JOURNAL OF CLINICAL MICROBIOLOGY, June 1979, p. 665-6720095-1137/79/06-0665/08$02.00/0

Vol. 9, No. 6

Presumptive Identification of Group A, B, and D Streptococcion Agar Plate Media

R. R. FACKLAM,* J. F. PADULA, E. C. WORTHAM, R. C. COOKSEY, AND H. A. ROUNTREEBureau of Laboratories, Bacteriology Division, Center for Disease Control, Atlanta, Georgia 30333

Received for publication 19 March 1979

Several presumptive tests were evaluated for their effectiveness in differentiat-ing streptococci. When the tests were combined into a battery and the resultingreactions were interpreted as patterns, the overall presumptive identification ratewas at least 97%. We used the hemolytic reaction, susceptibility to bacitracin andsulfamethoxazole plus trimethoprim (1.25 ug plus 23.75 ,ug), and standard CAMPreactions on sheep blood Trypticase soy agar, and bile-esculin and 6.5% NaCIagar tolerance tests with incubation in candle extinction jars. Subsequently, 98.9%of the group A; 95.3% of the group B; 100% of the beta-hemolytic non-group A, B,or D; 92.3% of group D enterococcal; 100% of the group D non-enterococcal; and92.8% of the viridans streptococci were presumptively identified. We then usedthe hemolytic reactions, susceptibility of bacitracin and sulfamethoxazole-plus-trimethoprim disks, CAMP disk reactions on sheep blood Trypticase soy agarand bile-esculin and 6.5% NaCI agar tolerance tests with incubation in normalatmosphere. Subsequently, 98.1% of the group A; 98.6% of the group B; 99.2% ofthe ,B hemolytic non-group A, B, or D; 97.5% of the group D enterococcal; 97.6%of the group D non-enterococcal; and 92.4% of the viridans strains were presump-tively identified.

Recent advances in the methodology for pre-sumptively identifying group B streptococci (2,9, 15) have led us to believe that a battery oftests could be designed to improve the presump-tive identification of group A, B, D enterococcal,D non-enterococcal, viridans, and beta-hemo-lytic non-group A, B, or D streptococcal strains.Although we have reported on a battery of testsused in presumptively identifying the above-mentioned categories of streptococci, the testswere a combination of broths, agar slants, andblood agar plates (7). In addition, one test re-quired 48 h of incubation (hippurate hydrolysis).A more convenient method would incorporateall the tests into one or two agar plate proceduresand limit the incubation period to 18 to 24 h.The most persistent source of error in our

battery of tests resulted from the susceptibilityof beta-hemolytic non-group A, B, or D strep-tococci to bacitracin. Approximately 8% of thesestreptococci are susceptible to bacitracin (7, 10).Because these strains are frequently mistakenfor group A streptococci, the procedures foridentifying these streptococci need to be im-proved.This report includes an evaluation of three

different agar formulas for the 6.5% NaCl toler-ance test, a comparison of results obtained withbile-esculin (BE) and 6.5% NaCl tests in tubes

and plates, and an evaluation of two differentbatteries of presumptive tests for identifying thestreptococci.

MATERIALS AND METHODSStreptococcal strains. Ail the streptococcal iso-

lates used in this study were from human specimenssubmitted to the Streptococcus Laboratory, Center forDisease Control, Atlanta, Ga., by federal, state, andcity public health departments during the past 2 years.Body sites from which they were obtained includethroat, skin, wounds, blood, and cerebrospinal fluid.

Identification procedures. All isolates were se-rologically grouped and evaluated for hemolytic andcatalase activity according to previously describedmethods (3). The group D streptococci were charac-terized by physiological tests described by Facklam(3) and Gross et al. (8). The viridans streptococci wereidentified in physiological tests described elsewhere(5).Media and reagents. Trypticase soy agar (BBL

Microbiology Systems) plates with 5% washed, defi-brinated sheep blood (SB-TSA) were prepared in ourlaboratory. These plates were used to determine thesusceptibility to bacitracin (0.04 U; BBL) and SXT(trimethoprim, 1.25 yg, plus sulfamethoxazole, 23.75ug; BBL) disks. These plates were also used to test thestreptococci for CAMP factor in the standard CAMP(2) and the CAMP disk tests (15).The CAMP disks were prepared as described by

W;lkinson (15). Filter paper disks were saturated withconcentrated fl-lysin, desiccated for 24 h in an evacu-

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ation chamber at room temperature, and stored at-20°C. On washed SB-TSA plates, CAMP disks pre-pared in this manner showed zones of ,B-lysin activityranging from. 13 to 18mm in diameter. Staphylococcusaureus strain SS-697 was used for the standard CAMPtest as well as for preparing the partially purified ,B-lysin.

Methyl green deoxyribonuclease (DNase) agar(MG-DTA; Difco) was prepared according to the man-ufacturer's instructions.

Modified BE medium was prepared as previouslydescribed (4), except that the medium was poured into15- by 100-mm petri dishes.

Modified heart infusion 6.5% NaCI agar (HIA-NaCI)was used to test for NaCI tolerance in both batteriesof tests. This medium contained the same ingredientsas previously described for modified heart infusionbroth (4), except that 1.5% agar was added. The agarwas poured into 15- by 100-mm plates.

Other NaCI media described by Qadri et al. (brainheart infusion-NaCI; 11) and Abshire (D streptococ-cus-enterococcus agar; 1) were prepared according tothe authors' instructions and poured into 15- by 100-mm petri dishes.

Testing. The inocula for all tests were taken fromovernight fresh cultures on SB-TSA. Ail strains werecoded to mask their identity when they were tested.Material obtained in a sweep through the pure culturewith an inoculating loop was used to inoculate thewashed SB-TSA plates. The inoculum was spreadevenly over one-half of the plate, and one streak wasmade on the other uninoculated half of the plate. Forthe standard CAMP test, a streak of the ,B-lysin-pro-ducing staphylococcus strain was then made perpen-dicular to the single streptococcus streak. The twostreaks were carefully placed 1 to 2 mm apart. For theCAMP disk test, the disk containing,8-lysin was placed1 to 2 mm from the end of the single streptococcusstreak. Bacitracin and SXT disks were placed approx-imately 50 mm apart on the section of the platecontaining the evenly spread inoculum. Another singlesweep of inoculum from the overnight culture wasued first to inoculate one-quarter of a BE plate andthen one-quarter of an HIA-NaCl plate. The inoculumwas carefully spread evenly enough not to be visibleon the HIA-NaCl plate to ensure that it did not appearas growth after incubation.The brain heart infusion-NaCl and D streptococcus-

enterococcus agars were inoculated in the same man-ner as the HIA-NaCl agar.The DNase medium was inoculated with a sweep

taken from the fresh overnight culture. A single streakapproximately 30 mm long was made on one-fourth ofa 15- by 100-mm petri 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 mediawere incubated in candle extinction jars at 35°C forthis first battery of tests.The second battery of tests, including bacitracin,

SXT, and CAMP disks, BE, HIA-NaCl, brain heartinfu8ion-NaCl, and D streptococcus-enterococcusagar, was incubated in a normal atmosphere at 35°C.

All the tests were read after overnight incubation(16 to 24 h). Any zone of inhibition of growth around

the bacitracin and SXT disks was interpreted as apositive reaction. CAMP reactions were consideredpositive when an arrowhead-shaped area of increasedlysis developed at the juncture of the streptococcaland staphylococcal streak in the standard CAMP testand when a crescent-shaped area of increased lysisformed between the streptococcal streak and the ,B-lysin-containing disk in the CAMP disk test. CAMPreactions were ranked as intermediate when areas ofincreased lysis developed in shapes other than thosedescribed above; these latter reactions were usuallyconsiderably smaller than the normal CAMP reac-tions. The BE reactions were considered positive whenthere was any blackening of the BE medium. All NaCltolerance tests were considered positive when anygrowth was visible, regardless of whether the indicatorchanged color. The DNase reaction was interpreted aspositive when a clear zone developed around the streakor stab growth of streptococci in the MG-DTA me-dium.

RESULTS

The reactions obtained with 710 strains ofstreptococci in the first battery of tests are listedin Table 1. Plates containing the test compo-nents were incubated in candle extinction jars.Almost all (98.9%) of the group A streptococciwere susceptible to bacitracin and resistant toSXT disks. None of the group A strains testedreacted positively in CAMP or BE tests, andnone tolerated NaCI media. IntermediateCAMP reactions interpreted as negative wererecorded for several group A strains. Almost allgroup A strains (186 of 190) reacted positively inDNase tests on MG-DTA media.Only group B streptococci reacted positively

in CAMP tests, with 96.7% of 215 strains beingpositive. Because the CAMP test is more specificthan bacitracin, SXT, or DNase procedures, re-sults obtained with the former were interpretedas more important in presumptively identifyingthe group B streptococci. None of the group Bstrains tested gave a positive BE reaction orgrew on 6.5% NaCl agar.Most (89.9%) beta-hemolytic non-group A, B,

or D strains tested were resistant to bacitracinand susceptible to SXT, and the others wereeither susceptible (5.6%) or resistant (4.4%) toboth bacitracin and SXT disks. All three pat-terns of reactions were considered presumptiveindicators of beta-hemolytic non-group A, B, orD streptococci because these patterns of reac-tions were more frequently observed with thebeta-hemolytic non-group A, B, or D strainsthan they were with group A or B strains. Thebeta-hemolytic non-group A, B, or D strainsreacted variably on MG-DTA media; 64.2% wereDNase positive. Only two of these strains werebacitracin susceptible and DNase positive,whereas the rest reacted negatively in one or

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IDENTIFICATION OF STREPTOCOCCI 667

TABLE 1. Reaction patterns of streptococci on seven presumptive tests incubated in an increased C02atmosphere

Test reactiona

Streptococci No. Presumptive interpreta-HEM BAC SXT CAMP MG- BE NaCI tionDTA

A ,-+ + - - 184 GroupA,B + - - - - - 4 Group ABi + + - + - - 1 ,B-not A, B, D',B - - - + - - 1 fl-notA, B, D

B fi - - + + - - 110 Group BBi - - + - - - 92 Group Bf, + - + + - - 4 Group Bfi - + + + - - 1 Group Bf - - - - - - 7 fl-not A, B, D

C/G/F ,B - + - + - - 97 f8-notA, B, Dfi - + - - - - 46 ,B-notA, B, D,B - - - + - - 3 ,B-not A, B, Df, + + - - - - 7 ,B-not A, B, D,B + + - + - - 2 fi-notA, B, D,B - - - - - - 4 ,B-notA, B, D

D enterococcus a/fl/y - - - - + + 26 D enterococcusa/fl/y - + - + + + 8 D enterococcusa/fl/y - - - - - + 2 D enterococcusa/y - + - - + - 1 D non-enterococcusa/y - + - - + - 2 D non-enterococcus

D non-entero- a/y - - - - + - 9 D non-enterococcuscoccus a/y - + - - + - 1 D non-enterococcus

Viridans a/y v + - - - - 81 Viridansa/y v - - - - - 9 Viridansa/y v + - - + - 7 D non-enterococcus

a + Susceptible to bacitracin or SXT disks, positive in CAMP or BE test, and grows on NaCi media; -,resistant to bacitracin or SXT disks, negative in CAMP or BE test, and does not grow on NaCi media; v,variable reactions, with some strains positive and some strains negative. HEM, Hemolysis; BAC, bacitracin.

b fi-not A, B, D is beta-hemolytic non-group A, B, and D.

both tests. However, fewer bacitracin-suscepti-ble strains of beta-hemolytic non-group A, B, orD streptococci than are usually encounteredwere found. If larger numbers of bacitracin-sus-ceptible group C and G streptococci were foundin clinical specimens, the usefulness of the baci-tracin test would be more questionable.

All of the enterococcal strains listed in Table1 reacted positively in BE and NaCi tolerancetests when tested in tubes, but two strains didnot react positively in BE tests when tested inplate BE medium, and three did not react posi-tively in NaCI tolerance tests when tested inplate NaCI medium. Even though the entero-cocci reacted variously to SXT disks, interpret-ing the SXT reaction in conjunction with resultsof the NaCI tolerance test led to a more accurateidentification. When positive BE and NaCl re-actions or negative (resistant) SXT and positiveNaCl reactions were obtained, the organisms

were presumptively identified as enterococci.Using these criteria, 36 of 39 (92.3%) enterococciwere presumptively identified. The fact that nei-ther criterion was adequate to identify thesestrains of enterococci prompted us to investigateother NaCl agar formulas. None of the entero-coccal strains were susceptible to bacitracin orreacted positively in CAMP tests.Only 10 strains of non-enterococcal group D

streptococci were tested in the battery of testslisted in Table 1. Ail reacted positively in BEand negatively in NaCl tolerance tests. Onestrain was susceptible to SXT and none wassusceptible to bacitracin or reacted positively inCAMP tests.Ninety of 97 (92.8%) of the viridans strains

were presumptively identified on the basis ofnegative BE and NaCl tolerance test reactions.The other seven strains (7.2%) were BE positiveand NaCl negative and were presumptively iden-

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tified as group D non-enterococci. Nine (9.3%)of the viridans strains were resistant to SXT.SXT susceptibility was not used as a differentialtest for group D streptococci because of thesmall number of these isolates tested. None ofthe viridans strains reacted positively in CAMPor DNase tests. Overall, 97.3% of the strainstested in this battery of tests were presumptivelyidentified correctly.The results of testing three different media to

be used in NaCI tolerance tests are shown inTable 2. NaCI tolerance was strongly consideredin differentiating the non-beta-hemolytic strep-tococci and was the most important test in dif-ferentiating BE-positive strains. Since all beta-hemolytic group A, B, and non-group A, B, or Dstreptococcal isolates did not react positively inBE tests, their growth on NaCl agar was notused as a differential criterion. None of the groupD non-enterococcal or viridans strains grew onbrain heart infusion-NaCI and HIA-NaCI agars,whereas 67 and 85% of the enterococcal strains,respectively, grew on these two media. All butone (97%) of the group D non-enterococcal and43% of the viridans strains grew on D strepto-coccus-enterococcus agar (Table 2). Overall,among the three media evaluated, HIA-NaClagar most clearly differentiated the non-beta-hemolytic streptococci.The reactions of 765 strains of streptococci

evaluated in the second battery of tests areshown in Table 3. None of these strains wasincluded in the first battery of tests. Whereas inthe first battery of tests (Table 1) samples wereincubated in an increased C02 atmosphere, inthe second battery samples were incubated innormal atmosphere. The reactions of the groupA streptococci to bacitracin and SXT disks weresimilar to those of the group A strains in thefirst battery of tests; i.e., 98.7% of the strainswere susceptible to bacitracin and resistant toSXT disks. One group A strain reacted positivelyin the CAMP disk test and was presumptivelyidentified as a group B streptococcus. One groupA strain was susceptible and one was resistantto both bacitracin and SXT disks. They werepresumptively identified as non-group A, B, orD beta-hemolytic streptococci, because 24.8 and6.8% of the beta-hemolytic non-group A, B, or Dstreptococci were susceptible and resistant, re-spectively, to both disks.Among the group B streptococci 98.6% of the

strains reacted positively in CAMP disk tests.These results are a slight improvement overthose with the standard CAMP test (97.2% ofthe group B strains positive) only because inter-mediate CAMP reactions were interpreted aspositive if the strain was resistant to bacitracin

TABLE 2. Evaluation of three formulas for NaCItolerance

No. of % PositiveStreptococci strains BHI- HIA- DSE

tested NaCl' NaCI agar'Group D enterococci 58 67 85 100Group D non-entero- 29 0 0 97

cocciViridans 159 0 0 43

<BHI-NaCl, Brain heart infusion-NaCI.b DSE, D streptococcus-enterococcus.

and SXT disks, and the hemolysis of thesestrains was typical of group B streptococci. AI-though two group B strains were resistant toboth SXT and bacitracin and were CAMP disknegative, these strains were identified as beta-hemolytic non-group A, B, or D streptococci,because 9 of 133 (6.8%) of the beta-hemolyticnon-group A, B, or D streptococci were CAMPdisk negative and resistant to SXT and bacitra-cin.Most (68%) of the beta-hemolytic non-group

A, B, or D streptococci were resistant to bacitra-cin, susceptible to SXT, CAMP disk negative,and BE negative. However, 25% of these strainswere susceptible to bacitracin and SXT and wereCAMP disk negative and BE negative. Also, themost common strains that were resistant to bac-itracin and SXT, and which reacted negativelyin CAMP disk and BE reactions were beta-he-molytic streptococci, non-group A, B, or D, (6.5%of these strains).Of the enterococcal strains tested, 94% reacted

positively as expected in BE and NaCl tolerancetests. The four enterococcal strains that did notreact positively in BE tests were presumptivelyidentified as enterococci because they were re-sistant to SXT and grew in the NaCl tolerancetest. This interpretation was possible becauseonly one viridans strain grew in the NaCl toler-ance test. Three enterococcal strains that failedto grow in the NaCl tolerance test were pre-sumptively, but erroneously, identified as groupD non-enterococcal streptococci. Eighty-onepercent of the enterococci were resistant to SXTdisks, whereas 84% of the viridans streptococciwere susceptible.

Forty-one of 42 group D non-enterococcalstrains were presumptively identified correctlyin the BE (positive) and NaCI tolerance (nega-tive) tests. All of the group D non-enterococcalstrains were Streptococcus bovis. The SXT pro-cedure was of no value in differentiating S. bovisfrom other streptococci; i.e., 23 strains were sus-ceptible and 19 strains were resistant to SXTdisks. One S. bovis strain was presumptively

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IDENTIFICATION OF STREPTOCOCCI 669

TABLE 3. Reaction patterns of streptococci on six presumptive tests incubated in normal atmosphere

Identification No. Presumptive interpreta-HEM BAC SXT CAMP BE NaCI

A f8 + - - - - 155 Group Afi + + - - 1 Group Bfi + + - - - 1 ,B notA, B, DbJf - - - - - 1 ,B-notA, B, D

B fi - - + - - 131 Group Bfi + - + - - 7 Group Bfi - + + - - 1 Group Bfi - - - - - 2 ,B-not A, B, D

C/G/F fi - + - - - 90 ,B-notA, B, DfiR + + - - - 33 f8-not A, B, Dfi - - - - - 9 fi-not A, B, Dfi + - - - - 1 Group A

D enterococci a/fi/y - - - + + 91 D enterococcusa/fi/y - + - + + 22 D enterococcusa/fi/y - - - - + 4 D enterococcusa/fi/y - - - + - 2 D non-enterococcusa/fi/y - + - + - 1 D non-enterococcus

D non-enterococci a/y - + - + - 22 D non-enterococcusa/y - - - + - 18 D non-enterococcusa/y + + - + - 1 D non-enterococcusa/y + - _ _ - 1 Viridans

Viridans a/y v + - - - 137 Viridansa/y v - - - - 21 Viridansa/y v - - + - 6 D non-enterococcusa/y v + - + - 6 D non-enterococcusa/y + - - - + 1 D enterococcus

See footnote a, Table 1.b See footnote b, Table 1.

identified as a viridans streptococcus because itdid not react positively in the BE test.Among the viridans streptococci evaluated

with the tests listed in Table 3, 92.4% werepresumptively identified correctly by negativereactions in BE and NaCI tolerance tests.Twelve strains were incorrectly identified asgroup D non-enterococci because they reactedpositively in BE tests. One strain (Streptococcusuberis) was incorrectly identified as an entero-coccus because it grew in the NaCI tolerancetest and was resistant to an SXT disk. Sixteenpercent of the viridans strains were resistant toSXT disks. The reaction to SXT disks by viri-dans streptococci was helpful but inconclusivein differentiating the organisms. Overall, 97% ofthe streptococci were correctly categorized bytheir reactions to the tests listed in Table 3.Table 4 shows the percentage of positive BE

and NaCI reactions on tubed (2-day) and plate(overnight) media. There was very little differ-ence between BE reactions of the group D en-terococcal and those of non-enterococcal strains

on BE plates and in BE tubes. Also there wasvery little difference between the NaCl brothand NaCl plate reactions of any of the strains.The greatest difference in results obtained withthe tube and plate media appeared in the BEreactions of the viridans streptococci. Substan-tially fewer viridans strains reacted positively inBE tests on BE plates than in those on BEtubes.

Since it was necessary to interpret the inter-mediate CAMP reactions as negative indicatorsof group B streptococci, intermediate CAMPreactions of the streptococci obtained with boththe standard and disk methods were evaluated(Table 5). The percentage of group A strepto-cocci which reacted intermediately in both typesof CAMP tests was high enough to introduce alarge error into the results of the battery of testsif the intermediate reactions were interpreted aspositive. However, the interpretation of inter-mediate CAMP reactions did not cause any dif-ficulty because the hemolysis of the group A, C,and G streptococci on the SB-TSA was much

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TABLE 4. Percentages ofpositive BE and 6.5%NaCI tests determined in tubes and plates

% Positive

Streptococci No. Tube PlatestestedBE NaCI BE NaCI

D enterococci 120 100 100 96.6 97.5D non-entero- 42 100 97.6

cocciViridans 169 20.7 2.3 7.1 0.6

TABLE 5. Intermediate CAMP reactionsNo. showing intermediate reaction/

total tested (%)Streptococci

Standard CAMP Disk CAMP testtest

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)

not A, B, DD enterococci 0/39 0/120D non-entero- 0/10 0/42

cocciViridans 0/97 0/171

more intense than the hemolysis of the group Bstreptococci. The uniquely characteristic hemol-ysis of the group B streptococci, used in con-junction with intermediate CAMP reactions,was interpreted to represent positive presump-tive identification of group B streptococci.

DISCUSSIONThe use of multiple tests to improve the pre-

sumptive identification of streptococci is not anew idea. Wallerstrom (14) described a multiple-test scheme for presumptive identification ofgroup A streptococci. He placed disks containingbacitracin, nucleate, and glucose on lawns ofbeta-hemolytic streptococci on blood agarplates. All the group A strains tested reactedpositively in the "triple test"; i.e., group A strainswere inhibited by bacitracin, the hemolysis wasstimulated by nucleate, and the hemolysis wasinhibited by glucose disks. However, only a fewnon-group A streptococci were tested, and oneof 12 group G strains reacted positively in thetriple test. Although there was some indicationthat group B streptococci could be differentiatedby the triple test, only two strains were tested.We previously described a battery of tests

with which to identify six categories of strepto-cocci presumptively: group A, group B, group Denterococcal, group D non-enterococcal, viri-dans, and beta-hemolytic non-group A, B, or Dstreptococci (7). Blood agar plates were used indetermining hemolysis and bacitracin suscepti-

bility. The hippurate hydrolysis and BE andNaCl tolerance tests were performed in tubescontaining the proper media.Two days of incubation were required before

95% of the streptococci could be accurately dif-ferentiated into the six categories. The largesterror in this presumptive identification was themisidentification of beta-hemolytic non-groupA, B, or D streptococci as group A streptococci.About 19% of these strains were misidentifiedbecause they were susceptible to bacitracin. Pol-lock and Dahlgren (10) warned that if bacitracinwas used as the major criterion for presumptiveidentification of group A streptococci, the poten-tial for misidentification was significant, espe-cially if the specimen was taken from the upperrespiratory tract or from a wound. Thus, it ap-pears pertinent that criteria for presumptiveidentification of the beta-hemolytic streptococcifrom these sources be improved.Darling (2) described a series of experiments

in which he attempted to standardize the CAMPtest for presumptive identification of group Bstreptococci. He found that the test worked bestif the streptococci were inoculated onto Trypti-case soy agar containing washed sheep erythro-cytes. The test material was incubated overnightin a candle extinction jar or in a normal atmos-phere. Under these conditions the standardCAMP reactions could be determined and groupB streptococci could be presumptively identifiedafter overnight incubation, as compared to 2days of incubation required for the hippuratehydrolysis test. Wilkinson modified the standardCAMP test by incorporating the ,B-lysin of theS. aureus into filter paper disks, thus eliminatingthe need for an actively growing culture for theCAMP reaction (15). Both Darling and Wilkin-son reported a 100% accurate identification ofgroup B streptococci by their CAMP tests after18 h of incubation.Recently, Gunn reported on the use of SXT

and bacitracin disks for presumptive identifica-tion of beta-hemolytic group A, group B, andnon-group A or B streptococci (9). He found thatwhen the disks were placed on an inoculum ofstreptococci on SB-TSA, group A streptococciwere susceptible to bacitracin and resistant toSXT disks, group B streptococci were resistantto both disks, and beta-hemolytic group C, G,and F streptococci were resistant to bacitracinbut susceptible to SXT disks. These reactionswere determined after overnight incubation inan atmosphere of 5% C02. Several strains ofgroup C, F, and G streptococci and one strain ofgroup A streptococci were susceptible to bothdisks. Gunn suggested that strains susceptible toboth disks should be interpreted as presumptive

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group A streptococci to prevent any misidenti-fication of group A streptococci. However, fromthe results of these studies it is apparent thatthis pattern of reactions is more indicative ofbeta-hemolytic streptococci, not group A, B, orD, than group A streptococci. Gunn also sug-gested that beta-hemolytic streptococci resistantto both bacitracin and SXT disks should bepresumptively identified as group B strepto-cocci. However, combining the results from Ta-bles 1 and 3 indicates that among the beta-hemolytic streptococci, 94.6% ofthe group B and2.6% of the non-group B strains were resistantto both disks. However, 97.4% of the group Band less than 0.5% of the non-group B strainsgave positive CAMP tests. Therefore, theCAMP reaction is more specific than the com-bined interpretation of the two disks.

It seemed reasonable that if the bacitracin,SXT, and CAMP tests could be performed onone plate, the presumptive identification wouldnot only be more convenient but also more ac-curate. It would also be more convenient if theBE and 6.5% NaCI media could be incorporatedinto plates and the incubation time could beshortened.

In the first battery of tests, samples wereincubated in candle extinction jars at 35°C be-cause both Darling (2) and Gunn (9) suggestedthat an atmosphere containing increased C02was beneficial in both the standard CAMP andSXT disk tests. In the second battery of tests,samples were incubated in normal atmosphereat 35°C because it may be more convenient forsome laboratories.Both batteries of tests were read after over-

night incubation (16 to 20 h). Although someviridans strains did not grow as well in normalatmospheres, this condition did not interferewith the presumptive identification. The totalnumber of correct presumptive identifications inthe two atmospheres was comparable: 97.3% incandle extinction jars and 97.0% in normal at-mosphere. We suggest that microbiologists usethe bacitracin, SXT, CAMP, BE, and NaCl tol-erance tests along with the hemolytic reactionsto make the same presumptive identificationsdescribed in our previous publication (7). Thebattery of tests should be interpreted as a whole;i.e., results obtained in individual tests shouldbe evaluated as they relate to those obtained inall tests run. Patterns of reactions will be com-parable to those described in Tables 1 and 3. Wesuggest that the streptococci can be presump-tively identified by the following criteria.

(i) Group A streptococci are beta-hemolytic,susceptible to bacitracin, resistant to SXT,CAMP reaction negative, 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 strains will havecomparable reaction patterns.

(iii) Group C, F, and G streptococci (beta-hemolytic, not group A, B, or D) wiil react inthree patterns to bacitracin and SXT. All strainsare beta-hemolytic and react negatively inCAMP and BE tests. Most strains are resistantto bacitracin and susceptible to SXT. An appre-ciable number of these strains may be suscepti-ble to both bacitracin and SXT. A few strainswill be resistant to both bacitracin and SXT.

(iv) Group D enterococcal streptococci varyin their hemolytic reaction; most strains arenonhemolytic, but alpha- and beta-hemolyticstrains are common. All strains are resistant tobacitracin and react negatively in CAMP tests.Three patterns of reactions from the SXT, BE,and NaCl tests are characteristic of enterococci.Most strains wiil react positively in BE andNaCl tolerance tests and be resistant to SXT.Some strains are susceptible to SXT and reactpositively in BE and NaCl tests. Some strainsare resistant to SXT and tolerant to NaCl (NaClpositive), but fail to give a positive BE reaction.

(v) Group D non-enterococcal streptococciare alpha- or nonhemolytic and are usually re-sistant to bacitracin. However, occasional strainsare sensitive, vary in their reactions to SXTdisks, and are BE positive and NaCl intolerant.

(vi) Viridans streptococci are alpha- or non-hemolytic and vary in their reaction to bacitra-cin and SXT. However, most strains are suscep-tible in SXT and negative in CAMP, BE, andNaCl tolerance tests.The results of these studies, using the above

interpretation, indicate that at least 97% of themedically important streptococci can be pre-sumptively identified. Streptococci that do notfit into one of the reaction patterns should bechecked for purity and retested. The retestingcan be performed on plates or in tubes as pre-viously described (7). Strains whose reactions inthe presumptive tests remain atypical should beserologically grouped and definitively identifiedin a reference laboratory.We suggest that the bacitracin, SXT, and

CAMP disks be stored at -20°C. (Placing themin the freezer of a regular refrigerator is satisfac-tory.) We had some difficulty maintaining theactivities of SXT and CAMP disks when theywere stored at 4°C for even as short a period as5 days, although they remain stable for 1 month(with daily use) when stored at -20°C.Accurate determination of hemolysis is the

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672 FACKLAM ET AL.

first and most useful step in identifying strepto-cocci. Bacteriologists should familiarize them-selves with typical hemolytic activities of thestreptococci. Although the hemolysis of group Astreptococci cannot be routinely differentiatedfrom that of group C or G streptococci, theformer differs from that of group B, D, and Fstreptococci. The zone of hemolysis surroundinggroup B streptococci is much smaller and thezone of hemolysis surrounding the group D en-terococcal streptococci is larger than that ofgroup A. Also, colonies of group D streptococciare much larger than those of other streptococci,and colonies of group F streptococci are muchsmaller than those of beta-hemolytic group A,B, C, D, and G streptococci. The zone of hemol-ysis surrounding these small colonies is quitelarge, nearly equivalent to the zones formedaround group A streptococci.We preferred to read the zones of inhibition

of growth around the SXT disks in the samemanner as we described for bacitracin (7). Anyzone of inhibition of growth around the bacitra-cin or SXT disks represents a positive result. Torequire certain zone sizes of growth inhibition, astandard inoculum must be used. However, intests to obtain a presumptive identification, re-quiring standardized inoculum would add anunnecessary element of complication.The CAMP test should be read in conjunction

with the hemolytic activity of the streptococci.Beta-hemolytic non-group B streptococci thatreact intermediately in CAMP tests are easilydifferentiated from group B streptococci whichreact weakly or intermediately in CAMP testsbecause of the differences in hemolytic activitydescribed above. Most group B streptococci areclearly positive in CAMP tests.The BE plate test is more difficult to interpret

than the tube test. Some enterococcal strainsreact very weakly; i.e., the blackening of themedium may not be apparent unless the plate isinspected in front of a white background. Weadvise reading the test in this manner and inter-preting any blackening of the medium as a pos-itive reaction, even though doing so may resultin a number of misidentifications of viridansstreptococci as group D non-enterococcal strep-tococci because ofweak BE reactions. This erroris medically less dangerous than misidentifyingenterococci because antibiotic susceptibilities ofthe group D non-enterococci are similar to thoseof the viridans streptococci (13).One reason that an occasional group D strep-

tococcus was negative in the BE plate test butpositive in the BE tube test was the incubationperiod. The plates were incubated for 18 to 20 h,whereas the tubes were incubated for 40 to 42 h.If the agar plate BE test had been incubated

longer, the reactions might have been more com-parable to those obtained with tubed media.The results with the 6.5% NaCl tolerance tests

were the most disappointing of those obtained.We strongly recommend that plates be carefullyinspected immediately after inoculation to in-sure that the inoculum is spread so as not to beread as growth. When the plates are prepared inthis manner, any growth interpreted as a positivereaction reflects an accurate indicator NaCl tol-erance.The CAMP disks are not available commer-

cially at present (November 1978). It is hopedthat in the future they will be made available orthat the media and disks will be commericallyavailable as a kit.

LMRATURE CITED1. Abshire, R. L. 1977. Evaluation of a new presumptive

medium for group D streptococci. Appl. Environ. Mi-crobiol. 33:1149-1155.

2. Darling, C. L. 1975. Standardization and evaluation ofthe CAMP reaction for the prompt, presumptive iden-tification of Streptococcus agalactiae (Lancefield groupB) in clinical material. J. Clin. Microbiol. 1:171-174.

3. Facklam, R. R. 1972. Recognition of group D streptococ-cal species of human origin by biochemical and physi-ological tests. Apple. Microbiol. 23:1131-1139.

4. Facklam, R. R. 1973. Comparison of several laboratorymedia for presumptive identification of enterococci andgroup D streptococci. Apple. Microbiol. 26:138-145.

5. Facklam, R. R. 1977. Physiological differentiation ofviridans streptococci. J. Clin. Microbiol. 5:184-201.

6. Facklam, R. R., and M. D. Moody. 1970. Presumptiveidentification of group D streptococci: the bile-esculintest. Apple. Microbiol. 20:245-250.

7. Facklam, R. R., J. F. Padula, L. G. Thacker, E. C.Wortham, and B. J. Sconyers. 1974. Presumptiveidentification of group A, 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 bovisand other group D streptococci from human sources byusing arginine and pyruvate tests. J. Clin. Microbiol. 1:54-60.

9. Gunn, B. A. 1976. SXT and Taxo A disks for presumptiveidentification of group A and B streptococci in throatcultures. J. Clin. Microbiol. 4:192-193.

10. Pollock, H. M., and B. J. Dahlgren. 1974. Distributionof streptococcal groups in clinical specimens with eval-uation of bacitracin screening. Appl. Microbiol. 27:141-143.

11. Qadri, S. M. H., C. W. Nichols, and S. G. M. Qadri.1978. Rapid sodium chloride tolerance test for presump-tive identification of enterococci. J. Clin. Microbiol. 7:238.

12. Smith, P. B., G. A. Hancock, and D. L. Rhoden. 1969.Improved medium for detecting deoxyribonuclease-pro-ducing bacteria. Appl. Microbiol. 18:991-993.

13. Thornsberry, C., C. N. Baker, and R. R. Facklam.1974. Antibiotic susceptibility of Streptococcus bovisand other group D streptococci causing endocarditis.Antimicrob. Agents Chemother. 5:228-233.

14. Wallerstrom, A. 1962. A simple biochemical "triple test"for preliminary identification of group A streptococci.Acta Pathol. Microbiol. Scand. 56:459-464.

15. Wilkinson, H. W. 1977. CAMP-disk test for presumptiveidentification ofgroup B streptococci. J. Clin. Microbiol.6:42-45.

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