CARBOHYDRATE FERMENTATION REACTIONS OF STAPHYLOCOCCI*

CARBOHYDRATE FERMENTATION REACTIONS OF STAPHYLOCOCCI* GEORGE H. CHAPMAN AND MERRITT H. STILES Clinicl Reserch Lbortory, New York, N. Y. A survey of the literture indictes tht wide vritions in technic nd in the incubtion period my hve been chiefly responsible for extreme differences in the proportion of stphylococci fermenting certin crbohydrtes, nd differences in the ssocition between crbohydrte fermenttion rections of stphylococci nd other properties of the cultures. Hoffstdt nd Youmns, Pinner nd Voldrich, Hbermn 4 nd others hve shown tht stphylococcl cultures my dissocite into vrints which differ in their fermenttive properties. Thus, it is possible tht, during the prolonged incubtion period ( to dys) often used in studying the fermenttion rections of stphylococci, dissocition of the cultures my occur which might ffect the fermenttion rections. The dvntges of shorter incubtion period do not pper to hve been recognized until Chpmn, Lieb nd Curcio 6 reported tht stphylococci which ferment mnnitol on the surfce of pltes of Bcto phenol red mnnitol gr usully produce detectble cid fter overnight incubtion. This brief intervl eliminted the influence of vrints hving fermenttive properties differing from those of the prent cultures. Thygeson 6 found tht this technic gve more relible results thn did previous methods. The results greed with those of the cogulse test in. per cent of 6 strins nd with the power to produce conjunctivitis-producing toxin in 7.4 per cent of 6 cultures. In the investigtion to be described the method ws pplied to the study of the fermenttion of other crbohydrtes by stphylococci nd the results were compred with certin rections of the cultures usully ssocited with pthogenicity. * Received for publiction, Mrch 6,. Downloded from https://cdemic.oup.com/jcp/rticle-bstrct//6//74 on April

FERMENTATION REACTIONS OF STAPHYLOCOCCI Technic The bse medium used in the fermenttion studies (Bcto phenol red gr bse) hd the following composition: grm Bcto beef extrct Bcto tryptose Sodium chloride Bcto gr Bcto phenol red. Enough wter ws dded to mke the totl volume cc. fter dding the crbohydrte solution. The rection ws djusted so tht fter steriliztion nd when redy for use the ph ws 7.4. Concentrted solutions of the crbohydrtes were filtered through Seitz filters nd dded to the cooled bse medium to mke. per cent concentrtion nd poured immeditely into Petri dishes. The poured pltes were set side in the refrigertor until the following dy to reduce running of growths cused by condenstion. The cultures were streked cross the surfce of the medi nd incubted t 7 C. After overnight incubtion the pltes were inspected for evidence of fermenttion, s indicted by the ppernce of yellow zones round the growths. If the results were negtive the pltes were returned to the incubtor nd re-exmined dily. The pltes were discrded fter dys becuse only n occsionl chnge occurred fter 4 hours, nd lter chnges were likely to hve been cused by the development of vrints in the cultures. Becuse there is generl greement tht the power of culture to cogulte humn or rbbit plsm, produce ornge pigment, hemolyze rbbit blood gr in hours nd produce ornge or violet growths on crystl violet gr* is closely ssocited with the pthogenic properties of the culture for mn nd rbbits, these rections were used s "in vitro" indictors of "pthogenicity." Cultures used Stphylococci recently isolted from persons suspected of hving chronic infection were clssified s follows: Pthogens (7 cultures): Producing ornge pigment. Hemolytic on rbbit blood gr fter overnight incubtion. Cogulting humn or rbbit plsm within hours. Producing violet or ornge growths on crystl violet gr. Degenerte pthogens ( cultures): Cogulting humn or rbbit plsm * It hs been clled to our ttention tht Epstein 7 described crystl violet gr rection, usully referred to s the "Epstein phenomenon," which is similr to the crystl violet gr rection of Chpmn nd Berens. Downloded from https://cdemic.oup.com/jcp/rticle-bstrct//6//74 on April

GEORGE H. CHAPMAN AND MERRITT H. STILES within hours, but lcking one or more of the other properties chrcteristic of pthogenic stphylococci. Non-pthogens ( cultures): Not cogulting humn or rbbit plsm. TABLE PROPORTION OP CULTURES FERMENTING DIFFERENT CARBOHYDRATES CABBOHYDBATB PATHOGENIC CULTUEEB, FEB CENT DEGENERATE PATHO GENS, PES CENT NON-PATHOGENIC CCL- TURES, FEB CENT Dextrose Levulose Scchrose Mltose Lctose d-glctose d-mnnose d-mnnitol Trehlose 6 6 7 7 7 4 7 7 4 4 4 7 7 4 Cellobiose Slicin Inulin i-inositol Dulcitol -Arbinose -Xylose Rffinose Rhmnose Melibiose Adonitol d-sorbitol Melezitose 4 4 4 4 6 7 7 7 7 Results obtined The crbohydrtes were fermented more esily by the pthogenic group, lthough there ws no close reltionship between the fermenttion nd in vitro rections of prticulr culture except in the cse of d-mnnitol, trehlose nd, to lesser extent, d-mnnose. All cultures fermented dextrose nd glycerol, nd ll except one fermented mltose, scchrose nd levulose. Lctose ws fermented by of the cultures. d-glctose ws fermented less esily thn lctose, cultures giving Downloded from https://cdemic.oup.com/jcp/rticle-bstrct//6//74 on April

TABLE ASSOCIATION AMONG REACTIONS OF DIFFERENT CABBOHYDBATES FEB CENT OF CULTURES SHOWING, () Lctose (b) d-glctose () (b) 7 Agreemenl Totl Prtil greement Totl Disgreement Totl () d-mnnose (b) d-mnnitol 7 6 () d-mnnose (b) Trehlose 7 7 7 () d-mnnitol (b) Trehlose 74 4 7, fermenttion within 4 hours;, fermenttion within 4 hours;, no fermenttion in dys. TABLE REACTIONS OF CULTURES WITH FERMENTATION REACTIONS DIFFERING FBOM THOSE OF THE MAJORITY OF CULTURES Rections of lctose, d-glctose, trehlose, d-mnnose, d-mnnitol nd melezitose re not considered STRAIN NUMBER 667 67 67 677 677 676 66 674 674 67 P H c V CARBOHYDRATES USUALLY FERMENTED i ID I CARBOHYDRATES USUALLY NOT FERMENTED ' f i m, rections occurring in 4 hours or lter. The three rections observed fter 4 hours re indicted by superscript numerls which denote the number of dys fter inocultion. s» I i CD o o < Downloded from https://cdemic.oup.com/jcp/rticle-bstrct//6//74 on April

4 GEORGE H. CHAPMAN AND MERRITT H. STILES negtive results but the distribution, like tht of lctose fermenttion with which it ws prllel in most instnces, ws unrelted to the in vitro tests of the cultures. d-mnnose, d-mnnitol nd trehlose were fermented more frequently by pthogenic type cultures (tble ). Only exceptionlly did culture ferment dextrin, i-inositol, melibiose, inulin, dulcitol, -rbinose, -xylose, rffinose, rhmnose, strch, donitol or d-sorbitol. Melezitose ws fermented by cultures but these included pthogens, degenerte pthogens nd non-pthogens. Cellobiose ws fermented by 4 pthogens. Slicin ws fermented by 4 pthogens nd non-pthogen. Certin ssocitions between the fermenttion of different crbohydrtes ppered to be significnt. There ws greement between lctose nd d- glctose fermenttion in cultures nd prtil greement in cultures (tble ). d-mnnitol nd trehlose showed similr rections in cultures nd closely relted results in. d-mnnose nd d-mnnitol rections greed in cultures nd were closely relted in nother (tble ). The two strins fermenting -rbinose lso fermented -xylose nd d-sorbitol. Two cultures (67 nd 677) which produced typicl colonies fermented crbohydrtes not fermented by most cultures. These ssocitions re detiled in tble. Delyed fermenttion (lter thn hours) occurred in of fermenters of d-mnnitol, of d-mnnose, 6 of trehlose, 4 of lctose, 6 of d-glctose nd in fermenters of rffinose. It occurred only once in ech of the following: scchrose, glycerol, cellobiose, strch, rhmnose, donitol, d-sorbitol, inulin nd slicin. With three exceptions, ll cultures tht fermented ny of the crbohydrtes did so within 4 hours. DISCUSSION A few pthogenic strins hd such vigorous fermenting power tht they fermented certin crbohydrtes not fermented by the mjority of strins. Conversely, the fermenttive power of few non-pthogenic cultures ws so reduced tht they filed to ferment certin crbohydrtes redily fermented by the mjority of strins. Although the power to ferment crbohydrtes ws more ctive in pthogenic thn non-pthogenic cultures, fermenttion rections were prllel with in vitro properties only in d-mnnose, d-mnnitol nd trehlose. The ssocition of fermenttion of d-mnnose, d-mnnitol nd trehlose, prticulrly the two ltter, is of considerble interest in view of the finding of mny investigtors tht fermenttion of d-mnnitol is chrcteristic property of pthogenic Downloded from https://cdemic.oup.com/jcp/rticle-bstrct//6//74 on April

FEBMENTATION EBACTIONS OF STAPHYLOCOCCI stphylococci. Fewer non-pthogens fermented trehlose thn d-mnnitol in hours. All highly pthogenic cultures fermented d-mnnitol nd trehlose within hours, while number of degenerte pthogens filed to ferment these two crbohydrtes. Becuse these three crbohydrtes give such contrsting results mong different strins, they should prove vluble in dissocition studies. Contrry to common opinion, lctose fermenttion ws found unrelted to the in vitro properties. Lctose ws fermented by per cent of pthogens, per cent of degenerte pthogens nd 7 per cent of non-pthogens. Chpmn, Berens, Nilson nd Curcio found tht slightly more pthogens thn non-pthogens fermented lctose under similr conditions ( nd per cent, respectively). CONCLUSIONS Most stphylococci, regrdless of their in vitro rections, ferment dextrose, levulose, scchrose, glycerol nd mltose within hours nd fil to ferment dextrin, cellobiose, slicin, inulin, i-inositol, dulcitol, -rbinose, -xylose, rffinose, rhmnose, strch, melibiose, donitol, d-sorbitol nd melezitose within dys, when tested on the surfce of tryptose, beef extrct gr with phenol red indictor. Pthogenic strins s group hd greter fermenting power thn the group of non-pthogenic strins, conforming to the generlly ccepted principle tht pthogenic strins of stphylococci show greter metbolic ctivity thn non-pthogens. Lctose nd d-glctose gve prllel results in of cultures but the rections were not ssocited with the cogulting, chromogenic, hemolytic nd crystl violet gr properties of the cultures. Fermenttion of d-mnnose, d-mnnitol nd trehlose ws more closely ssocited with these in vitro properties, the degree of correltion incresing in tht order. Two cultures, producing typicl growths, fermented crbohydrtes which were not fermented by 7 per cent of the cultures. The 4 cultures which fermented cellobiose nd 4 of the Downloded from https://cdemic.oup.com/jcp/rticle-bstrct//6//74 on April

6 GEOEGE H. CHAPMAN AND MEREITT H. STILES fermenters of slicin hd in vitro properties chrcteristic of highly pthogenic stphylococci. REFERENCES () HOFFSTADT, R. E., AND YOUMANS, G. P.: Stphylococcus ureus. Dissocition nd its reltion to infection nd immunity. Jour. Infect. Dis., : 6,. () HOFFSTADT, R. E., AND YOUMANS, G. P.: The genetic significnce of the dissocints of Stphylococcus ureus. Jour. Bct., 7:, 4. () PINNEB, M., AND VOLDRICH, M.: Derivtion of stphylococcus lbus, citreus nd roseus from Stphylococcus ureus. Jour. Infect. Dis., :,. (4) HABEBMAN, S.: Personl communiction. () CHAPMAN, G. H., LIEB, C. W., AND CTJBCIO, L. G.: The use of bromthymol blue gr nd phenol red mnnitol gr for the isoltion of pthogenic types of stphylococci. Amer. Jour, of Clin. Pth., Tech. Suppl., :,. (6) THYGESON, P.: Mnnitol fermenttion s n indictor of conjunctivl pthogenicity of stphylococci. Arch. Ophth., : 74,. (7) EPSTEIN, S.: Untersuchungen uber die Unterscheidung der Stphylokokken bei den verschiedenen Stphylokokkenerkrnkungen der Hut. Arch. f. Dermt. u. Syph., 7: 6, 4. () CHAPMAN, G. H., AND BEEENS, C: Crystl violet gr s differentil medium for stphylococci. Jour. Bct., : 47,. () CHAPMAN, G. H., BERENS, C, NILSON, E. L., AND CURCTO, L. G.: The differentition of pthogenic stphylococci from non-pthogenic types. Jour. Bct., :,. Downloded from https://cdemic.oup.com/jcp/rticle-bstrct//6//74 on April