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A NOTE ON A LYTIC PHENOMENON SHOWN BY GROUP B
STREPTOCOCCI
by K. CHRISTIE, N. E. ATKINS
AND E. MU NC H- PE TE HS EN
(B'rom the Commonwealth Serum Laboratories, I'niversity Bacteriology Depart-
ment and C.S.I.R. Animal Health Researeh Laboratx)ry. Melbourne).
Accepted for publication 11th July, 1944.)
Tn a recent outbreak of Scarlet Fever in a country disti'iet it was suspected
that the milk was tbe vehicle of infection. Five samples of the milk supplied to the
area were submitted to examination for haomolytie streptococci. After centrifuga-
tion, tlie mixed cream and sediment were stroked out on the surface of sheep blood
afjar plates which were inenbated over-nijclit at 37 C. From three of the samples
.streptoeocci were obtainetl whieh were apparently haemolytie ou tbe iii-iniary plates
but uon-haemolytic on sub-eultnre.
Examination of the primary plates revealed tbe presence of many .staphylo-
eocei of tbeafitype, that is their colonies were in tbe centre of a clear zone, which
in tnrn was Murronnded by a large darkened zone, wbere the stapbylocoeeal
i
toxin
had altered, bni not iysed, the sheep red cells. Wherever tliere were colonies of the
streptococcus within these zones of darkening, the colonies were surrounded iiy an
area of complete haemolysis, whilst elsewhere on the ])late.s they produeed no distinct
haemolysis
To confirm tliia ohserviitioii, a colony of
tlieae stui>liy (>t;occi wna grown by spot-
inoeuliitioii on the centre of
a
slieep blood
jigar plate. After L4 hours' incubation tho
atroptoeoeci were inofiilatod on to the darl;-
enod zone. Within two hours hiii.'nio].vsl3 was
visible aroinid the Rtrt'jitoeoccEiI growth.
Ill the ease of
n
st.iphyiococ
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198 R. CH RIS TIE , N. E. ATK INS
AXD
E . MtTNCH-PETERSEN
"i iioii-haeniolytie anaero bic stvaina of human o rigin (reeoivi'd th rough tlie courtesy of Miss
IL Btitler), none gavt> the roaetion when grown with the stapliylococcus, although aomo showed
;i narrow :nT i of iiicoHii)l('te lysiy. Fo rty -th roe stn iiiia from eases of siispofted and nini't('i.'n
from cast's of di fiiiitc liovinc mastiti s were tested . Of these, ali }mt three giivc tlie niai-tioii.
Scro logieal eX iimliKition tlicii revealed th at of all Ihe sti-aiiiN tested only those which belonged io
(rroiip B ]jaiH'e ielil gave the reaction, an d none belniigiiig to (irtui]> B failed io give H. Five
(jrou]:i B strains of liunian origin (tdiiaiis, vagina and skin lesion) also gave the positive reai'tioii,
Tliroc rep resen tative sti-iiins of each of the (iroiiis A,
V
and (i were teste
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GROUPB STREPTOCOCCI 199
incubation
for one
liour
at
37
C, the
cells were found
to be
lysed
in the
tube containing
the
undiluted filtriite,
bnt the
power
of the
filtrate
to
induce lysis
was
quickly lost
on
dilution.
An
undiluted sample which
had
been h eated
at 56 C. for 30
minutes eaused only
a
trace
of
lysis,
but
a sample lieated
at 50 C. for 30
minntes
and
then maintained
at
100
0. for
five minutes,
had a
iytic ])otency similartothatof theunheated filtrate. Asamjile heated forfive minutesat 100"C.
was asx>otent as the raw filtrate (seeTable 1). When an undiluted samjile of the filtratewas
allowed to act on the redcellsfor30 minutesat 37 C, lysis occurred immediately onadditionof
the
fi
toxin.
TABLE 1.
Results
of
haemolysis titrations, using streptoeoccal filtrate, staphylocoecal
i
toxin
and
sheep
red cells.
SeriesI. Ten
drops
of
each diluted preparation
of
streptoeoccal filtrate were added
to the
corresponding tube.
Ten
drops
of 3
toxin
(raw,
filtered toxin diluted
1: 10
with normal saline)
were added toeach tube. Two drops of a 10ji.c. suspension in salineof washed sheep redcells
were then added
and the
tubes inculiated
at
37
0. for 1
honr.
ScrictII. As above, exeept tha t the filtratewasfirst heatedto5(1 C.for 30minutes.
Sfries
III.
As in
series
I,
except that
the
filtrate
was
first heated
to 50 C. for 30
minutes,
iind then to100 C.forfive niiiiutes.
SeriesIV. As inseries^,except that the filtratewasfirst heated to10(1C .forfive minutes.
Scrien
V.
As in
series
I, but
witli saline substituted
for the /i
toxin.
Scries
VI.
As in
scries
I, bnt
with saline substituted
for the
streptoeoccal filtrate.
Initial dilutionof filtrate.
10/10 8/10 6/10 4/10 2/10
Series I
Series II
Series
III
Series IV
Series V _ .
Series
VI _ _ _ _ _
- --{-+ + Indicates comjilote lysis. -\-Indicates 25p.c. lysis.
Indicatesnolysis.
The qnantityof theagent present in the filtrate wasnotfound to beincreasedby incubation
for longer tiiau
i8
hours.
Th< staphylocot-ei most commonly found
in
animals
are
predominantly
(3
toxin-produeera
(Minett,
1936;
Cowan, 1938).
It is
only with these staphylococci that Gronp
B
strepto(;occi
produce the lysis described above. Not infreqnently staphylococci andGroupBstreptococciare
fonnd together
in the
bovine u dder; there
may be
some significant connection lietween th is
occurrence
and the
fact tha t toge ther they have
a
Iytic power which neither
has
independently.
The results obtained with
the
Group
B
streptococci
of
human origin give further evidence
of
the elose relationship of theanimal andhuman strains of this group, in spite (if the differences
in serological tyj)eand insome other characteristics establishedbySimmonsandKeogh (194 0).
The nejrative results ol>taiiied when horse blood was substituted for sheep
blood siijiuest that in routine tests for haemolytic streptococci, horse blood only
should be used, ijarticnlarly where Iitoxin-jn oducin^ stapliylococei are to be ex-
pected, as in milk samples.
The nuniber (tf members of the various rroups tested was not larfje, but the
resnlts obtained so far indicate that the phenomenon may provide a relatively
simple confirmatory or substitute test for Group B streptococci.
SUMMABY.
Strains of Group B streptococci, of aniinal and hnman orijzin. produce an
agent which will lyse sheep and ox. but not hnman, borse. rabbit or f^iinea-pij; red
cells, wh(?n these cells have been altered by staphyloeoccal itoxin.
The a^ent is extracellular, tlltrable, and thermostable.
Streptococci of liiiman and animal ori
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200 R. CPIRTSTIE, N. E . ATKTNS .\NDE . J ir 'N C H -PETER SEN
AcTcnn ivlctJgme nt. i. We are indebted to Professo r WoodrufF. of the rn iv e rs it r Itac-terio
logieal D epa rtmen t, for a(iviee and (riti( i.sni, to M iss M. Monsbourgh jiiid M r. H. A . Keddoine
for teclniU'al aasiatance, to Miss M. (iarhind for milk samples, aiul to Mr. R. T. Simmons for
providing strains reprcsentative of various groups of streptoeycei.
RE FE RE NCE S.
Christie, R. and Grnydon, J . J . (1SI41): Austr al. ,T. exp. Bio ., 19, p 9.
Cowaii, 8. T. (1938) : J . P ath . Bac t., 4(i, p. 31 .
Landsteiner, K. and von Raiiohenlnehler, R. (1909) : Z. Inimun. Porsch., 1, p. 4S9
Minett, F. C. (193{i) : J. Path. Bact., 42, p. 247.
Orcutt, M. L. and Howe, P. E. (1922) : J. exp. Med., 35, p. 409.
Simmons, R. T. and Keogh, E. V. (1940) : Austral. J. exp. Biol., 18, p. 1.^1.
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