A NOTE ON A LYTIC PHENOMENON SHOWN BY GROUP B

STREPTOCOCCI
by K. CHRISTIE, N. E. ATKINS AND E. M U N C H - P E T E H S E N
(B'rom the Commonwealth Serum Laboratories, I'niversity Bacteriology Department 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 centrifugation, 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 .staphyloeocei of tbe afi type, 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 spotinoeuliitioii on the centre of a slieep blood
jigar plate. After L'4 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<ruH producing
o toxin only and giving therefore au area of
partial haemolysis around a zone of clear
haemolysis no variation of the haemolysis
was caused Iiy growing the strojitocdCciiB iu
or near liie xoue of jiartial haemoly.sis, but iu
the dark zone around staphylococci producing (i toxin only, haemolysis was readily
limdiiced.
When the atrejitococcus was grown near
tlie e(lg<> of one of these uniform dark zones.
clearing of the medium could be seen at the
point nearest to the streptococcus colony.
suggesting that, an invisiMe agent, or the
products of such an agent, hiul I'oaclu'd the
rod cells already altered by the staphylococcal jS toxin (Fig. 1).
Fig. 1. The central streak is due to growth of
Under anaei-ohic conditions the effect was
staphylococ'ci on sheep blood sigar. The zone
more marked, and definite pitting of the
around it ia due to toxin. The lower streak
mc'diiim coiiid lie seen in position coinciding
shows a growth of Grou]) B streptococci with
with the clearing, iiulicattng that some conh!iein<i]ysis where it adjoins the stapliylococcal
stituent or constituents of the medium had
zone. The upper streak shows growth' of nonbeen altered to give diffusible siibatanceH.
Group B streptococci.
The pitting was too dee]> to \w due entirely
to migration of the haemoglobin liberated
tiy red cell lysis. Furthermore, the clear area waa glasay-clear and not faintlv turbid as is
u.'iually found when there is cell lysis only.
To demonstrate the lysis, sheep and' ox blood were found suitable. No lysis was ol.tdined
when horse, human, rabbit or guinea-pig blood was used.
A number of strains of strej.tococci were then tested. Of 55 from various huuian sources
(throat, sputum, urine, skin, tooth and noae), some haemolytic, others lum-haemolvtic :nid

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R. CHRISTIE, N. E. ATKINS AXD E . MtTNCH-PETERSEN

"i iioii-haeniolytie anaerobic stvaina of human origin (reeoivi'd through 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. Forty-throe stniiiia from eases of siispofted and nini't('i.'n
from cast's of di^fiiiitc liovinc mastitis were tested. Of these, ali }mt three giivc tlie niai-tioii.
Scrologieal eXiimliKition tlicii revealed that of all Ihe sti-aiiiN tested only those which belonged io
(rroiip B ]jaiH'e!ielil gave the reaction, and 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! representative sti-iiins of each of the (iroiiis A, V and (i were teste<l. None was positive.
\Vliere normal Iiaeinolynia (iccurred. aa with the Group A strains, the haemolytie ,'ire;i WA IH
mtire extensive where it overlapped the stitpliyloi-uccal zone than elsewhere in the medium. To
determine whether lysis, similar to that cauncd hy iioii-haeniolytic <jroiip li strains, wns oreiirring
i>nt lieiiig masked liy the ordinary stre]>toi-ni;'cal haemolysis, the boiled broth lec'liiU(|iie, (lescrilxMi
later, was used. The results were still negative. Xo (Irduj) B strain jirtKlut'ed suffifienl lysis by
itself to interfere with the rending iif the haemolysis imliieed within the st;iiiliyl<)c()ceiil zone.
Tilt' iiresence of the ai^tive iigent in the solid medium coukl be (.leteeteil fully imlf ii ceiitimelro
from ttie 8tri']toc'oec;il eoUinies, There was no cleteetiible differeuce in the degree to which tilt'separate strains jn'oduceil Ihe agent, although li! serologieal sub-typea were represented in tho
strains tested.
It is known that certain non-haeniolytif staphylocoeci, wlicn grown oil Iilood agar lu^ar a
staphyloeoccuB with a zone of fi toxin, prodnce lysis within this zone Just as do the (^iroup B
streptococci ((.'hristie and (Jraydoii, 1941). Some evidence lias been giveu to show that tlie active
agent from the stapliylncoccus is stapliylociicciil lipase, (Jroup B and other stre]ttoctK'oi were
therefore tested for !i]Kilytic activity by growing on cream bUiod agar (Orcutt and Howe, 1922)
anil on a Initter-fat neutral red iigar, lmt niint' could lie detected.
Many n()n-li,'Leni(jlytic air-lmrne organisms are known to cause lysis aronnd 0 haenmlytic
sta|iliylococci bnt whereas these cfintiiininants also canse similar lysis in tin; liiediiini close to
cdlonies of Cl. iirlcliii. the Group li streptococci do not do so. Furthermore, the cdntaniinaiils
near the stapliyloeoccal colonies prtxlnce a haemolytie zone with a sharply defined edge, whereas
the streptococci produce a zone with an Indefinite edge. Thus, there mnst be at least three
bacterial agents capable of causing lysis of red cells wliich have been treated with atajiliylococcnl
ji toxin, namely those from Group B stre])toeocei, and from non-liaemolytic stajiliylcicocci (which
are not identicil), and that from some air-borne contaminants (whidi again acts in a different
nianner),
(Jroup B streptococci are known to be non-fibrinolytic. Eight strains producing the above
lysis ('.i of animal and S of human origin) were tested agninst luimrin fibrin and found to be nonJilirinolytic. A strongly fihrinolytic streptococcus, not of (ironp B, was iiniible to iinluce the abovementiont'd lysis. Fibrinolysin was therefore ruled ont as the responsible lytic agent.
Since (iroiip B streptococci commonly occur in milk, take part in a reaction of an a]i]tarently
digestive nature, and are not lipolytic, tlieir ability t<) digest casein was examined in an iinsmc-essfnl effort to find a test for the lytie agent other than tlie above test witli slieep cella. 'I'ltey
were grown on agnr rendered slightly turt)i(l witli fat-free milk (4 p.c), and they showed only
fnint traces of clearing, much less tlinn wns shown by many streiitococci of other serological
gi'fUips; e.g. Str. faccdlls; wliicli does not jtrodnce the haemolytie reaction, gave large clear zoni's,
its digestive power for tlie casein being visible after two lionrs,
A strain of Gron]! B stre|,it(icoeci was grown in meat iiifnsittn hrotli for 18 hours and tlie
broth filtered through a Seitz KK pad. Tests proved the liltrate to be sterile. The initial pll
of tlie broth was 7*(i and the linal pll 7-0. A drop of the filtrate on the dark zone uroiind a
staphylococcns c{iiony caused lysis. Just visible after 111 minntes and (|uite definite after one hour.
Tlio clearing was most complete at tlie edge of the zone, Readjvistnient of the pT! of tlie liltrnte to
7-13 had no effect un the amount, of clearing it could produce.
Heating of the filtrate at 5(5 C. for 30 minutes, or at (t<t C. for 1(1 minntes, altered it sn tliat
it produced only :i trace of lysis and furtber lienting at 50 C, and 00 C, for 3(1 minutes ]ia4 no
more effect. On the other hand, a sample heated at 100 C. for l minntes gave almost as strung;
a reaction as the unlieated filtrate. Heated and uiilieated fresh broth gave no reaction. Tiie
unexpected positive result witli filtrate heated at 100 C. suggested tliat tlie samjdes, partly
inactivated by lieat at ,^6 C. or 60 V., would be re-activated by heating to 11MI C. On testing,
this was found to be the case. Tlie test was repeated with two other Group B strains, with similar
results,
Laiidsteiner and von R,iuelienbicliler (1909), using crude staphyloeoccal a toxin, found that
tills was inactivated when heated to G?i C. for ,30 minntes, with reactivation to one-i]narter of
the original titre after five minutes' further heating at 100 C. Tlie inactivation at (55 ('. was
due, not to destruction of the toxin, luit to the combination of tlie toxin witli some constitnent of
tho broth, a conibinaticm that was disrupted by lienting at 100 0. This jiheiiomenon seems to be
related to that fonnd with the streptococcal filtrate mentioned above.
It was fonnd possible to rejirodnce these findings in test-tnUe titrations, nsing Seitz-filtered
staphyloeoccal (i toxin and the streptoeoccal broth filtrate. Diluted samples of the filtrate were
addeii to constant volumes of the ii toxin, and then washed sheep red cella were added. After

GROUP B 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 heated 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 similar to that of the unheated filtrate. A samjile heated for five minutes at 100" C.
was as x>otent as the raw filtrate (see Table 1). When an undiluted samjile of the filtrate was
allowed to act on the red cells for 30 minutes at 37 C, lysis occurred immediately on addition of
the fi toxin.
TABLE 1.
Results of haemolysis titrations, using streptoeoccal filtrate, staphylocoecal (i toxin and sheep
red cells.
Series I. 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 to each tube. Two drops of a 10 ji.c. suspension in saline of washed sheep red cells
were then added and the tubes inculiated at 37 0. for 1 honr.
Scrict II. As above, exeept that the filtrate was first heated to 5(1 C. for 30 minutes.
Sfries III. As in series I, except that the filtrate was first heated to 50 C. for 30 minutes,
iind then to 100 C. for five niiiiutes.
Series IV. As in series ^, except that the filtrate was first heated to 10(1 C. for five 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 dilution of filtrate.
10/10
8/10
6/10
Series I
Series II
Series I I I
Series IV
Series V
Series VI

-!--{- + + Indicates comjilote lysis.

4/10

-\- Indicates 25 p.c. lysis.

2/10

.
_

Indicates no lysis.

The qnantity of the agent present in the filtrate was not found to be increased by 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 and Group B streptococci are
fonnd together in the bovine udder; there may be some significant connection lietween this
occurrence and the fact that together 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 the animal and human strains of this group, in spite (if the differences
in serological tyj)e and in some other characteristics established by Simmons and Keogh (1940).
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 Ii toxin-jn'oducin^ stapliylococei are to be expected, 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 (i toxin.
The a^ent is extracellular, tlltrable, and thermostable.
Streptococci of liiiman and animal ori<;in, belon^iiip; to Groups other than
Group B, have not been found to produce this agrent.

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R. CPIRTSTIE, N. E. ATKTNS .\ND E . Jir'NCH-PETERSEN

_ AcTcnnivlctJgme'nt.'i. We are indebted to Professor WoodrufF. of the rniversitr Itac-teriologieal Department, for a(iviee and (riti(^i.sni, to Miss M. Monsbourgh jiiid Mr. 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.
REFERENCES.
Christie, R. and Grnydon, J. J. (1SI41): Austral. ,T. exp. Bio!., 19, p 9.
Cowaii, 8. T. (1938) : J. Path. Bact., 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.