There is also abundant epidemiological evidencethat a given ample

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STUDIES ON HEMOLYTIC STREPTOCOCCI
I. METHODS OF CLASSIFICATION
ALICE C. EVANS'
UnitedStates Public Health Service, Washington, D. C.
Received for publication November 22, 1935
INTRODUCTION
That a satisfactory method for the classification of the hemo-
lytic streptococcihas not yetcomeintopractical use is illustrated
in the last edition (1934) of Bergey's Manual of Determinative
Bacteriology, by the description of six species, pyogenes, scarla-
tinae, puerperalis, erysipelatis, epidemicus and cuniculi, with no
distinctive characters other than that they are said to cause dif-
ferent diseases, and that, for some, specific serologic reactions are
claimed.
Ability to produce a specific disease is a character which may
beutilizedin bacterial classification, in so farasit may contribute
to the differentiation ofspecies. The factisnowwell established,
however, by the works of many investigators, and especially by
the agglutinin absorption studies of Williams and her collabora-
tors, and also those of Griffith, that a given serological type of
streptococcus may cause more than one kind of disease.
There is also abundant epidemiological evidence that a given
strain of streptococcus can cause more than one kind of disease,
in the manyreports to be foundin the literature ofmore than one
streptococcus disease occurring in the same epidemic. For ex-
ample, among 50 septic sore throat epidemics reported by Wil-
liams, cases of scarlet fever occurred in 18, and cases of erysipelas
occurred in 11. Hence it is obvious that an attempted differen-
tiation of streptococci into species on the basis of disease source
ends in confusion.
SeniorBacteriologist,National InstituteofHealth.
423
JOURNAL OF BACTERIOLOGY, VOL. 31, NO. 4
424 ALICE C. EVANS
Thompson and Megrail recently reported a study of 120
strains of streptococci, in which the correlation between type of
hemolysis on blood agar and certain biophysical, biochemical
and pathogenic properties of the strains was investigated. They
failedtofindatest whichwoulddifferentiate the hemolytic strep-
tococci into smaller groups of significant meaning. According
to Zinsser and Bayne-Jones (1934) the material for a natural
classification of streptococci is not yet available.
Much valuable information has been obtained, however, by
individuals, or by small groups working with one or more tests,
or working with strains from limited sources. To bring together
in a large collection representative strains from the collections
on which studies have been made, and to coordinate the work of
the manyinvestigatorsintoarationalclassification wastheobject
of the present study. The tests which other investigators have
found useful for classification were considered, and the strains
were also studied from a new point of view dependent on their
sensitivity to several races of streptococcus bacteriophage.
SOURCE OF CULTURES
Strains from the following sources have been studied:
Number
Animalsources: ofstrain
Various diseases .........................1.3.9
.......................
Suspected milk associated with epidemics 14
........................
Human sources:
Scarlet fever.................................................... 119
Puerperal fever 62
.................................................
Erysipelas 38
.................................
Epidemic sore throat............................................ 32
Rheumatism..................................................... 16
Acute suppurative diseases'..................................... 52
Other miscellaneous diseases 56
.................................
Normal throats................................................. 30
Intestinal and other miscellaneous sources....................... 15
Total......................................................... 573
The streptococci were isolated in distant parts of the world.
The writer is greatly indebted to the many investigators who re-
2Theacutesuppurative diseasesincludeempyema, osteomyelitis, peritonitis,
mastoiditis, meningitis, sinusitis, otitismedia, cystitis,adenitisandabscesses.
425
STUDIES ON HEMOLYTIC STREPTOCOCCI
sponded to requests for representative strains of their collections.
Without the cooperation which was so generously given, the
present studies could not have been made.
In determining the relationship between bacterial species and
specific diseases, it is of great importance that strains from dis-
tant localities should be compared. The data collected in the
present study show a number of instances in which an unusual
type of streptococcus was disseminated throughout a limited
area; and there are in the literature erroneous conclusions as to
the disease specificityofcertain types ofstreptococci whichwould
have been avoided if strains collected from wider areas had been
studied.
In anticipation of the forthcoming series of papers it may be
stated that several species with peculiar pathogenic properties
are recognizable. One is capable of causing erysipelas and no
other disease, in so far as our data show; another causes scarlet
fever, and apparently no other disease. The understanding of
their disease specificity has been confused, however, by the fact
that there are other species whichmay cause erysipelas, or scarlet
fever or various other kinds of infections. The results of this
study reconcile what appeared to be the opposing opinions of
those on the one haad, who contended that the streptococci of
scarlet fever and of erysipelas are disease specific; and of those,
on the other hand, who contended that the two diseases are
caused by the same organism.
CHOICE OF DIFFERENTIAL CRITERIA
In order to be practical, thecharactersutilizedforroutine iden-
tification ofstreptococcimustbe correlatedwithpathogenic prop-
erties; and in order to be most useful, they shouldbeassimple as
possible.
Some of the characters of streptococci which are of greatest
interest when their pathogenic properties are considered are of
little value for routine classification purposes, because differences
are quantitative, with every gradation between the extremes.
Thus, according to our present knowledge, one qualitative type
of leucocidin is produced in varying quantities by more than one
426 ALICE C. EVANS
species of streptococcus Likewise, according to Van Deventer,
there is only one immunological type of streptococcus when
judged by the fibrinolytic property. Todd reported that the
hemolysin produced by animal strains of hemolytic streptococci
differed serologically from that ofhuman strains, but Loewenthal
and Pradham found only quantitative differences.
Virulence for mice is another character in which differences are
quantitative, varying from the high degree generally possessed
by certain groups which cause spontaneous disease in animals,
to the low degree possessed characteristically by certain groups
of human strains, with gradations between the groups and within
each group.
On the other hand there appear to be qualitative differences in
theerythrogenictoxinproducedbydifferenthemolyticstreptococci
capable of causing human disease (Birkhaug; Dick and Dick;
Pilot and Dreyer; Trask and Blake; Hooker and Follensby;
Wadsworth and Coffey). As yet, however, the difficulty of ob-
taining a supply of suitable sensitive experimental animals re-
tards progress in the comparative study of toxins. Through
the courtesy of those investigators who have studied the different
types of streptococcus toxin the proffered scheme of classifica-
tion has been correlated with toxin production in a number of
strains.
THE IMPORTANCE OF SEROLOGICAL GROUPING IN A SCHEME OF
CLASSIFICATION
To be accepted as logical, the classification of hemolytic strep-
tococci cannot be discordant with the serological groupings which
have been worked out. The generous cooperation of those in-
vestigators who have made comprehensive studies of agglutinin
absorption and precipitin reactions has made it possible to cor-
relate serological grouping with the characters determined in the
present study, without a duplication of efforts. Further com-
parative studies will be necessary, however, to complete the de-
scription of characters of certain groups. Coordination of the
present study with the groupings according to agglutinin absorp-
tion reactions was made possible by the addition to our collection
427
STUDIES ON HEMOLYTIC STREPTOCOCCI
ofthe various type strains described byWilliams andher collabo-
rators; by Griffith; and by Mueller and Klise.
Coordination of the present study with the groupings accord-
ing to Lancefield's precipitin test was made possible by the addi-
tion to our collection of many strains which had been previously
studied by Lancefield (1933); by Edwards (1934); and by Plum-
mer. With very generous courtesy, Dr. Lancefield has typed a
few strains of our collection which represent phagological groups
not included in her previous studies.
The serological studies of the investigators mentioned have
yielded valuable information about the grouping of streptococci.
Lancefieldhas differentiatedthehemolyticstreptococciinto seven
groups according to precipitin reactions, and she is investigating
the further subdivision of the groups. The studies of Smith;
of Williams and her collaborators; of Mueller and Klise; and
of Griffith have agreed in showing that the agglutinin absorp-
tion reactions divide into many types the streptococci capable
of causing agiven disease. Forexample, the scarlet fever strains
studied by Griffith fall into 20 serological types.
It wouldbe illogical to regardas distinctspecies those serolog-
icaltypeswhich are indistinguishable except for agglutinative dif-
ferences. However, on account of the division of streptococci
into many types by agglutinin absorption reactions, that test is
most useful for tracing epidemics to their sources when time and
circumstances permit an exhaustive study. On the other hand,
the exigencies of an epidemic demand a more simple and speedy
test which will classify strains into groups harmonious with the
serological groups.
THE RELIABILITY OF FERMENTATION AND OTHER REACTIONS
The reliability of fermentation reactions for the differentiation
of hemolytic streptococci has been considered in recent reviews
of the literature. The Thomsons conclude that it would be dan-
gerous to classify streptococci primarily on the basis of sugar re-
actions, becausevariousinvestigators use differentmethodswhich
give different results. There is another aspect of the reliability
of fermentation tests for the classification of streptococci-con-
428 ALICE C. EVANS
cerned with the question whether differences in the fermentation
of a given carbohydrate should be regarded as belonging to the
category of species, variety or race differentiation. This con-
siderationisrecognizedbyOkell,whoconcludesthatfermentation
tests are useful in identifying particular individual strains, but
that they fail to supply a method of classification of pathological
importance. Williams thinks that fermentation reactions should
be used as a basis for the study of new strains until some other
methodis shown to be better. Thestudies of Ogura, ofEdwards
(1933) and of Minett showed that human strains of hemolytic
streptococci are characterized by the fermentation of trehalose
but not sorbitol, whereas animal strains generally ferment sorbi-
tol but not trehalose.
In our experience, after studying the correlation of fermenta-
tion reactions with other reactions and with the sources of the
strains, we have arrived at the conclusion that the ability to fer-
ment certain substances is of definite value for the classification
of hemolytic streptococci. Differences revealed by reactions in
the presence of those substances are so well correlated with other
distinguishing characters that they may be regarded as specific.
Final pH in glucose broth was proposed by Ayers, Johnson
and Davis as a character for the differentiation of streptococci.
Their observation that non-pathogenic bovine strains produce a
higher final acidity than pathogenic strains was confirmed and
amplified by Avery and Cullen, and by many subsequent inves-
tigators.
Ayers and Rupp devised another test which differentiates
"Streptococcus pyogenes" from Streptococcus mastitidis. They
found that the latterwill hydrolyze hippuric acid with the forma-
tion of benzoic acid and glycocoll. The benzoic acid is readily
detected in the medium by the addition of ferric chloride. This
observation has been confirmed by many investigators.
Capsule formation was not used as a differential test in this
study because many investigators (Rosenow; Ward and Lyons;
and others) have shown that a given strain may develop
capsules in one environment but not in another.
Colonial appearance was not considered in this study because
STUDIES ON HEMOLYTIC STREPTOCOCCI 429
it has been established by the observations of Cowanand many
subsequent investigators that a given strain may show variations
in type of colony.
The cultivation of streptococci on benzidine blood agar is ad-
vocatedbyTunniclifffor theidentification ofscarletfever strains.
She states, however, that some of the dissociants of scarlatinal
streptococci behave like erysipelas and sore throat strains in pro-
ducing a black color in this medium. In our experiments certain
erysipelas and sore throat strains produced a marked blackening
of the medium, in contrast to certain other strains which did not
produce this effect. Thescarlet feverstrains, however, were not
consistent in their behavior on this medium. Our results with
hemolytic streptococci from a great variety of disease sources,
representing every species in our collection, led to the conclusion
that the test lacks the precision necessary for a differential test.
Possibly the fact that old stock cultures were used in our studies,
whereas Tunnicliff apparently stuciied freshly isolated strains,
may account for the discrepancy in our results.
SENSITIVITY TO BACTERIOPHAGE AS A DIFFERENTIAL TEST
Bail was the first to suggest that sensitivity to bacteriophage
might serve as a means for the differentiation of closely related
bacteria. It has never been so used in any extensive classifica-
tion study, presumably because those races of phage which have
been subject to the most extended studies-the staphylococcus,
Salmonella, dysentery and cholera phages-are too broad in their
lytic capacities to permit differentiation of bacterial species.
Recently, however, Levine and Frisch studied the possibility of
classifying Salmonella according to the ability of the heat-killed
organisms to absorb bacteriophage. They were unable to dif-
ferentiate the organisms of the typhosus-enteritidis group and
those of the aertrycke-paratyphosu8 B groups from each other, al-
though the results of a previous study by these authors, and also
the investigations of Gough and Burnet, who worked with the
dysentery bacilli, indicate that the specific somatic antigen is a
complex labile polysaccharide which is responsible for both the
serologic character andthe phage susceptibilityofthebacteria.
430 ALICE C. EVANS
Using a different phagological technique from that employed
by Levine and Frisch, the writer has found that sensitivity to
several phages offers a key to species differentiation of strepto-
cocci, with phagological groupings in agreement with serological
groupings. In a previous publication (Evans, 1935), it was re-
ported that sensitivity to several races of phage in the nascent
stage, that is, in the presence of a sensitive strain, is a distinctive
character of hemolytic streptococci. Previous to that report,
Kendrick and Hollon had noted briefly that there is a definite
parallelism between the serologic and bacteriophagic reactions of
the fecal streptococci; and Lancefield (1932) had reported that
47 out of 56 strains of streptococci found to be sensitive to the
Clarkphage (B filtrate) were of animal origin andbelongedtoher
serological group C.
The technique described further on for the classification of
hemolytic streptococci according to phage sensitivity is reason-
ably simple. The phages retain their potency indefinitely when
kept in a refrigerator. Hence, the stock supply is always ready
for instant use, and the reactions may be determined overnight.
Four phage preparations are sufficient for the classification of
all hemolytic streptococci.
On the whole, the groupings made with the aid of sensitivity to
phage as a differential character agree with species which have
already been made on other bases. A few new groups have
emerged in this study, however, which appear to be so well de-
fined that they should be regarded as species not hitherto recog-
nized. Further, the study has shown that certain names which
have been applied to alleged groups with no other distinctive
character than the ability to produce a certain disease, do not
apply to distinct species and must be discarded or regarded as
synonyms in a logical scheme of classification.
CHARACTERS INCLUDED IN THE PRESENT STUDY
The present study includes the determination of the following
characters for all strains of the collection: Sensitivity to strep-
tococcusphages A, B, C and D;finalpHinglucosebroth;fermen-
tation of lactose, salicin, mannitol, trehalose and sorbitol; ability
STUDIES ON HEMOLYTIC STREPTOCOCCI 431
to grow on blood agar containing 10 per cent bile; ability to grow
on blood containing 40 per cent bile; and hydrolysis of sodium
hippurate.
The tests mentioned above served to classify the strains into
groups. Further studies, including the fibrinolytic reaction and
virulence for mice were made on representative strains of the
various groups. The ability to produce agglutinins in rabbits
was determined for some of the strains belonging to the species
Streptococcus equi and related groups; and the ability to produce
erysipelas in rabbits was determined for representative strains
derived from human erysipelatous lesions.
DIFFERENTIAL TESTS
The tests which were applied to all strains of the collection did
not prove to have equal differential value. The well-defined
groups are differentiated by the following characters: Sensitivity
to phages A, B, C andDinthenascentstate; sensitivity tophage
Bfiltrate; andfermentationreactionsinlactose, salicin, mannitol,
trehalose and sorbitol broths. These will be called the differen-
tial tests; and for convenience of discussion, the present scheme
of classification will be called the phagological classification,
though the5mentioned fermentationreactions are alsoimportant
in the scheme.
Ahighlyacidfinalreactioninglucosebrothmerelycorroborates
distinctions clearly made by the differential tests. The two
species which hydrolyze sodium hippurate, are clearly distin-
guished by the primary differential tests. Growth on 10-per
cent bile blood agar was found to have no differential value.
Growth on 40-per cent bile blood agar was found to be so well
correlated with the reactions of the differential tests that the
bacteriostatic action of bile merely adds confirmatory evidence.
PhageAwasfoundtobe sobroadinitsactivity thatitisuseful
only in the case of three groups to corroborate differences other-
wise shown by the differential tests. It was pointed out in an
earlierpaper (1935) thatbythe use of2strains ofphageC, certain
groups intermediary between S. pyogenes and S. epidemicus may
be distinguished. The use of both strains of phage C is recom-
mended for the differentiation of those species.
432
ALICE C. EVANS
EXPERIMENTAL PROCEDURES
The strains of streptococci included in this study are aerobic,
and produce a zone of clear hemolysis when smeared on blood
agar plates, (infusion agar containing 5 per cent of rabbit blood)
and incubated 48 hours before examination.
Sensitivtty to phage. The four races of streptococcus phage
used in these studies, A, B, C and D were described in an earlier
paper (Evans 1934) in which the histories of the strains of strep-
tococci used for substrata were also given. Lytic filtrates are
designated by a letter for the race of phage and a number which
designates the strain of streptococcus which served as a substra-
tum, as, for example, A/751.
It was stated in the earlier publication (1934) that the range
of activity of a given race of phage is influenced by the strain of
streptococcus which serves as the substratum for its multiplica-
tion. Hence it was necessary to choose for the propagation of
each race of phage a strain of streptococcus which would give it
such range of activity as would be most useful for classification
purposes. The two races ofphage, B and D, whichwere received
from other laboratories, were each propagated on the strain of
streptococcus which was used as a substratum by the preceding
investigators. For the two races ofphage isolated in this labora-
tory, A and C, a considerable time was spent in making an effort
to choose suitable streptococci for substrata. The effort was
withoutsuccessinthecaseofphageA. Theresultswithstrain751
for a substratum seemed promising, however, and all strains were
testedforsensitivitytonascentphage A/751. As alreadystated,
the range of activity of this phage proved to be too broad to be
useful for classification purposes.
In the case of phage C it was found that two strains of strep-
tococci, 594 and 646 would each impart to the phage a distinct
range of activity usefulfor the classification. Itwould be a com-
paratively easy matter now, after the classification studies have
been made, to select appropriate strains for use as substrata for
phage C. But the selections had to be made previous to the
classification studies, when the characters of relatively few of