CHARACTERISTICS OF A STRAIN OF STAPHYLOCOCCUS AUREUS GROWNIN VIVO AND IN VITRO

PAUL R. BEINING' AND E. R. KENNEDYBiology Department, The Catholic University of America, Washington, D.C.

Received for publication 15 October 1962

ABSTRACT

BEINING, PAUL R. (The Catholic Universityof America, Washington, D.C.) AND E. R.KENNEDY. Characteristics of a strain of Staphylo-coccus aureus grown in vivo and in vitro. J.Bacteriol. 85:732-741. 1963.-A comparativesurvey was conducted on the characteristics of astrain of Staphylococcus aureus after it had beengrown in vitro (VSB) and after it had beencollected from the peritoneal exudate of an in-fected guinea pig (GSB). Both VSB and GSBstrains gave the same results when studied in anextensive series of tests, including bound andsoluble coagulases, bacteriophage type, antibiotic-sensitivity pattern, the usual fermentation reac-tions, deoxyribonucleic acid base composition,and qualitative tests for hemolysins, deoxyribo-nuclease, ribonuclease, staphylokinase, staphylo-protease, lipase, and phosphatase. The in vivostrain differed significantly from the in vitrostrain in respiratory rate, agar gel diffusionstudies, agglutinability in tube tests, virulencetests in rabbits and mice, growth on tellurite-glycine agar, susceptibility to human -y-globulinin agar, and in the quantitative production ofdeoxyribonuclease, a-hemolysin, leucocidin, andhyaluronidase.

Since the work of Van de Velde (1894), thenotion has persisted that virulence can be in-increased by animal passage and is often pro-gressively lost during artificial cultivation. Theimpressive array of facts on the biochemical,serological, and virulence properties of Staphylo-coccus aureus, amassed from in vitro studies, maynot be reflecting the characteristics of true invivo staphylococci.The study of bacteria recovered from an in

vivo environment has been impeded by thetechnical difficulty of collecting large quantities

1 Present address: St. Joseph's College, Phila-delphia, Pa.

of bacteria free from extraneous material. Smith,Keppie, and Stanley (1953) established a work-able method for collecting and separating largeamounts of bacteria from the tissue fluid ofinfected guinea pigs. WVith this procedure, Gellen-beck (1962) showed that her in vitro strain ofS. aureus had a significantly lower exogenousrespiratory rate than did the same strain afterit had passed through a guinea pig.The purpose of the present investigation was

to conduct a comparative survey of the charac-teristics of a strain of S. aureus after it had beengrown in vitro (VSB) and after it had beencollected from the peritoneal exudate of aninfected guinea pig (GSB). The more pertinentbiochemical, serological, and virulence tests wereemployed in the survey.

MATERIALS AND IMETHODS

Source, preparation of suspensions, and main-tenance of S. aureus grown in vitro. The S. aureuswas obtained from Providence Hospital, Wash-ington, D.C., and represented a culture from asingle colony taken from a sheep-blood agar platethat had been streaked with pus from a freshfuruncle. Several slants, representing the firstpassage after isolation, were made, stored at 5 C,and used as stock cultures for all further prepara-tions. The staphylococei were cultured on Trypti-case Soy Agar (BBL) slants or in 10 ml of Trypti-case Soy Broth and incubated aerobically for 18hr at 37 C. The in vitro supernatant fluid was pre-pared by growing the organisms in broth culturefor 18 hr; the cells were removed by centrifuga-tion of the culture in the cold (2 C) at 10,000 X gfor 30 min. The sedimented bacteria were sus-pended with a glass rod in a small amount of 0.1M Veronal-buffered saline (pH 7.3); these bacteriawere called the in vitro-suspended cells (VSBcells). Cells and supernatant were stored at 5 C.

Inoculation and recovery of S. aureus fromguinea pigs. The inoculum for the injection ofguinea pigs was prepared by harvesting 24-hr

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cultures of VSB cells from five Trypticase SoyAgar slants and suspending the cells in 10 ml ofbuffer. This suspension represented approxi-mately 6 X 109 viable cells per ml. A maleguinea pig was injected, and the organisms re-covered and purified as reported previously(Gellenbeck, 1962).The guinea pig peritoneal supernatant fluid

was removed and immediately stored in 2-mlamounts at -20 C. The cells recovered from theanimal's body fluids were designated the GSBcells, and were stored at 5 C.

Biochemical and metabolic studies. Aerobicrespiratory rates of VSB and GSB cells in 0.0125M glucose and in buffer (endogenous) were de-termined by the procedure of Gellenbeck (1962).Minimal staphylococcal agar was made ac-

cording to the method of Burns and Holtman(personal communication) and contained (mg per100 ml): glucose, 100; Difco vitamin-free Casa-mino Acids, 500; thiamine HCl, 0.5; and agar,1,500.The -y-globulin was poliomyelitis-immune (hu-

man) globulin, purchased from Merck Sharp &Dohme, Philadelphia, Pa. Each ml of thismaterial contained 0.16 g of globulin and repre-sented a pooling of material from 2,000 donors.Sterile filter-paper strips (15 by 60 cm) weresoaked in the y-globulin and placed on mediumidentical with that used by Anderson (1956).Plates were incubated for 48 hr in an atmosphereof 30% CO2 in an anaerobic incubator (NationalAppliance Co., Portland, Ore.).

Fibrinolysis of heat-precipitated human fibrinby staphylokinase or by protease, when soybeantrypsin inhibitor (Nutritional Biochemicals Corp.,Cleveland, Ohio) was added to the medium, wasdetected by the procedure of Lack and Wailling(1954).

Titration of hyaluronidase and deoxyribo-nuclease activity was performed by the ACRAtest, according to the modification of Oakleyand Warrack (1951).

Preparation of antisera. Female albino rabbits(2 to 3 kg) were used for production of antisera.For sera against whole bacterial cells, suspensionsof live VSB and GSB cells and GSB cells killedwith Zephiran (Winthrop Laboratories, NewYork, N.Y.), according to the procedure ofMcCoy and Kennedy (1960), were injectedintravenously over a 16-day period. The periodconsisted of two series of six daily injections

with a 4-day rest separating the two series. Atotal of approximately 6.8 X 101 cells was givenin the 16-day period. Antisera were preparedfrom blood taken by cardiac puncture 5 daysafter the last injection and stored, withoutpreservative, at -20 C. For sera against solublestaphylococcal products, alum-precipitated sus-pensions of in vitro and in vivo supernatantfluids were used and injected by the intravenousroute. Three injections per week were given overa 6-week period. Antisera were collected 5 daysafter the last injection and stored at -20 C.To prepare a control rabbit antiserum, a

guinea pig was injected with Escherichia coli;its in vivo supernatant was collected in the samemanner as described previously for staphylococci.The control rabbit antiserum was preparedagainst this in vivo supernatant fluid of E. coliin the same manner as described above for theGSB supernatant.

Virulence studies: pathogenicity of in vivo andin vitro cells. VSB and GSB organisms in buffer(at a concentration of approximately 108 cellsper ml) were serially diluted, and 1 ml of thedilutions was injected intraperitoneally into fivealbino mice weighing 18 to 25 g. Examination ofmice ended 48 hr after inoculation. Portions (0.1ml) of the same dilutions were injected intra-dermally into depilated albino rabbits weighing2 to 3 kg. The site of injection was observed for4 days.

Pathogenicity of VSB and GSB organisms forrabbits was also determined by the intravenousroute. Samples of suspensions (which originallycontained approximately 7 X 107 organisms perml) were injected according to the schedule forimmunizing with particulate antigens. Injectionswere stopped when osteomyelitis, organ involve-ment, or death appeared.

Toxicity of in vivo and in vitro supernatantfluid: in mice. Portions (1 ml) of serial twofolddilutions of the supernatant fluids were injectedintraperitoneally into groups of five albino mice(18 to 25 g). The highest dilution producing deathin 50% of the animals within 3 days was takenas the end point of titration.In rabbits. Portions (0.1 ml) of the same serial

dilutions were injected intradermally; the firstdilution failing to give a dermonecrotic reactionwithin 4 days was considered the end point oftitration.

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TABLE 1. Respiratory response of Staphylococcusaureus strains VSB and GSB

Respiration (02 uptake)

At 60 min At 120 min

Substrate

Per- Per-cent- cent-

GSB VSB age GSB VSB agein- in-

crease crease

,uliters Mlziters ulliters I Iers

Glucose, 0.2 M. 210 92.6 78* 113 78.4 31*Endogenous

respiration(buffer). 1.35 5.75 76t 2.6 5.4 52t

* Percentage of increase with respect to GSB.t Percentage of increase with respect to V-SB.

RESULTS

Strains VSB and GSB exhibited the size, shape,and staining properties typical of staphylococci.Both strains were catalase-positive, showed he-molysis on rabbit and human red blood cellplates only, possessed bound and soluble co-agulases, and were bacteriophage type 80/81.Both strains were sensitive to chloramphenicol,oleandomycin, neomycin, nitrofurantoin, methen-amine mandelate, and novobiocin; both strainswere resistant to penicillin, dihydrostreptomycin,oxytetracycline, erythromycin, chlortetracycline,triple sulfa, sulfamethizole, sulfisoxazole, sulfa-diazine, and sulfathiazole. The two strains pro-duced brilliant golden-orange coloration withaerobic incubation on skim-milk agar plates;when incubated in 30% C02, both strains grewwell but all colonies were chalk-white.The possibility that passage through a guinea

pig caused the emergence of organisms with adifferent deoxyribonucleic acid (DNA) basecomposition was tested by several thermal de-naturation curves on both strains. There was aclose similarity between VSB and GSB strainsrelative to the thermal denaturation profile (Tm)and the per cent guanine plus cytosine (GCcontent). The Tm of VSB was 85.3 C and GCcontent 39%, whereas the GSB strain showed aTm of 85.5 C and 39.5% GC content. The slightdifference in Tm between the two strains iswithin the bound of experimental error and theconsistency of the apparatus used. The technicaldifficulty of obtaining enough GSB cells directly

from guinea pigs for the DNA extraction pro-cedure necessitated the passing of strain GSBonce into Brain Heart Infusion broth after re-moval from the guinea pig.The above characteristics, shared by the VSB

and the GSB strains, were considered to be theprinciple markers of this culture of S. aureus.Frequently, through the course of this investi-gation, stock cultures and subcultures weretested for these maikers to establish identitywith the original isolate used in the study.

Biochemical and metabolic studies. As Gellen-beck (1962) demonstrated with her strain, theaerobic respiratory rate of our GSB strain washigher than that of the VSB strain, although theendogenous respiratory rate of the VSB strainwas significantly higher than that of the GSBstrain. This relationship is shown in Table 1.

Studies with potassium tellurite-glycine agarindicated a significant difference in the metaboliccapacity of in vitro and in vivo strains. Thesurface of all plates was inoculated with oneloopful of bacterial suspension containing approxi-mately 103 organisms per ml, and readings weremade 24 hr later. Under certain defined experi-mental conditions, the GSB strain was completelyincapable of growing on potassium tellurite-glycine agar and of reducing tellurite within the24-hr period, whereas the VSB strain was ableto grow and reduce the tellurite. The ability ofstrain VSB, but not GSB, to grow on potassiumtellurite-glycine agar (final pH of 6.3 beforepouring the plate) and to reduce the tellurite isevident from Fig. 1; the right half of this figureshows that both strains grew equally well on acontrol Trypticase Soy Agar plate. The effect ofpH on the differential selectivity of potassiumtellurite-glycine agar for VSB, GSB, and S.epidermidis strains is recorded in Table 2. Themedium at pH 5.0 was inhibitory to all strains.However, at pH 6.3, all GSB isolates and S.epidermidis were inhibited by the medium, whilethe VSB strain grew well and reduced thetellurite. WVhen the pH was 7.2, a few of theGSB isolates began to reduce the tellurite andgrow on the medium. A pH of 9.6 was also lessfavorable to growth of the GSB strain; but, atthis pH, S. epidermidis began to grow and toreduce the tellurite. The majority of GSB isolateswere incapable of growing and reducing telluriteover the pH range used in this study. Since itwas possible that some factor in the in vivo super-

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FIG. 1. Differential growth of strains VSB and GSB within 24 hr on potassium tellurite-glycine agar (finalpH 6.3). Upper half of the tellurite-glycine agar plate on the left of the figure contained the VSB strain; thelower half of that plate was streaked with the GSB strain. The Trypticase Soy Agar plate on the right of thefigure was streaked in the same manner with VSB and GSB strains. All streaks were made with a loopful oforganisms fronm a suspension in Veronal-buffered saline containing approximately 108 organisms per ml.

TABLE 2. Effect of pH on the differential selectivityof potassium tellurite-glycine agar for

Staphylococcus epidermidis, VSB,and seven GSB isolates*

Substrate

Tellurite-gly-cine agar.....

Trypticase SoyAgar...

pH

5.06.37.29.6

Bacterial strains

S.epider-midis

0

0

0

2+

VSB

0

4+4+2+

4+ 4+

GSB GSBisolate isolate

a b

0

0

4+0

4+

0

0

4+2+

4+

* All GSB isolates represent distinct harvestsfrom different guinea pigs. The pH values are finalreadings made immediately before pouring plates.The 4+ indicates as much growth on tellurite-glycine agar as seen on the Trypticase Soy Agar-plate control for that isolate; 2+ would indicateapproximately one-half as much growth as thecontrol.

natant was acting as an inhibitor and preventingstrain GSB from metabolizing on this medium, apilot experiment was set up to determine theeffect of the supernatant fluid itself. Fresh GSBsupernatant was incapable of reducing telluriteand could not inhibit its reduction when mixedwith VSB isolates which were capable of reducingtellurite. As noted by Zebovitz, Evans, andNiven (1955), we also found that potassiumtellurite from various commercial sources differedin selective activity. The nature of inhibition ofgrowth of strain GSB in this medium is beinginvestigated further.Another manifestation of difference in the

metabolic capacity of in vivo and in vitro strainsis seen in Fig. 2. When S. epidermidis, VSB, andGSB were streaked at right angles to Py-globulin-impregnated filter paper, there was a differentialinhibition of growth due to the -y-globulin. Theconcentration of -y-globulin in the filter paper,apparently, was an important factor because thesame isolate (S. epidermidis, VSB, or GSB) wasinhibited to a slightly different degree on differentoccasions. However, when the three strains werestreaked on the same plate, the GSB strain was

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FIG. 2. Differential inhibition of growth of strainsGSB, VSB, and Staphylococcus epidermidis due to-y-globulin-impregnated filter paper in Andersonmedium. From left to right, the strains are GSB,VSB, and S. epidermidis, respectively.

always inhibited more than the VSB or S.epidermidis strains. When nine different isolatesof the three strains were tested, the average

distance of inhibition by -y-globulin was: strainGSB, 15 mm; strain VSB, 9 mm; S. epidermidis,7 mm. The y-globulin effect could not be demon-strated when filter-paper strips vere impregnatedwith guinea pig complement (BBL), normalrabbit serum, rabbit anti-GSB serum, or withanti-a-toxin serum.

Studies on the minimal staphylococcus agar ofBurns and Holtman (personal communication)indicated that the GSB strain is much lesscapable of metabolizing and growing than is theVSB strain. On the surface of minimal agar

plates, 1 ml of each strain (containing 100 to 300organisms per ml) was spread. The VSB straingrew almost as luxuriously on this medium as itdid on the Trypticase Soy Agar control medium,but only five colonies of the GSB strain grew on

the minimal agar as opposed to 200 colonies on

the control agar. Similarly, limited experimentsindicated that strain GSB grew less readily on

polymyxin B and phenylethyl alcohol agar thandid strain VSB; strain GSB, however, was lessinhibited on tetrazolium agar (Kennedy andBarbaro, 1952) plates than was the VSB strain.The reactions of strains VSB and GSB agreed

with the description of reactions of S. aureus inBergey's Mlanual (Breed, Murray, and Smith,1957) with respect to liquefaction of gelatin,failure to produce indole, reduction of nitrates,and tube fermentation tests with lactose, raf-finose, mannitol, dextrose, and sucrose. However,strain GSB did hydrolyze starch with the pro-duction of acid both aerobically and in 30%C02, whereas strain VSB was incapable ofhydrolyzing this polysaccharide.

Studies on diffusible cellular products. StrainsGSB and VSB were also compared as to theirqualitative and, where possible, quantitativeproduction of several extracellular, staphylo-coccal "elaborates" in vitro. Qualitatively, boththe GSB and VSB strains produced catalase,bound and soluble coagulase, a-hemolysin, a-hemolysin, leucocidin, deoxyribonuclease, ribo-nuclease, staphylokinase, staphyloprotease, lipase,and phosphatase.A significantly greater amount of a-hemolysin,

leucocidin, hyaluronidase, and deoxyribonucleasewas produced by those organisms which had beencultivated in the in vivo environment (Table 3).In conjunction with the titrations listed in Table3, a neutralization test was performed with aStaphylococcus antiserum (CPP 101 /63A), ob-tained from Wellcome Research Laboratories andknown to contain 480 units of anti-a-hemolysin.The antiserum completely neutralized the a-he-molvtic activity of undiluted GSB supernatant.

Serological studies. In Table 4 are listed the

TABLE 3. Qutantitative production of heinolysins,le ucocidin, deoxyribonuclease, and hyalutronidase

by VSB and GSB strains

Supernatant fluid

Soluble titration product EscherichiaGSB VSB coli

guinea pig

c-Toxin* ............ 102.4 0 0d-Toxin* ............ 20 0 06-Toxin* ........... 2 0 0Hyaluronidaset ... . 1,280 16 2Deoxyribonucleaset.. 1,024 4 32Leucocidint.64 1 Not

tested

* Units represent the reciprocal of the highestdilution giving 50% hemolysis.

t Units represent the reciprocal of the highestdilution to give a firm clot in the ACRA test.

tUnits represent the number of minimal leuco-cidal doses.

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TABLE 4. Comparison of strains GSB and VSB bythe tube agglutination test

Antiserum AntigenVSB GSB

Preimmunization sera. <1:4* <1:4*Normal rabbitserum.. <1:4 <1:4

Normal guinea-pigserum............... <1:4 <1:4

Anti-GSB (Zephi-ran-killed) .......... <1:10 1:320

Anti-GSB (living). .... 1:5,120 1: 320t* Figures represent the highest dilution of

serum giving 2+ or higher reading.t Thread agglutination.

TABLE 5. Cross-precipitation pattern in Oudintubes* of VSB, GSB, and Escherichia coliguinea-pig supernatant fluids againsthomologous and heterologous antiserat

External reactant (zones)

E. coilInternal reactant GSB VSB guinea-

super- super- pignatant natant super-

natant

Anti-GSB supernatant.... 5 0 3Anti-E. coli guinea pigsupernatant ............ 3 0 3

Normal rabbit serum 0 0 0

* Recording after 5 days at 25 C.t Final dilution of serum was 1:2.

agglutination titers of strains VSB and GSB inthe tube agglutination test. In addition to thedifference in titer of the two strains, only theGSB cells exhibited "thready" agglutination inantisera against the living GSB organism. Theresults imply that a different major antigeniccomponent(s) is present on the surface of theGSB and VSB strains; the results could alsoimply that killing with the detergent Zephirandestroyed a major antigen(s) more pertinent tothe VSB than to the GSB strain.

Studies on the antigenic cellular "elaborates"of strains VSB and GSB were made by the Oudintechnique. Table 5 gives the cross pattern ofprecipitation of VSB and GSB supernatant fluidsagainst their homologous and heterologous anti-sera. E. coli guinea pig supernatant and theantiserum prepared against it were included inthe Oudin tests as a control to determine the

FIG. 3. Composite graph showing the concentrationof areas and number of zones for VSB supernatant(0), GSB(-), and Escherichia coli (X) guinea pigsupernatant fluids against antiserum to GSB super-natant in Oudin columns. Recording made after 5days at 25 C. Final dilution of serum was 1:2.

number of zones of precipitation due to non-specific guinea pig protein in the in vivo super-natant. Antisera to VSB supernatant producedno reaction with the three supernatant fluids and,therefore, was not included in the table. Figure 3is a composite graph of the tracings of a photron-reflectometer measuring the number of zones andconcentration of areas of the same external re-actants vs. anti-GSB supernatant. From Table 5and Fig. 3, it is evident that a minimum of twoprecipitation bands are produced by staphylo-coccal soluble products in the GSB supernatant.Presumably, these soluble products are notpresent in the VSB supernatant. It is also evidentthat three nonspecific zones are produced byguinea pig soluble antigens carried in the in vivosupernatant fluids.

Tracings of the reaction of VSB and GSBsupernatant fluids and controls vs. anti-GSBsupernatant by the Ouchterlony technique aredepicted in Fig. 4. A minimum of two andpossibly three bands peculiar to staphylococcalproducts is evident around the fourth antigenwell. The antigens used in the fourth and thirdwell represent fresh isolates taken immediatelybefore the Ouchterlony plate was set up, whereasthe antigens in wells one and two were isolated afew months prior to the test. It is possible thattheir staphylococcal elements were destroyed instorage. As in the Oudin tubes, the Ouchterlonyplates showed that staphylococcal products were

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FIG. 4. Reaction in Ouchterlony plate of anti-GSBsupernatant with various antigens. The serum (anti-GSB supernatant) was in the center wall. The fol-lowing antigens were in the numbered wells: (1)GSB supernatant, isolate a; (2) GSB filtered suspen-sion; (3) VSB supernatant; (4) GSB supernatant,isolate b; (5) guinea pig complement; and (6)Escherichia coli guinea pig supernatant.

in the GSB but not in the VSB supernatantfluids. Figure 4 also portrays well the reaction ofidentity due to guinea pig protein in the GSBsupernatant, complement, and E. coli guinea pigsupernatant fluids.

Virulence studies. To compare the lethal proper-ties of the VSB and GSB strains, groups of fivemice were injected intraperitoneally with thebacterial cells or their supernatant fluids. Noneof the mice could be killed with the supernatantof VSB, whereas in 2 days five of ten mice in-jected with a 1:5 dilution of the GSB supernatantwere killed. When living cells of the two strainswere injected intraperitoneally into mice, 2 X 109VSB cells and only 9 X 107 GSB cells were

needed to insure 100% mortality.In the course of this work and another problem

under investigation in this laboratory, a total of39 rabbits were injected intravenously with theliving VSB and GSB strains. Of 20 rabbits re-

ceiving the GSB strain, 19 developed an acuteosteomyelitis within 10 days. Each rabbit hadreceived, at most, 3.4 X 108 cells by this time.Debilitation, incontinence, and death would havefollowed if chloramphenicol had not been ad-

ministered. Autopsy of dead animals showedmultiple abscesses in the liver, spleen, and cardiacmuscle but not in the kidneys. Of the 19 rabbitsreceiving the VSB cells, 9 developed an osteo-myelitis, but not before 3 to 4 weeks; by thattime, these animals had received two to threetimes more cells than did the rabbits receivingthe GSB cells.To contrast the invasive and dermonecrotic

abilities of the VSB and GSB strains, a few rabbitswere injected intradermally with 0.1 ml of thecells or their supernatant fluids. The site ofinjection with the VSB supernatant fluid ex-hibited only a slight erythema by 24 hr; thislesion did not progress, and all traces of it haddisappeared by 4 days. However, the injection ofGSB supernatant produced an intense spreadingnecrosis of the skin which, within 48 hr, hadprogressed to a brown punched-out ulcer (2 cmin diameter), a reaction typical of the dermo-necrotizing toxin. This effect was completelyneutralized in rabbits by known antidermo-necrotic serum and also by human y-globulinwhen passively administered. Within 24 hr, theintradermal injection of 2 X 106 live GSB cellsproduced a blanched area (4 cm in diameter)which progressed by 4 days to a raised, induratedlesion with a punched-out center. In time, thepunched-out area became necrotic and resembledthe effect of the necrotizing toxin. Wlhen 5.2 X 107VSB cells were injected intradermally, only avery slight area of erythema developed in 24 hr;within 4 days, this intensified and was followedby the appearance of yellow pus and softening inthe center of the lesion, the pattern of a typicalfuruncle.

DISCUSSIONGellenbeck (1962) reported that staphylococci

recovered from an infected animal differed sig-nificantly in respiratory rate when comparedwith the same culture grown in vitro. The presentwork shows that the two strains, VSB (in vitro)and GSB (in vivo), differed significantly incharacteristics other than respiratory rate. Anextensive series of tests applied to VSB and GSBwere equivocal. These procedures included theusual characteristics of taxonomic significanceand the usual tests employed in clinical work todetermine potential pathogenicity. The in vivocells differed from the in vitro cells in at leasteight particular ways. The in vivo strain quanti-

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tatively produced more deoxyribonuclease, a-he-molysin, leucocidin, and hyaluronidase than didthe in vitro strain. The GSB cells differed fromVSB cells in agglutinability and in virulencepotential. Strain GSB failed to grow on tellurite-glycine agar; strain VSB grew well. Human'y-globulin markedly inhibited growth of GSBorganisms, whereas VSB cells were inhibited to alesser degree.The dramatic difference in amount of a-he-

molysin, leucocidin, hyaluronidase, and deoxy-ribonuclease in the GSB supernatant fluid overthat in the VSB supernatant is significant. Toour knowledge, no single artificial medium ex-hibits the same efficiency. Studies are in progressto determine how many and how much of thevarious staphylococcal products can be found inthe VSB supernatant fluid after manifold concen-tration of this fluid. Whether the presence ofthese cellular products has any causal relation to,or is merely concurrent with, the property ofvirulence is still uncertain. However, Anderson(1956) found a direct relationship between thenumber of flocculation lines from soluble productsof staphylococci and the severity of the staphylo-coccal lesion.Although both VSB and GSB strains were

bacteriophage-type 80/81, tube-agglutinationtests indicated a marked difference in aggluti-nability of the two strains when tested in the sameantiserum. Antisera prepared against Zephiran-killed GSB organisms agglutinated GSB cellswell (titer, 1:320) but agglutinated VSB cellspoorly (titer, 1:10). Antisera to living GSBantigen yielded strong, granular agglutination ofthe VSB antigen in dilutions as high as 1:5,120,but the same antisera gave "thready" aggluti-nation of GSB cells in dilutions as high as 1:320."Thread" agglutination in the enteric organismsis a manifestation of 0-inagglutinability (Stuart,Feinberg, and Feinberg, 1948) and is frequentlyassociated with the virulence of the organism. Itis possible that strain GSB could represent aserotype distinct from strain VSB, but it is justas possible that, along the lines of the finding ofBrodie, Guthrie, and Sommerville (1958), strainVSB may represent a less pathogenic (moreagglutinable) member of the same serotype asstrain GSB. Recently, Pereira (1961) reportedthat most newly isolated strains of S. aureuspossessed one of the antigens 13, 17, or 18; oncontinuous subculture in vitro, the variation of

antigen 13 -) 3 and 17 -- 1 was very often ob-served. The VSB strain was never subcultured invitro as often as Pereira found necessary toeffect this antigenic variation. Since GSB anti-serum was directed against the in vivo strain, theagglutination results may be detecting the sameprogressive antigenic fluctuation. Pereira foundno change in phage type in a strain before andafter antigenic variation.The virulence studies do not allow the con-

clusion that the VSB strain is avirulent and theGSB strain virulent, but they do indicate thatthe degree and possibly the type of virulence ofstrain GSB is quite distinct from that of strainVSB.The inability of the VSB supernatant fluid to

induce a pathological situation by the intra-peritoneal or intradermal routes may simplyreflect the fact that enough soluble products werenot produced by this method. Death of micefollowing intraperitoneal injection of the twostrains may have been brought about by an over-whelming administration of foreign protein, butmore likely a definite number of cells (alwaysfewer of the GSB strain) are needed to guaranteeenough preformed ca-hemolysin. A different typeof virulence seems to be in evidence with theintravenous injection of live cells into rabbits.The fact that we were able to induce death inrabbits by this route faster and with fewer cellswhen the GSB strain was used would seem to usto be convincing evidence of the greater virulenceor the distinctness in type of virulence of theGSB strain. Smith and Dubos (1956) came to asomewhat similar conclusion. In working withinfections in mice, they found the difference invirulence between coagulase-positive and co-agulase-negative strains to be quantitative ratherthan qualitative; in fact, they found that variousstrains could be arranged in a continuous spec-trum with many intermediate degrees of viru-lence.

Hoeprich, Croft, and West (1960) and Burnsand Holtman (1960) recommended the use oftellurite-glycine agar and a minimal agar, re-spectively, as a means of isolating potentiallypathogenic staphylococci and of excluding non-pathogenic strains. Our work confirms this onlyif the nonpathogenic strain belongs to the S.epidermidis group. On the other hand, we findthat these two media selectively favor the growthof the less virulent strain (VSB) over the more

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virulent strain (GSB). This disparity in ourfindings and theirs is cause for serious concern,in that the use of certain media may actuallyprevent one from isolating staphylococci whichresemble strain GSB. The fact might imply anew approach in the isolation of the more virulentstaphylococcal colonies from pathological situ-ations. One such approach could be a quasireplica plating on enriched, as well as tellurite-glycine and minima], agar. Strains, which wouldnot grow on the latter media, could be pickedfrom the enriched medium. The application ofthe replica-plating technique of Runnels andWilson (1960) would be a step in the right di-rection if we could avoid the incubation in beef-infusion broth before plating on the indicator orselective media. Even one passage in artificialmedium seems to accelerate the emergence of aless virulent population in a strain grown origi-nally in vivo.The results described here indicate that human

y-globulin exhibits a greater bactericidal effectin agar against GSB than it does against VSBand S. epidermidis strains. To the best of ourknowledge, this phenomenon has not been re-ported to date. Staphylococci could not be culti-vated from the area of inhibition. We could notdemonstrate this bactericidal effect with rabbitsera prepared against GSB living cells, GSBsupernatant fluid, or with commercial horse anti-a-toxin. The effect does not seem to be due tothe human-serum antistaphylococcal factor, de-scribed by Yotis and Ekstedt (1959), because theinhibitory effect was greater against the co-agulase-positive than against the coagulase-nega-tive strains. It seems possible that the differentialinhibitory effect of human y-globulin may becorrelated with the kind and the frequency ofstaphylococcal infection. For example, the kindof staphylococcal infection may manifest itself(in order of increasing severity and decreasingfrequency) as a single self-limiting furuncle,multiple and chronic furunculosis, osteomyelitis,generalized septicemia with or without organinvolvement, and as a systemic, lethal infectionwhich is rapidly fulminating. The present workshows that, at least in animals, the strain withthe greater (systemic) virulence potential wasmore susceptible to the bactericidal action of'y-globulin than was the strain with the lesservirulence.

ACKNOWLEDGMENTSG. P. Gladstone, University of Oxford, Oxford,

England, performed the leucocidin titrations.Viola Mae Young and C. H. Zierdt, Clinical

Microbiology Service, Pathology Department,Clinical Center, National Institutes of Health,determined the bacteriophage types. S. Falkowand I. R. Ryman, Walter Reed Army Instituteof Research, assisted in the guanine plus cytosinedeterminations. The help of these persons is verygratefully acknowledged.

LITERATURE CITEDANDERSON, K. 1956. A survey of toxicity in staphy-

lococci. J. Clin. Pathol. 9:257-260.BREED, R. S., E. G. D. MURRAY, AND N. R. SMITH.

1957. Bergey's manual of determinativebacteriology, 7th ed. The Williams & WilkinsCo., Baltimore.

BRODIE, J., W. GUTHRIE, AND T. SOMMERVILLE.1958. An attenuated variant of Staphylococcusaureus with specific immunizing properties.Brit. J. Exptl. Pathol. 39:199-202.

BURNS, J., AND D. F. HOLTMAN. 1960. Biochemicalproperties of virulent and avirulent staphylo-cocci. Ann. N.Y. Acad. Sci. 88:1115-1124.

GELLENBECK, S. M. 1962. Aerobic respiratorymetabolism of Staphylococcus aureus from aninfected animal. J. Bacteriol. 83:450-455.

HOEPRICH, P. D., G. F. CROFT, AND L. M. WEST.1960. Tellurite reduction as an indicator ofpotentially pathogenic staphylococci. J. Lab.Clin. Med. 55:120-128.

KENNEDY, E. R., AND J. F. BARBARO. 1952. Theinhibitory effect of triphenyltetrazolium onsome strains of micrococci. J. Bacteriol.63:297-298.

LACK, C. H., AND D. G. WAILLING. 1954. A studyof 435 strains of Staphylococcus aureus withreference to factors which may contribute topathogenicity. J. Pathol. Bacteriol. 68:431-443.

McCoy, K. L., AND E. R. KENNEDY. 1960. Autoge-nous vaccine therapy in staphylococcic infec-tions. J. Am. Med. Assoc. 174:35-38.

OAKLEY, C. L., AND G. H. WARRACK. 1951. TheACRA test as a means of estimating hyalu-ronidase, deoxyribonuclease and their anti-bodies. J. Pathol. Bacteriol. 63:45-55.

PEREIRA, A. T. 1961. Antigenic loss variation inStaphylococcus aureus. J. Pathol. Bacteriol.81:151-156.

RUNNELS, J. L., AND J. B. WILSON. 1960. Isolationof staphylococci by replica plating. J. Bacte-riol. 80:727-728.

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VOL. 85,1963 PROPERTIES OF IN VIVO- AND IN VITRO-GROWN S. AUREUS

SMITH, H., J. KEPPIE, AND J. STANLEY. 1953. Amethod for collecting bacteria and theirproducts from infections in experimentalanimals, with special reference to Bacillusanthracis. Brit. J. Exptl. Pathol. 34:471-477.

SMITH, J. M., AND R. J. DUBOS. 1956. The behaviorof virulent and avirulent staphylococci in thetissues of normal mice. J. Exptl. Med. 103:87-108.

STUART, C. A., R. FEINBERG, AND R. FEINBERG.1948. An unusual type of 0 agglutination. J.Bacteriol. 56:419-422.

VAN DE VELDE, H. 1894. Etude sur le mecanisme

de la virulence du Staphylocoque pyogene.Cellule 10:401-460.

YOTIS, W. W., AND R. D. EKSTEDT. 1959. Studieson staphylococci. I. Effect of serum andcoagulase on the metabolism of coagulasepositive and coagulase negative strains. J.Bacteriol. 78:567-574.

ZEBOVITZ, E., J. B. EVANS, AND C. F. NIVEN, JR.1955. Tellurite-glycine agar: a selectiveplating medium for the quantitative detectionof coagulase-positive staphylococci. J. Bac-teriol. 70:686-690.

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