INFECTION WITH CORYNEBACTERIUM
PYOGENES
W
MAN*
DOROTHY O. BALLARD, M.T. (A.S.C.P.), ALBERT -B;J UPSHER, M.D.,
AND DOROTHY D. SEELY, M.T. (A.S.C:P:)'
From the Departments of Pathology and Bacteriology, DepartmentafHealth,
Hospital, Kansas City, Missouri
v
Municipal
.' ',
The occurrence of Corynebacterium pyogenes infection in man is app$r£titly
very rare. Many investigators, in reporting work on C. pyogenes as an animal
pathogen, have pointed out the possibility«of the organism being pathogenic for
man. In our review of the literature, wehave found only one reference of a
proved case of human Corynebacterium pyogenes infection. 4 ' 8 In this instance;
Halbron and Forgeot and co-workers isolated the organism from several abscesses
in a sheep herder. The patient first developed an abscess in the thoracic region.
During the last six months of his life, other abscesses appeared in the left maxilla,
right arm and upper regions of the right leg. The abscesses were followed by
formation of fistulas and it was believed that several bones were involved: Experimental inoculation of the isolated organism was not fatal for rabbis 1 "guinea
pigs, or mice.
. . .
-.ji.i-.--"--•';
Since the original descriptions of C. pyogenes by Lucet,14 Grips,6 I$unnemann ?
and Glage,6 the organism has been isolated from calves, cattle, pigs and goats in
cases of pneumonia, summer mastitis, endometritis, pyometraj''abscesses,
polyarthritis, nephritis and granulomatous lesions. Brown and Orcutt 3 and
Rolle16 made comprehensive studies of C. pyogenes. Merchant" studied several
species of Corynebacterium as pathogens in domestic animals and Lovell*0-13
recently investigated the ability of the organism to produce toxin and stimulate
the production of antitoxin.
•• , i , i'' : |i '•• •••<
T h e purpose of this p a p e r is t o r e p o r t a case of h u m a n Corynebacterium
pyogenes
infection, occurring as a complication after frostbite of the feet- The report
includes a description of the organism, with its cultural characteristics, pathogenicity and serologic reactions.
JiiiJMj
R E P O R T O F CASE
A 37 year old white male, a truck driver, was admitted to the hospital oil January 19,
1943, complaining of "frostbite of the feet." He had been subjected to extreme cold for
forty-eight hours prior to his admission.
The past history revealed the occurrence of phlebitis in 1938 and Wernicke's disease in
1939.
.. ...r.
On admission, the patient was well developed and not acutely ill. Physical examination
was largely negative. The eyes reacted slowly to light, but well to accommodation. The
reflexes were normal. The feet were red, swollen, painful to pressure and no blanching
was seen on deep pressure. The distal portions of all the digits were dark in color and
a large number of blisters was seen over the dorsal aspects of the toes, j The temperature
was 102.4 F., the pulse rate 100, the rhythm regular and the blood pressure 126/60.
The erythrocyte count was 4,100,000. The leukocyte count was 13,450, with 79 per cent
polymorphonuclears, 18 per cent lymphocytes, 2 per cent monocytes and 1 per cent eosino* Received for publication, October 8,1946.
209
<
210
BALLARD, TJPSHER AND SBELY
phils. The blood Wassermann and Kahn tests were negative. The blood sugar was 96
mg. per 100 c c ; the blood creatinine, 1.3 mg.; and the nonprotein nitrogen, 24 mg. The
urinalysis was negative except for 2 plus albumin.
The patient's feet were carefully cleaned and sprayed with sulfathiazole. They were
wrapped in sterile cotton and the patient was placed in a Sanders' bed. Pour gm. of sulfathiazole was administered by mouth daily. Inspection of the feet three days after admission revealed considerable improvement with return of color to all portions except the toes,
in which there appeared to be definite evidence of gangrene. On January 25, the patient's
temperature suddenly became elevated to 103 P. from previous readings of 99 F. and 100
F. A skin rash accompanied the fever. Inspection of the feet failed to show further significant changes. X-ray films of the chest were negative. Sulfathiazole was discontinued
since it was believed that the patient was sensitive to the drug. On January 28, administration of sulfathiazole was again started and the patient again reacted unfavorably. On
February 1, amputation of all the toes of the left foot and four toes of the right foot was
performed. The pathologic examination revealed sections of skin, supporting tissues and
necrotic bone, all of which showed a rather intense inflammatory reaction characterized
by large numbers of polymorphonuclear leukocytes, edema, fibrin and cell debris, with
evidence of gangrene. No organisms were identified in the sections.
On February 9, the temperature curve was diurnal in type. Inspection of the feet
showed the operative incisions to be satisfactorily granulating and no infection was visible.
The patient continued to have daily elevations of temperature, up to 103 F. and 104 F.
Culture of the blood, drawn on February 16, revealed Corynebacterium pyogenes. At this
time, the white blood cell count was 16,000, with 73 per cent polymorphonuclear leukocytes,
19 per cent lymphocytes, 4 per cent monocytes and 4 per cent eosinophils. The blood
chemical findings were normal. The urinalysis was negative. The patient was given
sulfanilamide, 6 gm. daily, and several days later a blood level of 5.5 mg. per 100 cc. was
recorded. No unfavorable reaction was apparent. The fever began to abate and the
patient appeared improved. Inspection of the patient's feet showed evidence of infection
at the operative sites and in the region of the heels. About the heels, small sinus tracts
had developed with seropurulent drainage. Cultures taken from the feet revealed the
identical organism as that isolated from the blood. On March 10, the patient's hemoglobin
had dropped to 55 per cent and the red blood cell count to 3,000,000. He received several
blood transfusions. The patient's local condition did not improve. The temperature
curve returned to normal and the sulfanilamide was gradually reduced to small doses.
X-ray films of the feet showed minimal destruction of the os calcis of the left foot. There
was no evidence of peripheral arterial disease. Clinically, a low grade infection was still
present in the incisional areas and heels, and the sinus tracts in the heels continued to drain.
Repeated blood cultures were all negative. The patient was discharged from the hospital
on May 1, and was able to walk with the aid of crutches. He returned to the hospital for
plastic repair of the extremities. On October 20, 1944, cultures from the areas of the feet
previously treated still revealed C. pyogenes present.
BACTERIOLOGIC FINDINGS
The organism was first isolated from a routine blood culture taken on February
16, 1943. Fifteen cc. of blood was drawn and inoculated into mediums by
distributing 5 cc. into brain broth, 5 cc. into brain heart infusion broth, 1 cc. into
thioglycolate broth and 2 cc. each into plain agar and dextrose agar pour plates.
In three days, the flask of brain heart infusion showed evidence of growth in the
form of two or three individual colonies among the blood cells in the bottom of the
flask. Microscopic examination showed pleomorphic, small gram-positive
bacilli. The bacilli varied in length from 0.3 to 2 microns. For the most part,
they took a uniform stain, but a few showed barred forms. They were arranged
INFECTION WITH
Corynebacterium pyogenes
211
in clumps or in palisades, like diphtheroids. On the fourth day, one deep colony
with a zone of hemolysis, approximately 2 mm. wide, appeared in the dextrose
agar pour plate. With Gram's stain, the organisms of the colony showed the
same microscopic characteristics. Subsequent blood cultures taken February
24, March 2 and April 5,1943, were negative.
On February 25, pus from a draining sinus on the patient's right foot was
cultured. It showed a heavy growth of the same gram-positive bacillus and a
light growth of hemolytic Staphylococcus aureus. On April 27, cultures were
taken of pus from a draining sinus in each foot. Both cultures showed findings
identical to the previous culture from the right foot.
Repeated cultures from the sinuses of the feet were taken during the next
eighteen months, when the patient visited the out-patient clinic. Cultures of
the patient's feet taken on August 2, September 9 and October 20, 1944, still
showed the presence of the predominant organism as Corynebacterium pyogenes,
in association with hemolytic Staphylococcus aureus.
Morphology. Further subcultures on mediums containing blood or blood
serum showed the same pleomorphism as originally seen. The organism appeared as short, thin gram-positive bacilli, or cocco-bacilli, often in palisade
arrangement similar to that of diphtheroids. On staining a twenty-four hour
culture, most of the organisms showed gram-positive characteristics, but occasionally some appeared gram-amphophilic. Individual bacilli frequently exhibited irregularities of density in staining, producing a similarity to the barred
forms of C. diphtheriae. They varied in size from 0.3 micron in the coccoid form,
to 2 microns in the longer form. They showed little evidence of clubbing and
were nonmotile.
Cultural characteristics. The organism seemed to grow equally well aerobically
and anaerobically. One of the predominant characteristics was its ability to
hemolyze blood, producing a zone of hemolysis when grown on human or rabbit
blood agar. The colonies, when grown on blood agar, appeared in twenty-four
hours as small pin point colonies, with zones of hemolysis, approximately 2 mm.
wide. They were similar to the colonies of beta-hemolytic Streptococcus. In
forty-eight hours, the colonies appeared slightly larger and opaque, with a dry
granular surface and larger „zone of hemolysis. As the colonies aged, they
became medium-sized with increased opacity and granularity. The zones of
hemolysis increased to 5 or 8 mm. in width.
The organism reproduced well only on, or in, mediums containing blood or
blood serum. There was no growth on plain agar. There was no pigment production. It grew in plain broth only in association with the hemolytic Staphylococcus aureus. The morphology and growth was excellent on Loemer's medium.
It grew in thioglycollate broth with added blood serum. For growth in any of
the fermentation broths, it was necessary to add blood serum.
Fermentation tubes were employed using 0.1 per cent bromcresyl blue and 1
per cent sugar and an enrichment of blood serum. The organism showed
fermentation with acid production from dextrose, sucrose, lactose, arabinose,
trehalose, xylose, maltose and levulose, while mannite and inosite were not
212
BALLARD, UPSHER AND SEELY
fermented. The following chart gives the sugar fermentations of our strain and
of various other strains of C. pyogenes, as reported by Bergey,2 Merchant16 and
Lovell.13
Subcultures of the organism isolated from the blood and sinuses of the feet of
the patient were sent in March, 1943, to the National Institute at Bethesda,
Maryland, for identification. On April 19, 1943, the Institute reported the
identity of the organism in these cultures as Corynebacterium pyogenes.
SEROLOGIC
REACTIONS
The patient's serum taken on August 2 and September 9, 1944, was tested for
antitoxin content according to Lovell's12 method with known Corynebacterium
TABLE l
REACTION OP DIFFERENT STRAINS OF C. pyogenes WITH ^RESPECT TO FERMENTATION IN
SUGAR MEDIUMS, AS REPORTED BY VARIOUS AUTHORS
OUR STRAIN
Dextrose.
Sucrose..
Lactose. . .
Arabinose.
Mannite. .
Trehalose,.
Inosite..
Xylosei
Litmus milk.
Maltose..
Levulbse.1
+
+
+
MERCHANT'S STRAIN
+
+
+
+ (slow)
+
+
+
+
+
+
+
— (few strains
+)
+
+
— (few strains
•+)
+ (slow)
+ (slow)
acid, slight
coagulation
LOVELI/S STRAIN
+
acid, slight
coagulation
+
(few strains +) coagulation
and digestion
coagulation
and digestion
+
+ •:
+
pydgenes'tbxixi, These serums failed to show any antitoxin present other than
in* the undiluted state, which is within the range of normal as determined by
Lovell 'in: his work on human serums. Unfortunately, serums previously
obtained from the patient were discarded before antitoxin studies could be
carried out.
A rabbit,1 was first inoculated intravenously on May 10, 1943, with 1 cc. of a
forty-eight hour culture of the organism isolated from the patient. The animal
died during the third night. Cultures taken from the rabbit were negative and
microscopic examination of the rabbit's tissues showed no specific lesions relating
to the inoculated organisms.
Several animals were inoculated October 27, 1944, with a forty-eight hour
culture of the organism isolated from pus from the patient's foot on October 20.
The animals were first bled and then inoculated as follows: one rabbit, 1 cc.
intraperitoneally; one rabbit, 1 cc. intravenously; one guinea pig, 0.5 cc. intra-
INFECTION WITH
Corynebacterium pyogenes
213
peritoneally; one guinea pig, 0.5 cc. subcutaneously; 2 mice, each 0.2 cc. intraperitoneally; one mouse, 0.2 cc. subcutaneously. The animals developed no
particular symptoms, nor did any of the animals die. Three weeks after inoculation, the animals were again bled and killed, but no abnormalities were observed
on autopsy. Tissue sections taken for microscopic study failed to reveal specific
lesions.
Antitoxin determinations were run on the serums obtained before, and three
weeks after, the inoculation of the animals with the isolated organism. The
antitoxin determinations were made according to Lovell's12 method, with some
modification.
Dr. Lovell supplied the known Corynebacterium -pyogenes toxin and antitoxin,
which had been found to have the following values: dried C. -pyogenes toxin,
TABLE 2
PROTOCOL O F P R I M A R Y T I T R A T I O N
AMOUNT OF C.
pyogenes ANTITOXIN ADDED:
1:50 DILUTION
OR i U. PER
0.5 cc.
O F C. pyogenes
AMOUNT OF C.
pyogenes D R I E D
TOXIN ADDED:
0.5 cc.
0.5 cc.
dilution 1:2
0.5 cc.
0.5 cc.
dilution 1:4
0.5 cc.
0.5 cc.
dilution 1:8
continue serial dilutions
of toxin, as above
0.5 cc.
plus
1 PER CENT
RABBIT CELLS
0.06 GM. PER 10
CC.SALINE
0.5 cc.
0.5 cc.
Control I . .
Control I I .
T O X I N AND A N T I T O X I N
0.5 cc. saline
1.0 cc. saline
Incubate 1
hour a t room
temperature
1 CC.
1 cc.
1 cc.
Incubate
1 hour a t
37 C .
1 cc.
1 cc.
1 cc.
approximately 0.006 gm. = 1 Lh dose; C. pyogenes antitoxin, 1 cc. = 25 " x "
units. A primary titration was done, using a fixed amount of antitoxin in each
tube and varying the amount of toxin to determine what dilution of toxin would
give a 2 plus hemolysis of 1 cc. of 1 per cent rabbit cells with J "x" unit in 0.5 cc.
of antitoxin.
That dilution of toxin which showed a 2 plus hemolysis with J "x" unit of
antitoxin was then used to titrate the various animal serums. The animal
serums were heated forty-five minutes at 56 C. Then each serum was titrated
making serial dilutions from 1:2 to 1:2048, and using 0.5 cc. quantities; to each
dilution was added 0.5 cc. of determined toxin dilution. Tubes were incubated
one hour at room temperature; 1 cc. 1 per cent rabbit cells was added to each
tube, and then the tubes were incubated one hour at 37 C. The 2 plus hemolysis
end point was read. The following controls were used: control I, saline and
rabbit cells; control II, J "x" units antitoxin, saline and rabbit cells; control III,
duplicate 2 plus hemolysis tube of \ " x " units antitoxin, dilution of toxin
214
BALLARD, UPSHER AND SEELY
determined in primary titration and rabbit cells. Two normal rabbit serums in
serial dilutions were also run as controls.
None of the serums of animals taken before inoculation showed any titer.
Only the serum of the rabbit which had been inoculated intraperitoneally with
1 cc. of a forty-eight hour culture of the organism showed a titer. This serum
showed a content of C. pyogenes antitoxin of 512 "x" units per cc. This demonstrated that our organism was capable of inducing formation of specific antibody,
which in turn could be titrated with a known C. pyogenes toxin. Thus the
organism was identified serologically as Corynebacterium pyogenes.
C. pyogenes isolated from animal infections, has been found to be pathogenic
for rabbits, mice, rats and guinea pigs. 3 ' 1 3 , 1 5 But the strain isolated by Forgeot
et. al. i was not fatal for laboratory animals. It is possible that strains vary a
great deal in virulence. It may be postulated that this strain of the organism
which we isolated lost some virulence because of the chronicity of the infection,
the chemotherapy employed before cultures were taken, or because of the serologic studies made.
COMMENT
The present report offers evidence that Corynebacterium pyogenes is pathogenic
for man. The organism invaded tissue already weakened and damaged by
frostbite and produced an infection having both acute and chronic phases. It
produced further local tissue damage and bone necrosis. It was hazardous as a
blood stream invader, since the septicemia which occurred in this case, offered
opportunities for metastatic lesions to occur. Presumably, however, the sulfonamides acted to prevent the establishment of other foci. That the organism can
involve bone was shown by the x-ray findings.
The necrotic lesions, the sinus formation and the involvement of the bone in
this case appeared to be similar to the types of lesions which were produced in
the patient described by Forgeot et. al.4. Both of these human strains are similar
in the types of lesions produced and in their inability to cause a fatal infection in
laboratory animals.
This strain of the organism was able to grow and produce localized areas of
degeneration. The failure to demonstrate lesions in inoculated animals is
possibly due to the low virulence of our strain, or to virulence that was altered
by previous chemotherapy. A notable feature of the infection in this human
case was its long chronicity, extending over many months.
SUMMARY
An instance is reported of a human infection with Corynebacterium pyogenes
which constitutes the second such infection reported in the literature. In
the present infection the tissue showed previous circulatory damage. There was
also a septicemia. Serologic examination showed production of toxin by the
organism and the ability of the latter to stimulate formation of antibody.
Acknowledgments. The authors wish to express their thanks to Dr. I. A. Merchant,
Iowa State College, Ames, Iowa, for supplying us with cultures of C. pyogenes used for
INFECTION WITH
Coryriebacterium pyogenes
215
comparison, and t o D r . Reginald Lovell, Royal Veterinary College, London, for supplying
us with t h e C. pyogenes toxin a n d antitoxin used in our determinations.
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1. BALOZET. C , AND RECEVEUR, P . : Contribution a l'£tude des Corynebact&ries pyogenes.
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Baltimore: T h e Williams and Wilkins Co., 1939, p . 796.
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J. E x p e r .
Med., 32: 219-248, 1920.
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Bact., 49: 329-338,1939.
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production. J . P a t h , and Bact., 46: 339-355, 1937.
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