shigella - journal of bacteriologyjb.asm.org/content/37/3/315.full.pdf · the cultural and...

THE CULTURAL AND ANTIGENIC PROPERTIES OFSHIGELLA SONNEI

JOHN H. GLYNN AND D. HUGH STARKEYDepartment of Bacteriology and Immunity, McGill University and the Royal

Victoria Hospital, Montreal

Received for publication August 19, 1938

The present study aims to define the taxonomic position of thatgroup of dysentery bacteria commonly known as Shigella para-dysenteriae, var. sonnei (Bergey, 1934). Most of the materialon which this work is based has been recently isolated in thislaboratory but a number of type strains of the species from TheNational Collection of Type Cultures in London, England, havebeen included for comparison. Also included for comparativepurposes, are a number of strains of closely related members ofthe Genus Shigella which are mannitol-fermenters; namely, thegroups known as Flexner, Dispar and Alkalescens.

ORIGIN OF STRAINS

A total of eighty-eight strains were studied. These included:42 strains of Shigella sonnei isolated in this laboratory either

within three months of the initiation of this work or while thework was in progress. All of these strains were recovered fromthe stools of patients with intestinal complaints;

12 strains of Shigella sonnei obtained from the National TypeCollection at the Lister Institute, London;

2 strains of Shigella alkalescens obtained from Professor E. G.D. Murray both of which were original strains described by SirFrederick Andrewes (1918);

3 strains of Shigella dispar isolated in this laboratory;8 type strains of Shigella dispar obtained from the National

Collection of Type Cultures at the Lister Institute, London.4 strains of Shigella dispar obtained from Professor Murray

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JOHN H. GLYNN AND D. HUGH STARKEY

and originally described by him (1918) as his Class 4. At thattime he identified them as the type described by Kruse (1907) asType E, although he states (p. 387) "Class 4 is certainly equiv-alent to Kruse E.... The strains comprising Class 4 (Kruse E)would fall into the group comprised by Andrewes' B. dispar."Kruse E is at present generally held to be the same as Shigellasonnei (Gardner (1929) Gay (1935)).

5 strains of Shigella paradysenteriae (flexner) obtained fromProfessor Murray, representing the types V, W, X, Y and Z ofAndrewes and Inman (1919) each of which is described in theirmonograph.

A. CULTURAL PROPERTIES

1. Colony formThe colony form was studied on plain agar, on Chandelier and

on MacConkey plates. In general the Sonne strains producecolonies which are slightly larger, more granular and more opaquethan those of the Flexner, Alkalescens and Dispar strains. Allplates were kept in a moist jar for three weeks at 370C.

a. Shigella sonnei colony. On original isolation all strainsshowed a uniform colony. At twenty-four hours incubationthey were rather flat, slightly granular and opaque, about 3 to4 mm. in diameter, with a smooth raised central zone grading outto a thin slightly irregular edge. The whole colony was lactose-negative. The colonies continued to increase in size and tospread peripherally until discarded and have reached three cen-timeters in diameter.On further incubation, secondary colonies or papillae appeared

on the surface of most, but not all, of the original colonies.These papillae, or daughter colonies, consisted of raised, smooth,entire, rounded outgrowths on the surface which, soon after theirappearance, caused the medium immediately beneath them toshow the lactose-positive change. These daughter colonies wereusually 1 to 2 mm. in diameter within forty-eight hours aftertheir appearance; some continued to increase in size while othersdid not. The length of time required for these papillae to appearvaried from strain to strain but could be correlated roughly with

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PROPERTIES OF SHIGELLA SONNEI

the time required for the individual strain to ferment lactose inpeptone water medium.Only one strain (out of fifty-four) failed to show secondary

colonies. In this strain the centre of the colony became pro-gressively more heaped up and irregular and eventually the entirecolony went lactose-positive. Repeated platings of this strainfailed to change its behavior.Attempts were made to get these secondary papillae to breed

true by carefully fishing them from the parent colony and re-plating through 10 to 12 generations. Each generation began asa lactose-negative colony upon which lactose-positive papillaeappeared at about the same interval as on original isolation.The twelfth generation did not differ from the first generationeven on a percentage basis. Similarly, the few original primarycolonies which showed no lactose-positive papillae (althoughmost other colonies of these same strains on the original platedid show numerous papillae) were replated up to twenty genera-tions, alternating solid and fluid media, without changing thegeneral appearance of any generation. The colonies alwaysstarted as lactose-negative and most of them later developedlactose-positive papillae.A second colony form appeared on plating out from older

cultures or from cultures grown in 1:10,000 Brilliant-GreenPeptone Water. This is a flat, entire colony with a bevellededge, not granular and with a moist shiny surface, smaller thanthe ordinary colony, increasing little in size with further incuba-tion and not developing papillae. These colonies do not breedtrue.The two colony types gave identical fermentation reactions.b. Shigella dispar colony. These strains showed colonies in

every way similar to the granular type of the Sonnei colonies-including the formation of small, smooth secondary papillaewhich became lactose-positive.

c. Shigella alkalescens colony. These strains showed a some-what smoother granular type of colony and they also laterdeveloped secondary papillae. These secondary papillae, how-ever, always remained consistently lactose-negative.

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d. Shigella paradysenteriae (Flexner's bacillus). Secondarypapillae were less frequently encountered in the Flexner type.When they did appear they also remained consistently lactose-negative. The Flexner colony, in general, was the smoothestof all.

2. Microscopic appearanceSmears from the various colonies showed approximately the

same morphology; all were gram-negative rods. The only differ-ence noted was that the old strains seemed to contain morefilamentous forms than freshly isolated strains.

S. MotilityMotility was checked on all strains by observing hanging drop

preparations on eight- and on eighteen-hour cultures in beef-infusion broth at room temperature and at 370C. In addition,each strain was tested for migration through semisolid agar atroom temperature and at 370C. All strains were uniformlynon-motile.

4. Biochemical reactionsThe reactions shown in table 1 were found to be the most

useful in separating the groups. The sugar media consisted of1 per cent of sugar in 1 per cent peptone water with Andradeindicator. The tubes were kept at 370C. for sixty days. Atfirst, two methods of controlling evaporation were used: eitherkeeping the tubes filled to a mark with sterile distilled water, orwaxing the cotton stoppers with paraffin. After twenty-threestrains had shown identical results by both methods, the waxedplug method was adopted for the rest of the work in order toavoid the occasional contamination.The litmus milk was kept in a tube with a waxed plug.Indole was tested for by treating two-day and five-day growths

in 1 per cent peptone water with Ehrlich's reagents.Some of the type strains were put through the full examination

only once, but most of the recently isolated strains were re-examined and the tests were repeated for all strains which showed

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PROPERTIES OF SHIGELLA SONNEI 319

results not conforming with the general results. In these repeattests, the only difference found was that two strains which failedto ferment lactose in the first sixty-day trial, did ferment lactoseon retest in 27 and 32 days respectively.As shown in table 1, both Shigella sonnei and Shigelka dispar

are sharply distinguished from Shigella alkalescens and Shigellaparadysenteriae (flexner), and, incidentally, from all other Shigella,in the late fermentation of lactose. The Sonne group may be

TABLE 1The most useful reactions for separating the mannitol-fermenting groups of

dysentery bacteria

r 04CLASSIFI- Na N 1 ETMUBMXK/CATION SP LITMUS______L

Sonne 54 A A A A A 0 0 00 Neutral to A4-32 2-32 Very about 2 daysdays days rare- after Lactose

ly ADispar 15 A A A AA A A' 0 + Neutral to A

2-19 1-6 about 2 to 8days days days

Alkales- 14 A 0 0 A A A A A + Neutral to verycens 3-17 alkaline 3 to

days 10 daysFlexner Com- A var. 0 A var. 0 var. 0 var. var., A changing

piled to alkaline

All strains non-motile, gelatin negative, salicin negative.A = acid; 0 = no change during 60 days incubation; var. = variable, depending

on the particular strain tested.

separated from the dispar group since the latter ferment xyloseand sorbitol and produce indole.

Shigella alkalescens stands distinctly apart from the others inthe production of alkalinity in litmus milk, a feature of diagnosticsignificance, with the fermentation of dulcitol.The reactions of the Flexner group are not based on the five

strains used in this study alone, but compiled from the literatureas well as our own experience with other Flexner strains in thepast.

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In the fermentation of mannitol, all of these groups are related.As Murray (1918), Andrewes and Inman (1919), and Gardner(1929) point out, this is the one important fermentable substanceof taxonomic value which has stood the test of time.

It is apparent from the data given in table I that completeidentification of Shigella sonnei by cultural tests alone cannot bemade within a time short enough to be of clinical value. Thedistinguishing changes in lactose and litmus milk often take twoor three weeks to develop. Thus, for rapid diagnosis any man-nitol-fermenting dysentery organism must be subjected toserological tests.

B. ANTIGENIC PROPERTIES

The serological properties of the groups were studied by theuse of agglutination, agglutinogenic power and the agglutininabsorption reaction.

MethodsFrom an original plate the smoothest colony was selected and

large agar plates inoculated. Incubation was for eighteen hoursin order to avoid the occurrence of secondary papillae (lactose-positive variants) which, in our experience, never appeared underforty-eight hours. The growth was scraped from the surface ofthe plate with a spade and suspended in 0.85 per cent NaCl towhich 0.3 per cent of formalin was added. Such suspensionsremained stable, having no tendency to sediment spontaneouslyas "rough" strains would be expected to do. They were stand-ardized by comparison with standard opacity tubes by Brown'smethod.

Rabbits were immunized by the intravenous injection of 500million, 1000 million and 2000 million formalized bacteria onthe first, sixth and eleventh days respectively. The animalswere bled on the seventeenth day and the serum obtainedusually reached a titre of between 5,000 and 40,000.

For agglutination tests, the antigen suspensions were stand-ardized to a final concentration of 400 million bacteria in eachtubt, the total volume of fluid being 2.0 cc. The range of serum

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dilutions in each test was from 1 in 40 to 1 in 80,000. Lowerdilutions were avoided because experience had shown that normalrabbit serum often agglutinated various dysentery bacteria inlower dilution but that the titre rarely exceeded 1 in 40. Theend-point selected in each test was the greatest dilution of serumwhich gave complete agglutination and a clear supernatant fluidafter twenty-four hours incubation at 370C.

For agglutinin absorption tests the antigens were standardizedto a final concentration of 135,000 million bacteria per cc. ofundiluted serum. Absorptions were done in a final 1-in-10dilution of serum in a total volume of 10 cc; incubated for 5hours at 370C.; then left in the ice box overnight before centri-fuging. The absorbed serum was then tested not only againstthe homologous strain but also against several others.At the beginning of this study an agglutinating serum was

prepared in rabbits for all of the strains available at that time.Twenty-seven serums in all were prepared against twenty-sevendifferent strains. Of these, 14 were S. sonnei, 5 were S. para-dysenteriae (flexner), 4 were S. alkalescens and 4 were S. dispar.Later, additional strains were added to the series, so that ourresults are finally based on tests involving twenty-seven serumsand eighty-eight strains. 54 of these strains were finally classi-fied as S. sonnei, 14 as S. alkalescens, 15 as S. dispar and 5 weretype strains of S. paradysenteriae (flexner).Table 2 shows the result of cross agglutinations between 14

serums and 22 strains of S. sonnei.The existence of two groups seems indicated by the table;

neglecting, for the moment, differences in agglutinability ofstrains-a factor not controlled in this type of experiment butcapable of being checked by absorption tests.The first group included 13 strains isolated in this laboratory

and 3 National Type Collection strains. Nine other NationalType Collection strains as well as 17 other strains isolated in thislaboratory are not listed in the table but have been shown to fallinto this group. Each strain is agglutinated to full titer by aserum produced from any other strain. This result is in agree-ment with that of many previous investigators who contend that

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the sonnei group is antigenically homogeneous, (Kruse (1907),Hilgers (1920), Fraser, Kinlock & Smith (1926), Kerrin (1928),Nelson (1930) and Koser, Reiter, Bortniker and Swingle (1932)),

TABLE 2Cro08 agglutinations of SO strains of Shigella sonnei with rabbit agglutinating serums

prepared from 14 of these trains

AMWIIS|UM

eq Iq

AIMGZN

eq C.q

Nat. type col. 2318. 10 10 10 10 10 10 10 10 10 10 10 10 10 10Nat. type col. 2182........... 10 10 10 10 10 10 10 10 10 10 10 10 10 10Nat. type col. 268.......... .. 10 10 10 10 10 10 10 10 10 10 10 10 10 10B593 ..................... 10 10 10 10 10 10 10 10 10 10 10 10 10 10CMH683..................... 10 10 10 10 10 10101010 10 10 10 10 10CMH218 ..................... 10 10 10 10 10 10 10 10 10 10 10 10 10 10B314 ..................... 10 10 10 10 10 10 10 10 10 10 10 10 10 10CMH212 ..................... 10 10 10 10 10 10 10 10 10 10 10 10 10 10CMH207 ..................... 10 10 10 10 10 10 10 10 10 10 10 10 10 10CMH232 ..................... 10 10 10 10 10 10 10 10 10 10 10 10 10 10A3252 ..................... 10 10 10 10 10 10 10 10 10 10 10 10 10 10B637 ..................... 10 10 10 10 10 10 10 10 10 10 10 10 10 10CMH8122-b .................. 10 10 10 10 10 10 10 10 10 10 10 10 10 10CMH8187-lb ................. 10 10 10 10 10 10 10 10 10 10 10 10 10 10CMH8187-2a ................. 10 10 10 10 10 10 10 10 10 10 10 10 10 10CMH8222-lb ................. 10 10 10 10 10 10 10 10 10 10 10 10 10 10B217..................... 5 3 2 4 2 3 2 2 2 3 2 1010B597..................... 4 2 2 4 2 3 2 2 2 2 3 2 1010CMH8122-a.................. 5 2 3 3 2 3 3 2 2 3 13 2 1010CMH8187-la................. 3 3 2 4 3 2 2 2 2 2 2 2 1010CMH8207.................... 2 2 2 2 3 2 2 2 2 2 2 2 1010CMH8222-la................. 5 3 2 4 2 3 2 2 2 2 3 2 1010

Numbers refer to the end point tube (complete agglutination) in a serieswhere 10 represents the titre for the homologous strain.

though others (Sonne (1915), Murakami (1927), Leuchs andPlockmann (1927)) suggest the existence of two serological va-rieties. Since it is apparently the most common type of thesonnei organism isolated, we propose calling this group Shigelasonnei, Type I.

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The second group includes 6 strains isolated in this laboratory.An additional 6 strains, also isolated here but not listed in thetable, fall into this group as well. These strains are agglutinatedto not more than a small fraction of the titre by any serum ofType I. However, a serum prepared from two of these strains,B217 and B597, will agglutinate all strains of both types to fulltitre. Since this group is less commonly found than the formerwe propose calling it Shigella sonnei, Type II. It should berecorded that this Type II is probably more distinctive of thespecies Shigella sonnei than Type I as judged by taxonomicstandards-a point which will be further supported by laterevidence.

In order to check these results and to control the factor ofvariation in- agglutinability of strains, absorption tests werecarried out.Table 3 shows the result of some of these absorptions. The

table is condensed from a large series of absorptions in which theresults illustrated were characteristic of the entire series.These absorptions confirm the results of straight agglutination

tests as to the existence of two types of Shigelka sonnei. Theyalso warrant additional statements regarding the antigenicstructure of the two types.Type II evidently contains both Type I and Type II antigens

in considerable amounts because, not only will its serum agglu-tinate both types to full titre, but it will itself completely absorbthe agglutinins from serum of both types.Type I, on the other hand, apparently contains the Type I

antigen in predominance, with a relatively small amount ofType II antigen, since its serum will agglutinate Type II toonly a fraction of its titre, and it will absorb not more than 50to 75 per cent of the agglutinins from a Type II serum.The question might arise whether we are dealing here with

a smooth-rough type of variation such as Johnston and Kaake(1932) concluded was the explanation of the two types describedby Johnston and Brown (1930). In its original definition thesmooth-rough variation was related to differences in colony form,kind of growth in broth and salt sensitiveness and more recently

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324 JOHN H. GLYNN AND D. HUGH STARKEY

to loss of an antigenic component. Although the Sonne colonyis never quite as smooth as the Flexner colony even on primaryisolation it grows with uniform turbidity in broth, does notsediment spontaneously in saline and the two colony forms wedescribe are not related to the immunological types by agglu-tination.

TABLE 3Reciprocal absorption between two Type I strain of Shigella sonnei (nat. type col.

*318 and B593) and two Type II trains (B217 and B597)

Homologous ab- Jsorptions Type I {

Heterologousabsorptions

Homologous ab- fsorptions Type II

SERUM

N.T.C.2318N.T.C.2318B593B593

N.T.C.2318N.T.C.2318B593B593B217B217B597B597

B217B597

ABSORBINGSTRAIN

B593N.T.C.2318N.T.C.2318B593

B217B597B217B597N.T.C.2318B593N.T.C.2318B593

B597B217

ABSORB-ING DOSEOF UN-DILUTEDSERUM

millionsper cc.

135,000135,000135,000135,000

135,000135,000135,000135,000135,000135,000135,000135,000

135,000135,000

TTRE FORHOMOLOGOUS

STRAIN

Ua-AftersUnbed absorp-Iobe tion

5,120 05,120 0

40,00040,000

5,1205,120

40,00040,0002,5002,5002,5002,50

2,5002,500

64080

00

1,280640

1,2801,280640

1,280

00

REDUC-TION OFHOMOLO-GoUSTITRE

.pa cont

1001009999

100100969950507550

100100

Referring to the clinical records we find that in some instancesdifferent colonies of the same original stool plate fall into differentserological types. Thus, in case CMH 8122, colony a is Type IIwhile colony b is Type I. In case CMH 8187, colony la is TypeII while colonies lb and 2a are Type I. In case CMH 8222colonyla is Type II while colonylb is Type I. These observa-tions might suggest that we are dealing with a diphasic variationwhich has hitherto been considered to apply only to flagellatedspecies of bacteria. (See however, Takita (1937).)

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Our data at present do not permit a decision on these pointsand to pursue them further would unnecessarily prolong thispaper and would deviate from our original purpose.

Tentatively, we suggest that Shigelkl sonnei contains two pre-dominant antigens which we call Type I and Type II. TheType II is the more distinctive Sonne bacillus in that it containsa considerable amount of both antigens while the Type I seemsto lack some antigen present in Type II. In other words, ananti-Type II serum will agglutinate all types of Sonne strainsthat we have encountered but an anti-Type I serum will fail toagglutinate some of them except in fractions of the serum titre.Having determined the major antigens in Shigella sonnei we

next studied the minor antigens which relate it to the othermannitol-fermenting groups of dysentery bacteria.

Table 4 is condensed from a larger series to illustrate theserelationships. a

It is apparent from this table that Shigella dispar is itselfantigenically heterogeneous; this confirms the observations ofForsyth (1933) and of Watanabe (1935). The Flexner antigens(Shigella paradysenteriae) are omitted from the table since it iswell known that they are heterogeneous. Shigella alkalescensappears to be homogeneous and it may be remarked that 10additional strains not listed in the table were also agglutinatedto full titre by the four Alkalescens serums.The Sonne Type I antigen is agglutinated to a slight extent

by Flexner, Alkalescens and Dispar serums. It is a curious factthat, in a larger series comprising 42 strains of Sonne, every oneshowed some agglutination with Flexner V, W, X and Z serumsbut uniformly failed to be agglutinated by a Flexner Y serum.In contrast to this the Alkalescens antigens were uniformlyagglutinated by V, W, X and Y serums and failed to be agglu-tinated by a Flexner Z serum.The Sonne Type II antigens showed less obvious relation to

either Flexner, Alkalescens or Dispar. Of the two antigens il-lustrated, B597 appears to be the more nearly a pure Shigellasonnei antigen in that it fails to react with any of these serumswhile B217 reacts with both Flexner Z and Alkalescens serums-aproperty of this strain not disclosed by any of our previous tests.

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The results of table 4 again confirm our views that the Type IISonne bacillus is the more typical of the species in that its antigensare more redominantly Shigella sonnei in character. If it con-tains the antigens common to the Flexner, Alkalescens andDispar groups, these are not brought into evidence by this typeof test.

Reciprocal absorption tests failed to add to the informationgiven in table 4.

DISCUSSION

If the dysentery group as a whole be divided primarily on abasis of mannitol fermentation we find that the non-mannitol-fermenters, Shigella dy8enteriae, Shigella ambigua, (Schmitzbacillus) stand sharply apart from the others by reason of a num-ber of properties; cultural, antigenic and pathogenic. The nextsubdivision, among the mannitol fermenters themselves, is notso clear cut and overlapping occurs in many of the properties ofeach species. The most striking and most reliable feature whichcan be used to separate the Sonnei-Dispar group from the Flexnergroup is the fermentation of lactose, but this may be delayed aslong as three or four weeks. Because the individual strains ofthe Flexner group are so variable in themselves it is impossibleto distinguish between Flexner and Sonne or Dispar on culturalgrounds alone until sufficient time has been allowed for the fer-mentation of lactose. Serologically also, enough overlappingoccurs to require a complete series of agglutinations to the fulltiter of the serum before reliance can be placed on this type ofinformation.

It is, of course, just this similarity of properties among themannitol-fermenting dysentery organisms that justifies con-sidering them more closely related to one another than is therelationship of any one of them to either Shiga or Schmitz.We believe that Alkalescens belongs in the mannitol-fermenting

group of dysentery organisms in spite of its indefinite patho-genicity. From a taxonomic viewpoint, pathogenicity mustbe regarded as only one of the properties of a species, not neces-sarily more important per se than a combination of other proper-

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ties. For similar reasons we have excluded from our study suchspecies as have been described under the name of "Coli Anaero-genes." On closer examination some of these strains are eithertrue Sonne or Dispar or they produce small quantities of gas,often late, and thus appear to be more nearly related to theEscherichiae (Coli-Aerogenes group) than to the dysentery group.(Topley and Wilson 1936)).The occurrence of secondary papillae on colonies is not confined

to members of the Genus Shigella; it is occasionally found alsoamong other genera in Eberthelka and Salmonella. But in theGenus Shigella it reaches a marked development. Less com-monly seen in the Shiga and Schmitz species, it becomes morefrequent in the Flexner and Alkalescens species and is the rulerather than the exception in Sonne and Dispar. So much is thisso, that the phenomenon of late lactose fermentation in Sonneand Dispar seems dependent on the production of these papillae.They are, in fact, variants of the original strains with new fer-menting powers. We have consistently failed to get the variantsto breed true; they also revert to the lactose-negative state onfresh media and the fermentation of this sugar occurs only inageing cultures. The failure to secure a pure culture of lactose-positive Sonne has prevented our using it for antigenic analysis.While we feel certain of the presence of at least two major

antigens in Shigella sonnei, we fine no indication that they areassociated with a rough-smooth variation. If "roughness" bedefined as a rough colony which does not remain in suspension inthe presence of electrolytes, these antigens are both smoothantigens. If, on the other hand, "roughness" is defined as thequantitative loss of some antigenic component, we find that ourType II possesses an antigenic component not present in ourType I; but both serological types are independent of the varia-tion observed in colony form. We have not investigated thepossibility of a diphasic variation.From the standpoint of practical diagnosis only the Type II

antibody can be relied on to detect all strains of Shigella sonrei;the Type II antibody will agglutinate all strains to full titrewhile the Type I antibody will agglutinate only the Type I

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antigen to full titre and the type II antigen to a fraction of itstiter.The minor antigens of Shigella sonnei which show cross re-

actions with Shigella paradysenteriae, Shigella alkalescens andShigella dispar again emphasize the close taxonomic relationshipof these four species. It is interesting that Type II, which isregarded as the more distinctive, shows fewer cross reactions withthe other three than does Type I.We have searched for, and failed to find, any cultural or bio-

chemical property by which Type I strains might be distinguishedfrom Type II strains. All of our Type II strains have been re-cent isolations but an even greater number of recent strains be-long to Type I. Indeed, both types have occurred at the sametime on the same original plate from the same stool specimen.

SUMMARY AND CONCLUSIONS

1. Shigella sonnei is closely related culturally and antigenicallyto three other members of the mannitol-fermenting species ofdysentery organisms; namely the Shigella paradysenteriae (Flex-ner's bacillus), Shigella alkalescens and Shigella dispar.

2. The most outstanding distinguishing property of Shigellasonnei, the late fermentation of lactose, is due to the appearancein ageing cultures of variants with new fermenting powers.These variants are seen in the original colony as secondary papil-lae arising directly from the mother colony only after the colonyhas aged.

3. These variants are not permanent. When transferred tofresh medium, they immediately revert to the lactose-negativestate and, eventually, these transfers in turn produce lactosepositive variants. This succession of events may be continuedindefinitely.

4. At least two major antigens are present in Shigella sonnei-both of which are present in all strains but to varying degrees.Shigella sonnei Type II is the more representative type sinceboth antigens are abundantly represented in it; but it is leastfrequently encountered. Shigella sonnei Type I is most fre-

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330 JOHN H. GLYNN AND D. HUGH STARKEY

quently found and contains mainly Type I antigen with a rela-tively small amount of Type II antigen.

5. Minor antigens in Shigella 8onnei are common to Shigelladispar, Shigella alkalescens, Shigella paradysenteriae.

6. On a basis of cultural and antigenic properties the Sonnegroup differs sufficiently from the closely related Flexner, Disparand Alkalescens groups to warrant recognition as a separatespecies of the Genus Shigella rather than as a variant of Shigellaparadysenteriae. This confirms the species Shigelik sonnei(Weldin).

REFERENCES

ANDREWES, F. W. 1918 Dysentery bacilli. Lancet, 1, 560-563.ANDREWES, F. W. AND INMAN, A. C. 1919 A Study of the serological races of the

Flexner group of dysentery bacilli. Spec. Rep. Ser. No. 42, Med.Research Committee, London.

FORSYTH, W. L. 1933 Bacterium diepar (Andrewes) and its association withdysentery. Jour. Trop. Med. & Hyg., 36, 65-69.

FRASER, A. M., KINLOCK, J. P. AND SMITH, J. 1926 Sonne dysentery in Aberdeen.Jour. Hyg., 25, 453460.

GARDNER, A. D., DUGEON, L., BULLOCH, W. AND O'BRIEN, R. A. 1929 Thedysentery group of bacilli,-A System of Bacteriology in Relation toMedicine, Medical Research Council, London, 4, Ch. 3, 159-253.

GAY, F. P. 1935 The enteric group of bacteria-genus Shigella and bacillarydysentery, Agents of Disease and Host Resistance, Baltimore, Ch. 31,669-688.

HILGERS, W. E. 1920 Ueber die Rasse E (Milchzuckerrasse) der Pseudody-senterie. Zeitschr. f. Immunitatsf., 1, orig. 30, 77-94.

JOHNSTON, M. M. AND BROWN, A. 1930 B. dysenteriae Sonne infection. Can.Pub. Health J., 21, 394 402.

JOHNSTON, M. M. AND KAAKE, M. J. 1932 Studies on B. dysenteriae Sonne.Can. Pub. Health J., 23, 159-166.

KERRIN, J. C. 1928 An investigation of Bacillus dysenteriae (Sonne Type III).Jour. Hyg., 28, 4-8.

KOsER, S. A., REITER, D. O., BORTNIKER, E., AND SWINGLE, E. L. 1932 A studyof Bacterium dysenteriae, Sonne type. Jour. Prev. Med., 4, 477-501.

KRUSE, W. 1907 Neue Untersuchungen Qber die Ruhr. Deutsch. med. Woch-enschr. 23, 292-296, 338-342.

LEUCHB, J. AND PLOCKMANN, E. 1927 Zur Frage der Ruhrerreger. Centralbl. f.Bakt. O., 104, 347-355.

MURAKAMI, K. 1927 Ueber Milch gerinnende Arten von Pseudodysenterie-bazillen. Centralbl. f. Bakt. O., 101, 377-383.

MURRAY, E. G. D. 1918 An attempt at classification of Bacillus dysenteriaebased on an examination of the agglutinating properties of fifty-threestrains. Jour. of R. A. M. C., 31, 257-271, 353-398.

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PROPERTIES OF SHIGELLA SONNE4 331

NELSON, R. L. 1930 Sonne dysentery. Jour. Bact., 20, 183-201.SONNE, C. 1915 Ueber die Bakteriologie der giftarmen Dysenteriebazillen.

Centralbl. f. Bakt. O., 75, 408-456.TAKITA, JUNGO 1937 A new type of antigenic variation occurring in the Flexner

group of dysentery bacilli. Jour. Hyg., 37, 271-279.TOPLEY, W. W. C. AND WILSON, G. S. 1936 Principles of Bacteriology and

Immunity. Ed. II, London & Baltimore.WATANABE, K. 1935 Serologische Untersuchung von Dysenteriebazillen. Zeit-

schr. f. Immunitatsf., 84, 156-164.

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