variants of hemolytic streptococci

8/8/2019 Variants of Hemolytic Streptococci

http://slidepdf.com/reader/full/variants-of-hemolytic-streptococci 1/17

VA R I A N T S O F H E M O LY T I C S T R E P T O C O C C I ; T H E I R R E L A -T I O N T O T Y P E - S P E C I F I C S U B S TA N C E , V I R U L E N C E ,

A N D T O X I N .

BY E. W. TO DD,* M.D ., AND R. C. LAN CE FIE LD , PI~.D

(From the Hospital o f The Rockefeller Institu te for Medical Research.)

(Received for publication, August 7, 1928.)

In previous comm unicat ions one of us (1) descr ibed three sub-s tances which can be extracted f rom hemolyt ic s treptococci: (1)Thenudeoprotein P i s common to a l l s t ra ins of hemolyt ic s t reptococciand is serological ly re la ted to the nucleoproteins of pneumococci andof green streptococci. (2) The non-protein substance C,which appearsto be a carbohy drate , i s found in a l l s t ra ins of hemolyt ic st reptococcibu t is species-specific and serologically distinct fro m th e car boh ydr atefract ions of pneumoco cci and green s t reptococci . (3)The type-specific fraction M, which is probably protein in nature , has n ot beenisolated from oth er species of microo rganism s; i t occurs in serologicallydis t inguishable forms which serve to different ia te hemo lyt ic s t repto-cocci into types.

One of us (2) has previously descr ibed two forms of hemolyt ic

s t reptococci dis tinguishable by the morp hology of their colonies .Th e general appearanc es of these colonies, when g rown on a specialmed ium and viewed by ref lected l ight, are the same as those whichdis t inguish the rough and smoo th var iet ies of o ther bacter ia but thete rms "R " and " S" have no t been used and the co lon ies have beendes ignated "m at t " and "g lossy" to avo id confus ion which wouldotherwise cer ta inly ar ise f rom the c i rcumstance that the rough, ormat t , colonies are the virulent ty pe whi le the smoo th, or glossy, formsare rela t ively avirulent. I t i s the purpose of th is paper to show thatthe type-specif ic substance M is present in the potent ia l ly virulentorganisms compris ing the mat t var ie ty of colony and that i t i s notpresent in the avirulent var iant cocci which form glossy colonies .

* Bri t i sh M edical Research Counci l Fel low.

751



752 VARIANTS O]~ ItEN[OLYTIC STREPTOCOCCI

I t wi ll also be shown that f i lt ra tes f rom both m at t and glossy culturesof hemolytic streptococci contain skin-reactive toxin.

Some Characteristics of the Type-Specific Substance M.

Th e type-specific substan ce :M is prep ared by a modification ofPorges ' (3) method. The bacter ia l bodies are extracted with N/20HC1 in sal t solut ion a t the temperature of boi l ing water ; and af terneutraliza tion and clarification by centrifuging the resu lting clearslightly yellow fluid is used as an antigen for precipitin reactions.Bacter ia l extracts prepared in this mann er con tain the type-specif icsubstance M and in add i t ion they also contain small quant i t ies of thenon-type-specific fractions, P a nd C, which ma y cause som e pre-

cipi ta tion with sera prepared against any s t ra in of hemolyt ic s t repto-coccus. To avoid the appearance of these confusing precipi ta tes theserum may be absorbed with any heterologous s t ra in of hem olyt ics t reptococcus and by this means a specif ic ant i -M serum is obtainedwhich will only precipi ta te in the presence of the ho mologous Msubstance.

In the or iginal work (1) which led to the recogni t ion of the type-specific substan ce M thir teen strains were used which Dochez, Averyand Lancefield (4) had classified, some years earlier, into types byagglut inat ion and protect ion tes ts. Ten of these s t ra ins yie ldedtype-specific precipi ta t ing substances which different ia ted th em intotypes corresponding to those or iginal ly determined. The remainingthree strains failed to yield any type-specific substan ce althou gh theyhad b een classified by ag glutina tion and prote ction in the earlier w ork.I t was suggested that prolonged cul t ivat ion in the labo ratory hadcaused these three strains to lose their type-specific characteristics.In the present communicat ion i t wil l be shown that m at t cul turescontaining the type-specific substance can, by various means, bereduced to the glossy form in which th e type-specif ic substance is nolonger present .

The Preparation of An ti- M Serum.

As the type-specific substan ce M, after separation from th e bacterialcel l , does not produce any demonstrable ant ibody, when injectedinto animals , the only ant igen avai lable for prepar ing an t i -M serum



E . W . T O D D A N D R . C. L A N C E F I E L D 753

i s a s u sp e n s i o n o f b a c t e r i a i n t h e m a t t f o r m . M a t t c o c ci c o n t a i nt h e t h r e e s u b s t a n c e s P, C a n d M a n d s e r a p r e p a r e d w i t h t h e s e o r g a n -

i s m s , t h e r e f o r e, c o n t a i n a n t i b o d i e s t o e a c h o f t h e t h r e e s u b s t a n c e s .O n t h e o t h e r h a n d , c o cc i i n t h e g l o ss y f o r m c o n t a i n t h e t w o s u b s t a n c e sP a n d C b u t a r e d e v o i d of t h e t y p e - s p e c i fi c s u b s t a n c e M ; c o n s e q u e n t l y,

a n t i b a c t e r i a l s e r a p r e p a r e d w i t h g l o s s y s t r a i n s c o n t a i n a n t i b o d i e s t oP a n d C b u t d o n o t c o n t a i n a n y t y p e - s p e c i f ic a n t i b o d y.

Anti-M sera were prepared by inoculating rabbits intravenously with 16 hourcultures of ma tt cocci grown in tryptic broth. Immu nizatio n was commen cedwith four injections on consecutive days of 1 cc. of heat-killed culture, followed aweek later by four doses of 2 cc. of the same vaccine. During the 3rd week therabbits received four doses of 0.5 cc. of living culture and in the 4th week this

dose was doubled. A final series of doses of 2 cc. of living culture w as givenduring the 5th week and the animals were bled 10 days after the last injection.The sera of animals immunized in this way usually contained a satisfactoryquan tity of antibody to the type-specific substance M but with some strains it wasnecessary to continue immunization with 5 cc. and even 10 cc. of living culturebefore useful sera could be secured. A few strains hav e been encountered which,although th ey were in the ma tt fo rm and mo derately virulent for mice (0.001 cc.or 0.0001 cc.), produced o nly traces of type-specific antibo dy in ra bbits even afterintensive immunization. Twelve rats were immunized with a strain (NewYork V El4) which had previously failed to produce more than traces of type-specific antibody in the sera of twelve immunized rabbits. The rats, which re-mained perfectly weU during imm unization, received the following intraperitonealdoses: 1st week 3 doses of 0.25 cc. of heat-killed culture ; 2rid week 4 doses of 0.5cc. of heat-killed culture; 3rd week 4 doses of 1.0 cc. of heat-killed culture; 4thweek 3 doses of 1.0 cc. of living culture; 5th week 4 doses of 2.0 cc. of livingculture; 6th week 4 doses of 2.0 cc. of living culture. Seven of the imm unizedrats yielded mode rately good anfi-M sera; the remaining five sera contained tracesof type-specific antibody. As rats appear to be able to tolerate relatively largerdoses of culture tha n rab bits it is possible that they m ay be mo re suitable for thepreparatio n of anti-M serum; b ut the small yield of serum makes this metho d im-practicable for routine purposes.

The Absence of the Type-Specific Substance M from Organisms WhichForm Glossy Colonies.

I t h a s a l r e a d y b e e n s t a t e d t h a t t h e t y p e - s p e ci f i c s u b s t a n c e M i sf o u n d i n H C 1 e x t r a c t s o f m a t t h e m o l y t i c s t r e p t o c o c c i a n d t h a t i t i s

n o t f o u n d i n s i m i la r e x t r a c t s p r e p a r e d f r o m t h e g l o s sy v a r i a n t s .



754 VA R I A N T S O F H E M O LY T I C S T R E P T O C O C C I

This is demonst rated b y the following experiment:Four type-specific anti-M sera were prepared by immunizing rabbits with four

mat t strains of hemolytic streptococci belonging to different serological types andby subsequently removing the non-type-specific antibodies from the sera by ab-sorption with heterologous strains.

Table I gives the precipitin reactions of the four sera with HC1 extracts pre-pared from cultures of the homologous cocci (1) in the matt form and (2) in theglossy variant form.

TA B L E I .

Precipitin Reactions of Type-Specific Anti-M Sera with Extracts of theHomologous Strains (1) i n the Ma tt Form, (2) in the Glossy Form.

Vo l u m e s ofextracts*

0.40.10 . 0 2 5

Strain $43

1 2

Extract Extractfrom frommatt glossyform form

%÷+

Strain $23

1 2

Extract Extractf rom f romm a t t glossyform form

~-+++

Strain C203

1 2Extract Extract

from frommatt glossyform form

+ +

Strain London

1 2Extract Extract

from f r o m

matt glossyform form

+4- --

* These volumes were made up to 0.4 cc. with saline, and 0.1 cc. of serum wasadded to each tube.

I t will be seen from Table I t hat the extract prepared from the mat tform of each strain gave a good precipitin reaction wi th the homologousantiserum; on the other hand, the extract prepared from the glossyform of each strain gave a negat ive precipitin reaction with the singleexception of Strain C203 which gave a fain tly positive reaction.Although this experiment seems to show that each of the four strainslost its type-specific substance in the process of degradation to theglossy vari ant form, yet it will be seen from the results of experimentswith highly concentrated extracts th at, in reality, only one strain hadcompletely lost its type-specific substance. High ly concent rated ex-trac ts were prepared from each of the four glossy vari ants referredto in Table I in the following manne r: The centr ifuged deposit from9 liters of broth culture was extracted with HC1; and, after concen-tration by alcoholic precipitation, the precipitate was redissolved in



E. W. TODD AND R. C. LAN CEFIE LD 755

5 cc. of sal t solution. Precipi t in tests , with the con centrated ex-tract p repared from Strain $23 were negat ive showing that this s t rainwas com pletely devoid of type-specific substance. Precipi t in tests ,wi th the o ther th ree concent ra ted ex t rac t s and the i r homologousspecif ic ant i-M sera, were weakly posi t ive showing tha t three of tbeglossy cultures retain ed traces of type-specific substance. Themin ute am oun ts of specif ic substance remaining in these cul tures canbe judged from the fol lowing f igures--9 l i ters of broth cul ture wereused in preparing extracts from the glossy forms--50 cc. of bro th cul-tu re were used in prepar ing ex t ract s f rom the m at t fo rms- -180 t imesmo re cul ture was, therefore, used in the prepara t ion of the glossy ex-t rac t s than in the prepara t ion of the mat t ex t rac t s and , in sp i te o f

these disproport ionate quanti t ies , the lat ter extracts contained thelarger quanti ty of the type-specif ic substance M. I t ap pears fromthese experiments that hemolyt ic s treptococci are rarely degraded tothe poin t at which type-specif ic substance com pletely disappears .

Some Characteristics of Matt Cultures of Hemolytic Streptococci.

Twenty -eight s t rains of hemo lyt ic s treptococci were examined im-med iately af ter isolat ion from pathological condit ions in the hum anbody. The sources of these cul tures included cases of puerperalsepticemia, pleural effusion, scarlet fever, pneumonia and sinusitis;s t rains were also isolated from the depths of enucleated tonsi ls andfrom thro at swabs. In twen ty-one cases the cul tures when freshlyisolated, were entirely co mpo sed of ma tt colonies; in five cases bothma tt and glossy colonies were seen on the plates; and in two cases thecultures were ent irely glossy, but as bot h th e glossy s trains were ob-tained from throat swabs and were accompanied by other bacter iathere was no evidence that the hemolyt ic s treptococci were playinga pathogenic r61e. Table II gives the source and character of thecultures.

There is , therefore, some evidence that cul tures freshly isolatedfrom human sources are usual ly of the matt variety and this s tate-me nt part icular ly applies to diseases such as sept icemia in which thestreptococci are the undoubted causal agent .

I t is f requently found that matt s t rains of hemolyt ic s treptococci ,isolated from hum an lesions and un dou btedly pathog enic for man , are



756 VAR IAN TS O F ]EE~[OLYTIC STR EPT OCO CCI

r ~

g

o

~g

o

r..) N

S

88

. ~ "~ ~" ,,.~ .-- ., ,.~ ~ "~



~.o W. ~ODD AND R, C. LANCEYIELD 757

o ~

~ 8

°~

8

B¢J

8O

R



75 8 VARIANTS OF HEMOLYTIC STREPTOCOCCI

avi rule nt for mic e (~.L.9. 0.5 cc. or 1.0 cc.) . Such a cul ture en-t i re ly comp osed of m at t colonies wi l l be referred to in th is pap er as t hema t t a t t enu a ted fo rm because i t i s av i ru l en t fo r mice ye t possessesthe colony character is t ics and the speci f ic substance of the v i rulentfo rm. At t em pts to inc rease the v i ru l ence o f ma t t a t t enu a ted cu l -tu re s , by mouse passage, h ave a lways been success fu l a lthough someof the s t r a ins t e s t ed have r equ i red ve ry many passages be fo re themax imal v i rulence of 0 .000001 cc . has been a t ta ine d a nd in som ecases the v i rulen ce has ne ver r i sen above 0 .0001 cc . even af ter 80 or90 consecu t ive passages th rough mice .

Vi ru lence is t he o n ly qua l i ty wh ich d i s tingu i shes the ma t t v i ru l en tfo rm f rom the ma t t a t t enu a ted va r i e ty a s these co lon ies a re iden t ica l

in appearance, and serological examinat ion of HCI extracts does notshow an y s ignif icant d i fference in the qu ant i ty of type-specif ic sub-s t ance wh ich can be ex t r ac t ed f rom equa l vo lumes o f the two cu l tu res.F rom these expe r imen t s i t appea r s tha t t he ma t t fo rm, wh ich isa lways po ten t i a l ly v i ru l en t , may occur in , a t l ea s t , two sepa ra t evar ie t ies character ized by qua nt i ta t ive d i fferences in v i rulence formice; and i t i s probable that , by sui table passage exper iments , addi-t ional forms can be ob ta ined dis t inguishable by di fferent degrees ofvi rulence for o th er species of animals .

Methods of Converting the Matt Form to the Glossy Variant.

The degree of ease wi th w hich glossy v ar iants can be obta ined f romdifferent s t ra ins var ies enormo usly. In some cases great d i ff icul tyi s exper i enced in ma in ta in ing l abora to ry s tock cu l tu res o f t he ma t t ,o r po ten t i a l ly v i ru l en t , fo rm as th ey spon taneous ly change to theglossy var ia nt even when s tored in b lood broth in the ice box. Inthese cases i t i s necessary to resor t to f reque nt mouse passages top reven t the to t a l lo s s o f t he ma t t fo rm. On the o the r hand , ma t ts t r a ins have been enco un te red which do no t show any t endenc y tochange to the g lossy va r i an t a f t e r r epea ted subcu l t iva t ions on aga r.In t e rm ed ia t e be tween these ex t r emes a re s t r a ins wh ich deve lop asmal l propor t ion of g lossy colonies af ter repeated subcul t ivat ions onagar. Wh en one of the g lossy colonies der ived f rom these s t ra ins issubcu l tu red in b ro th and r ep la t ed on aga r a mix tu re o f ma t t a ndglossy colonies usual ly appears but occas ional ly a pure g lossy cul ture



E, W. TODD AND R. C. LANCEFIELD 759

ma y be obtained by this method . By rep eated select ion of glossycolonies and subcul t ivat ion in broth a cu l ture composed ent ire ly ofglossy colonies can of ten be ob tained and in some instances a purecul ture of the glossy var iant can be secured by the s imple process ofrepeated subc ul t ivat ion on agar s lants.

Griff i th (5) and o thers (6-8) hav e shown that smoo th pneumo coccican be converted to the rough form by cul t ivat ion in the homologousant i -S serum. We have appl ied this technique to hemolyt ic s t repto-cocc i and have found tha t the cu l t iva t ion o f ma t t s t ra ins in und i lu tedhomologous anti -!V[ serum of high t i ter is the quickest and mo stre l iable method of obtaining glossy var iants , and with some highlystable mat t s t ra ins this is the only method by which we have been

able to secure the glossy form. Her e again there are wide differencesbetween individual s t ra ins; in some cases , a few t ransfers in serumsuff ice to convert a vi rulent mat t cul ture , containing abundant Msubstance, in to the glossy avirulent var iant devoid of any specif icsubstance; in other cases , af ter as man y as 90 t ransfers in high t i terserum traces of the type-specific substanc e M can stil l be de tected inconcentrated bacter ia l extracts , a l though the colonies appear to beglossy and the organisms have los t their v irulence for mice. At te mp tshave been mad e to r id these cul tures of the remaining t races of type-specif ic substance by al ternately cul t ivat ing the cocci in immuneserum, pla t ing out , se lect ing the m ost glossy colonies and again sub-cul tur ing in immune serum; one s t ra in was subjected to this t reat-me nt thi r ty t imes af ter 90 previous consecut ive t ransfers in immu neserum but a t the end of the exper iment i t s t il l re ta ined t races of thespecific substance.

Some Characteristics of Glossy Cultures of Hemolytic Streptococd.

The glossy var iant is avirulent for mice in comparison with thema tt v i rulent cul ture f rom which i t i s der ived and at t emp ts to ra isethe virulence of the var iants by mouse passage have usual ly beenunsuccessful . I t i s, however, possible to obtain glossy cul turesw hiehare par t ia l ly virulent for mice. A s t ra in which, in the m at t v i rulentform , killed mice regularly in doses of 0.000001 co. or 0.0000001 ce.was cul t ivated in the ho mologous ant i -M serum and, af ter 55 t rans-fers in 50 per cent serum, a pure cul ture of the glossy var ian t was ob-



760 VARIANTS OF HEMOLYTIC STREPTOCOCCI

ta ined which did not co nta in an y type-speci fic substance , yet th isglossy cul ture was suff ic ient ly v i rulent to k i l l mice regular ly in doses of0.01 cc. and occa sionally in doses of 0.001 cc. M ore prolon ged culti-va t ion in an t i -M se rum d id no t cause any fu r the r dec rease in v i ru lence .The pa r t i a l ly v i ru l en t g lossy cu l tu re was passed th rough twen ty - f ivemice in t r ape r i tonea l ly bu t t he v i ru l ence r ema ined unchanged andthere was no reapp earan ce of type-speci f ic substance . This appearsto be a n except ional s t ra in as t he M.I~.D. of th e m ajo r i ty o f g lossystrains is 0.5 cc. or 1.0 cc.

Dur ing the p rocess o f convers ion from ma t t t o g los sy var ious typeso f co lon ies appea r, wh ich ma y poss ib ly r ep resen t i n t e rmed ia t e fo rmsor ma y be du e to ind iv idua l co lon ies con ta in ing a mix tu re o f m a t t

and glossy cocci . We have observed tha t d i fferent s t ra ins somet imesshow pecul iar i t ies in the morphology of thei r colonies which are sos t r ik ing that the s t ra in can be recognized e i ther in the mat t or g lossyform. In addi t io n to these s t ra in pecul iar i t ies o the r var ie t ies ofco lon ies appea r du r ing the g radua l change f rom mat t t o g los sy.Gri ff i th (9) has noted that in spi te of the apparent ly d iverse appear-ances of s t reptococcal colonies three forms can general ly be dis-t ingu i shed . Two o f h i s fo rms appea r to co r re spond to ou r ma t t a ndglossy colonies and the th i rd i s character ized by a sof t consis tency, awhi t i sh opaque ra i sed cen te r and a th in t r ans lucen t marg in . In aprevious co mm unicat io n one of us (10) descr ibed a s imi lar form ofco lony, wh ich d i ff e red in the impo r t an t r e spec t o f be ing tough in -s t ead o f wa te ry ; bu t fu r the r obse rva t ion has shown tha t co loniescha rac te r i zed by a f l a t marg ina l zone su r round ing a cen t r a l eminencemay occur in two fo rms , co r re spond ing to the ma t t o r t o the g los sys t a t e . The ma t t fo rm o f th i s co lony i s opaque and o f tough con-s i s t ency wi th a cen t r a l em inence su r rounded b y a f i at marg ina l zone ;the g los sy fo rm has a simi l ar con tour bu t i s so f t and w a te ry. Their regular shape of these colonies causes d i ff icul ty in observing thel ight- ref lec t ing chara cter of thei r surfaces but the m at t and glossyforms can general ly be dis tinguished by other character is t ics . Theseobse rva t ions seem to ind ica t e tha t t h i s t h i rd typ e o f co lony i s no t ad i s t inc t en t i ty sepa ra t e f rom the ma t t and g lossy fo rms ; bu t we havefai led to determ ine the s ignif icance of these ver y character is t iccolonies.



~E. W. TO DD AN D iZ. C . LA NC EF IE LD 761

The class if ication of colonies is fur ther complicated b y the occasionalappearance o f pseudog lossy fo rms . Whe n a mat t cu l tu re i s sp readon a pla te the colonies in c lose proxim ity to each other ma y prese ntthe typ ica l ma t t appearance b u t wide ly separa ted co lon ies in the samecu l tu re may be g lossy in appearance . I f one o f the l a t t e r co lon iesis se lected and spread on a f resh pla te a pure cul ture of typical mat tcolonies ma y resul t . Pseudog lossy colonies are general ly larger thantrue mat t or t rue glossy colonies but they so near ly resemble thetrue glossy form that they are l iable to cause confusion.

Owing to these var ia t ions in the appearance of colonies we havebeen unable to re ly ent i re ly on the colony form as a guide to thecharacte r of cul tures . The cr i ter ia we have used to determine whe n

a cul ture is completely degraded from the mat t s ta te are: (1) thatconcentrated HC1 extracts of glossy cul tures should not cause anyprec ip i t a t ion when mixed wi th pure homologous an t i -M se rum(absorbed with a heterologous s t ra in to remove the ant ibodies to Pand C) ; (2) that the resul t of the abo ve tes t should remain u nchan gedaf ter the cul ture has been passed through a mouse.

I t wi l l be seen in the detai led descr ipt ion of exper iments that wehave only been able to secure one s t ra in in this completely degradedstate .

Reversion of Glossy Cultures to the Matt Form.

Dawson and Avery (11) have shown tha t many s t ra ins o f Rpneumococc i can be rever ted to the S fo rm by repea ted mouse pas -sages or by cul t ivat ionin vitro in ant i -R serum. Griff i th (I2) hasshown tha t R pneumococc i f r equen t ly rever t when they a re mixedwith large doses of heat-ki l led S pneumococci and inoculated sub-cu taneous ly in to mice .

We have a t t emp ted to rever t g lossy hemoly t i c s t r eptococci to thema t t fo rm by each of these th ree methods .

1. Mouse Passage.--Passage experiments have been done with glossy cul-tures derived from five different strains ($3 , $23, I-Ienson, $43 , C203) but nodefinite evidence has been obtained that reversion can be achieved by this method.In two ca ses, $3 (te n passages) and I-Ienson (twenty-five passages), the glossycharacter of the cultures was judged entirely b y the appearance of the colonies asno anti-M serum was available for these strains.



E . W. T O D D A N D R . C . L A N C E F I E L D 763

of pure anti-P se rum on glossy cultures of four strains was tested. High titeranti-P serum was prepared by immunizing rabbits with purified nucleoproteinextracte d from hem olytic streptococci. The glossy varia nt forms of four strains($43, $23, C203, London) we re cultivate d for twelve transfe rs in a 10 per ce ntdilution of this serum. This treatm ent did not alter the colony forms of the cul-tures although the virulence of two strains ($23 and London) was definitely in-creased. The quan tities of type-specific substance, howe ver, which could beextracted from the cocci of all four strains remained unchanged.

3. gubcutaneous Inoculation of M ice w ith Glossy Cultures in Combination withthe Homologous Matt Cocci Kille d by Hea t.--Afew experiments have been donewith one of our glossy strains (Henson) in an att emp t to reve rt this culture to thema tt form by the technique devised by Griflith for the reversion of R pneumoc occito the S form. A hea vy suspension of the matt culture was prepare d by heating,at 60°C. for 30 minutes, the deposit from 50 cc. of culture, conce ntrated to 2 cc.

0.5 cc. of the heat-killed suspension was mixed with 0.05 cc. of living glossy cul-ture and injected subcutaneously into a mouse. Cultures from the lesion in themouse contained both matt and glossy colonies although controls indicated thatthe matt organisms of the heated suspension were dead. Unfortu nately, thistechnique, so successful with pneumococci, is not altogether satisfactory forhemolytic streptococci as it has been found impossible to avoid ulceration whenlarge numbers of heat-killed matt organisms are combined with glossy culturesand even small doses of glossy culture alone frequently cause ulceration. Ma ttcolonies isolated from these open ulcers must be viewed w ith suspicion since theymay arise from contaminating cocci, but it seems probable that further workwith this technique may yield convincing evidence of the reversion of the glossycocci to the matt form.

S o f a r a s a n y c o n c l u s i o n s c a n b e d r a w n f r o m t h e l i m i t e d n u m b e r o f

o b s e r v a t i o n s r e c o r d e d i t s e e m s t h a t t h e g l o s s y v a r i a n t i s a h i g h l ys t a b le f o r m b u t t h a t r e v e r si o n m a y o c c u r u n d e r c e r t a i n c o n d i t i o ns .

A Comparison of the Toxigenicity of Ma tt and Glossy Cultures of theSame Stra in .

T h e m e t h o d u s e d f o r c o m p a r i n g t h e t o x i g e n i c i t y, t h e v i r u l e n c e f c rm i c e a n d t h e c o l o n y a p p e a r a n c e o f m a t t a n d g l o s s y c u l t u r e s o f t ~ es a m e s t r a i n w a s a s fo l l ow s :

Young broth cultures of the different forms of each strain were sown in 50 cc.of tryptic digest broth. After 16 hours incubation the virulence and colonyappearance of a sample taken fr om each culture were determined; a nd the flaskswere then returned to the incubator. The cultures were filtered after 4 days ofincubation and the filtrates were tested by injecting 0.1 cc. of diluted filtrate into



76 4 VARIANTS O F HEMOLYTIC STREPTOCOCCI

-g

~.~ o

I

r I ~ I i

I

v

I I

1 1 I I I I I I I I I

~ o ~

a. J

~9

~9

~9

~9



E. W. TO DD AND R. C. LANCEFIELD 765

the skin of one, or more, known positive reactors. Table II I gives the dimensionsof the reactions which followed the injection of active filtrate and of the samefiltrate after heating in boiling water for 2 hours. Measurements, wh ich aregiven in ram. representing the average diameter of the skin reactions, w ere taken24 hours after injection. The figures in brackets give the virulence for m ice ofthe cultures from which the filtrates we re prepared.

The two strains isolated from cases of bronchopneumonia produced weak toxicfiltrates in comparison with the scarlet fever strains and were therefore used ingreater concentration.

I t wi l l be seen f rom Table I I I tha t the f i l t ra tes f rom the d i fferentfo rms o f each s t r a in caused approx ima te ly equa l r eac t ions and tha tno corre la t ion could be es tabl ished betwee n vi ru lence and toxigenic i ty.

DISCUSSION.

I t m ay be s ta ted as a broad genera l iza t ion tha t the type-speci f ic sub-s tance M is prese nt in the potent ia l ly v i ru lent organism s compris ingthe ma t t va r i e ty o f co lony and tha t i t is no t p re sen t i n t he av i ru l en tvar ian t cocci which form glossy colonies. This i s analogous to theinvar iable presence of the soluble speci f ic subs tance S in v i ru lentcul tures of pneumo cocci an d i t s absen ce f rom avi ru lent l~ cul tures ,but here the analogy breaks down as far as v i ru lence for mice i s con-cerned, s ince i t i s poss ib le to prepare mat t cul tures of hemolyt ics t reptococci which conta in large quant i t ies of the type-speci f ic sub-s tance M and yet a re avi ru lent for mice .

One of the most s t r ik ing character is t ics of hemolyt ic s t reptococciis the d i ff icul ty which has a lways been exper ienced in secur ing h ighlyvi ru lent cul tures of a la rge num ber of s t ra ins . This is und oub ted lydue in par t to the fac t tha t the g lossy var iant i s a h ighly s tableavi ru lent form bu t even when we exclude th is var ia nt form a nd conf ineour a t t en t ion to t he ma t t o r po ten t i a l l y v i ru l en t va r i e t i e s we a res t il l unable to secure h ighly v i ru lent cu l tures wi th any degree of ease .The behav io r o f ma t t a t t enua ted cu l tu re s unde rgo ing mouse pas sageis f reque nt ly capr ic ious ; v i ru lence genera lly r i ses to a mod era te levelaf ter the in i t ia l ten , or twenty passages and i t may then increasesuddenly, or i t may gradual ly increase af ter many more passages , ori t ma y remain for an indefin i te p er iod in a s ta te of mediocr i ty. Thisis in contras t to pneumococci which appear to be e i ther rough andaviru lent for mice or smooth and of maxima l v i ru lence for th is



766 VARIANTS OF HEMOLYTIC STREPTOCOCCI

species. The virulence of pneu moco cci appears to be intimate lyassociated with the presence or absence of the soluble specific sub-stance S; in the case of he moly tic streptococci, however, virulence isnot ent i re ly depen dent on the presence or absence of the type-specificsubstance M ; some addi t ional unkn own factor is operat ive . Glossyvar iants , wh en ful ly degraded, contain no type-specif ic substance andare avirulent for mice; mat t organisms occur in two forms equal lyr ich in type-specific substan ce--on e of these forms is no mo re virulentfor mice than the glossy var iant , the other is h ighly virulent.

I t i s possible that th is contras t between pneumo cocci and hemo lyt ics t reptococci ma y be p ar t ly due to differences in bacter ia l s t ructure .In the case of pneum ococc i the soluble specific substance S is disposed

in a capsular layer over the surface of the organism; but microscopicexaminat ion of hemo lyt ic s treptococci gives no info rmat ion as to thesituation of the type-specific substance M in the bacterial bodies;and i t i s possible that the dis t r ibut ion of th is substance throughoutthe organ isms ma y rend er i t less accessible and therefor e less suscep-tible to external influences.

Certa in s t ra ins of hemolyt ic s t reptococci exhibi t an unexpectedstabi l ity in al l forms. Cul tures which are par t ia l ly degraded so thatthey contain only smal l quant i t ies of type-specif ic substance m ay bepassed through a number of mice without any accumulat ion oftype-specif ic substance and without any al terat ion of vi rulence.Conversely, when the virulence of a mat t s t ra in has become es-tabl ished i t i s d i ff icul t to reduce the cu l ture to the m at t a t tenu atedsta te and at the same t ime to avoid convers ion to the glossy var iantform.

No relation ship could be established between toxigenicity andvirulence; in some instances highly virulent ma t t cul tures produ cedweak toxic f i lt ra tes an d the glossy var iant avirulent forms were equal lytoxigenic; in other ins tances re la tively avirulent ma t t s t ra ins pro-duced highly toxic fil trates.

I t ap pears therefore that vi rulence is not determine d by toxigenici tyand is not ent i re ly dependent on the presence or absence of type-specif ic substance a l though cul tures which have los t their typespecificity are inv ariably avirulen t.

An u nkn own factor d etermines whether hemo lyt ic s t reptococci ,



E. W. TODD AND R. C. LANCEFIELD 76 7

which conta in thei r fu l l quota of type-speci f ic substance , are v i rulento r a t t enua ted .

SUMMARY.

Hemolyt ic s t reptococci , when f reshly isola ted f rom pathogenicles ions , form character is t ic mat t colonies and conta in the type-specific substance M.

Two var ie t ies of mat t cul tures , equal ly r ich in type-speci f ic sub-s tance , can be dis t inguished by the vi rulence of the organisms formice : (1 ) t he ma t t v i ru l en t va r i e ty, (2 ) the ma t t a t t enu a ted va r i e ty.

The mat t forms of hemolyt ic s t reptococci can be degraded to ath i rd var ie ty which forms glossy colonies and is a lways re la t ivelyavirulent . This i s accomplished by prolonged cul t ivat ion on ar t i -f ic ia l media , by se lect ion of colonies or by cul t ivat ion in hom ologousant i -M serum. In th e process of degradat ion the cocci lose themajo r par t of thei r type-speci f ic substance b ut complete d isappearanceof type-specific substance rarely occurs.

The glossy var iant form, when ful ly degraded, i s h ighly s table ; bu tglossy cul tures which have re ta ined some type-speci f ic substance canoccasional ly be rever ted to the or ig inal mat t form.

Toxic f i lt ra tes f rom m at t and glossy cul tures are approx imat elyequal in skin react iv i ty.

No re la t ionship ap pears to exis t between vi rulence and toxigenic i ty.

BIBLIOGRAPHY.

1. Lancefield, R. C.,J. Exp. Med., 1928, xlvii, 91,4 69, 481.2. Todd, E. W., Brit. J. Exp. Path., 1928, ix, 1.3. Porges, O., Wien. klin. Woch., 1905, xviii, 691.4. Dochez, A. R., Avery, O. T., and Lancefield,R. C., J. Exp. Meal.,1919, xxx,

179.5. Griffath, F., Rep. Pub. Health and Med. Subj., Ministry of ttealtk, No. 18,

1923, 1.6. Reimarm, H. A., J. Exp. ]fled.,1925, xli, 587.7. Amoss, H. L., J. Exp. Med., 1925, xli, 649.8. Dawson, M. H., Y. Exp. Med.,1928, xlvii, 577.9. Griffith, F., J. Hyg., 1927, xxvi, 363.

10. Todd , E. W., Brit. J. Exp. Path., 1927, viii, 289.11. Dawson, M. H., and Avery, O. T., Proc. Soc. Exp. Biol. and Med., 1927,

xxiv, 943.12. Griffith, F., J. Hyg., 1928, xxvii, 113.

variants of hemolytic streptococci

Documents