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1THS

A STUDY OF THE FIBRINOLYTlC
PROPERTIES OF LANCEFlELD'S
GROUP c STREPTOCOCCI

Thesis for the Degree of M. S.
MICHiGAN STATE COLLEGE

Leander F. 'Williams
194l

 

 

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A STUDY OF THE FIBRINOLYTIO PROPERTIES OF

LANCEFIELD'S GROUP 0 STREPTOCOCOI

by
LEANDER FARNHAM ¥ILLIAMS

A THESIS

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

MASTER OF SCIENCE

Department of Bacteriology
1941

THESIS

 

TABLE OF CONTENTS

IntrOdUCtion o o o o o 0

History 9 o o o o o o 0

Materials and Experimental Procedures

For Biochemical Work

Procedure for Fibrinolytic Tests

Experimental . . . . . .

O 0 O O O 0

Influence of Composition of the Medium

on Fibrinolysin Production .

Influence
Influence
Influence
Influence
Influence

Comment

of

of

of

of

of

Attempts to Increase

Fibrinolytic Test

Physical Treatment of Cultures .

Peptone . .
Dextrose .
BUffer o o

BlOOd o o o

Incubation Time

Sensitivity of the

Cultivation in the Presence of

Fibrin

13%230

Page

14
15
15
17
l9
19
19

20

21

TABLE OF CONTENTS

(cont.)

Page
Amount of Fibrin in the Test . . 21

Source of Fibrin . . . . . . . . 21
Relation of Fibrinolytic Ability to Bio-
chemical Characteristics . . . . 24
Fibrinolytic Properties of Hemolytic
Streptococci from Different
Sources . . . . . . . . . . 25
Discussion . . . . . . . . . . . . . . . . . . . 28
Summary . . . . . . . . . . . . . . . . . . . . . 51

Bibliography 0 o o o o O O o o o o o o o o o o o 52

A Study Of the Fibrinolytic Properties of Lancefield's

Group C Streptococci

Introduction

 

Since Lancefield (6) began to identify hemolytic
streptococci serologically in 1955 there has been great
interest in cultures which have fallen into her group C.
The probable reason for this is that streptococci from
both man and animals fall into this group so that it is
of interest to the medical worker as well as the veter-
inarian. There have been various attempts to separate
group G into “human" and "animal" types by biochemical
and serological methods but these have not proved a1-
together successful. The present study was undertaken
for the purpose of determining the possible use of the
fibrinolytic test in separating "human" type and
"animal“ type strains of group 0.

History

Much emphasis has been placed on the fermentation
of sorbitol and trehalose as an aid in the differentia-
tion of this important serological group. Lancefield
(6) in 1935 found that of forty—nine strains which
were classified serologically as group C and which were
isolated from lower animals, forty-five fermented sorb-

itol but not trehalose and four strains fermented

neither of these carbohydrates. Her cultures were
isolated from guinea pigs, cows, rabbits, horses, fowl,
and foxes.

Dimock and Edwards (5) in 1955 demonstrated that

Streptococcus equi, though serologically a member of

 

Lancefield's group C, was a definite entity. They

based their belief on the observation that Str. egui

 

was the only member of group C which would produce
"strangles' in horses. This organism has a biochemical
pattern of its own in that it does not ferment lactose,
sorbitol, or trehalose. Dimock and Edwards also pro-
posed to divide streptococci of Lancefield's group C
which were derived from animal sources into four types
on the basis of their ability to ferment lactose, sorb-
itol and trehalose; namely, types A, Bl, B2 and equi.
Type A ferments lactose and sorbitol but not trehalose.
Type B ferments trehalose only and type 82 ferments

lactose and trehalose but not sorbitol. Str. egui does

 

not attack any of these substances. This classification
has been adopted by many but it has several shortcomings.
In a follow up of this work, Edwards (4) in 1955 stated
that these types set up by himself and Dimock fall into
the same serological group and can be differentiated by
biochemical reactions only. He also stated that their
type A cultures were probably strictly animal parasites
and that they had not been found in human disease. He

found that streptococci isolated from animals might be

of the "human“ type and cause human disease while human
beings could harbor streptococci of the animal type.
Brown (1) in 1957 in an extensive study of the
hemolytic streptococci found in milk, studied two
hundred and fourteen strains of Lancefield's group C
streptococci of animal origin and found that all
fermented sorbitol but not trehalose. He stated that
the animal strains of group C streptococci including

Str. egui would not lyse fibrin. All of his fibrin-

 

olytic tests were made with human plasma. The strains
that he isolated which were of human origin all lysed
human plasma. In the summary of his paper he recom-
mended the fibrinolytic test using human plasma to show
which organisms are to be regarded as pathogenic for man.
In 1957 Plastridge and Hartsell (11) described a
new species of streptococci found in bovine mastitis
which was biochemically similiar to Lancefield's group
B streptococci but which was serologically different.
They named this species Streptococcus pseudo-agglactiae.
They found that this organism represented about five
percent of the cultures isolated from experimental
herds being tested for chronic streptococcic mastitis.
This organism usually produced an acute or mild mastitis
Of short duration. They showed that it yielded extracts
which reacted serologically with Lancefield's group C
antisera.

Sherman and Niven (12) in 1958 studied the hemo-

lytic streptococci of milk and applied the fibrinolytic
test of Tillet and Garner in which they used human
plasma. In their work they found that of eleven "animal
pyogenes“ five partially lysed the plasma clot in 24
hours while six did not. Of eight strains of “human"
type group C streptococci, six caused complete lysis of
the plasma and two only a partial lysis in twenty-four
hours. Sherman and Niven incubated their fibrinolytic
tests at 57'C. and also 55°C. and found that the 'animal"
type group C cultures would not lyse human fibrin at 55°
C. while the "human“ type group C cultures at 55°C.
would give results comparable to those obtained at 57°C.
and suggested this as a criterion for differentiating
between these two types.

Tillet and Garner (15) in first describing the
fibrinolytic test noted that three strains out of eight-
een derived from animal sources were capable of lysing
human plasma. One strain was isolated from a cow and
the other two were from a rabbit and a guinea pig. The
serological reaction of the cultures which they used at
that time was not studied. They found that animal
strains would not lyse rabbit plasma while some human
strains would do so after twenty hours incubation.

Madison (7) while working on the fibrinolytic
property of streptococci derived from lower animals
found that the majority of the strains which he tested

would lyse horse plasma in preference to human plasma.

He suggested the specificity of the strain for the
plasma of the type host from which the strain was
isolated. Serological identification of the cultures
he used was lacking.

Tillet (16), while doing further work on the
fibrinolytic reaction and correlating the biochemical
and serological reactions with the ability of strepto-
cocci to lyse human plasma, found only one out of
twenty-one cultures of Lancefield group C cultures that
was able to lyse human fibrin. He stated that the
determination of the presence or absence of fibrin-
olysin in the cultures is‘a helpful procedure in se-
parating human pathogenic strains from others which
probably do not cause human disease. In a review of
the literature, Tillet (17) stated that fibrinolysis is
not necessarily associated with any biochemical char-
acteristics but appears to be related to the disease
producing capacity of the strain when isolated.

Alice Evans (5) in 1956 after studying Str. egui

 

and related strains concluded that there were waves of
human infections due to trehalose fermenting strains
of group C organisms, thereby suggesting a possible con-
nection between such infections and bovine mastitis
caused by strains of this group.

Planet (9) in 1955 first found that strains of
streptococci from horses would act on horse fibrin but
not on human fibrin and also that a human strain would

not act on horse fibrin.

Materials and Experimental Procedures
For Biochemical Work

Blood Broth: Blood broth was prepared by adding 5
percent sterile defibrinated ox blood to beef infusion
proteose peptone broth containing 0.5 percent sodium
chloride and adjusted to pH 7.5.

Litmus Milk: This was prepared by adding 2 percent
Of a saturated aqueous solution of litmus to fresh skim
milk.

Carbohydrate Broths:*

Solution A
Serum (ox serum not sterile) 90 ml.
15 percent sodium hydroxide 10 ml.

Put in flask in boiling water

bath for 20 minutes.

Solution B
Distilled water 900 m1.
Proteose peptone 10 grams
Sodium chloride 5 grams
Beef extract 5 grams

Dissolve ingredients, add solution A to 8,
add 10 m1. of Andrade's indicator and adjust reaction
to pH 7.4. Divide the resulting broth into three por-
tions, add 1 percent lactose, sorbitol, trehalose

respectively, tube and sterilize by autoclaving at 10

* Formula developed in the Department of Animal Diseases,

Storrs Agricultural EXperiment Station, Storrs, Conn.

pounds pressure for 20 minutes.

Broth for the Determination pf Final pH:

 

Difco Proteose Peptone 10 grams

Meat extract 4 grams

Sodium chloride 5 grams

Dextrose 10 grams

Distilled water ' 1000 m1.
pH 7.0

Tube in 5 ml. amounts and sterilize at 15 pounds
pressure for 15 minutes.

Sodium Hippurate Broth: This was prepared accord-

 

ing to the method of Coffey and Foley as published in
the American Journal of Public Health Volume 27,
number 10, pages 972 to 974, 1957.
Procedure for Fibrinolytic Tests

M3333: The media found to be best suited for the
production of fibrinolysin by streptococci of animal
origin was 1 percent proteose peptone beef infusion
broth containing 0.5 percent sodium chloride and
adjusted to pH 7.2.

Anti-coagulant: A 2 percent solution of sodium

 

oxalate was used as an anti-coagulant. One milliliter
of the solution was mixed with 10 ml. of freshly drawn
blood. This procedure was used for horse and human
plasma. For rabbit plasma 1.5 ml. of the solution

was used per 10 ml. of blood.

Coagulant: Coagulation was accomplished by adding

 

0.25 ml. of a 0.25 percent solution of calcium
chloride in 0.85 percent saline to the plasma-culture
mixture. This coagulated oxalated plasma within a
period of 56 hours after it is drawn.

Method 22 Activation: After the cultures were in-

 

cubated at 57°C. for 18-24 hours they were then immedi-
ately chilled and stored in a refrigerator for 5 hours
at 4°C.

Description pf the Test: To 0.1 ml. of oxalated

 

plasma, 0.9 ml. of physiological saline and 0.5 ml. of
"activated" broth culture were added. Then 0.25 ml.
of the calcium chloride solution was added and the in-
gredients mixed well. The tubes were then placed in a
water bath or incubator at 57'C. Under these conditions
the clot will form in approximately 15 minutes and the
cultures may begin lysing within a period of 15 minutes
after they have clotted. The tests were arbitrarily
terminated after 24 hours incubation.
Experimental

There appeared to be two ways in which cultures of
group C streptococci might be induced to lyse fibrin;
namely, by using media more suitable for growth, or by
finding some means of making the test more sensitive.
Our assumption was that all hemolytic streptococci
might be fibrinolytic and that those which did not lyse
fibrin failed to do so because they did not produce

enough fibrinolysin to reveal a reaction or that the

Culture

Original

History of Cultures

Table 1

Remarks

 

 

 

 

 

 

 

 

 

 

 

Where Obtained Sburce
Number Designation
Group A
1 Str. Dr.H.L.Kulp Human Low grade
scarlatinae Univ.of Conn. scarlet fever
Group A
2 Wilhelm Dr.U.L.Kulp Human Skin lesion
Univ.of Conn.
Gr_5up A
5 Ottenhiemer Dr.H.L.Kulp Human Human lesion
Univ.of Conn.
Group A
4 Elliot Dr.V.L.Ku1p Human Abcess
Univ.of Conn.
Group A
5 Str. Dr.H.L.Kulp - Originally ob-
hemolyticus Univ.of Conn. tained-Lederle
Group A
6 Upton Dr.H.L.Kulp Human Arm infection
Univ.of Conn. '
Group A
7 Gatchell Dr.H.L.Ku1p Human Hand infection
Univ.of Conn.
_ Group A
8 Cleveland Dr.U.L.Kulp Human Infection of
Univ.of Conn. human cervix
Mastitis
9 N49 Dr.A.H.Stable- Bovine Group 1
forth England type 1a*
Mastitis
10 8102 Dr.A.H.Stable- Bovine Group 1
forth England type lb
Mastitis
ll G19 Dr.A.H.Stable- Bovine Group 1

 

 

forth England

 

 

type 1c

 

 

Table 1 (cont.)

History of Cultures

 

 

 

 

 

 

 

 

 

 

 

§:;:::° Dgzigiziion Ihere Obtained Source Remarks
Mastitis
12 G2 Dr.A.V.Stab1e- Bovine Group 1
forth England type 1d
Hastitis
l5 SS4 Dr.A.U.Stable- Bovine Group 1
forth England type 2a
Mastitis
14 3101 Dr.A.U.8tab1e- Bovine Group 1
forth England type 5a
Mastitis
15 3107 Dr.A.H.8table- Bovine Group 1
forth England type 5b
Mastitie
16 ,8104 Dr.A.U.Stab1e- Bovine Group 1
forth England type 5c
Maetitis
17 0903 Dr.R.C.Lance- Human Group 1
field type 4A
Rockefeller I.
Mastitis
18 H568 Dr.R.C.Lance— Human Group 1 type4b
field Blood of new
Rockefeller I. born baby
Mastitis
19 G42 Dr.F.U.Stewart Bovine Group 1
Australia type 4c
20 H-11-2 Univ. of Conn. Bovine Str. uberis
type 6
Group C
21 2901 Dr.J.M.Sherman Human Isolated from
Cornell Univ. human feces
Group C
22 H46A Dr.J.M.Sherman - Originally

 

 

Cornell Univ.

 

 

from Dr. R000
Lancefield 2L

 

 

Culture

Original

Table 1 (cont.)

History of Cultures

 

 

 

 

 

 

 

 

 

 

 

 

 

Number Designation Where Obtained Sou::e Remarks
Group C
25 Shirley Univ. of Conn. Bovine Mastitis
Group C
24 1 Dr.P.R.Edwards Equine Infection of
Univ. of Kty. Os uteri-
__ mare
Group C
25 2 Dr.P.R.Edwards Equine Infection of
' Univl of Kty. Os uteri-
mare
Group C
26 5 Dr.P.R.Edwards Equine Infection of
Univ. of Kty. Os uteri-
mare
Group C
27 4 Dr.P.R.Edwards Equine Mare with
Univ. of Kty. lymphagitis
-Str. egui
Group C
28 5 Dr.P.R.Edwards Equine hip joint of
Univ. of Kty. foal—died s.
sgpticemia
Group C
29 Tuttle 55-5 Univ. of Conn. Bovine Mastitis
Group C
50 Strick. 1-4 Univ. of Conn. Bovine Mastitis
Group C
51 Sm. 67-4 Univ. of Oonn. Bovine No other
evidence of
Mastitis
Group 0
52 Sm.75-2 Univ. of Conn. Bovine Mastitie
Group C
55 Eck. 8-4 Univ. of Conn. Bovine Mastitis

 

 

 

 

 

 

Culture

Original

Table 1 (cont.)

History of Cultures

 

 

 

 

 

 

 

 

 

 

 

Number Desigpation Hhere Obtained Source Remarks
54 Eck. 51-5 Univ. of Conn. Bovine GroupC
Mastitis
Group C
55 B958 Univ. of Conn. Bovine No other
evidence of
Mastitis
56 A.S. 19-2 Univ. of Conn. Bovine Group C
Mastitis
_ Group C
57 Horse Univ. of Conn. Equine Uterine ex-
udate-foaled
5wks. early
58 Dye. I Dr.K.Diern- Bovine Group C
hoffer Str. dys-
Germany =galactiae
59 Dye. II Dr.K.Diern- Bovine Group C
hoffer Str. dys-
Germany __ga1actiae
40 Cl Dr.R.C.Lance- Cheese Group D
field
Rockefeller I.
41 C5 Dr.R.C.Lance- Cheese Group D
field
Rockefeller I.
42 Yale Dr.U.L.Kulp Unknown Group E
Univ.of Conn. History
lost
45 Ioodworth Dr.U.L.Kulp Human Group E
Univ.of Conn. Human
infection
44 M. I. Dr.H.L.Kulp Human Group E
Univ. of Conn. Throat

 

 

 

 

infection

 

 

History of Cultures

Table 1 (cont.)

Culture

 

 

 

 

 

 

 

 

Original bt d R I!
Number Designation Where 0 sins Source emar s
45 K129 Dr.R.C.Lance- Bovine Group E
field From certified
Rockefeller I. milk
46 K151 Dr.R.C.Lance- Bbvine Group E
field From certified
Rockefeller 1. milk
47 J148A Dr.R.C.Lance- Monkey Group G
field Normal throat
Rockefeller I.
48 H460 Dr.R.C.Lance- Human Group G
field Human vaginal
Rockefeller I. swab, normal
Group K
49 Turner Unknown Unknown From Queen
Charlotte
Hospital
50 Viridans 1 Dr.H.L.Kulp Unknown Streptococcus
Univ.of Conn. viridans
51 Viridans 2 Dr.l.L.Ku1p Unknown Streptococcus
Univ.of Conn. viridans
52 Viridans 5 Dr.I.L.Kulp Unknown Streptococcus
Univ.of Conn. viridans

 

 

 

 

 

 

* Mastitis Streptococci Group 1 as described by Stable-
forth (14) belong in Lancefield's Group B and may be
designated as Str. agalactiae.

test was not sufficiently sensitive.

Influence of Composition of the Medium

on Fibrinolysin Production

First an attempt was made to find a medium which
would produce the largest amount of cells because it
has been shown by Madison and Taranik (8) that the
production of cells is connected with the production
of lysin. Tillet (17) stated that, with strains which
elaborate relatively small amounts of fibrinolysin,
experience has shown that the extent of multiplication
of streptococci - which may be limited in unfavorable
media - and the age of the culture may be important
factors in determining the results of fibrinolytic
tests with individual strains. Madison and Taranik (8)
have shown that the rate of fibrinolysin production and
the rate of test tube proliferation of streptococci are
parallel during the logarithmic phase of growth and
that there is an apparent quantitative linkage between
enzyme secretion and cell division. They further state
that during the static phase of growth a fairly rapid
destruction or inactivation of the lytic factor takes
place.

The marked influence of the composition of the
culture medium on the amount of growth and agglutin-
ability of mastitis streptococci, and on the phase of

streptococcus cultures was shown by Plastridge,Banfie1d

and Williams (10) and by Dawson, Hobby and Olmstead (2)
respectively. These observations suggested that the
different ingredients used in preparing medium might
also influence the production of fibrinolysin.

Influence pf Peptone: Peptone was the first ingre-

 

dient studied and was found to be one of the two most
important factors in the production of fibrinolysin.
Table 2 shows the influence of different commercial
peptones on the production of fibrinolysin.

Three different plasmas were used in making the
fibrinolytic tests. lOne of these was highly suscepti-
ble, one partially so and the third was not attacked
by streptofibrinolysin. It will be noted that Fair-
child's Peptone, Bacto Peptone and Difco Proteose Pep-
tone gave fairly uniform results, while Difco Nee-pep-
tone and Bacto-Tryptone were the least favorable. While
Witte's Peptone was satisfactory it seemed to contain
some substances which tended to make the clot soft and
to one not experienced in this technique it might be
interpreted as partial lysis. Difco Proteose Peptone
was chosen for use because uniform results were obtained
upon repeated tests.

Influence pf Dextrose: Tillet and Garner (15) add-

 

ed 0.05 pereent dextrose to their medium to enhance the
production of the fibrinolysin. They found that it
materially aided fibrinolysin production by Lancefield

group A streptococci. Dr. F. R. Smith, in personal

Effect of Peptone

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2
Culture 25 21 22
Plasma H1 32 B5 H R H R
Bacto p5
12 6 O 12 O 1 1
Peptone
Difco p p
Proteose 12 6 O 12 6 l 1
Peptone
Difco
Neo- 24 0 0 24 0 l 1
peptone
Bacto p -
24 6 O 12 O l 24
Tryptone
Uitte's p ,
l2 6 O 5 O 1 l
Peptone
Armour's
24 0 0 12 O 1 24
Peptone
Fair- p .
child's 2 6 O 12 O l 24
Peptone J
l- H-Human plasma
2- R-Rabbit plasma
5- B-Bovine plasma
4- Numbers-time required for lysis in hours
5- p-partial lysis in 24 hours

16

correspondence, remarked that he had found that dextrose
could not be used in the culture medium because of the
excess acid produced by his cultures. Although his
paper (15) did not give the serological classification
of the cultures with which he worked it would seem from
his data that his cultures might have been Lancefield's
group 0.

As shown in table 5, the addition of 0.05 percent
dextrose to the culture medium caused culture 20 to
produce so much acid that the clot would not form upon
the addition of calcium chloride. When the dextrose
content reached 0.5 percent none of the culture-plasma
mixtures would clot. Cultures grown in the presence of
2 percent dextrose and then neutralized to pH 6.6 would
allow the culture-plasma mixture to clot. If the cult-
ures produced fibrinolysin subsequent lysis occurred
normally. The fibrinolytic test did not act more rapid-
ly or seem more sensitive when the cultures were grown
in the presence of 2 percent dextrose with subsequent
neutralization than when grown without dextrose in the
medium.

Influence pf Buffer: Buffers were added to the

 

medium containing 0.05 percent dextrose broth in the form
.Of either dipotassium phosphate or calcium carbonate in
quantities large enough to keep the pH within the criti-
cal range (pH 6.0-7.1) for clot formation, but the lysin

was not appreciably more active than that which was

endpaso go LpSOam ampwm 0.6 mg op Umpwshwm Esflvou *

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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+ + I o I o I o + + + + Hm
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semen pOHo %mumaq poao semen pone mamas pone semen pone semen pone qustpm
CNN am an $6 $05 mo

 

 

Omoupxea Mo Gowpmmpaoonoo

m mapde

53% E. semen. ,3 pasta

 

produced in the absence of dextrose. Neither the
presence of the phosphate nor the carbonate in the
culture medium appeared to affect the fibrinolytic test.

Influence pf Blopg: The influence of salt upon the

 

medium was not studied but 0.5 percent sodium chloride
was added to the medium so that blood might be added
and its effect studied. Defibrinated ox blood can be
added to the medium and the hemolytic properties in
blood broth determined at the same time that the cult-
ure is grown for the fibrinolytic test. The presence
of hemolyzed blood cells in the medium did not interfere
with the test.

The addition of blood to the medium in which the
organisms were stored helped to retain the fibrinolytic
properties of the cultures.

Influence 33 Incubation Time: Madison and Taranik

 

 

(8) have shown that after the Optimum growth period of
12 to 18 hours at 57.0. a rapid destruction or inactiva-
tion of the lytic principle takes place. In preliminary
experiments the longer a culture was left at 57°C. after
the 18-24 hour period of incubation the greater was the
decrease in potency of the lysin. In the instance of
one particularly potent strain no lysis could be demon-
strated after 96 hours.

9232223: Due to the marked influence of the compo-
sition of the medium upon the fibrinolytic activity of

the culture it was deemed advisable to use media of the

20

following composition. To a beef infusion base 1 per-
cent Difco Proteose Peptone and 0.5 percent sodium
chloride were added and the reaction was adjusted to
pH 7.2. This medium was selected as best suited for

fibrinolysin production by the group C organisms.

Attempts to Increase Sensitivity of the

Fibrinolytic Test

The other possibility which lay open was finding
a way to make the test itself more sensitive.

Physical Treatment pf Cultures: Table 4 gives the

 

 

results of a comparative study of the influence of
activation, shaking, shaking and activation, and no
physical treatment of the culture. Shaking was under-
taken because of the possibility of increasing the
metabolic products of the organism by shaking the
culture during incubation. A shaking machine was
placed in a 57’C. incubator and the medium was inocu-
lated and immediately placed in the machine. The cult-
ures were shaken for eight hours and at the end of that
time taken out and tested for ability to lyse human and
horse fibrin. These shaken cultures were then "acti-
vated' by rapid chilling and storage at 4°C. for 5
hours and another fibrinolytic test made. Activation
of the cultures was the most successful physical treat-
ment of those used to prepare streptococcus fibrinolysin
from Lancefield group C organisms. Upon testing addi-

tional'strains Of Lancefield's group C streptococci for

20A

.0 macaw camp

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21

fibrinolysin production it was found in some instances
that the only way in which the presence of fibrinolysin
in the culture could be demonstrated was thrOugh acti-
vation. Shaking, and shaking and activation proved to
be of no value.

Cultivation $3 the Presence pf Fibrip: In pre-

 

 

 

liminary work the effect of growing cultures of fibrin-
olytic streptococci in the presence of normal washed
fibrin was studied. This was done by collecting sterile
fibrin, washing and drying it. Shreds of the dried
fibrin were dropped into the culture medium at the time
of inoculation. No visible evidence of lysis of the
fibrin took place.

increase the sensitivity of the test was to reduce the
amount of citrated plasma used. It was found that only
0.1 m1. of plasma was necessary for the test. The ad-
vantage of this was to make the same amount of plasma
serve for twice as many tests as in the original pro-
cedure described by Tillet and Garner. This is es-
pecially helpful where the source of supply of human
plasma is limited. By reducing the plasma content of
the culture-plasma mixture it was hoped that a more
rapid reaction would take place but this was not true.
The use of less than 0.1 ml. of plasma in the test re-
sulted in a clot that was not firm.

Source 23 Fibrin: Because Madison and others have

shown that some streptococci showed little or no
evidence of the production of fibrinolysin when human
fibrin was used, it seemed desirable to determine if
some other suitable fibrin might be found to demon-
strate fibrinolysin production by Lancefield's group C
streptococci. Oxalated plasma from man, cow, horse,
rabbit, sheep, chicken and goat was tried. Lance-
field's group C streptococci would lyse only plasma
from human, horse, and sometimes rabbit. Table 5 shows
the fibrinolytic activity of various strains of strepto-
cocci in the presence of the three fibrins. The first
group lysed human fibrin and under certain conditions
also rabbit fibrin but upon repeated tests they have
consistantly failed to attack horse fibrin.

The second group attacked only horse or rabbit
plasma but never human. The strains which attacked
human or horse plasma have never varied so this would
seem to be a constant characteristic for this group.

On the basis of these observations it would appear that
the ability to lyse human or horse plasma might be a
legitimate basis for dividing group C cultures into
"animal" and "human" types.

The third group showed no evidence of attacking
either human or animal plasma by the methods used.

Oxalated human and horse plasma, which has been
dried while frozen and sealed in a vacuum, is suitable

for this work. Plasma so stored has been usable for a

25

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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period of one year.

Relation of Fibrinolytic Ability to

Biochemical Characteristics

As shown in table 5 in attempting to correlate the
biochemical characteristics of group O streptococci
with their fibrinolytic activity, no definite bio-
chemical pattern was found to be produced by any fibrin-
olytic type of organism. The only characteristic which
showed a correlation with the fibrinolytic ability of
the organism was the amount of acid produced in l per-
cent dextrose broth. Those organisms which attacked
human plasma produced an average pH of 6.26 in l per-
cent dextrose broth while the final pH for the organ-
isms that attacked horse plasma averaged 5.1..

No correlation between ability to ferment sorbitol
and trehalose and fibrinolytic prOperties was found.
Three cultures attacked both sorbitol and trehalose.

It was observed that the action of all group C
streptococci cultures in blood broth was not similar.
All of the cultures in group C produced at least a
small zone of hemolysis on ex blood agar while some
produced zones of hemolysis comparable to Lancefield's
group A streptococci.

The results obtained indicate that if the Lance-
field group O cultures are to be divided into "human”

and "animal" types it would be better to divide them

on the basis of their fibrinolytic activity rather than
by any other single characteristic. The "human“ type
should be designated by their ability to attack human
fibrin only. The “animal“ type should include both of
the other two groups. It appears that group C organ—
isms which produce little or no demonstrable lysin
should be grouped with the “animal“ type since the
writer has never seen a culture which has produced
partial lysis only of "human" fibrin in 24 hours. A
further reason for classifying these organisms as
”animal" type was the fact that they closely resembled
this type in the amount of acid produced in 1 percent

dextrose broth.

Fibrinolytic Properties of Hemolytic Streptococci

From Different Sources

The last part of this study concerns itself with
the fibrinolytic properties of hemolytic streptococci
in relation to the serological type and to the source l
of the strain. The results are summarized in table 6. A

Culture numbers 1 to 8 represent Lancefield's
serological group A, and were taken from human infec-
tions. They were from patients affected with septi-
cemia, scarlet fever or low grade skin infections.
None of these cultures lysed either horse or rabbit 5
fibrin. However all of the eight cultures lysed human I

fibrin. Several of the cultures were not particularly

Comparison of Serology and Fibrinolytic Activity

Table 6

Fibrinolysis

I
Culture Lancefield s Source Horse Rabbit Human
Number Sero. group

hrs.
1 hrBo
hr.
hrs.
hrs.
1hr.
hrs.
hrs.

Human
Human
Human
Human
Human
Human
Human
Human
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Human
Human
Bovine
Str.uberis Bovine
Human
Bovine
E uine
E uine
E uine

0000000000

A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
B
B

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0
2 hrs. 1 hrs.
hrs. 1hr.
O 1 hrs.
1 hrs. 0
O O
0 hrs.

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Comparison of Serology and Fibrinolytic Activity

Table 6 (cont.)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

- ibr‘ 1
Culture Lancefield's Seurce Horge Hggbizisfluman
Number Sero. group
2] C Equine 2Ehrs. O 0
28 C Equine lBhrs. O O
29 C Bovine lBhrs. O O
fiO C Bovine 5Ehrs.’ O__ O
§l C Boving_ lBhrs. O__ O
_22 0 Bovine O O 0
5} 0 Bovine O O O
54 0 Bovine O 0 O
55 0 Bovine 25hrs. 0 O
56 C Bovine 0 O O
)7 C Equine lnhrs. 24hrs. O
58 0 Bovine lBhrs. lBhrs. O
59 C Bovine 18hrs. 18hrs.§fl O
#10 D Cheese 0 0 O
41 D Cheese 0 O 0
£2 E Unknown 0 O __ O
Eég E Human O 0 O
54 E Human O O O
45 E Bovine? O O 0
—E6 E Bovine? O - 0 O
47 G Monkey 0 3hrs. hrs.
48 G Human O O 18hrs.
39 K Unknown 0 0 O
50 Str.viridans Unknown 0 O O
51 Str.viridans Unknown 0 O 0
52 Str.viridans Unknown 0 O O

 

 

 

 

 

 

 

active in lysing human fibrin and required from 18 to
2% hours to show evidence of lysis.

The Lancefield group B cultures (numbers 9 to 19)
were the serological types of Streptococcus agalactiae
described by Stableforth (14) in 1937. None of these
cultures attacked any of the three plasmas used. It
was further noted that, although Madison (7) suggested
that there might be a specificity of the organism for
the plasma of the host from which the organism was
isolated, bovine mastitis streptococci including S35;
agalactiae, group C streptococci of bovine origin and

Str. uberis all failed to attack or fibrin.

 

The Lancefield group C cultures lend themselves to
three divisions discussed in detail previously.
Group D, E, and K cultures and also three strains

of Streptococcus viridans failed to attack any of the

 

three fibrins used.

Group G cultures reacted similarly to the “human"
type group A and group C cultures which attacked only
human or rabbit fibrin and not that from horses. These
cultures required a longer time to lyse human plasma.
than did the group A cultures.

It may be seen from table 6 that the only sero-
logical type of hemolytic streptococcus cultures which
attacked horse plasma was Lancefield group C.

. Discussion

The fibrinolytic test definitely has its place in

the routine testing of Lancefield's group C strepto-
cocci. It is the only test used which gave an indica-
tion as to which of these cultures might affect the
health of human beings. In making fibrinolytic tests

it is necessary to use a suitable medium for growth of
the culture because various ingredients used in the
medium affect the production of fibrinolysin, the most
important of these are the basic broth, the peptone,

and fermentable carbohydrate content. In making fibrin-
olytic tests on group C streptococci it was necessary to
omit the carbohydrate since it resulted in the produc-
tion of.so much acid that the pH of the inoculum lower-
ed the pH of the plasma sufficiently, unless it was
neutralized, to prevent coagulation upon addition of the
normal amount of calcium chloride. It appears that this
factor led many of the earlier investigators of this
problem to believe that certain cultures were lytic

when actually they produced little or no lysin.

Horse fibrin was the only fibrin used which the
“animal” type group C cultures lysed consistently. The
group C cultures which lysed neither horse nor human
fibrin appear to be of the "animal' type because of
their biochemical properties and because the “animal"
type in general produced a weaker lysin than the “human"
type of this group. It is possible that these cultures
did not produce enough lysin to yield a reaction, by

the methods used.

Physical variations of the original Tillet and
Garner technique were tried such as adding sterile
fibrin to the media in which the cultures were grown.
Test cultures were grown while being agitated because
it was thought that this might increase the amount of
metabolic products produced by certain strains of
organisms. Neither of these procedures was of any
value in the production of fibrinolysin. “Activation“
by rapid chilling the culture after growth and storing
at 4'0. for five hours was the only physical treatment
which gave a more rapid and clear cut reaction.

In testing various types of streptococci it was
found that the ''animal" types of group C were the only
ones which would attack horse plasma. There was no in-
dication by either the group A or group G cultures,
which produce a large amount of lysin for human fibrin,
that this lysin would attack horse fibrin. It would
seem logical then that organisms which belong in the
“animal" type of group C produce a different type of
lysin than is produced by group A, the "human” type of
group C, and group G. Ability of group C cultures to
lyse human and horse fibrin would seem to offer a more
rational basis for division of the group C cultures

than any method suggested previously.

Summary

1. The lytic properties of group C streptococci
were enhanced more by growing in a medium containing
Difco Proteose Peptone than in any of the other six
commercial peptones studied.

2. The presence of dextrose in the culture medium
resulted in the production of too much acidity for this
to be used in the fibrinolytic test unless the culture
was first neutralized.

5. It would seem advisable to regard “human“ type
group C cultures as those which attack human fibrin and
to regard as “animal" type group C those which attack
either horse fibrin or neither human nor horse fibrin.

4. It was found necessary to run fibrinolytic
tests with both human and horse fibrin to differentiate
the types of Lancefield's group C streptococci.

5. Biochemical reactions were of no help in sepa-
rating those cultures which lysed human fibrin from
those which lysed horse fibrin or those which lysed
neither, except in determining the final pH in a l per-
cent dextrose broth.

6. Horse fibrin was 1ysed by cultures of Lance-
field's group C of the "animal" type only, and not by
group A or group G cultures.

7. Bovine mastitis streptococci, that is S35;

agalactiag, Str. dysgalactiae, and Str. uberis, failed

 

to lyse human, horse, rabbit, sheep, goat, ox, or

 

”-31-. . ‘ \ A .. n

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. 4 . .~ _.. .~ -:'-=""-,_'~'~‘ -..- -‘

SUmmary (cont.)

chicken fibrin.
8. No simple method other than "activation" was
found which would aid in making the fibrinolytic test

more sensitive or easier to interpret.

Acknowledgements

The author wishes to express his appreciation to
the following people: To Dr. C. S. Bryan of Michigan
State College for his supervision of this project and
the many kindnesses and courtesies extended by him:
To Dr. Hard Giltner of Michigan State College for his
suggestions and help in preparation for this Master's
Degree thesis: To Dr. I. N. Plastridge of the Storrs
Agricultural Experiment Station for his suggestions
and help on this project: Lastly to my wife for her
hard work and patience in typing and correcting this

manuscript.

 

(1)

(2)

(5)

(4)

(5)

(6)

(7)

(5)

(9)

BIBLIOGRAPHY

 

Brown, J. H.
1957 SIGNIFICANCE OF THE HEMOLYTIC STREPTOCOCCI
FOUND IN MILK. Cornell Vet. 27:110-121.

Dawson, M. H., Hobby, G. L. and Olmstead, M.
1958 VARIATIONS IN THE HEMOLYTIC STREPTOCOCCI.
Jouro Of Info D130 62:158-168.

Dimock, H. H. and Edwards, P. R.
1955 HEMOLYTIC STREPTOCOCCI OF HORSES AND OTHER
ANIMALS AND THEIR RELATION TO THE STREPTO-
COCCI OF MAN. Kty. Agr. Eth. Sta.
Research Bul. 558,55p.

Edwards, P. R.
1955 THE SEROLOGICAL DIFFERENTIATION OF HEMO-
LYTIC STREPTOCOCCI OF HUMAN AND ANIMAL
ORIGIN. Kty. Agr. Expt. Sta. Research
BUlo 556,18po

Evans, A. C.
1956 STUDIES ON HEMOLYTIC STREPTOCOCCI
III STREPTOCOCCUS EQUI AND RELATED STRAINS.
Jour. of Bact. 52:541-556.

Lancefield, R. C.
1955 A SEROLOGICAL DIFFERENTIATION OF HUMAN AND
OTHER GROUPS OF HEMOLYTIC STREPTOCOCCI.
Jour. of Expt. Med. 57:571-595.

Madison, R. R.
195# FIBRINOLYTIC STREPTOCOCCI FROM LOWER
ANIMALS. Soc. Eth. Biol. and Med.
Pro. 52: 44A-445.

Madison, R. R. and Taranik, Jeanette D.
1957 DYNAMICS OF FIBRINOLYSIN PRODUCTION BY
STREPTOCOCCI. Soc. EXpt. Biol. and Med.
Pro. 56:1-5.

Planet, Nelson
1955 SUR L'ACTION FIBRINOLYTIQUE DES STREPTO-
COQUES HEMOLYTIQUES D'ORIGINE EQUINE.
Comp. Rend. Soc. Biol. 120:169-172.

(lO)Plastridge, U. N.,Banfield,L. F. and Williams, L. F.

1940 AGGLUTINABILITY OF MASTITIS STREPTOCOCCI.
Jouro 0f Info D180 66:202-211.

(11)

(12)

(15)

(14)

(15)

(16)

(17)

Bibliography (cont.)

Plastridge, U. N. and Hartsell, S. E.
1957 BIOCHEMICAL AND SEROLOGICAL CHARACTER-
ISTICS OF STREPTOCOCCI OF BOVINE ORIGIN.
Jour. of Inf. Dis. 61:110-121.

Sherman, J. M. and Niven, C. F.
1958 THE HEMOLYTIC STREPTOCOCCI OF MILK.
Jour. of Inf. Dis. 62:190-201.

Smith, F. R., Hankinson, C. L., and Mudge, C. S.
1956 FIBRINOLYTIC ACTIVITY OF BETA HEMOLYTIC
STREPTOCOCCI FROM COW'S MILK. Soc. EXpt.
Biol. and Med. Pro. 54:266-270.

Stableforth, A. U.
1957 SEROLOGICAL TYPES OF STR. AGALACTIAE
(STREPTOCOCCUS GROUP B) IN THIS AND
OTHER COUNTRIES. Jour. Path. and Bact.
45:265—277.

Tillet, U. S. and Garner, R. L.
1955 THE FIBRINOLYTIC ACTIVITY OF HEMOLYTIC
STREPTOCOCCI. Jour. of Expt. Med.58:

Tillet, '0 So
1955 THE FIBRINOLYTIC ACTIVITY OF HEMOLYTIC
STREPTOCOCCI IN RELATION TO THE SOURCE
OF STRAINS AND TO CULTURAL REACTIONS.
Jour. of Bact. 29:111-150.

Tillet, W. 30
1959 THE FIBRINOLYTIC ACTIVITY OF HEMOLYTIC
STREPTOCOCCI. BECt. Rev. 2:161-216.

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