JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1987, p. 1845-18500095-1137/87/101845-06$02.00/0Copyright 1987, American Society for Microbiology

Comparison of Physiologic Tests Used To Identify

Vol. 25, No. 10

Non-Beta-Hemolytic Aerococci, Enterococci, and StreptococciSUSAN S. FERTALLY't AND RICHARD FACKLAM2*

Emory University, Atlanta, Georgia 30322,1 and Bacterial Reference Laboratory, Bacterial Disease Division, Centers forDisease Control, Atlanta, Georgia 303332

Received 20 April 1987/Accepted 19 June 1987

Twenty-one reference strains and 88 clinical isolates of Aerococcus, Enterococcus, and Streptococcus specieswere tested for reactions in the Rapid Strep (RS) and modified conventional tests. We conclude that some butnot all of the tests in the RS system could be used to substitute for conventional tests. RS tests for hydrolysisof arginine, esculin, L-pyrrolidonyl-naphthylamide, production of acetyl methyl carbinol (Voges-Proskauer),and fermentation of arabinose, lactose, mannitol, raffinose, and sorbitol were satisfactory substitutes forconventional tests. However, the RS tests for hydrolysis of hippurate and starch and fermentation of inulinwere not satisfactory substitutes for conventional tests. We also conclude that, of the five test tubeVoges-Proskauer tests, the Coblentz modification is the most discriminatory.

The taxonomy of the Streptococcus genus is undergoing adrastic change. Based on DNA homology studies, the genusis being split into three genera: Enterococcus, Lactococcus,and Streptococcus (2, 4, 6, 9, 12, 15, 16). Additional speciesare being described and new descriptions of older specieshave been formed. Investigators using DNA homology stud-ies to differentiate species have used a combination ofconventional bacteriologic tests as well as experimentalminiaturized-dehydrated tests to phenotypically describe thespecies. The two systems most often used are the API 50CHsystem, which is a system containing 49 carbohydrate sub-strates, and the API 20 Strep system, which is identical tothe Rapid Strep system distributed in the United States. TheAPI 50CH and API 20 Strep systems can be obtained fromAPI Systems S.A., la Balme-Les-Grotes, France. There isvery little information available to correlate the results ofconventional tests and miniaturized-dehydrated tests. Insome instances, conventional tests are not available withwhich to make this comparison.The Rapid Strep (RS) system can be used to identify

Streptococcus species by determining seven-digit profilenumbers from the test results and then matching profilenumbers with the manufacturer's index (8). One laboratoryhas suggested that the tests in the RS system can be used toreplace conventional tests and an ordinary differentiationscheme can be based on those tests together with a minimalnumber of conventional tests (3).We realize that the tests in the RS system are not

specifically designed to correlate with test tube (conven-tional) tests. However, with the changing of nomenclatureand new descriptions of old and new species of gram-positive cocci, users of the RS system, as well as othersimilar products, will be tempted to correlate RS test resultsto those test results used to describe new species. Thus,users will want to know which tests are reliable and whichare not.

This study was designed to compare various conventionaltests with results obtained in the RS system. Results ob-

* Corresponding author.t Present address: Coral Springs Medical Center, Coral Springs,

FL 33065.

tained with modifications of the conventional tests are alsocompared with those of the basic tests.

MATERIALS AND METHODSStrains. Reference strains for most Aerococcus, Entero-

coccus and Streptococcus species were selected as controlstrains (Table 1). Clinical isolates for most species were alsoincluded in this study (Table 1).

Cultures of strains were retrieved from a -70°C freezer.Each was streaked for isolation to Trypticase soy agarprepared with 5% sheep blood (BBL Microbiology Systems,Cockeysville, Md.). The plates were incubated in a candleextinction jar at 35°C for 18 to 20 h. Slowly growingorganisms and small-colony producers were incubated for 48h. At the end of the incubation period, a single colony waspicked and subcultured to another blood plate. The inoculumwas streaked across the entire surface of the plate to obtainheavy growth. This same colony was also inoculated into 5ml of Todd-Hewitt broth. Inoculated plates were incubatedin a candle extinction jar at 35°C, and the tubes wereincubated aerobically at 35°C for 24 h.Media and reagents. The RS system was purchased from

Analytab Products, Division of Sherwood Medical, Plain-view, N.Y.Methyl red and Voges-Proskauer (VP) broth (Difco Lab-

oratories, Detroit, Mich.) was prepared according to themanufacturer's instructions, divided into various amounts,and placed into capped tubes. The amounts were 0.3 ml in 13by 100-mm tubes, and 1.0, 2.0, and 2.5 ml in 16- by 125-mmtubes. The various VP reagents were made by the method ofMacFaddin (13). Coblentz reagent A is 5% alpha-naphthol in95% ethyl alcohol and reagent B is 0.3% creatine in 40%potassium hydroxide. Barritt's reagent A is 5% alpha-naphthol in absolute ethanol and reagent B is 40% potassiumhydroxide. O'Meara's single reagent is 0.3% creatine in 40%potassium hydroxide. Barry and Feeney's reagents are 0.3%creatine and Barritt's VP reagents A and B (1).Sodium hippurate broth and ferric chloride reagent,

esculin agar, Moeller decarboxylase medium with arginine,carbohydrate fermentation broths for acid production inarabinose, mannitol, sorbitol, lactose, trehalose, inulin, andraffinose, and 1 and 2% starch agars were prepared by themethods of Facklam and Carey (7). Medium and reagents for

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1846 FERTALLY AND FACKLAM

TABLE 1. Strains of Aerococcus, Enterococcus, andStreptococcus species used in this study

Reference strain No. ofOrganism identification csolatesisolates

Aerococcus sp. CDC SS-928 5Enterococcus avium ATCC 14025 3E. avium variant None 3E. casseliflavus ATCC 25788 1E. durans ATCC 11576 6E. faecalis ATCC 29212 5E. faecium CDC SS-442 4E. faecium variant None 3E. gallinarum NCDO 2313 2E. hirae NCDO 1258 3E. malodoratus NCDO 846 0Streptococcus CDC SS-1019 4

acidiminimusS. bovis ATCC 9809 4S. bovis variant CDC SS-1189 6S. constellatus ATCC 27823 3S. equinis NCTC 10389 0S. intermedius ATCC 9895, 27335 5S. intermedius variant ATCC 15912 3S. mitis ATCC 9811 5S. mutans NCTC 10449 3S. salivarius CDC SS-908 6S. sanguis ATCC 10556 8S. sanguis Il ATCC 10557 4

ATCC, American Type Culture Collection; CDC SS, Centers for DiseaseControl stock strain; NCDO, National Collection of Dairy Organisms; NCTC,National Collection of Type Cultures.

the rapid hippurate test were prepared by the methods ofHwang and Ederer (10). Niven's arginine broth for strep-tococci (14), and Falkow's decarboxylase medium base(GIBCO Diagnostics, Madison, Wis.) with 1% L-arginine(13) were prepared. L-Pyrrolidonyl-g-naphthylamide (PYR)agar was made with Todd-Hewitt broth (BBL), 0.7% agar,and 0.01% L-pyroglutamic acid P-naphthylamide (SigmaChemical Co., St. Louis, Mo.). The medium was autoclavedand poured into 15- by 100-mm plates. PYR reagent, p-dimethylaminocinnamaldehyde (Sigma), was suspended in asolution of 2.5 ml of sodium dodecyl sulfate, 2.5 ml of glacialacetic acid, 5.0 ml of 2-methoxyethanol, and 90 ml ofdistilled water. The reagent was stored at 4°C in a darkcontainer.Commercially prepared carbohydrate fermentation me-

dium anaerobic base and Andarade base for base control,arabinose, mannitol, sorbitol, lactose, inulin, and raffinosewere purchased from Carr-Scarborough (CS) Microbiologic-als, Inc., Stone Mountain, Ga. PYR broth, esculin broth,and Moeller decarboxylase media with arginine were alsopurchased from CS.

Inoculation and incubation. The inoculum for the RSsystem was prepared from Trypticase soy agar-sheep bloodagar as described previously (8). After preparation of the RSinoculum, the same swab was used to inoculate the PYR, 1and 2% starch agars, and a blood agar plate for purity. The2.0-ml VP broth was inoculated with a sterile swab to aturbidity of a McFarland no. 4 standard or greater for theCoblentz method. Two of the 0.3-ml VP broths were inocu-lated with the same swab to the same turbidity as theCoblentz test for the 2- and 5-h Barry and Feeney tests. The1.0-ml VP broth for O'Meara's VP method, the 2.5-ml VPbroth for the Barritt VP method, and the remaining tubedmedia (carbohydrate fermentation, esculin, and hippurate

broths) were inoculated with 2 drops of growth from theTodd-Hewitt broth. The Moeller decarboxylase tests forarginine hydrolysis were overlaid with sterile mineral oil. Allcaps were closed tightly.

All tests were incubated aerobically at 35°C. The agarplates were incubated in a candle extinction jar. The RS stripwas incubated according to the manufacturer's directions.The Coblentz VP test was incubated for 6 h, Barry andFeeney methods were incubated for 2 and 5 h, respectively,the O'Meara VP was incubated for 24 h, and Barritt'smethod was incubated for 3 days. The rapid hippurate testwas incubated for 2 h, and the conventional hippurate testwas incubated for 7 days. The PYR broth was incubated for1 h, and the PYR agar was incubated for 24 h; the starchagars were incubated for 48 h. Niven's arginine broth wasincubated for 48 h, and the remaining arginine tests, theesculin broth and agar, and all of the carbohydrate mediawere incubated for 7 days.Performance of the tests. Reagents were added to the RS

strip and the tests were read in the manner described by themanufacturer. The Coblentz VP method was performed byadding 0.6 ml of Coblentz reagent A followed by 0.2 ml ofCoblentz reagent B to each tube. The tubes were looselycapped, shaken vigorously for 5 s, and slanted on the tabletop over a white sheet of paper. The angle of the slant wassuch as to expose as much of the broth to air as possible,while leaving some broth in the butt of the tube. Thisamounted to 10 to 15 mm of the butt remaining filled with thebroth. Care was taken not to disturb the broth before thereading was taken. This is especially important for thedetection of weak reactions. A positive reaction was re-corded as a pink or red color at the surface of the broth. Astrongly positive reaction resulted in diffusion of a deep redthroughout the broth. Faint pink around the surface edges ofthe broth in the slanted area of the tube was recorded as aweakly positive reaction. Positive tests were removed fromweakly positive or negative reactions to eliminate any reflec-tions of red. Negative control strains were always includedin the test runs and weakly positive tests were comparedwith these. Most positive reactions were apparent within 15min. Final test results were read after 1 h.The Barry and Feeney 2- and 5-h modifications of the VP

test were performed by adding 2 drops of 0.5% creatine,followed by 2 drops of Barritt VP reagent A and 2 drops ofBarritt VP reagent B. Each tube was shaken vigorously for 5s. The tubes were placed upright in a rack. Readings weretaken at 5 and 15 min, and a final reading was taken at 30min. Pink to red was recorded as positive, very light pinkwas recorded as weakly positive, and no color productionwas recorded as negative. After the 30-min period, allnegative reactions were shaken again for 5 s. In a few cases,this action hastened a positive reaction. O'Meara's VP testwas performed by adding 1.0 ml of O'Meara single VPreagent to each test. The tubes were shaken vigorously for 5s and then placed in a rack. Readings were taken hourly forup to 4 h. Pink to red was recorded as a positive reaction,faint pink was considered weakly positive, and colorless wasrecorded as negative. Barritt's VP method was performed byadding 0.6 ml of Barritt reagent A followed by 0.2 ml ofBarritt reagent B to each test. The same procedure describedabove for the Coblentz VP method was followed. Readingswere taken in the same fashion.The PYR agar test was performed by adding a drop of

PYR reagent to the growth on the PYR plate. A positiveresult was the development of a pink to red which, in somecases, intensified with time. Negative results were recorded

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TABLE 2. Positive reactions of Aerococcus, Enterococcus, and Streptococcus species in five modifications of the VP test

% Positive reactions

Expected results Barry and Barry andRS Coblentz Feeney Feeney Barritt

2h 5h

Positive (n = 73) 97.3 95.8 95.8 95.8 94.5E. avium, E. astium variant, E. cassefifla'us,

E. durans, E. faecalis, E. faecium, E. faecium variant, E. gallinarum,E. gallinarum-like, E. hire, E. malorderatus,

S. bovis, S. bovis variant, S. constellatus, S. equinus, S. iniermedius,S. mutans, S. salivarius

Negative (n = 35) 0 2.9 8.5 8.5 20.0AerococcusS. acidiminimus, S. intermedius variant, S. mnitis, S. sanguis,

S. sanguis Il

as the development of yellow, orange, or coral. The orangeand coral faded in intensity with time. The performance andinterpretation of all other tests in this study have beendescribed previously (5, 7).

RESULTS AND DISCUSSION

The O'Meara VP method was discontinued after testing 55strains because only 27% of the expected positive strainswere positive in this test. The expected positive reactions byeach bacterial species studied in this communication arethose reactions obtained and reported by authors describingthe same species (2, 4, 5, 9, 11, 12, 15, 16).

All 73 strains expected to give positive VP reactions wereconsistently positive by other methods with four exceptions(Table 2). One Streptococcus bovis variant strain was VPpositive with RS but VP negative by the other methods; onevariant strain of E. faecium and one strain of Streptococcussalivarius were VP negative by all methods. These strainswere typical of their species in all other physiologic charac-teristics. Enterococcus avium strains were consistently VPpositive, although this species has been reported to be VPnegative by some investigators (2) and positive by others (3).One E. avium variant strain failed to give a positive reactionin the Barritt modification test but was positive in all otherVP tests.Of the 35 strains expected to give negative reactions in the

VP tests, S. acidominimus, S. intermedius variant, S. mitis,and S. sanguis II strains consistently gave negative reactionsin all tests. One Aerococcus sp. strain gave a positivereaction in Barrett's VP test and was negative in ail other VPtests. Of the nine S. sanguis strains tested, none, one, three,three, and four strains gave positive reactions in the RS,Coblentz, Barry and Feeney 2- and 5-h, and Barritt VP tests,respectively.

If the E. avium and E. avium variant strains are consideredpositive for the VP reaction, then RS VP is very discrimina-tory with expected positive and negative reactions. None ofthe expected negative strains was positive by this method,and a high percentage of positive reactions with expectedpositive strains was observed. The RS VP correlates bestwith the Coblentz conventional VP test, which was also themost discriminatory of the conventional VP modificationsused in this study. The disadvantages of the Coblentzmethod are the 6-h incubation and the relatively largevolume ofVP broth used for the test. Our 2-h modification ofthe Barry and Feeney test gave equivalent positive testresults with their 5-h test; however. more of the expected

negative test results were positive with this modificationthan with Coblentz or RS. The advantages of this test werethe shorter incubation time, the small volumes of VP brothand reagents needed, the rapidity with which the testsbecame positive, and the ease of reading the positive reac-tions. The Barritt VP method required a 3-day incubationtime and was the least discriminatory of the VP testscompared in this study.RS enzymatic test results given in Table 3 were compara-

ble to conventional test results with the majority of strains;however, discrepancies were observed with some tests. Inaddition, some of our results differed from those reported inthe literature. The positive reactions in RS arginine com-pared well with both the Facklam and Carey and CS-prepared Moeller decarboxylase methods. Only six strainsgave positive reactions with Niven's arginine test. Resultswith Falkow's arginine method were equivalent to theMoeller RS decarboxylase method except for certain strainsof E. casseliflavus, S. sanguis, and S. constellatus whichfailed to give positive reactions.The RS arginine test was not quite as discriminatory with

expected negative strains as were the two Moellerdecarboxylase tests. Niven's and Falkow's methods wereeven less discriminatory with expected positive strains.Possible explanations for the low percentage of positiveresults with Niven's method include the following: themedium contains 0.3% D-arginine, and the majority ofstreptococci apparently metabolize only the L-(+) forms ofamino acids; the formulation of the medium may not havecontained adequate nutrients to support the growth of thestreptococci; and the concentration of ammonia liberatedmay have been too low to detect the Nessler reagent (14).Falkow's arginine medium contains 0.5% L-arginine, de-pends on a shift in the pH for interpretation, and does notneed an oil overlay, but the caps must be tightened.Falkow's method was easiest to use because neither an oiloverlay nor the further addition of reagents was required,but the low percentage of positive results was discouraging.These results may have been due to the interpretation of thefaded-out, yellow-purple colors as negative. This faded coloris said to be due to cadaverine production and is read as apositive reaction according to MacFaddin (13).The positive reactions with all methods of determining

esculin hydrolysis were comparable. Tests with the CSesculin medium gave the highest percentage of positiveresults with expected positive strains but also gave an 18%false-positive result with strains that should have beennegative. These positive reactions with expected negative

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TABLE 3. Comparative results of RS with conventional tests for determining hydrolysis of arginine, esculin, hippurate, PYR, and starch

No. of % Positive No. of % Positive resultsTest Test variation' strains results with strains with expected

tested expected positive tested negative resultsresultsArginine RS 54 94 54 6

Facklam and Carey 94 0CS-Moeller's 93 2Niven's 13 0Falkow's 83 0

Esculin RS 90 96 18 ilFacklam and Carey 98 0CS-anaerobic 99 17

Hippurate RS 28 57 80 1Rapid 86 1Facklam and Carey 100 0

PYR RS 49 98 59 2This paper 100 29CS 98 3

Starch RS 16 75 90 12This paper-1% 100 0Facklam and Carey-2% 100 0

a Facklam and Carey, Formulation described in reference 7; CS, Carr-Scarborough formulation.

strains are inexplicable and constitute the disadvantage ofusing this method.We used the conventional hippurate test described by

Facklam and Carey as the reference procedure for measur-ing hippurate hydrolysis; thus, this test was completelydiscriminatory for expected positive and negative strains.Only 57% of the expected positive reactions were observedwith the expected RS hippurate and all but one of theexpected negative strains were negative. This low-percentage positive rate was most likely due to the instruc-tions of the manufacturer for interpretation of results. Thedirections state to "wait 10 minutes" before reading, apositive test is dark blue or violet, and a negative is colorlessor pale blue. A color chart provided with the kit approxi-mates what shades of blue the positive and negative reac-tions should be. The only blue observed was with a stronglypositive hippurate test, and other shades such as iilac wereinterpreted as negative.The RS and CS PYR tests were quite satisfactory in

discriminating between expected positive and negativestrains and were comparable to each other. Twenty-ninepercent of expected negative strains were positive by ourmodified agar method. This simplified method of PYR agarand reagent preparation may explain these results. We madeno attempt to purify the reagents purchased from Sigma. Itcan be concluded that homemade PYR agar may not havethe specificity of the commercially prepared products.The RS starch test depends upon the production of acid

for determination of a positive test. The agar method de-scribed by Facklam and Carey was read as positive when acomplete zone of clearing with no residual starch aroundcolonies was observed. The RS starch test gave a highpercentage of positives with expected negative strains (Table3). The species that produced acid from RS starch but didnot hydrolyze starch with the method described by Facklamand Carey were one strain of E. faecium, two variant strainsof E. faecium, five strains of E. faecalis, and three strains ofS. salivarius (Table 3). Since S. bovis is the only group DStreptococcus species that hydrolyzes starch by the conven-

tional method (7), that other group D species give positivestarch reactions in the RS system would complicate differ-entiation procedures.The expected production of acid from arabinose, lactose,

mannitol, sorbitol, and trehalose in the RS system closelycorresponded with the conventional tests used in this study(Table 4). Acid production for al tests in the RS system wasread at 24 h.Very poor comparison was observed between the RS

inulin test and three other methods. The percentage ofpositive results for inulin was the lowest observed for all testmethods used in this study. Inulin is one of the critical testsused for differentiation of the viridans group streptococci (5,7). Failure of the RS inulin test to correlate with conven-tional inulin tests would lead to erroneous identification ofviridans group species. Most of the RS inulin test errorswere observed for the S. salivarius and S. sanguis strainswhich often failed to give positive reactions. The inulin testwas the most difficult test in this study to read, and werecorded weak reactions as negative.Good correlation was observed between RS and the

conventional test described by Facklam and Carey forfermentation of lactose. One strain of S. constellatus gave apositive reaction in each of the anaerobic and Andradefermentation media for lactose. A positive reaction by astrain of S. constellatus in lactose broth would lead to anerror by changing the identification fromn S. constellatus to S.intermedius.Colman and Ball (3) implied that the test for the presence

of P-galactosidase in the RS system should correlate with theacidification of lactose in the RS as well as conventionaltests. Of the 93 strains that produced acid in the RS lactosetest, 66 (70%) were positive in the test for P-galactosidase.Three of the 15 strains negative for lactose in the RS systemwere positive in the RS 3-galactosidase test. All threepositive results were found with strains of S. acidominimus.These results imply either a very poor correlation betweenthe two tests or that more than one mechanism is beingmeasured in the two tests.

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TABLE 4. Comparison of the RS with three conventional methods of determining carbohydrate fermentations

No. of 17 Positive No. of V( Positive resultsCarbohydraite Test base" strains results with strainso7 sitive results

tested ~~expected positiveteednatersistested ~~~~resuitstetdngtvrsusArabinose RS 21 95 87 1

Facklam and Carey 100 1Anaerobic 100 1Andrade 10( 1

lnulin RS 27 52 81 2Facklam and Carey 96 0Anaerobic 100 1Andrade 100 1

Lactose RS 95 96 13 0Facklam and Carey 100 0Anaerobic 97 8Andrade 100 8

Mannitol RS 38 100 70 7Facklam and Carey 100 0Anaerobic 100 3Andrade 100 9

Raffinose RS 48 84 60 4Facklam and Carey 98 0Anaerobic 100 10Andrade 100 7

Sorbitol RS 24 96 85 2Facklam and Carey 100 0Anaerobic 100 2Andrade 100 1

Trehalose RS 98 97 il 45Facklam and Carey 100 0

Facklam and Carev. heart infusion base with bromcresol purple indicator (7); anaerobic. anaerobic base with broomthymol blue indicator (CS); Andrade,Andrade's base with Andrade's indicator (CS).

There was complete agreement in the expected positivereactions with all tests for mannitol fermentation (Table 4).

Six strains failed to give positive reactions in the RSraffinose tests but gave positive reactions in all three con-ventional tests. Several positive reactions for raffinose fer-mentation were observed in the anaerobic and Andrademedium formulations among the expected negative strains(Table 4). Colman and Ball (3) reported that the RS test forcx-galactosidase and the acidification of raffinose in the RSand conventional systems should correlate. Of the 45 strainstested that were RS raffinose positive, 41 (91%) were alsoa-galactosidase positive. However, 19 of the 63 RS raf-finose-negative strains (30%) were also cx-galactosidase pos-itive. When the x-galactosidase test results were comparedwith the conventional raffinose acidification test describedby Facklam and Carey, we found that 84%c of the conven-tional raffinose-positive strains were also c-galactosidasepositive and 30% of the conventional raffinose-negativestrains were also a-galactosidase positive. These results, likethose for lactose fermentation and ,-galactoside. are inex-plicable.One strain of S. mnutans failed to give a positive reaction in

the RS sorbitol test but did give positive reactions in all othersorbitol tests. Two Aerococcus strains gave positive reac-tions in RS and anaerobic base sorbitol broth but not in thesorbitol broth formulation of Facklam and Carey. OneAerococcuus strain also gave a positive reaction in Andrade'ssorbitol medium.

The fermentation of trehalose is not important in thedifferentation of the group D and viridans group strepto-cocci. Three strains failed to give a positive RS trehalose testbut did in the medium described by Facklam and Carey. Ofthe six Aerococcus strains tested, all were positive in the RStrehalose test, but only one was positive in the conventionalmedium.An interesting observation with variant S. intermedius

strains was the apparent correlation of reactions with someof the biochemical tests. These variant strains were VPnegative and raffinose positive, whereas typical S. interme-dius strains gave the reverse reactions. The discovery ofvariant strains with apparent consistent reactions differingfrom type strains may be useful in resolving the heterogene-ity of the genospecies (15).The anaerobic carbohydrate fermentation media required

a back-titration of base and test with 1% aqueous brom-thymol blue solution in 3% of the tests performed. The testwas then compared with the base to determine the acid ornegative reactions. The RS, Facklam and Carey, andAndrade fermentation tests did not require any extra stepsbefore interpretation of the reactions.Our results with the leucine aminopeptidase test in the RS

system are in agreement with those of Colman and Ball (3).Aerococcus strains were consistently negative in this test,whereas all strains of Enterococcus and Streptoc(ccus givepositive leucine aminopeptidase reactions.

Caution should be used when RS inulin results are the

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basis for identification to species, since this RS test had thelowest discriminatory value of all tests in that system. TheRS hippurate test was somewhat difficult to interpret withour study organisms. The RS starch test gave more positiveresults with expected negative strains than did the conven-tional agar method. If an investigator is relying on the inulin,starch, and hippurate tests to aid in the differentiation ofstreptococci, we recommend that conventional tests be usedto confirm these particular test reactions. We concluded thatthe most desirable VP method with the highest discrimina-tory factor and correlation to the RS system is the CoblentzVP method. For all of the conventional tests compared withthe RS system, the methodologies discussed in reference 7closely correspond to the expected positive and negativereactions.

LITERATURE CITED1. Barry, A. L., and K. L. Feeney. 1967. Two quick methods for

Voges-Proskauer test. Apple. Microbiol. 15:1138-1141.2. Collins, M. D., D. Jones, J. A. E. Farrow, R. Kilpper-Balz, and

K. H. Schleifer. 1984. Enterococcus avium nom. rev., comb.nov.; and E. casseliflavus nom. rev., comb. nov.; E. gallinarumcomb. nov.; and E. malodoratus sp. nov. Int. J. Syst. Bacteriol.34:220-223.

3. Colman, G., and L. C. Ball. 1984. Identification of streptococciin a medical laboratory. J. Apple. Bacteriol. 57:1-14.

4. Coykendall, A. L., and K. B. Gustafson. 1985. Deoxyribonucleicacid hybridizations among strains of Streptococcus salivariusand Streptococcus bovis. Int. J. Syst. Bacteriol. 35:274-280.

5. Facklam, R. R. 1977. Physiological differentiation of viridansstreptococci. J. Clin. Microbiol. 5:184-201.

6. Facklam, R. R. 1984. The major differences in the American andBritish Streptococcus taxonomy schemes with special referenceto Streptococcus milleri. Eur. J. Clin. Microbiol. 3:91-93.

7. Facklam, R. R., and R. B. Carey. 1985. Streptococci and

aerococci, p. 154-175. In E. H. Lennette, A. Balows, W. J.Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinicalmicrobiology, 4th ed. American Society for Microbiology,Washington, D.C.

8. Facklam, R. R., D. L. Rhoden, and P. B. Smith. 1984. Evalua-tion of the Rapid Strep system for the identification of clinicalisolates of Streptococcus species. J. Clin. Microbiol. 20:894-898.

9. Farrow, J. A. E., and M. D. Collins. 1985. Enterococcus hirae,a new species that includes amino acid assay strain NCDO 1258and strains causing growth depression in young chickens. Int. J.Syst. Bacteriol. 35:73-75.

10. Hwang, M., and G. M. Ederer. 1975. Rapid hippurate hydrolysismethod for presumptive identification of group B streptococci.J. Clin. Microbiol. 1:114-115.

11. Kilpper-Balz, R. 1984. Relatedness of Streptococcus milleri withStreptococcus anginosus and Streptococcus constellatus. Syst.Apple. Microbiol. 5:494-500.

12. Knight, R. G., D. M. Shlaes, and L. Messineo. 1984. Deoxyri-bonucleic acid relatedness among major human enterococci.Int. J. Syst. Bacteriol. 34:327-331.

13. MacFaddin, J. F. 1980. Biochemical tests for identification ofmedical bacteria, 2nd ed., p. 308-320. The Williams & WilkinsCo., Baltimore.

14. Niven, C. F., Jr., K. L. Smiley, and J. M. Sherman. 1941. Thehydrolysis of arginine by streptococci. J. Bacteriol. 43:651-660.

15. Schieifer, K. H., and R. Kilpper-Balz. 1984. Transfer of Strep-tococcus faecalis and Streptococcus faecium to the genusEnterococcus nom. rev. as Enterococcus faecalis comb. nov.and Enterococcus faecium comb. nov. Int. J. Syst. Bacteriol.34:31-34.

16. Schieifer, K. H., et ai. 1985. Transfer of Streptococcus lactis andrelated streptococci to the genus Lactococcus gen. nov. Syst.Apple. Microbiol. 6:183-195.

17. Szewczuk, A., and M. Mulczyk. 1968. Pyrrolidonyl peptidase inbacteria: the enzyme from Bacillus subtilis. Eur. J. Biochem.8:63-67.

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