Presumptive identification of group A, B, and D streptococci on agar plate media

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0095-1137/79/06-0665/08$02.00/0

Presumptive Identification

of

Group

A,

B,

and D

Streptococci

on

Agar Plate Media

R.R. FACKLAM,* J. F. PADULA, E. C. WORTHAM, R. C.COOKSEY,AND H. A. ROUNTREE

BureauofLaboratories,BacteriologyDivision,Centerfor Disease Control, Atlanta, Georgia 30333

Received forpublication19March1979

Severalpresumptivetests wereevaluated for their effectivenessin differentiat-ingstreptococci. When thetestswerecombinedinto abatteryand theresulting

reactions wereinterpretedaspatterns,theoverall presumptive identificationrate

was atleast 97%. We usedthehemolytic reaction, susceptibilitytobacitracinand

sulfamethoxazoleplustrimethoprim (1.25 ugplus23.75,ug),and standard CAMP

reactions on sheep blood Trypticase soy agar, and bile-esculin and 6.5% NaCI

agartolerancetestswithincubation in candle extinction jars. Subsequently,98.9%

ofthegroupA; 95.3% of thegroupB; 100%of the beta-hemolyticnon-group A,B,

orD; 92.3%ofgroup Denterococcal; 100% ofthe group D non-enterococcal; and 92.8% of the viridans streptococci were presumptivelyidentified. We then used the hemolytic reactions, susceptibility of bacitracin and sulfamethoxazole-plus-trimethoprim disks, CAMP disk reactions onsheep blood Trypticase soy agar and bile-esculin and 6.5% NaCI agar tolerance testswith incubation in normal atmosphere. Subsequently, 98.1% of thegroup A; 98.6% of the group B; 99.2% of the,Bhemolytic non-groupA,B,orD; 97.5% of thegroup Denterococcal; 97.6% of the group Dnon-enterococcal; and 92.4% of the viridans strainswere presump-tively identified.

Recentadvances in themethodologyfor pre-sumptively identifyinggroupB streptococci (2, 9, 15) have led us to believe that abattery of

testscould bedesignedtoimprove the

presump-tive identification ofgroupA,B,Denterococcal, D non-enterococcal, viridans, and beta-hemo-lyticnon-groupA,B,orDstreptococcal strains. Althoughwehavereportedon abatteryoftests used in presumptively identifying the above-mentioned categories ofstreptococci, the tests

were acombination ofbroths, agarslants, and

blood agar plates (7). In addition, one test re-quired48hof incubation(hippurate hydrolysis).

A more convenient method would incorporate

all thetestsintoone or twoagarplateprocedures andlimit the incubation periodto18to24h.

The most persistent source of error in our batteryoftestsresulted from the susceptibility ofbeta-hemolytic non-group A, B,or D strep-tococcitobacitracin.Approximately8% of these streptococciaresusceptibletobacitracin(7, 10). Because these strains are frequently mistaken

for group A streptococci, the procedures for

identifying these streptococci need to be im-proved.

This report includes an evaluation of three

differentagarformulasfor the 6.5%NaCl

toler-ancetest, acomparisonof resultsobtainedwith

bile-esculin (BE) and 6.5% NaCl testsin tubes

and plates, and an evaluation oftwo different batteries ofpresumptivetestsfor identifying the streptococci.

MATERIALS AND METHODS

Streptococcal strains. Ail the streptococcal

iso-latesusedin this study were from human specimens

submitted to theStreptococcusLaboratory,Center for

Disease Control, Atlanta, Ga., by federal, state, and

citypublichealth departments during the past2years.

Body sites from which they were obtained include

throat, skin, wounds, blood, andcerebrospinal fluid.

Identification procedures. All isolates were

se-rologically grouped and evaluated for hemolytic and

catalase activity according to previously described

methods (3). The group D streptococciwere

charac-terized by physiological tests described by Facklam

(3) and Grossetal.(8). The viridans streptococciwere

identified in physiological testsdescribed elsewhere

(5).

Media and reagents. Trypticase soyagar (BBL

Microbiology Systems) plates with 5%washed,

defi-brinatedsheepblood(SB-TSA)werepreparedinour

laboratory.Theseplateswere usedto determinethe

susceptibility to bacitracin (0.04 U; BBL) and SXT

(trimethoprim, 1.25 yg, plus sulfamethoxazole, 23.75

ug;BBL) disks. Theseplateswerealso usedtotestthe

streptococci for CAMP factorin thestandardCAMP

(2) and the CAMP disktests(15).

The CAMP disks were prepared as describedby

W;lkinson(15). Filter paper disksweresaturatedwith

concentratedfl-lysin,desiccatedfor24hinan

evacu-665

on February 7, 2020 by guest

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ation chamber at room

temperature,

and stored at

-20°C.On washed SB-TSAplates,CAMPdisks

pre-paredin thismannershowedzonesof,B-lysin activity

ranging from. 13 to 18 mm in diameter.Staphylococcus

aureusstrain SS-697wasused for the standard CAMP

test aswellasforpreparingthepartially purified

,B-lysin.

Methyl green deoxyribonuclease (DNase) agar

(MG-DTA;Difco)wasprepared accordingtothe

man-ufacturer'sinstructions.

Modified BE mediumwasprepared aspreviously

described (4),exceptthat the mediumwaspouredinto

15-by 100-mmpetridishes.

Modified heart infusion 6.5% NaCI agar(HIA-NaCI)

wasusedtotestfor NaCItoleranceinboth batteries

oftests.Thismedium contained thesameingredients

as previously described for modified heart infusion

broth (4), except that 1.5% agarwasadded.The agar

waspoured into15-by100-mmplates.

OtherNaCImedia describedby Qadrietal.(brain

heartinfusion-NaCI; 11) and Abshire (D

streptococ-cus-enterococcus agar; 1)werepreparedaccordingto

theauthors'instructions andpouredinto 15-by

100-mmpetridishes.

Testing.Theinocula foralltestsweretaken from

overnight fresh culturesonSB-TSA.Ail strainswere

coded to mask theiridentitywhen theyweretested.

Material obtained inasweepthroughthe pureculture

with an inoculating loop was used to inoculate the

washed SB-TSA plates. The inoculum was spread

evenlyoverone-halfof theplate,andonestreakwas

madeonthe other uninoculatedhalfof theplate.For

the standard CAMP test,astreak of the

,B-lysin-pro-ducingstaphylococcusstrainwasthenmade

perpen-dicular to the single streptococcus streak. The two

streakswerecarefully placed1 to2mmapart.For the

CAMP disk test, the diskcontaining,8-lysinwasplaced

1 to 2mmfrom theend of thesinglestreptococcus

streak. Bacitracin and SXT diskswereplaced

approx-imately 50 mm apart on the section of the plate

containingtheevenly spread inoculum.Anothersingle

sweep ofinoculum from the overnight culture was

ued firsttoinoculate one-quarter ofaBEplateand

then one-quarter ofanHIA-NaClplate.Theinoculum

wascarefullyspread evenly enoughnot tobevisible

ontheHIA-NaCl plateto ensurethat it didnotappear

asgrowthafter incubation.

The brain heart infusion-NaCl and D

streptococcus-enterococcusagarswereinoculated in thesame

man-nerastheHIA-NaClagar.

TheDNasemedium was inoculated withasweep

takenfromthefreshovernight culture.Asingle streak

approximately30 mmlongwasmadeonone-fourth of

a15-by100-mmpetri dish containing MG-DTA

me-dium. A stab was made into the agar along the streak. The agar plates containing the bacitracin, SXT,

standard CAMP, BE, HIA-NaCl, and DNase media

wereincubated in candle extinction jars at 35°C for

thisfirst battery of tests.

The second battery of tests, including bacitracin,

SXT,and CAMP disks, BE, HIA-NaCl,brain heart

infu8ion-NaCl, and D streptococcus-enterococcus

agar,wasincubated in a normalatmosphereat35°C.

All thetests wereread afterovernight incubation

(16 to 24h).Any zone of inhibition of growth around

the bacitracin and SXT disks was interpreted as a

positive reaction. CAMP reactions were considered

positive whenanarrowhead-shapedareaof increased

lysis developed atthejuncture of the streptococcal

andstaphylococcalstreak in the standard CAMP test

and when a crescent-shaped area of increased lysis

formed between the streptococcal streak and the ,B-lysin-containing disk in the CAMP disk test. CAMP

reactionswererankedasintermediate when areas of

increasedlysisdevelopedinshapes other than those

described above; these latter reactions were usually

considerably smaller than the normal CAMP

reac-tions. The BE reactionswereconsidered positive when

therewasanyblackeningof the BE medium.All NaCl

tolerance tests were considered positive when any

growthwasvisible,regardlessofwhether the indicator

changed color. The DNase reaction was interpreted as

positive whenaclearzonedeveloped around the streak

orstab growthofstreptococci in the MG-DTA

me-dium.

RESULTS

The reactions obtained with 710 strains of

streptococci in the first battery of tests are listed in Table 1. Plates containing the test

compo-nents were incubated in candle extinctionjars.

Almost all (98.9%) of the group Astreptococci

were susceptible to bacitracin and resistant to

SXT disks. None of the groupAstrainstested

reacted positively in CAMP or BE tests, and none tolerated NaCI media. Intermediate CAMP reactions interpreted as negative were recordedfor several group Astrains. Almostall

group Astrains(186of190) reactedpositively in

DNasetests onMG-DTA media.

Onlygroup B streptococcireacted positively

inCAMP tests,with 96.7% of215strains being

positive.BecausetheCAMPtestismorespecific thanbacitracin, SXT,orDNaseprocedures, re-sults obtained withtheformerwereinterpreted asmoreimportantinpresumptively identifying

the group Bstreptococci. None of thegroup B

strains tested gave a positive BE reaction or

grew on6.5%NaClagar.

Most(89.9%) beta-hemolyticnon-group A,B, orDstrains testedwereresistanttobacitracin and susceptible to SXT, and the others were either susceptible (5.6%) orresistant (4.4%) to both bacitracin and SXT disks. All three

pat-terns ofreactionswereconsideredpresumptive

indicatorsofbeta-hemolytic non-group A,B,or

D streptococci because these patterns of

reac-tions were more frequently observed with the beta-hemolytic non-group A, B, or D strains

thantheywerewith group A or B strains. The

beta-hemolytic non-group A, B, or D strains

reactedvariablyonMG-DTA media; 64.2% were

DNasepositive. Only two of these strains were bacitracin susceptible and DNase positive, whereas the rest reacted negatively in one or

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TABLE 1. Reaction patternsof

streptococci

onseven

presumptive

testsincubated in anincreased

C02

atmosphere

Test reactiona

Streptococci No. Presumptive

interpreta-HEM BAC SXT CAMP _DTAMG- BE NaCI tion

A ,-+ + - - 184 GroupA

,B + - - - 4 Group A

Bi + + - + - - 1 ,B-not A,B,D'

,B - - - + - - 1 fl-notA,B, D

B fi - - + + - - 110 Group B

Bi - - + - - - 92 Group B

f, + - + + - - 4 Group B

fi - + + + - - 1 Group B

f - - - 7 fl-notA,B,D

C/G/F ,B - + - + - - 97 f8-notA,B,D

fi

- + - - - - 46 ,B-notA,B, D

,B - - - + - - 3 ,B-not A,B, D

f, + + - - - - 7 ,B-not A,B,D

,B + + - + - - 2

fi-notA,

B, D

,B - - - 4 ,B-notA,B, D

Denterococcus a/fl/y - - - - + + 26 D enterococcus

a/fl/y

- + - + + + 8 Denterococcus

a/fl/y - - - + 2 D enterococcus

a/y - + - - + - 1 D non-enterococcus

a/y - + - - + - 2 Dnon-enterococcus

D non-entero- a/y - - - - + - 9 Dnon-enterococcus

coccus a/y - + - - + - 1 Dnon-enterococcus

Viridans a/y v + - - - - 81 Viridans

a/y v - - - 9 Viridans

a/y v + - - + - 7 Dnon-enterococcus

a +

Susceptible

tobacitracin orSXTdisks, positive in CAMPorBE test,and grows on

NaCi

media; -,

resistant to bacitracin or SXTdisks, negativein CAMP orBE test, and does not grow on NaCi media; v,

variablereactions,withsomestrainspositive and some strains negative. HEM,Hemolysis;BAC,bacitracin.

b

fi-not

A,B, D is

beta-hemolytic

non-group

A,B,andD.

bothtests. However, fewer

bacitracin-suscepti-ble strains ofbeta-hemolyticnon-groupA,B,or D streptococci than are usually encountered werefound. Iflargernumbers of bacitracin-sus-ceptiblegroupC andG streptococciwerefound inclinicalspecimens, the usefulness of the baci-tracintestwould bemorequestionable.

All of the enterococcal strains listedinTable 1 reacted positivelyin BE andNaCi tolerance

testswhen tested in tubes, but two strainsdid

not react positivelyin BE tests when testedin

plateBEmedium,andthree did not react posi-tively in NaCI tolerance tests when tested in plate NaCI medium. Even though the entero-cocci reactedvariouslytoSXTdisks, interpret-ing the SXT reactioninconjunctionwith results of theNaCI tolerancetestledto a more accurate identification. When positive BE and NaCl

re-actionsornegative(resistant) SXTand

positive

NaCl reactions were obtained, the organisms

were presumptively identified as enterococci. Usingthesecriteria,36of39(92.3%) enterococci werepresumptivelyidentified. The fact that nei-ther criterion was adequate to identify these strains of enterococcipromptedustoinvestigate other NaClagarformulas. None of the entero-coccal strains weresusceptible to bacitracinor

reactedpositivelyinCAMPtests.

Only 10strains ofnon-enterococcal group D streptococci weretested in the batteryoftests listed in Table 1. Ail reacted positively in BE and negatively in NaCl tolerance tests. One

strain was susceptible to SXT and none was

susceptibletobacitracinorreactedpositivelyin

CAMPtests.

Ninety of 97 (92.8%) of the viridans strains were presumptively identified on the basis of negative BEandNaCl tolerancetestreactions. The othersevenstrains(7.2%)wereBEpositive and NaClnegativeandwerepresumptively

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668 FACKLAM ET AL.

tified as group D non-enterococci. Nine (9.3%)

of the viridans strains were resistant to SXT.

SXTsusceptibilitywas notusedas adifferential

test for group D streptococci because of the

small number of these isolates tested. None of

the viridansstrains reactedpositivelyinCAMP

or DNase tests. Overall, 97.3% of the strains

tested inthis batteryoftests werepresumptively

identified correctly.

The resultsof testing three different mediato

be used in NaCI tolerance tests are shown in Table2.NaCItolerancewasstronglyconsidered indifferentiatingthe non-beta-hemolytic strep-tococciandwasthemostimportanttestin dif-ferentiating BE-positivestrains. Sinceall beta-hemolyticgroupA,B,and non-group A,B,orD streptococcal isolatesdidnot reactpositivelyin

BE tests, their growth on NaCl agar was not

usedas adifferentialcriterion. None of the group D non-enterococcal orviridans strains grew on

brain heartinfusion-NaCIandHIA-NaCI agars,

whereas67and85%of the enterococcalstrains,

respectively, grew on these two media. All but one(97%) ofthe group D non-enterococcaland 43% of the viridans strains grew on D

strepto-coccus-enterococcus agar (Table 2). Overall,

among the three media evaluated, HIA-NaCl

agar most clearly differentiated the

non-beta-hemolyticstreptococci.

The reactions of 765 strains of streptococci evaluated in the second battery of tests are

shown in Table 3. None of these strains was

includedin the firstbatteryoftests.Whereas in

the firstbattery oftests (Table 1) sampleswere

incubated in an increased C02 atmosphere, in

the second battery samples were incubated in

normalatmosphere.The reactions of the group

Astreptococcitobacitracinand SXT diskswere similar to those of the group A strains in the

first battery of tests; i.e., 98.7% of the strains

were susceptible to bacitracin and resistant to

SXT disks.One group A strain reactedpositively

inthe CAMP disk test and was presumptively

identifiedas agroupB streptococcus.Onegroup

A strain wassusceptible and one wasresistant

to both bacitracin and SXT disks. They were

presumptively identified as non-group A, B, or

Dbeta-hemolyticstreptococci,because 24.8 and

6.8% of thebeta-hemolytic non-group A,B, orD

streptococci were susceptible and resistant, re-spectively,toboth disks.

Among thegroup Bstreptococci 98.6% of the

strains reacted positively in CAMP disk tests.

These results are a slight improvement over

those with the standard CAMP test (97.2% of

thegroup Bstrains positive) only because

inter-mediate CAMP reactions were interpreted as

positive if the strain was resistant to bacitracin

TABLE 2. Evaluationofthreeformulas forNaCI tolerance

No. of % Positive Streptococci strains BHI- HIA- DSE

tested NaCl' NaCI agar'

GroupDenterococci 58 67 85 100

Group D non-entero- 29 0 0 97

cocci

Viridans 159 0 0 43

<BHI-NaCl, Brain heart infusion-NaCI.

bDSE,Dstreptococcus-enterococcus.

and SXT disks, and the hemolysis of these

strains was typicalof group Bstreptococci.

AI-though two group B strains were resistant to both SXT andbacitracin andwere CAMPdisk negative, these strains were identified as beta-hemolytic non-group A, B, or D streptococci,

because 9 of 133 (6.8%) of the beta-hemolytic

non-groupA,B, or DstreptococciwereCAMP

disknegativeandresistanttoSXTand bacitra-cin.

Most (68%) of the beta-hemolytic non-group A,B,orDstreptococciwereresistant to bacitra-cin, susceptible to SXT, CAMP disk negative, andBEnegative.However,25%of thesestrains weresusceptibletobacitracinandSXTandwere CAMP disknegative andBEnegative. Also,the

most commonstrainsthatwereresistantto

bac-itracinandSXT, and which reacted negatively inCAMP diskand BEreactionswere beta-he-molyticstreptococci,non-groupA,B,orD,(6.5% of thesestrains).

Oftheenterococcal strains tested, 94%reacted positivelyasexpectedin BEandNaCltolerance

tests.Thefourenterococcalstrainsthatdid not

reactpositivelyin BE tests werepresumptively

identified as enterococci because they were

re-sistant toSXT and grew in the NaCltolerance

test. This interpretation was possible because

onlyoneviridans strain grew in theNaCl

toler-ancetest.Threeenterococcalstrains thatfailed

to grow in the NaCl tolerance test were pre-sumptively,buterroneously, identified as group

D non-enterococcal streptococci. Eighty-one

percentof theenterococci were resistant to SXT

disks, whereas 84%of the viridans streptococci weresusceptible.

Forty-one of 42 group D non-enterococcal

strains were presumptively identified correctly

intheBE (positive) andNaCI tolerance

(nega-tive) tests.All of the group D non-enterococcal

strains wereStreptococcus bovis. The SXT

pro-cedurewasofnovalue indifferentiatingS. bovis

from otherstreptococci; i.e.,23strains were

sus-ceptible and 19 strains were resistant to SXT

disks. One S. bovis strain was presumptively

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TABLE 3. Reactionpatternsof streptococcionsixpresumptivetestsincubated in normalatmosphere

Identification No. Presumptive

interpreta-HEM BAC SXT CAMP BE NaCI

A f8 + - - - - 155 Group A

fi + + - - 1 Group B

fi + + - - - 1 ,B notA,B,Db

Jf - - - 1 ,B-notA,B,D

B fi - - + - - 131 Group B

fi + - + - - 7 GroupB

fi - + + - - 1 GroupB

fi - - - 2 ,B-notA,B,D

C/G/F fi - + - - - 90 ,B-notA,B,D

fiR

+ + - - - 33 f8-notA,B,D

fi - - - 9 fi-notA,B,D

fi + - - - - 1 GroupA

D enterococci a/fi/y - - - + + 91 D enterococcus

a/fi/y - + - + + 22 Denterococcus

a/fi/y

- - - - + 4 Denterococcus

a/fi/y - - - + - 2 D non-enterococcus

a/fi/y - + - + - 1 Dnon-enterococcus

Dnon-enterococci a/y - + - + - 22 Dnon-enterococcus

a/y - - - + - 18 D non-enterococcus

a/y + + - + - 1 D non-enterococcus

a/y + - _ _ - 1 Viridans

Viridans a/y v + - - - 137 Viridans

a/y v - - - - 21 Viridans

a/y v - - + - 6 Dnon-enterococcus

a/y v + - + - 6 Dnon-enterococcus

a/y + - - - + 1 D enterococcus

See footnotea,Table1.

bSee footnote

b,

Table 1.

identifiedas aviridansstreptococcusbecause it

didnot reactpositivelyinthe BEtest.

Among the viridans streptococci evaluated

with the tests listed in Table 3, 92.4% were

presumptively identified correctly by negative reactions in BE and NaCI tolerance tests. Twelve strains were incorrectly identified as

group D non-enterococci because theyreacted

positively in BE tests. One strain (Streptococcus uberis) wasincorrectly identifiedas an

entero-coccus because it grew in the NaCI tolerance

test andwasresistant to anSXT disk. Sixteen

percentof theviridans strainswereresistantto

SXT disks. The reactionto SXTdisksby viri-dansstreptococci washelpful but inconclusive indifferentiatingtheorganisms. Overall,97% of the streptococci were correctly categorized by theirreactionstothetestslisted in Table 3.

Table4shows thepercentage ofpositive BE andNaCI reactionsontubed (2-day) andplate (overnight) media. There wasvery little differ-encebetween BE reactionsofthe group D en-terococcal and those of non-enen-terococcal strains

onBE plates and in BE tubes. Also therewas very little difference between the NaCl broth and NaCl plate reactions ofanyof thestrains.

Thegreatestdifference in results obtained with

the tube andplate media appeared in the BE reactions of the viridansstreptococci. Substan-tiallyfewerviridans strains reacted positively in

BE tests on BE plates than in those on BE

tubes.

Sinceitwas necessary tointerpret the inter-mediate CAMP reactionsasnegativeindicators

of group B streptococci, intermediate CAMP

reactionsof the streptococci obtained with both the standard and disk methodswere evaluated (Table 5). The percentage of group A strepto-cocci which reactedintermediatelyin bothtypes of CAMPtests washigh enoughtointroducea largeerrorinto theresultsof thebatteryoftests if theintermediate reactionswereinterpretedas positive. However, the interpretation of inter-mediateCAMP reactions didnot causeany dif-ficulty because thehemolysisof thegroupA,C, and G streptococci on the SB-TSA was much

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670 FACKLAM

TABLE 4. Percentagesofpositive BE and

6.5%

NaCI tests determined in tubes and plates %Positive Streptococci _testedNo. Tube Plates

BE NaCI BE NaCI

Denterococci 120 100 100 96.6 97.5

D non-entero- 42 100 97.6

cocci

Viridans 169 20.7 2.3 7.1 0.6

TABLE 5. Intermediate CAMP reactions

No.showingintermediate reaction/ total tested (%) Streptococci

Standard CAMP DiskCAMP test test

Group A 48/190 (25.3) 31/158(19.6)

Group B 7/215 (3.3) 5/141 (3.5)

Beta-hemolytic, 3/159 (1.9) 7/133 (5.3)

notA,B,D

Denterococci 0/39 0/120

D non-entero- 0/10 0/42

cocci

Viridans 0/97 0/171

moreintensethan thehemolysisof thegroup B streptococci.Theuniquelycharacteristic hemol-ysis of the group B streptococci, used in con-junction with intermediate CAMP reactions, wasinterpreted torepresent positive presump-tive identification ofgroupBstreptococci.

DISCUSSION

Theuseofmultipletests toimprovethe pre-sumptive identification ofstreptococci isnot a newidea. Wallerstrom (14) describeda multiple-test scheme for presumptive identification of

group Astreptococci.Heplaced disks containing

bacitracin, nucleate, and glucose on lawns of beta-hemolytic streptococci on blood agar plates. All the group A strains tested reacted positively in the "triple test"; i.e.,group Astrains wereinhibitedby bacitracin, the hemolysiswas stimulated by nucleate, and the hemolysis was inhibited byglucose disks. However, onlyafew

non-group Astreptococci were tested, and one

of 12 group G strainsreacted positively in the

tripletest.Although therewas someindication

thatgroup B streptococci could bedifferentiated

by the tripletest, only two strains weretested. We previously described a battery of tests withwhichto identify sixcategories of

strepto-cocci presumptively: group A, groupB,group D

enterococcal, group D non-enterococcal, viri-dans, and beta-hemolyticnon-group A,B, or D streptococci(7). Bloodagarplates were used in determininghemolysis and bacitracin

suscepti-bility. The hippurate hydrolysis and BE and NaCl tolerance tests were performed in tubes containing thepropermedia.

Twodaysof incubationwererequiredbefore 95%of thestreptococci could be accurately dif-ferentiated into the sixcategories. The largest

errorin thispresumptive identificationwasthe

misidentification of beta-hemolytic non-group A,B,orDstreptococciasgroup Astreptococci. About 19% of these strains were misidentified becausetheyweresusceptibletobacitracin. Pol-lock andDahlgren (10) warned that ifbacitracin wasusedasthemajor criterion for presumptive identificationofgroupAstreptococci, the poten-tial for misidentification was significant, espe-cially if the specimenwastaken from theupper respiratorytract orfromawound. Thus, it

ap-pears pertinent that criteria for presumptive

identification of thebeta-hemolytic streptococci from thesesourcesbeimproved.

Darling (2) describeda series of experiments inwhich heattemptedtostandardize the CAMP test for presumptive identification ofgroup B streptococci.Hefound that thetestworked best if thestreptococciwereinoculatedonto

Trypti-casesoy agarcontainingwashedsheep

erythro-cytes.Thetestmaterialwasincubatedovernight

inacandleextinctionjarorinanormal atmos-phere. Under these

conditions

the standard CAMP reactions could be determined andgroup Bstreptococcicould bepresumptively identified after overnight incubation, as compared to 2 days of incubation required for the hippurate hydrolysistest.Wilkinson modified the standard CAMP test byincorporating the,B-lysin of the

S.aureusinto filterpaper

disks,

thus

eliminating

theneedforanactively growingculture for the CAMP reaction (15). BothDarlingand Wilkin-son reported a 100% accurate identification of

groupBstreptococciby their CAMPtestsafter

18hof incubation.

Recently, Gunn reported on theuse ofSXT and bacitracin disks forpresumptive identifica-tion of beta-hemolytic group A, group B, and

non-group A orBstreptococci (9). Hefoundthat

whenthe disks were placedon an inoculumof streptococci on SB-TSA, group A streptococci were susceptible to bacitracin and resistant to SXTdisks,group Bstreptococciwereresistant toboth disks, and beta-hemolytic group C, G, and Fstreptococci were resistanttobacitracin but susceptible to SXT disks. Thesereactions were determined afterovernight incubation in an atmosphere of 5% C02. Several strains of

groupC, F, and G streptococci andone strainof

group A streptococci were susceptible to both

disks. Gunnsuggestedthatstrainssusceptibleto bothdisks should be interpretedaspresumptive

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IDENTIFICATION OF STREPTOCOCCI

group Astreptococci to prevent any misidenti-fication of group Astreptococci. However, from the results of these studies it is apparent that this pattern of reactions is more indicative of beta-hemolytic streptococci, notgroupA, B,or D, than group A streptococci. Gunn also sug-gested thatbeta-hemolyticstreptococciresistant to both bacitracin and SXT disks should be presumptively identified as group B strepto-cocci.However,combining the results from Ta-bles 1 and 3 indicates that among the beta-hemolyticstreptococci, 94.6% of the group B and 2.6%of the non-group B strains were resistant to both disks. However, 97.4% of the group B and lessthan 0.5% of the non-group B strains gave positive CAMP tests. Therefore, the CAMP reaction ismorespecific thanthe com-binedinterpretationof thetwodisks.

It seemed reasonable that if the bacitracin,

SXT, and CAMP tests could be performed on

oneplate,thepresumptive identification would notonlybemoreconvenient butalso more ac-curate. Itwould also be moreconvenient if the BEand 6.5%NaCImedia couldbeincorporated into plates and the incubation time could be shortened.

In the first battery of tests, samples were incubated incandle extinctionjarsat35°C

be-causebothDarling(2) and Gunn (9) suggested

that an atmosphere containing increased C02 wasbeneficialinboth the standard CAMP and

SXT disk tests. Inthe secondbattery oftests,

sampleswere incubated in normal atmosphere at35°Cbecause it may bemoreconvenient for somelaboratories.

Both batteries oftests were read after over-night incubation (16 to 20 h). Although some viridans strains didnotgrow aswell in normal atmospheres, this condition did not interfere with the presumptive identification. The total numberof correctpresumptive identifications in thetwo atmosphereswascomparable:97.3% in candle extinction jars and 97.0% in normal at-mosphere. We suggest thatmicrobiologists use thebacitracin,SXT, CAMP, BE,and NaCl

tol-erance testsalongwith thehemolyticreactions

to make the same presumptive identifications described in our previous publication (7). The batteryoftestsshould beinterpretedas a

whole;

i.e., results obtained in individual tests should beevaluatedasthey relatetothose obtainedin

all tests run. Patterns of reactions will be

com-parabletothose describedinTables1and3.We suggest that the streptococci canbe presump-tively identifiedbythefollowingcriteria.

(i) GroupAstreptococciarebeta-hemolytic, susceptible to bacitracin, resistant to SXT, CAMP reactionnegative,and BE

negative.

(ii) Group B streptococci are beta-hemoltyic, variably susceptible to bacitracin and SXT (al-though most strains are resistant to both disks), CAMP reaction positive, and BE negative. Oc-casional nonhemolytic group B strainswillhave comparable reaction patterns.

(iii) Group C, F, and G streptococci (beta-hemolytic, not group A,

B,

or D) wiil react in three patterns to bacitracin and SXT.Allstrains are beta-hemolytic and react negatively in CAMPand BE tests. Most strains are resistant tobacitracin andsusceptible to SXT. An appre-ciable number of these strains may be suscepti-ble to both bacitracin and SXT. A few strains willbe resistant toboth bacitracin and SXT.

(iv) Group D enterococcal streptococci vary in their hemolytic reaction; most strains are nonhemolytic, but alpha- and beta-hemolytic strains are common. All strains are resistant to bacitracin and reactnegatively in CAMP tests. Three patterns ofreactions from the SXT, BE, and NaCl tests arecharacteristicof enterococci. Most strains wiil react positively in BE and NaCl tolerance tests and be resistant to SXT. Some strains are susceptible to SXT and react positively in BE and NaCl tests. Some strains areresistanttoSXT and tolerant toNaCl (NaCl positive), but fail to give a positive BEreaction. (v) Group D non-enterococcal streptococci arealpha- or nonhemolytic and areusually re-sistant tobacitracin. However, occasional strains are sensitive, vary in their reactions to SXT disks,and are BEpositive andNaCl intolerant. (vi) Viridans streptococci are alpha- or non-hemolytic and vary in their reaction to bacitra-cin and SXT.However,moststrains are suscep-tible in SXT and negative in CAMP, BE, and NaCl tolerance tests.

The results of thesestudies, using the above interpretation,indicate that atleast 97% of the medically important streptococci can be pre-sumptively identified. Streptococcithat do not fit into one of the reaction patterns should be checked forpurity and retested. The retesting canbeperformed onplates orin tubes as pre-viouslydescribed(7). Strains whose reactions in thepresumptivetestsremainatypicalshould be serologically groupedanddefinitivelyidentified inareferencelaboratory.

We suggest that the bacitracin, SXT, and

CAMPdisks be stored at-20°C. (Placingthem

inthe freezerof aregular

refrigerator

is satisfac-tory.) Wehad some

difficulty

maintaining the activities of SXT and CAMP disks whenthey werestoredat4°C foreven asshortaperiodas 5days,althoughtheyremainstable for1month (withdailyuse)when storedat-20°C.

Accurate determination of hemolysis is the

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(8)

firstandmostusefulstep inidentifying

strepto-cocci. Bacteriologists should familiarize

them-selves with typical hemolytic activities of the

streptococci.Althoughthehemolysisofgroup A

streptococci cannot be routinely differentiated

from that of group C or G streptococci, the

former differs from that of group B, D, and F streptococci. Thezoneofhemolysissurrounding group B streptococci is much smaller and the zone ofhemolysissurroundingthe group D en-terococcal streptococci is larger than that of group A. Also,colonies of groupDstreptococci aremuchlarger than those of other streptococci, and colonies ofgroup F streptococci are much smaller than those ofbeta-hemolytic group A, B,C, D, and Gstreptococci. Thezoneof hemol-ysis surrounding these small colonies is quite large, nearly equivalent to the zones formed

aroundgroupAstreptococci.

We preferred toread the zones of inhibition

of growth around the SXT disks in the same

manner as wedescribed forbacitracin (7). Any

zoneofinhibitionofgrowth aroundthe

bacitra-cin orSXTdisksrepresentsapositiveresult.To

require certain zonesizes ofgrowthinhibition,a standard inoculum must be used. However, in

tests toobtain apresumptiveidentification,

re-quiring standardized inoculum would add an unnecessary element ofcomplication.

TheCAMPtestshouldbe read inconjunction with thehemolytic activityofthestreptococci. Beta-hemolytic non-group B streptococci that

react intermediately in CAMP tests are easily

differentiated fromgroup Bstreptococciwhich

reactweakly or intermediately in CAMP tests

because of thedifferencesinhemolytic activity describedabove. MostgroupBstreptococci are clearly positiveinCAMPtests.

TheBEplatetest is moredifficulttointerpret than the tube test. Some enterococcal strains

react very weakly; i.e., the blackening of the

mediummay not be apparent unless theplateis

inspected in front of a white background. We advisereadingthe test in this manner and inter-preting anyblackeningofthe mediumasa

pos-itive reaction, even though doingso may result

in a number of misidentifications of viridans

streptococci asgroupDnon-enterococcal

strep-tococcibecauseofweak BEreactions.Thiserror

ismedically lessdangerous than misidentifying enterococci because antibioticsusceptibilitiesof thegroupDnon-enterococciaresimilartothose oftheviridansstreptococci (13).

Onereasonthatanoccasional groupD

strep-tococcus was negative inthe BE plate test but

positive intheBEtubetestwastheincubation

period. The plateswereincubatedfor 18 to 20h,

whereasthetubes wereincubatedfor 40 to 42 h.

If the agar plate BE test had been incubated

longer,

thereactionsmighthavebeenmore

com-parabletothose obtainedwith tubed media.

The results with the 6.5%NaCl tolerancetests

were themost

disappointing

ofthose obtained. Westronglyrecommend thatplatesbecarefully inspected immediately after inoculation to

in-surethat theinoculumisspreadso asnot tobe

readasgrowth.Whentheplatesarepreparedin thismanner, anygrowth interpretedas a

positive

reactionreflectsanaccurateindicatorNaCl

tol-erance.

The CAMP disksare notavailable

commer-cially

at present (November 1978). It is

hoped

that in thefuturetheywill bemadeavailableor

that the media and diskswill be commerically

available as a kit.

LMRATURE CITED

1. Abshire, R. L. 1977. Evaluation ofanewpresumptive medium forgroupDstreptococci. Appl. Environ. Mi-crobiol.33:1149-1155.

2. Darling, C.L. 1975.Standardization andevaluationof theCAMP reactionfor the prompt, presumptive iden-tification ofStreptococcusagalactiae (Lancefieldgroup B)inclinical material. J. Clin.Microbiol. 1:171-174. 3. Facklam,R. R. 1972.Recognitionof groupD

streptococ-calspeciesofhumanorigin bybiochemicaland

physi-ologicaltests.Apple.Microbiol.23:1131-1139. 4. Facklam,R. R.1973.Comparison of severallaboratory

mediaforpresumptive identificationof enterococciand groupDstreptococci.Apple.Microbiol.26:138-145. 5. Facklam, R. R. 1977. Physiological differentiation of

viridansstreptococci.J.Clin.Microbiol. 5:184-201. 6. Facklam,R.R.,andM. D.Moody. 1970.Presumptive

identificationof groupDstreptococci: thebile-esculin

test.Apple.Microbiol. 20:245-250.

7. Facklam, R. R.,J. F. Padula,L. G.Thacker, E. C. Wortham, and B. J. Sconyers. 1974.Presumptive identificationof groupA,B,and D streptococci.Appl. Microbiol. 27:107-113.

8. Gross, K. C., M. P. Houghton, and L. B. Senterfit. 1975. Presumptive speciation of Streptococcus bovis and othergroup D streptococci from human sourcesby using arginineandpyruvatetests.J. Clin.Microbiol.1: 54-60.

9. Gunn, B. A. 1976. SXT and Taxo A disks for presumptive identification ofgroupAandBstreptococci inthroat cultures.J. Clin.Microbiol. 4:192-193.

10.Pollock, H. M., and B.J.Dahlgren. 1974. Distribution ofstreptococcal groups in clinical specimens with eval-uationofbacitracinscreening. Appl. Microbiol. 27:141-143.

11. Qadri, S. M. H., C. W. Nichols, and S.G. M.Qadri. 1978.Rapid sodiumchloridetolerancetest for presump-tiveidentificationofenterococci.J.Clin.Microbiol.7: 238.

12. Smith, P. B., G.A.Hancock,and D.L.Rhoden. 1969. Improved mediumfordetecting deoxyribonuclease-pro-ducing bacteria.Appl.Microbiol.18:991-993. 13. Thornsberry, C., C. N. Baker, and R. R. Facklam.

1974. Antibiotic susceptibility of Streptococcus bovis andother group Dstreptococci causing endocarditis. Antimicrob.AgentsChemother. 5:228-233.

14. Wallerstrom,A. 1962. Asimplebiochemical "tripletest" forpreliminaryidentificationofgroup Astreptococci. ActaPathol. Microbiol. Scand.56:459-464.

15. Wilkinson, H.W. 1977.CAMP-disktest for presumptive identificationofgroup Bstreptococci.J.Clin.Microbiol. 6:42-45.

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