6
Vol. 22, No. 5 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1985, p. 772-777 0095-1137/85/110772-06$02.00/0 Copyright C 1985, American Society for Microbiology Incidence and Characterization of Beta-Hemolytic Streptococcus milleri and Differentiation from S. pyogenes (Group A), S. equisimilis (Group C), and Large-Colony Group G Streptococci JODY LAWRENCE, DAVID M. YAJKO, AND W. KEITH HADLEY* Department of Laboratory Medicine, University of California, San Francisco General Hospital Medical Center, San Francisco, California 94110 Received 7 May 1985/Accepted 12 August 1985 The biochemical characteristics of 172 clinical isolates of group A, C, F, or G or "nongroupable" beta-hemolytic streptococci were examined. Among these isolates, 91 were identified as beta-hemolytic strains of Streptococcus milleri. The remaining isolates included 20 Streptococcus pyogenes, 21 Streptococcus equisimilis, 37 large-colony group G streptococci, and 3 unidentified nongroupable isolates. A majority (84%) of the S. milled strains possessed Lancefield group antigen (3 A, 27 C, 41 F, and 5 G), whereas 15 S. milleri strains (16%) were nongroupable, Serological tests did not differentiate S. milleri isolates with group A, C, or G antigen from S. pyogenes (group A), S. equisimilis (group C), or large-colony group G streptococci. Biochemical tests which were found useful for differentiation included the Voges-Proskauer test, hydrolysis of pyroglutamic acid and beta-D-glucuronide, bacitracin susceptibility, and acid production from ribose. S. milleri represented 56% of the group C, 100% of the group F, and 83% of the nongroupable beta-hemolytic streptococci isolated in our clinical laboratory, whereas the incidence of S. milleri among group A and group G streptococci was estimated to be low. The role of beta-hemolytic S. milleri as a cause of human infection remains obscured by the failure to routinely differentiate S. milleri from other beta-hemolytic streptococci. Streptococcus milleri is considered an important cause of purulent disease in humans (28). In one study, S. milleri was the streptococcus most often isolated from abscesses of internal organs (28). Various infections in which S. milleri has been implicated include brain abscesses (10, 23, 28, 33), liver abscesses (5, 25, 28), appendicitis (30), peritonitis (25, 28), and endocarditis (25, 28). Although most strains of S. milleri are nonhemolytic, it is estimated that 25% are beta-hemolytic and may possess Lancefield group A, C, F, or G antigen (2). Accurate identification of beta-hemolytic strains of S. milleri is ham- pered by the practice of identifying beta-hemolytic strepto- cocci solely by their Lancefield group antigen. If emphasis is placed on serological criteria for identification, beta- hemolytic S. milleri with group A, C, or G antigen may not be distinguished from Streptococcus pyogenes (group A), Streptococcus equisimilis (group C in humans), and the large-colony group G streptococci. Differentiation of S. milleri from these organisms is required before its patholog- ical role can be accurately determined. The taxonomic status of the species S. milleri is somewhat controversial. The name S. milleri was first proposed in 1956 by Guthof (16) for a group of streptococci isolated from dental abscesses and other oral lesions. These strains were nonhemolytic and lacked Lancefield group antigen. Related organisms have been variouisly characterized as Streptococ- cus anginosus (1, 12, 36), minute hemolytic streptococci (4, 22), group F streptococci (21, 22), Streptococcus MG (24), Streptococcus intermedius (17, 18), Streptococcus constel- latus (17, 18), Streptococcus MG-intermedius (11), Strepto- coccus anginosus-constellatus (11), and Streptococcus intermedius-MG-anginosus group (32). In 1972, Colman and Williams (8) presented a classification system which was based on transformation studies, cell wall analysis, physio- * Corresponding author. logical characterization, and computer-assisted numerical analysis of the streptococci. In this system, Guthofs strains of S. milleri, indifferent streptococci described by Ottens and Winkler (26), Streptococcus MG, and minute hemolytic streptococci were placed within a single species which Colman and Williams characterized anew but placed under the old name S. milleri (8). Subsequent studies by Ball and Parker (2) and Poole and Wilson (29, 31) contributed to the current description of S. milleri: (i) alpha hemolytic, beta hemolytic, or nonhemolytic, (ii) serologically heteroge- neous, possessing one of the Lancefield group A, C, F, or G antigens or no group antigen, (iii) often enhanced by and variously requiring increased CO2 for growth, and (iv) gen- erally associated with the biochemical pattern of acid pro- duction from sucrose, salicin, trehalose, and lactose; hydro- lysis of arginine and esculin; production of acetoin from glucose (Voges-Proskauer [VP] reaction); and resistance to bacitracin and nitrofurazone. Although the term S. milleri is well established in Euro- pean nomenclature, a different taxonomy for this group of organisms is used by Facklam at the Centers for Disease Control (12). This latter system uses only species names which are already in the Approved Lists of Bacterial Names (34). Facklam's nomenclature emphasizes hemolysis and acid production from lactose in separating organisms that would be included under S. milleri by other investigators (11, 12). In Facklam's system, non-beta-hemolytic S. milleri strains are divided into the lactose-positive species S. intermedius and the lactose-negative species S. constellatus, whereas minute beta-hemolytic strains are classified sepa- rately according to their Lancefield group antigen as subspe- cies of S. anginosus (12). From a genetic standpoint, sepa- ration of these organisms into different species may not be justified. In the DNA hybridization studies of Welborn et al. (37), the American Type Culture Collection type strains of S. intermedius and S. constellatus as well as two clinical 772 on March 19, 2020 by guest http://jcm.asm.org/ Downloaded from

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Vol. 22, No. 5JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1985, p. 772-7770095-1137/85/110772-06$02.00/0Copyright C 1985, American Society for Microbiology

Incidence and Characterization of Beta-Hemolytic Streptococcusmilleri and Differentiation from S. pyogenes (Group A), S.

equisimilis (Group C), and Large-Colony Group G StreptococciJODY LAWRENCE, DAVID M. YAJKO, AND W. KEITH HADLEY*

Department of Laboratory Medicine, University of California, San Francisco General Hospital Medical Center, SanFrancisco, California 94110

Received 7 May 1985/Accepted 12 August 1985

The biochemical characteristics of 172 clinical isolates of group A, C, F, or G or "nongroupable"beta-hemolytic streptococci were examined. Among these isolates, 91 were identified as beta-hemolytic strainsof Streptococcus milleri. The remaining isolates included 20 Streptococcus pyogenes, 21 Streptococcusequisimilis, 37 large-colony group G streptococci, and 3 unidentified nongroupable isolates. A majority (84%)of the S. milled strains possessed Lancefield group antigen (3 A, 27 C, 41 F, and 5 G), whereas 15 S. milleristrains (16%) were nongroupable, Serological tests did not differentiate S. milleri isolates with group A, C, orG antigen from S. pyogenes (group A), S. equisimilis (group C), or large-colony group G streptococci.Biochemical tests which were found useful for differentiation included the Voges-Proskauer test, hydrolysis ofpyroglutamic acid and beta-D-glucuronide, bacitracin susceptibility, and acid production from ribose. S.milleri represented 56% of the group C, 100% of the group F, and 83% of the nongroupable beta-hemolyticstreptococci isolated in our clinical laboratory, whereas the incidence of S. milleri among group A and groupG streptococci was estimated to be low. The role of beta-hemolytic S. milleri as a cause of human infectionremains obscured by the failure to routinely differentiate S. milleri from other beta-hemolytic streptococci.

Streptococcus milleri is considered an important cause ofpurulent disease in humans (28). In one study, S. milleri wasthe streptococcus most often isolated from abscesses ofinternal organs (28). Various infections in which S. millerihas been implicated include brain abscesses (10, 23, 28, 33),liver abscesses (5, 25, 28), appendicitis (30), peritonitis (25,28), and endocarditis (25, 28).Although most strains of S. milleri are nonhemolytic, it is

estimated that 25% are beta-hemolytic and may possessLancefield group A, C, F, or G antigen (2). Accurateidentification of beta-hemolytic strains of S. milleri is ham-pered by the practice of identifying beta-hemolytic strepto-cocci solely by their Lancefield group antigen. If emphasis isplaced on serological criteria for identification, beta-hemolytic S. milleri with group A, C, or G antigen may notbe distinguished from Streptococcus pyogenes (group A),Streptococcus equisimilis (group C in humans), and thelarge-colony group G streptococci. Differentiation of S.milleri from these organisms is required before its patholog-ical role can be accurately determined.The taxonomic status of the species S. milleri is somewhat

controversial. The name S. milleri was first proposed in 1956by Guthof (16) for a group of streptococci isolated fromdental abscesses and other oral lesions. These strains werenonhemolytic and lacked Lancefield group antigen. Relatedorganisms have been variouisly characterized as Streptococ-cus anginosus (1, 12, 36), minute hemolytic streptococci (4,22), group F streptococci (21, 22), Streptococcus MG (24),Streptococcus intermedius (17, 18), Streptococcus constel-latus (17, 18), Streptococcus MG-intermedius (11), Strepto-coccus anginosus-constellatus (11), and Streptococcusintermedius-MG-anginosus group (32). In 1972, Colman andWilliams (8) presented a classification system which wasbased on transformation studies, cell wall analysis, physio-

* Corresponding author.

logical characterization, and computer-assisted numericalanalysis of the streptococci. In this system, Guthofs strainsof S. milleri, indifferent streptococci described by Ottensand Winkler (26), Streptococcus MG, and minute hemolyticstreptococci were placed within a single species whichColman and Williams characterized anew but placed underthe old name S. milleri (8). Subsequent studies by Ball andParker (2) and Poole and Wilson (29, 31) contributed to thecurrent description of S. milleri: (i) alpha hemolytic, betahemolytic, or nonhemolytic, (ii) serologically heteroge-neous, possessing one of the Lancefield group A, C, F, or Gantigens or no group antigen, (iii) often enhanced by andvariously requiring increased CO2 for growth, and (iv) gen-erally associated with the biochemical pattern of acid pro-duction from sucrose, salicin, trehalose, and lactose; hydro-lysis of arginine and esculin; production of acetoin fromglucose (Voges-Proskauer [VP] reaction); and resistance tobacitracin and nitrofurazone.Although the term S. milleri is well established in Euro-

pean nomenclature, a different taxonomy for this group oforganisms is used by Facklam at the Centers for DiseaseControl (12). This latter system uses only species nameswhich are already in the Approved Lists ofBacterial Names(34). Facklam's nomenclature emphasizes hemolysis andacid production from lactose in separating organisms thatwould be included under S. milleri by other investigators (11,12). In Facklam's system, non-beta-hemolytic S. milleristrains are divided into the lactose-positive species S.intermedius and the lactose-negative species S. constellatus,whereas minute beta-hemolytic strains are classified sepa-rately according to their Lancefield group antigen as subspe-cies of S. anginosus (12). From a genetic standpoint, sepa-ration of these organisms into different species may not bejustified. In the DNA hybridization studies of Welborn et al.(37), the American Type Culture Collection type strains of S.intermedius and S. constellatus as well as two clinical

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CHARACTERIZATION OF BETA-HEMOLYTIC S. MILLERI

isolates of beta-hemolytic group F streptococci (i.e., S.anginosus) were all shown to be closely related genetically.These data support the inclusion of these organisms under asingle species. Farrow and Collins (15), using optimumrenaturation conditions, confirmed the results of Welborn etal. (37). Kilpper-Balz and Schleifer (19) and Kilpper-Balz etal. (20) obtained similar results, except that when morestringent renaturation conditions were used, the DeutscheSammlung fur Mikroorganismen type strains of S. anginosusand S. constellatus hybridized less than 50% to each other.Thus, although considerable DNA studies have already beendone, the question of the taxonomy of S. milleri is stillunresolved.According to conventional identification schemes, the

streptococci are initially separated by their ability tohemolyze erythrocytes. Beta-hemolytic streptococci aresubsequently identified by serological methods, whereasnon-beta-hemolytic streptococci are differentiated to thespecies level by physiological tests (11, 13). This dichoto-mous approach can pose a problem with regard to theidentification of S. milleri. Although non-beta-hemolytic S.milleri strains are accurately identified by the physiologicalmethods used to differentiate the viridans streptococci,beta-hemolytic S. milleri strains are likely to be differenti-ated by serological tests only as group A, group C, group F,group G, or nongroupable streptococci. Consequently, beta-hemolytic S. milleri strains with group A, C, or G antigenmay be mistaken for other species of beta-hemolytic strep-tococci which have been traditionally associated with theseLancefield group antigens (i.e., S. pyogenes, group A; S.equisimilis, group C in humans; and the large-colony groupG streptococci).

In this study, clinical isolates of beta-hemolytic strepto-cocci were biochemically characterized in an effort to deter-mine practical methods for differentiating beta-hemolytic S.milleri from S. pyogenes, S. equisimilis, and the large-colonygroup G streptococci. A detailed identification of theseisolates provided an estimate of the relative incidence of S.milleri among various serological groups of beta-hemolyticstreptococci isolated in our clinical laboratory.

MATERIALS AND METHODS

Bacterial strains. The beta-hemolytic streptococci used inthis study were clinical isolates from patients at SanFrancisco General Hospital. The streak-stab method (13) wasused in determining hemolysis on Trypticase soy agar plates(BBL Microbiology Systems, Cockeysville, Md.) containing5% sheep blood. The cultures were examined after 18 to 24h of incubation at 35°C in 5% CO2

Isolates were serologically screened with the Streptexrapid latex testing system (Wellcome Reagents Div., Bur-roughs Wellcome Co., Research Triangle Park, N.C.) fordetecting Lancefield groups A, B, C, D, F, and G. Thenumber of isolates which were further characterized bio-chemically was limited by including only a sampling of thegroup A and group G isolates that were morphologicallytypical of S. pyogenes and the large-colony group G strep-tococci, respectively. However, all minute-colony isolateswith group A or group G antigen were tested. In addition, allisolates of group C, group F, and nongroupable (i.e., notgroup A, B, C, D, F, or G) streptococci obtained during thisstudy were included. Ultimately, 172 clinical isolates ofgroup A, C, F, or G or nongroupable beta-hemolytic strep-tococci were characterized by using a battery of biochemicaltests.

Biochemical tests. The following tests were incubated at35°C in an ambient atmosphere, unless stated otherwise.Acid production from glucose, inulin, lactose, mannitol,melibiose, raffinose, salicin, sorbitol, sucrose, trehalose, andribose was tested with bromocresol purple pH indicator inheart infusion broth base (Difco Laboratories, Detroit,Mich.) containing 1% (wt/vol; final concentration) carbohy-drate (all from Difco, except for ribose, which was fromSigma Chemical Co., St. Louis, Mo.) as described byFacklam (11). Esculin hydrolysis was determined in heartinfusion broth medium containing 0.03% esculin (Difco) butnot bromocresol purple pH indicator. The inoculum for eachtest was 1 drop from an 18- to 24-h culture in Todd-Hewittbroth (GIBCO Diagnostics, Madison, Wis.) supplementedwith pyridoxal hydrochloride (final concentration, 0.001%;Sigma). The carbohydrate broths were read for acid produc-tion over a 7-day incubation period. Esculin hydrolysis wasread after 5 days by adding 2 drops of 1% ferric ammoniumcitrate solution.The method used for detecting the production of acetoin

from glucose was adapted from previously described meth-ods for rapid VP testing (3, 7). A tube containing 0.2 ml ofmethyl red-VP broth (GIBCO) was inoculated with a loopfulof growth from an overnight culture on blood agar andincubated for 4 to 6 h. After incubation, 1 drop each of thefollowing solutions was added: 0.5% creatine (Sigma), alpha-naphthol (Analytab Products, Plainview, N.Y.), and 40%KOH. The tube was shaken and observed for the develop-ment of a pink-red color within 30 min.Four rapid tests were used for the detection of preformed

enzymes. These included a chromogenic substrate test forthe hydrolysis of pyroglutamic acid (PYR) and fluorogenicsubstrate tests for the hydrolysis of N-acetyl-beta-D-glucosaminide (NAG), beta-D-glucuronide (P-GUR), andbeta-D-galactoside (,B-GAL). The method used filter paperdisks impregnated with substrate. A loop or wooden stickwas used to inoculate the disks with colonies taken from anovernight culture on blhrod agar. A pyroglutamyl-naphthylamide derivative (0.5 mg/ml in methanol; EY Lab-oratories, San Mateo, Calif. l was used in the PYR test. Afterthe disk was inoculated, 1 di op of fast garnet dye (0.1 mg/ml;EY Laboratories) was added. The hydrolysis of PYR wasindicated by the development of a pink-red color within 10min at room temperature. An adaptation of the methoddescribed by Slifkin and Gil (35) was used to detect thehydrolysis of fluorogenic 4-methylumbelliferyl conjugates(Sigma) of NAG, P-GUR, and 1-GAL. The disks wereimpregnated with 1-mg/ml solutions of the individual sub-strates in 0.05 M phosphate-buffered saline, pH 6.5. Thedisks were inoculated and incubated at 35°C for 30 min. Onedrop of a saturated sodium bicarbonate solution was added,and the disks were observed immediately for fluorescenceunder a long-wave (366-nm) UV lamp.

Isolates were tested for susceptibility to bacitracin withTaxo A disks (0.04 U of bacitracin; BBL MicrobiologySystems) (13).

RESULTSOf the 172 clinical isolates of beta-hemolytic streptococci

characterized in this study, 91 proved to be biochemicallyidentical to S. milleri. The remaining isolates included 20 S.pyogenes (group A), 21 S. equisimilis (group C), 37 large-colony group G streptococci, and 3 unidentified isolates.Based on serological agglutination tests, a majority (84%) ofthe S. milleri strains were found to have Lancefield groupantigen (3 A, 27 C, 41 F, and 5 G). However, 15 S. milleri

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774 LAWRENCE ET AL.

TABLE 1. Origins of 172 clinical isolates of beta-hemolytic streptococci from human sourcesNo. of isolates of:

Source Total Total S. Inilleri group S. py'ogenes S. eqoi.iinilis Large-colony UnidentifiedgroupA group C group 6 nongroupablestreptococci S. /nilleni A C F G Nongroupable group A group C streptococci streptococci

Blood 13 3 1 2 4 1 5Other fluids' 10 5 2 3 1 1 3Respiratory tractUpper 10 5 1 1 1 1 1 2 1 2Lower 20 17 1 7 5 2 2 1 1 1

Wounds 34 17 4 10 3 4 5 8Abscesses 47 29 5 14 1 9 9 2 6 1Genital tract 5 3 1 2 2Miscellaneous' 5 1 1 1 3Unspecified 28 11 1 6 3 1 1 8 8

' Peritoneal, synovial, and pleural.b Includes urine, breast colostrum, newborn gastric aspirate, and eye.

strains (16%) did not react with any of the antisera to groupA, B, C, D, F, or G. These strains are described here asnongroupable. Table 1 shows the distribution of isolates bygeneral site of isolation. Since not all isolates of S. pyogenesand the large-colony group G streptococci were included inthe study, it was not possible to determine the incidence ofbeta-hemolytic S. milleri in various sites or specimens.However, beta-hemolytic S. milleri strains were frequentlyrecovered from wounds and abscesses and from both theupper and lower respiratory tracts.The biochemical reactions of the organisms are summa-

rized in Table 2. Isolates identified as S. milleri characteris-tically produced small to pinpoint colonies on 5% sheepblood agar after 24 h of incubation in 5% CO2. Variations incolony morphology and subtle differences in beta hemolysiswere observed among these strains. Microscopic examina-tion of various cultures of S. milleri confirmed their produc-tion of beta hemolysis as defined by Facklam and Carey (13).

S. milleri is usually considered a member of the viridansgroup of streptococci. Although carbohydrate tests are usedfor identifying species of viridans streptococci (11), thesetests were not very useful in differentiating beta-hemolytic S.milleri strains from other beta-hemolytic species of strepto-cocci (Table 2). S. milleri isolates were distinguished fromother beta-hemolytic streptococci biochemically by beingpositive in the VP test, negative for the hydrolysis of PYRand ,B-GUR, and negative for acid production from ribose(Table 3). Some variation in the other biochemical reactionswas found when the S. milleri strains were divided accordingto their serological reactions (Table 2). Of the S. milleristrains with group C antigen, 15 (56%) were susceptible tobacitracin, whereas all other S. milleri strains were resistant.In addition, an association was observed in the group Cstrains between bacitracin susceptibility and a lack of acidproduction from trehalose. Only 4 of 15 (27%) of the baci-tracin-susceptible strains but 10 of 12 (83%) of the bacitracin-resistant strains of S. milleri with group C antigen producedacid from trehalose. Two isolates of S. milleri with group Gantigen were unique among the isolates in this study in thatthey produced acid from raffinose and melibiose.The biochemical patterns for S. equisimilis (group C) and

the large-colony group G streptococci were virtually identi-cal. With few exceptions, the strains in both groups werecharacterized by acid production from ribose, hydrolysis ofboth 3-GUR and NAG, and a negative VP test. Thus, S.equisimilis and the large-colony group G streptococci could

only be differentiated from one another by their Lancefieldgroup antigen. Approximately one-third each of S.equisimilis and the large-colony group G streptococci (33and 27%, respectively) was susceptible to bacitracin, butnone hydrolyzed PYR. In contrast, all strains of S. pyogeneswere susceptible to bacitracin and hydrolyzed PYR.

Although S. pyogenes, S. equisimilis, and the large-colonygroup G streptococci generally produced larger coloniesthan did S. milleri, colony size was not a definitive charac-teristic. Several isolates of group C streptococci producedcolonies of only moderate size, yet they were identified as S.equisimilis because they produced acid from ribose, hydro-lyzed ,B-GUR, and were negative in the VP test. Threestrains of group G streptococci which were biochemicallymost consistent with the large-colony form appeared mor-phologically similar to S. milleri.Three isolates remained unidentified by the methods used

in this study. All three were nongroupable in the Streptexsystem. One strain, isolated from a throat specimen, pro-duced a biochemical pattern similar to that described byFacklam (11) for Streptococcus sanguis II. S. sanguis II,however, is described by Facklam (11) as not being betahemolytic. The remaining two isolates, one from a handabscess and the other from a throat specimen, producedunusual but identical biochemical patterns. These twostrains do not fit any of the 28 phenons of streptococcidescribed by Bridge and Sneath (6) and may represent a newspecies of streptococcus.

In terms of serological identification, the incidence ofbeta-hemolytic S. milleri among the clinical isolates ofbeta-hemolytic streptococci in our study was as follows: S.milleri represented 27 of 48 (56%) isolates of group Cstreptococci, all isolates of group F streptococci, and 15 of18 (83%) isolates of nongroupable streptococci. The preciseincidence of beta-hemolytic S. milleri among clinical isolatesof group A and group G streptococci was not determined inthis study because not all isolates with group A or G antigenwere included. However, this incidence is estimated to below, since only three isolates of S. milleri with group Aantigen and five isolates of S. milleri with group G antigenwere found as a result of examining all clinical isolates ofgroup A and group G streptococci for colony morphology.

DISCUSSIONThe results of this study indicate that Lancefield serolog-

ical grouping does not provide sufficient information for

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CHARACTERIZATION OF BETA-HEMOLYTIC S. MILLERI 775

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776 LAWRENCE ET AL.

TABLE 3. Differentiation of beta-hemolytic S. milleri from otherbeta-hemolytic streptococci

Reaction" for:

Hydrolysis of: AcidStreptococci VP Bacitracin production

PYR ,B-GUR test susceptibility fromPYRp-GUR ~~~~~~~ribose

Group AS. milleri - - +S. pyogenes + v - +

Group CS. milleri - - + vS. equisimilis - + - v +

Group GS. milleri - - +Large-colony - + - v +

NongroupableS. milleri - - +Unidentified -

Group FS. milleri - - +

a -, Negative; +, positive; v, variable.

species identification of beta-hemolytic streptococci ofgroups A, C, and G isolated from humans. It also shows thatthe conventional biochemical tests used for identifying spe-cies of viridans streptococci do not distinguish beta-hemolytic S. milleri strains from other beta-hemolytic spe-cies of streptococci.The isolates identified in this study as S. milleri conform in

most aspects to the description of this species given byColman and Williams (8) and by Ball and Parker (2). Theseinvestigators noted that esculin hydrolysis and acid produc-tion from lactose, raffinose, and melibiose were less commonamong beta-hemolytic strains than among non-beta-hemolytic strains of S. milleri. However, our data and thoseof Poole and Wilson (29) suggest that these reactions mayvary among beta-hemolytic S. milleri in relation to theirLancefield group antigens (Table 2). We found that a major-ity of beta-hemolytic S. milleri strains with group C or Gantigen were positive for esculin hydrolysis and acid produc-tion from lactose. In addition, most of these strains hydro-lyzed NAG. The opposite was true for beta-hemolytic S.milleri strains with group A or F antigen or with no groupantigen. Poole and Wilson (29) found a similar pattern foresculin hydrolysis and acid production from lactose amongthe beta-hemolytic streptococci which they identified as S.

milleri. In both studies, acid production from trehalose wasless common among beta-hemolytic S. milleri strains withgroup C antigen than among the majority of beta-hemolyticS. milleri. We also found that over half (56%) of our S.

milleri isolates with group C antigen were susceptible tobacitracin, whereas all of our other S. milleri isolates wereresistant. Acid production from raffinose was found onlyamong S. milleri isolates with group G antigen. Two of ourfive (40%) and 46% of Poole and Wilson's (29) minute-colony-forming beta-hemolytic group G isolates producedacid from raffinose. These strains are biochemically similarto a subset of S. milleri described by Ball and Parker (2) as

producing acid from raffinose, melibiose, and less oftenmannitol.

The serological and biochemical heterogeneity observedamong the S. milleri strains in this and previous investiga-tions (2, 8) suggests the possibility that S. milleri mayrepresent more than one species. DNA hybridization studies(15, 37) support the inclusion of S. intermedius, S. constel-latus, and the beta-hemolytic group F streptococci (S.anginosus) under a single species (i.e., S. milleri). However,genetic evidence is still needed to verify the additionalinclusion of minute beta-hemolytic strains with Lancefieldgroup A, C, or G antigen or with no group antigen under S.milleri. Hybridization studies may also determine whetherthere is any taxonomic significance associated with bacitra-cin susceptibility among minute beta-hemolytic group Cstreptococci or with acid prodution from raffinose and meli-biose among minute beta-hemolytic group G streptococciand among the strains described by Ball and Parker (2). Inthe absence of further genetic data, the inclusion of thesestrains under S. milleri is based on their serological andphysiological similarities to accepted members of this spe-cies.

All of our S. milleri isolates were positive in the VP test (4to 6 h) and were differentiated from S. pyogenes, S.equisimilis, and the large-colony group G streptococci onthis basis. Our data agree with the findings of Bucher andvon Graevenitz (7). In studies by Ball and Parker (2) andPoole and Wilson (29), although the vast majority of S.milleri strains were positive in the VP test, a few werenegative when a method requiring 5 days of incubation wasused. In addition, Ball and Parker (2) found that 11% of theS. pyogenes strains thus tested produced a positive VPreaction. This suggests that the value of the VP reaction as adifferential test may depend on the method used and thatadditional tests may be necessary to differentiate beta-hemolytic S. milleri strains from other species of beta-hemolytic streptococci. Our data indicate that several testscan be used as alternatives or supplements to the VP test forthis purpose.Although only three isolates of beta-hemolytic S. milleri

with group A antigen were found, all three strains werenegative in the PYR test. In contrast, all S. pyogenes strainswere positive. Thus, the PYR test may serve as a rapidmethod for differentiating these organisms. Our limited datasuggest that the bacitracin test may also be used for thispurpose. However, the PYR test has the advantage ofdifferentiating between S. pyogenes and the bacitracin-susceptible strains of either S. equisimilis or the large-colonygroup G streptococci, since all strains of the latter are PYRnegative (14; J. Lawrence, D. M. Yajko, and W. K. Hadley,Program Abstr. 24th Intersci. Conf. Antimicrob. AgentsChemother., abstr. no. 19, 1984).We found that serological testing is necessary to differen-

tiate between S. equisimilis and the large-colony group Gstreptococci. As evident from this study and from previousdescriptions of these organisms (9, 27), S. equisimilis and thelarge-colony group G streptococci have virtually identicalbiochemical patterns. Recent DNA-DNA hybridization testshave shown that S. equisimilis, large-colony group G strep-tococci, and group L streptococci actually represent a singlespecies (15).The clinical significance of S. milleri has been examined

by several investigators (25, 28-31, 33). However, since inmost clinical laboratories beta-hemolytic isolates of S. mil-leri are not distinguished from other species of streptococci,the incidence and pathogenicity of S. milleri may be under-estimated. In the present study, a high incidence of S. milleriwas observed among the clinical isolates examined. The

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Page 6: Incidence Characterization of Beta-Hemolytic Streptococcus ... · be distinguished from Streptococcus pyogenes (group A), Streptococcus equisimilis (group C in humans), and the

CHARACTERIZATION OF BETA-HEMOLYTIC S. MILLERI

occurrence of streptococcal group A, C, and G antigensamong beta-hemolytic strains of S. milleri demonstrates thatcorrect identification of beta-hemolytic streptococci to thespecies level requires a combination of serological andbiochemical tests. The rapid biochemical tests describedhere have been found to be useful for this purpose.

ADDENDUM IN PROOF

After this manuscript was submitted for publication, K. L.Ruoff, L. J. Kunz, and M. J. Ferraro published similarfindings (J. Clin. Microbiol. 22:149-151, 1985). They foundthat S. milleri accounted for 75% of group C, 15% of groupG, 100% of group F, and 75% of nongroupable beta-hemolytic streptococci of clinical origin. No S. milleri withgroup A antigen were found in their study.

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