-1 25.0 50.0 3.13 25.0 12.5 12.5
15-2 6.25 25.0 12.5 25.0 6.25 6.25
15-3 12.5 50.0 6.25 12.5 3.13 3.13
15-6 1.56 6.25 12.5 25.0 3.13 3.13
15-9 25.0 50.0 3.13 6.25 12.5 12.5
15-10 25.0 50.0 12.5 12.5 12.5 12.5
15-12 25.0 50.0 6.25 25.0 12.5 12.5
19-8 25.0 50.0 12.5 50.0 6.25 6.25
8A-1 0.19 0.78 1.56 3.13 12.5 12.5
OSU-l 6.25 50.0 12.5 12.5 6.25 6.25
'NYs-a 25.0 50.0 12.5 25.0 12.5 12.5
L-z 12.5 50.0 I 6.25 12.5 6.25 6.25

Micrograms per milliliter inhibiting growth of or-
ganisms.

TABLE III

47

RESULTS or IN VITRO TESTS FOR SENSITIVITY or SIX-
TEEN STRAINS or PASTEURELLA MULTOCIDA To
AUREOMYCIN, CHLOROMYCETIN, AND

 

DIHYDROS TREPTOMY CIN1

 

 

 

 

 

 

 

 

Culture stififititcin Aureomycin Chloromycetin
Number

4 hrs.2 18 hrs. 4 hrs. 18 hrs. 4 hrs. 18 hrs.
5-11a 1.56 6.25 0.19 0.39 0.39 0.39
5-11b 3.13 6.25 0.78 1.56 0.78 0.78
49—50-

800 1.56 3.13 0.78 1.56 3.13 3.13
50-51-

110 3.13 6.25 0.78 1.56 0.78 0.78
50-51-

111 3.13 6.25 3.13 3.13 1.56 1.56
651 1.56 3.13 0.39 0.78 0.39 0.39
49-50-

812 3.13 3.13 0.39 0.78 0.78 0.78
49-50-

638 1.56 3.13 0.78 0.78 1.56 1.56
1184 0.39 50.0 0.39 3.13 6.25 6.25
49-50-

829 3.13 6.25 3.13 3.13 3.13 3.13
49—50-

618 1.56 6.25 0.78 0.78 1.56 1.56
49-50-

681 1.56 3.13 0.19 0.39 0.39 0.39
49-50-

557 0.78 3.13 0.39 0.78 0.39 0.39
OSU-lb 6.25 50.0 6.25 12.5 0.78 0.78
NYS-3b 0.19 0.78 0.39 0.78 0.39 0.39
L-169 50.0 50.0 0.39 3.13 6.25 6.25

Micrograms per milliliter inhibiting growth of or-
ganisms.

Hours of exposure to antibiotic.

48
Similar effectiveness was noted for aureomycin against

S. choleraesuis and S. typhimurium at the 4-hour incubation.

 

 

However, there was a decided variation in the sensitivities of
the salmonellae at-the end of 18 hours' incubation.- There was
a one- to twofold difference between the time periods needed

to sensitize most of the organisms. _P_. multocida was very

 

susceptible to aureomycin at the end of 4 hours of incubation,
and showed less than a onefold average variation after 18 hours'
incubation. These fluctuating end points are quite conceivable
when one considers the instability of aureomycin in solution at
room temperature. Bliss and Chandler (1948) and Price _e_t_ fl.
(1948) found that low concentrations of aureomycin deteriorated
rapidly even in distilled water at 25 degrees centigrade. Chandler
and Bliss (1948) studied growth curves of bacteria subjected to
low concentrations of aureomycin and found the curves declined
rapidly for 24 hours. After this period of time, the growth
steadily increased.

Dihydrostreptomycin sulfate is less effective, generally,

against S. choleraesuis than either Chloromycetin or aureomycin

 

at both 4- and 18-hour incubations. However, dihydrostreptomycin

was as effective as Chloromycetin and aureomycin against S.

49

typhimurium at the 4-hour incubation. The 18-hour incubation

 

indicated a great fluctuation of the end point, inasmuch as there
was an average threefold difference from the 4-hour results.
Dihydrostreptomycin was more effective against _P_. multocida
strains than against the salmonella strains. At 4 hours' incu-
bation, all the pasteurella strains were susceptible, except
strain L-l69. This organism appeared to be a highly resis-
tant strain, as 50.0 micrograms per milliliter of dihydrostrep-
tomycin did not suppress its growth even slightly. The growth

of three strains of g. multocida, L-l69, O.S.U.-1b, and 1184,

 

similarly was not suppressed by 50.0 micrograms per milliliter
after 18 hours of incubation. Sensitivity tests on the other
pasteurellae indicated an average onefold variation between the
two time intervals.

The variation of the sensitivities of the organism at 18
hours was due to the development of a streptomycin-resistant
variant within the bacterial population, and not to deterioration
of the antibiotic, for it had been found to be relatively stable.
Miller (1948) stated that susceptible strains of bacteria easily
developed resistance to streptomycin, noting that they tolerated

up to 50,000 micrograms per milliliter when transferred twice

50
to streptomycin medium. He further revealed that bacterial
mutation initiated the development of these variant strains,
which consisted of streptomycin-resistant and streptomycin-
dependent organisms. The organism used in this experiment
appeared to be a resistant strain, as no organisms required
streptomycin to remain viable, which is an essential criterion

for the streptomycin-dependent strains.

Morphology

All antibiotic-treated organisms, stained with Cram and
methylene-blue stains, were observed under a binocular micro-
scope and were found to be morphologically the same as the
untreated organisms. Miller (1948) and Paine and Finland (1948)
observed no morphological variation in organisms that were

either resistant to or dependent upon streptomycin.

Biochemical Reactions

The results obtained for both periods of incubation with
the normal organisms and organisms subjected to the antibiotics
were identical. In the cases of aureomycin and Chloromycetin

subinhibitory concentrations, the antibiotic evidently was not in

51
sufficient amounts to inhibit all of the organisms present. This
probability was suggested by the work of various workers Show-
ing the difficulty in producing an aureomycin- or Chloromycetin-
resistant organism.

However, dihydrostreptomycin is one of the antibiotics
to which organisms can easily develop resistance if not in suf-
ficient quantities to be bactericidal. Assuming that the organisms
have developed resistance to a sublethal dose of the antibiotic, it
is easy to understand why the biochemical reactions are the
same as those of untreated organisms. Miller (1948) and Paine
and Finland (1948) studied the effects of streptomycin-dependent
and streptomycin-resistant strains on the various biochemical
reactions. Both papers concluded that dependent and resistant
strains reacted biochemically in a manner identical to that of
normal organisms. Berkman gt a_1.. (1948) stated that strepto-
mycin does not interfere with the carbohydrate metabolism of
dependent or resistant strains, even though the resistant strains
adsorb streptomycin onto their cell surface, as do the suscep-

tible strains .

52

In Vivo Re sults

Thus far, it has been observed that organisms subjected
in vitro to a subinhibitory concentration of an antibiotic showed
no observable effect morphologically or metabolically. Obser-
vation has shown suppression without inhibition of growth. In
vivo investigations were carried out in an attempt to find out
if antibiotic-affected organisms were.more susceptible to the
host's defenses than normal untreated organisms.

Organisms that were found to be highly sensitive to the

antibiotic were selected for the experiment. S. typhimurium

 

strain SF-368 was utilized for Chloromycetin and aureomycin
experiments because it was highly sensitive and was found to
be pathogenic for mice. One-tenth of the culture diluted 10-4,
injected intraperitoneally, killed the mice in six days. An un-
diluted culture (0.1 cc) was also inoculated intraperitoneally into
mice. All mice succumbed within a 36-hour period. S. m-
murium strain SF-368 was sensitive to dihydrostreptomycin after
4 hours‘ incubation, but after 18 hours' incubation the culture

had become resistant to 50 microgramps per milliliter. There-

fore, S. typhimurium strain 13-5 was selected for the tests with

 

53
dihydrostreptomycin, for the organism was more susceptible at
the termination of the 18-hour period. This organism, undiluted,
was found to kill mice in 36 hours. All animals were injected
intraperitoneally with 0.1 cubic centimeter of the culture.

S. choleraesuis was selected for this part of the study

 

because it is pathogenic for mice. S. choleraesuis strain 15-6

 

was selected for the study on Chloromycetin and dihydrostrepto-

mycin, while S. choleraesuis strain 15-9 was found to be suit-

 

able for tests with aureomycin. These organisms, undiluted,
killed inoculated mice in 84 hours (see Table IV).
One strain of pasteurellae was used for the experiments

with all three antibiotics. E.mu1tocida strain 49—50-638 was

 

sensitive to all the antibiotics and killed control mice in 24

hours.

Chloromjcetin. Chloromycetin, in subinhibitory concen-

 

trations, was 100 percent effective against S. choleraesuis strain

15-6 and 2. multocida strain 49-50-638 for both the 4-hour and

 

18-hour periods (see Table V). However, against S. typhimurium

 

strain SF-368 Chloromycetin only seemed to delay death of the

mice. The first death occurred in the 18-hour series at the

 

54
TABLE IV

BROTH, ORGANISM, AND ANTIBIOTIC CONTROLS

 

 

 

 

 

 

 

 

 

 

 

 

1:? Organism Culture Vehicle Antibiotic
(“13‘ Mice Number
’j, 3 §_- typhimurium SF-368 B.H.I.*
": 3 S. typhimurium SF-368 B.H.I.
3 S. typhimurium 13-5 B.H.I.
.WE’J 3 S. typhimurium 13-5 B.H.I.
‘ m. 3 S. choleraesuis 15-6 B.H.I.
3 S. choleraesuis 15-6 B.H.I. l
3 S. choleraesuis 15-9 B.H.I. ‘
3 S. choleraesuis 15-9 B.H.I.
3 E. multocida 49-50-638 B.H.I.
3 g. multocida 49-50-638 B.H.I.
3 B.H.I.
3 B.H.I.
3 M.A.B.**
3 M.A.B.
3 B.H.I. Chloromycetin
3 M.A.B. Dihydm’ .
streptomycm
3 B.H.I. Aureomycin

 

 

* Brain Heart Infusion Broth.
** Mycin Assay Broth.

TABLE IV (Continued)

55

 

 

 

Hours Concentration No. No. Percent
Incubation of Antibiotic Injected / Survived Survived
In Vitro (ug/ml)

4 3 / 0 0
18 3 / o 0
4 3 / 0 0
18 3 / 0 0
4 3 / 0 0
18 3 / 0 0
4 3 / 0 0
18 3 / ° 0
4 3 / 0 0
18 3 / 0 0
4 3 / 3 100
18 3 / 3 100
4 3 / 3 100
18 3 / 3 100

100 3 / 3 100

100 3 / 3 10°

100 3 / 3 100

 

 

56
TABLE V

EFFECT OF SUBINHIBITORY CONCENTRATIONS OF
DIHYDROSTREPTOMYCIN SULFATE, AUREOMYCIN,
HYDROCHLORIDE, AND CHLOROMYCETIN UPON
SALMONELLA TYPHIMURIUM, SALMONELLA
CHOLERAESUIS, AND PASTEURELLA
MULTOCIDA INOCULATED

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

INTO MICE
No.
of Organism Culture Antibiotic
. Number
Mice
4 S. typhimurium SF-368 Chloromycetin
4 S. typhimurium SF-368 Chloromycetin
4 S. choleraesuis 15-6 Chloromycetin
4 S. choleraesuis 15-6 Chloromycetin
4 g. multocida 49-50-638 Chloromycetin
4 a. multocida 49-50-638 Chloromycetin
4 S. typhimurium 13-5 Dihydrostreptomycin
4 S. typhimurium 13-5 Dihydrostreptomycin
4 S. choleraesuis 15-6 Dihydrostreptomycin
4 S. choleraesuis 15-6 Dihydrostreptomycin
4 B. multocida 49-50-638 Dihydrostreptomycin
4 _P_. multocida 49-50-638 Dihydrostreptomycin
6 S. typhimurium SF-368 Aureomycin
6 S. typhimurium SF—368 Aureomycin
6 S. choleraesuis 15-9 Aureomycin
6 S. choleraesuis 15-9 . Aureomycin
6 _11. multocida 49-50-638 Aureomycin
6 E. multocida 49-50-638 Aureomycin

 

 

 

 

 

TABLE v (Continued)

57

 

 

Hours

Subinhibitory

 

. . No. No. Percent

Incubation Concentration 111.“:th / Survived Survived

In Vitro (ug/ml) J
4 1.56 4 / 0 O
18 1.56 4 / 0 0
4 1.56 4 / 4 100
13 1.56 4 / 4 10°
4 0.78 4 I 4 10°
18 0.78 4 / 4 10°
4 3.13 4 / 4 100
13 12.5 4 / 4 10°
4 0.78 4 / 4 10°
13 3.13 4 / 4 100
4 0.78 4 / l 35
18 1.56 4 / 4 10°
4 1.56 6 / 0 0
13 6.25 6 / 0 O
4 1.56 6 l 2 33
13 3.13 6 / 0 0
4 0.39 6 / 3 5°
18 0.39 6 / 3 50

 

 

58
end of the third day, and the last mouse died on the tenth day.
In the 4-hour series, the first mouse died on the ninth day, and
the last mouse died on the eleventh day. Salmonella was iso-
lated from the heart blood agar culture plates.

Studies by Eagle gt a_1_. (1950) on host reaction to peni-
cillin-treated organisms might well be applied to the different
reactions obtained by S. typhimurium strain SF-368 as com-

pared to S. choleraesuis strain 15-6 and B. multocida strain

 

49-50-638. Group A and Group G hemolytic streptococci, and
also Type I and Type III pneumococci were used by Eagle gt 31.
Mice showed little or no protection against these organisms.
They investigated the length of time it took the four bacterial
species in vitro and in vivo to recover from the effect of peni-
cillin, and to begin multiplication. The bacteria were found to
recover more rapidly in vitro than in vivo. Group A and Group
B hemolytic streptococci required 6 to 8 hours to recover and
resume multiplication in the host. During this recovery period,
the viable organisms decreased progressively. It was noted
that 98.6 to 99.6 percent of the viable organisms were appar-
ently killed by the host, indicating that organism‘s damaged by

penicillin were more susceptible to the host than were the

59
untreated bacteria. These organisms showed a decrease in
virulence in later experiments. The reactions of Type I and
Type II pneumococci differed from the streptococci. The pneu-
mococci required 4 hours to recover in vivo before resuming
multiplication, and the decrease in number of viable organisms
ranged from 45 to 65 percent. There was no appreciable evi-
dence of enhanced susceptibility of organisms damaged by peni-
cillin to the host, as these organisms demonstrated only a“
slight decrease in virulence.

The bactericidal properties of Chloromycetin (Seligmann

and Wassermann, 1949) exerted on S. typhimurium strain SF-

 

368, S. choleraesuis strain 15-6, and g. multocida strain 49-

 

 

50-638 might well have affected these organisms like it did
those studied by Eagle gt _al. (1950). The Chloromycetin-affected

S. typhimurium strain SF-368 organisms resembled the Type I

 

and Type III pneumococci used by Eagle _e_t_ _a_l_., in that the
organisms were more susceptible to the host reactions than
were normal organisms. Though the organism was more sus-
ceptible to the host, the degree of susceptibility was not suf-
ficient to enable the host to combat the remaining viable or-

ganisms, with the result that the mice began to die on the

60
third day. The last mouse of this series died on the eleventh
day.

S. choleraesuis strain 15-6 and _P_. multocida strain 49-

 

 

50-638 appeared to be the type of organisms that become highly
susceptible to host reactions after being subjected to Chloromy-
cetin. The mice were well and alive at the end of the 30-day
period. These mice were further used to detect evidence of
resistance, on the part of the host, to subsequent inoculations

of untreated organisms of known pathogenicity.

Dihydrostreptomycin. The general reaction of dihydro-

 

streptomycin appeared to increase the susceptibility of S. typhi-

murium strain 13-5, S. choleraesuis strain 15-6, and E. multo-

 

c_i_c_1g strain 49-50-638 to the body defenses of the host. There
was lOO-percent survival of the mice inoculated with dihydro-
streptomycin-treated S. typhimurium strain 13-5 and S. _c_1'_19_1_-
eraesuis strain 15-6 organisms. The same results were ob-
tained with 18-hour E. multocida strain 49-50-638, but the or-
ganisms incubated for 4 hours apparently were little affected
by the action of dihydrostreptomycin.

The mice which were protected 100 percent from infec-

tions with the various organisms were evidently aided by the

61
damaging effect of dihydrostreptomycin upon those organisms.
As in the Chloromycetin-affected organisms, the dihydrostrep-
tomycin-treated organisms were more susceptible to the host
defense. It is also quite logical to assume that there was a
decrease in virulence of the organism, as was shown by the
Group I and Group II hemolytic streptococci used in the ex-
periments by Eagle 2 gt. (1950). Another possibility is that
the organisms might have become dependent upon dihydrostrep-
tomycin, and that without the antibiotic the organisms could not

remain viable.

Aureomycin. Aureomycin hydrochloride demonstrated

 

less effect upon the organisms tested in vivo than did the other

two antibiotics. Organisms treated with S. Lyphimurium strain

 

SF-368 destroyed their respective hosts within 15 days from
the day of inoculation. The first death was observed on the
fourth day, and the last death was on the fifteenth day. Treated

S. choleraesuis strain 15-9 followed a similar course in de-

 

stroying its hosts. The noticeable difference between the two
salmonellae was that S. choleraesuis strain 15-9 did not kill
its hosts as rapidly as the antibiotic-treated S. typhimurium

strain SF-368. The first death was observed on the seventh

62
day, and the last death was recorded on the thirty-second day.
This prolonged survival time of the infected mice seemed to
indicate that, although aureomycin deteriorates rapidly in vitro,
the organisms were affected sufficiently to enable the defense
of the host to respond effectively. Another reason for the pro-
longed survival time could have been a decrease in virulence
of the organism for the host resulting from the action of the
antibiotic before deteriorating.

Aureomycin-treated _P_. multocida strain 49-50-638 was

 

only 50-percent effective in killing its hosts (Table IV). The
organisms that killed their respective hosts did so within a
2-day period. The first death was recorded on the second day
after inoculation, and the last death occurred on the fourth day.
All mice surviving the fourth day remained alive and well un-
til the termination of this experiment. These results could
have been due to one of the many uncontrollable variables in
the in vivo experiments, including individual susceptibility of
the host.

All mice surviving injection with antibiotic-treated or-
ganisms were challenged with a lethal dose of the untreated

organism. All mice survived a 10-day test period. This

63
indicates that there might have been an effective immunological
response by the host to the antibiotic-treated organism. Normal
control mice received a subsequent inoculation of the same lethal
dose given to the recovery mice. All mice in the control group

receiving S. choleraesuis strain 15-6 were dead by the end of

 

the sixth day, as were the mice receiving S. typhimurium strain

 

13-5. The mice in the third series, receiving 0.1 cubic centi-

meter of a 10“3 dilution of R. multocida strain 49-50-638 died

 

on the fifth day (see Table'VI).

Unfortunately, the in vivo data are not sufficient to justify
definite conclusions as to the effect of a subinhibitory concen-
tration of an antibiotic on an organism. Many more mice would
have to be used. Antibody production in response to treated
organisms would have to be checked by serological tests to
determine the titer of the antibodies produced. This would
necessitate serological tests at different time intervals, follow-
ing the inoculation of the antibiotic-treated organism. The
results would demonstrate that the antibiotic-treated organisms
retain their ability to stimulate antibody production.

This would suggest that organisms so treated could serve

as effective immunizing agents.

 

.n “pm—cs...

 

64

TABLE VI

IMMUNITY ESTABLISHED BY ORGANISMS TREATED WITH

SUBINHIBI TORY CO NCEN TRATIONS OF

THREE ANTIBIOTICS

 

 

Initial Inoculum

 

 

No.
1V13ifce §$::: Antibiotic h::::ison
In Vitro

4 15-6 Chloromycetin 4
4 15—6 Chloromycetin 18
4 49-50-638 Chloromycetin 4
4 15-6 Dihydro streptomycin 4
4 15-6 Dihydrostreptomyc in 18
3 13- 5 Dihydro streptomycin 4
4 13 - 5 Dihydro streptomycin 18
1 49-50 -638 Dihydrostreptomycin 4
4 49-50-638 Dihydrostreptomycin 18
3 49-50-638 Aureomycin 4
3 49-50-638 Aureomycin 18
6 Control

6 Control

6 Control

 

 

65

TABLE VI (Continued)

 

 

 

Challenge Dilution No. No. Percent
Culture Injected Survived Survived

-4

15-6 10 4 / 4 100
-4

15-6 10 4 / 4 100
-3

49-50-638 10 4 / 4 100
-4

15-6 10 4 / 4 100
-4

15-6 10 4 / 4 100
-4

13-5 10 3 / 3 100
-4

13-5 10 4 / 4 100
-3

49-50-638 10 1 / 1 100

49-50-638 10’3 4 / 4 100

49-50-638 10’3 3 / 3 100

49-50-638 10'3 3 / 3 100
-4

15-6 10 6 / o o
-4

13-5 10 6 / 0 0
-3

49-50-638 10 6 / 0 0

 

 

Further investigation along these lines might prove

profitable.

66

SUMMARY

The in vitro sensitivity of fourteen strains of Salmonella

typhimuriurg thirteen strains of Salmonella choleraesuis, and

 

sixteen strains of Pasteurella multocida to aureomycin, chloro-
mycetin, and dihydrostreptomycin was tested.

There was a slight variation in the sensitivity of the
strains within the same‘species to the same antibiotic, and to
all three antibiotics at the end of 4 hours.

The sensitivities of all organisms to Chloromycetin was
constant at the termination of 18 hours. There was a decided
fluctuation in the sensitivities of all the organisms to aureomy-
cin and dihydrostreptomycin. Also, decided variation of sensi-
tivities between strains of the same Species to the same anti-
biotic was noted at 18 hours.

The order of activity of the three antibiotics determined
from the sensitivity tests was: (1) Chloromycetin, (2) aureo-
mycin, and (3) dihydrostreptomycin. This order was generally
valid for all three species of organisms.

A comparison of the biochemical reactions of normal

Cultures of all the organisms and those organisms subjected

68

separately with subinhibitory concentrations of each of the three
antibiotics showed no observable difference.

Morphological studies were made on all organisms sep-
arately subjected to a subinhibitory concentration of each anti-
biotic. There was no observable difference between the treated
and normal organisms.

A few selected strains of S. typhimurium and S. choler-

aesuis, and one selected strain of _P_. multocida were subjected

 

to subinhibitory concentrations of an antibiotic and then inocu-
lated into mice.
All mice inoculated with S. choleraesuis strain 15-6 and

_P_. multocida strain 49-50-638, both of which had been treated

 

with Chloromycetin for 4 and 18 hours, survived. S. typhimurium
strain SF-368, subjected to the same antibiotic, killed all mice
at both time intervals.

S. choleraesuis strain 15-6 and S. typhimurium strain

 

13-5 which had been treated with dihydrostreptomycin for 4 and
18 hours and the 18-hour-treated B. multocida strain 49—50-638

did not kill mice. I_’. multocida strain 49-50-638 which had been

 

treated with dihydrostreptomycin for 4 hours killed 75 percent

of the mice in the series.

69

Aureomycin-treated S. choleraesuis strain 15—9 and S.

 

typhimurium strain SF-368 treated for 4 and 18 hours killed

 

mice. Fifty percent of the two series of Inice inoculated with

_P_. multocida strain 49-50-638 for the two time periods survived.

 

Immunity responses of the mice surviving injections with
the antibiotic-treated organisms were checked by challenging the
mice with a lethal dose of the normal cultures.

Mice surviving an injection with S. choleraesuis strain
15-6 treated with Chloromycetin received an 0.1 cubic centimeter
of the untreated culture, which was diluted 10-4. All mice were
alive and well at the end of 10 days. Mice surviving injections
with E. multocida strain 49-50-638 treated with Chloromycetin,

survived a challenge of the normal organism diluted 10-3.

 

Mice inoculated with dihydrostreptomycin-treated S. _t_y_-

phimurium strain 13-5 survived 0.1 cubic centimeter of a 10-

 

dilution of the untreated organism. Similarly, mice receiving

S. choleraesuis strain 15-6 subjected to dihydrostreptomycin

 

-4
survived 0.1 cubic centimeter of a 10 dilution of the normal
organism. Mice initially inoculated with dihydrostreptomycin-

treated Ii. multocida strain 49-50-638 survived 0.1 cubic centi-

 

meter of a 10”3 dilution of the organism.

70
Mice inoculated with aureomycin-treated E. multocida
-3
strain 49-50—638 also recovered when challenged with a 10

dilution of the normal organism.

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