Francisco, California logical characterization, and computer-assisted numerical analysis of the streptococci. In this system, Guthofs strains

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1985, p /85/ $02.00/0 Copyright C 1985, American Society for Microbiology Vol. 22, No. 5 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 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 hampered by the practice of identifying beta-hemolytic streptococci solely by their Lancefield group antigen. If emphasis is placed on serological criteria for identification, betahemolytic 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 pathological 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 Streptococcus anginosus (1, 12, 36), minute hemolytic streptococci (4, 22), group F streptococci (21, 22), Streptococcus MG (24), Streptococcus intermedius (17, 18), Streptococcus constellatus (17, 18), Streptococcus MG-intermedius (11), Streptococcus 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. 772 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 heterogeneous, 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) generally associated with the biochemical pattern of acid production from sucrose, salicin, trehalose, and lactose; hydrolysis 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 European 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 ofbacterial 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 separately according to their Lancefield group antigen as subspecies of S. anginosus (12). From a genetic standpoint, separation 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

2 VOL. 22, 1985 CHARACTERIZATION OF BETA-HEMOLYTIC S. MILLERI 773 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 a single species. Farrow and Collins (15), using optimum renaturation conditions, confirmed the results of Welborn et al. (37). Kilpper-Balz and Schleifer (19) and Kilpper-Balz et al. (20) obtained similar results, except that when more stringent renaturation conditions were used, the Deutsche Sammlung fur Mikroorganismen type strains of S. anginosus and S. constellatus hybridized less than 50% to each other. Thus, although considerable DNA studies have already been done, the question of the taxonomy of S. milleri is still unresolved. According to conventional identification schemes, the streptococci are initially separated by their ability to hemolyze erythrocytes. Beta-hemolytic streptococci are subsequently identified by serological methods, whereas non-beta-hemolytic streptococci are differentiated to the species level by physiological tests (11, 13). This dichotomous approach can pose a problem with regard to the identification of S. milleri. Although non-beta-hemolytic S. milleri strains are accurately identified by the physiological methods used to differentiate the viridans streptococci, beta-hemolytic S. milleri strains are likely to be differentiated by serological tests only as group A, group C, group F, group G, or nongroupable streptococci. Consequently, betahemolytic S. milleri strains with group A, C, or G antigen may be mistaken for other species of beta-hemolytic streptococci which have been traditionally associated with these Lancefield group antigens (i.e., S. pyogenes, group A; S. equisimilis, group C in humans; and the large-colony group G streptococci). In this study, clinical isolates of beta-hemolytic streptococci were biochemically characterized in an effort to determine practical methods for differentiating beta-hemolytic S. milleri from S. pyogenes, S. equisimilis, and the large-colony group G streptococci. A detailed identification of these isolates provided an estimate of the relative incidence of S. milleri among various serological groups of beta-hemolytic streptococci isolated in our clinical laboratory. MATERIALS AND METHODS Bacterial strains. The beta-hemolytic streptococci used in this study were clinical isolates from patients at San Francisco General Hospital. The streak-stab method (13) was used in determining hemolysis on Trypticase soy agar plates (BBL Microbiology Systems, Cockeysville, Md.) containing 5% sheep blood. The cultures were examined after 18 to 24 h of incubation at 35 C in 5% CO2 Isolates were serologically screened with the Streptex rapid latex testing system (Wellcome Reagents Div., Burroughs Wellcome Co., Research Triangle Park, N.C.) for detecting Lancefield groups A, B, C, D, F, and G. The number of isolates which were further characterized biochemically was limited by including only a sampling of the group A and group G isolates that were morphologically typical of S. pyogenes and the large-colony group G streptococci, respectively. However, all minute-colony isolates with group A or group G antigen were tested. In addition, all isolates of group C, group F, and nongroupable (i.e., not group A, B, C, D, F, or G) streptococci obtained during this study were included. Ultimately, 172 clinical isolates of group A, C, F, or G or nongroupable beta-hemolytic streptococci were characterized by using a battery of biochemical tests. Biochemical tests. The following tests were incubated at 35 C in an ambient atmosphere, unless stated otherwise. Acid production from glucose, inulin, lactose, mannitol, melibiose, raffinose, salicin, sorbitol, sucrose, trehalose, and ribose was tested with bromocresol purple ph indicator in heart infusion broth base (Difco Laboratories, Detroit, Mich.) containing 1% (wt/vol; final concentration) carbohydrate (all from Difco, except for ribose, which was from Sigma Chemical Co., St. Louis, Mo.) as described by Facklam (11). Esculin hydrolysis was determined in heart infusion broth medium containing 0.03% esculin (Difco) but not bromocresol purple ph indicator. The inoculum for each test was 1 drop from an 18- to 24-h culture in Todd-Hewitt broth (GIBCO Diagnostics, Madison, Wis.) supplemented with pyridoxal hydrochloride (final concentration, 0.001%; Sigma). The carbohydrate broths were read for acid production over a 7-day incubation period. Esculin hydrolysis was read after 5 days by adding 2 drops of 1% ferric ammonium citrate solution. The method used for detecting the production of acetoin from glucose was adapted from previously described methods for rapid VP testing (3, 7). A tube containing 0.2 ml of methyl red-vp broth (GIBCO) was inoculated with a loopful of growth from an overnight culture on blood agar and incubated for 4 to 6 h. After incubation, 1 drop each of the following solutions was added: 0.5% creatine (Sigma), alphanaphthol (Analytab Products, Plainview, N.Y.), and 40% KOH. The tube was shaken and observed for the development 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 for the hydrolysis of pyroglutamic acid (PYR) and fluorogenic substrate tests for the hydrolysis of N-acetyl-beta-Dglucosaminide (NAG), beta-d-glucuronide (P-GUR), and beta-d-galactoside (,B-GAL). The method used filter paper disks impregnated with substrate. A loop or wooden stick was used to inoculate the disks with colonies taken from an overnight culture on blhrod agar. A pyroglutamylnaphthylamide derivative (0.5 mg/ml in methanol; EY Laboratories, San Mateo, Calif. l was used in the PYR test. After the disk was inoculated, 1 di op of fast garnet dye (0.1 mg/ml; EY Laboratories) was added. The hydrolysis of PYR was indicated by the development of a pink-red color within 10 min at room temperature. An adaptation of the method described by Slifkin and Gil (35) was used to detect the hydrolysis of fluorogenic 4-methylumbelliferyl conjugates (Sigma) of NAG, P-GUR, and 1-GAL. The disks were impregnated with 1-mg/ml solutions of the individual substrates in 0.05 M phosphate-buffered saline, ph 6.5. The disks were inoculated and incubated at 35 C for 30 min. One drop of a saturated sodium bicarbonate solution was added, and the disks were observed immediately for fluorescence under a long-wave (366-nm) UV lamp. Isolates were tested for susceptibility to bacitracin with Taxo A disks (0.04 U of bacitracin; BBL Microbiology Systems) (13). RESULTS Of the 172 clinical isolates of beta-hemolytic streptococci characterized in this study, 91 proved to be biochemically identical to S. milleri. The remaining isolates included 20 S. pyogenes (group A), 21 S. equisimilis (group C), 37 largecolony group G streptococci, and 3 unidentified isolates. Based on serological agglutination tests, a majority (84%) of the S. milleri strains were found to have Lancefield group antigen (3 A, 27 C, 41 F, and 5 G). However, 15 S. milleri

3 774 LAWRENCE ET AL. J. CLIN. MICROBIOL. TABLE 1. Origins of 172 clinical isolates of beta-hemolytic streptococci from human sources No. of isolates of: Source Total Total S. Inilleri group S. py'ogenes S. eqoi.iinilis Large-colony Unidentified groupa group C group 6 nongroupable streptococci S. /nilleni A C F G Nongroupable group A group C streptococci streptococci Blood Other fluids' Respiratory tract Upper Lower Wounds Abscesses Genital tract Miscellaneous' Unspecified ' 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 group A, B, C, D, F, or G. These strains are described here as nongroupable. Table 1 shows the distribution of isolates by general site of isolation. Since not all isolates of S. pyogenes and the large-colony group G streptococci were included in the study, it was not possible to determine the incidence of beta-hemolytic S. milleri in various sites or specimens. However, beta-hemolytic S. milleri strains were frequently recovered from wounds and abscesses and from both the upper and lower respiratory tracts. The biochemical reactions of the organisms are summarized in Table 2. Isolates identified as S. milleri characteristically produced small to pinpoint colonies on 5% sheep blood agar after 24 h of incubation in 5% CO2. Variations in colony morphology and subtle differences in beta hemolysis were observed among these strains. Microscopic examination of various cultures of S. milleri confirmed their production of beta hemolysis as defined by Facklam and Carey (13). S. milleri is usually considered a member of the viridans group of streptococci. Although carbohydrate tests are used for identifying species of viridans streptococci (11), these tests were not very useful in differentiating beta-hemolytic S. milleri strains from other beta-hemolytic species of streptococci (Table 2). S. milleri isolates were distinguished from other beta-hemolytic streptococci biochemically by being positive in the VP test, negative for the hydrolysis of PYR and,b-gur, and negative for acid production from ribose (Table 3). Some variation in the other biochemical reactions was found when the S. milleri strains were divided according to their serological reactions (Table 2). Of the S. milleri strains with group C antigen, 15 (56%) were susceptible to bacitracin, whereas all other S. milleri strains were resistant. In addition, an association was observed in the group C strains between bacitracin susceptibility and a lack of acid production from trehalose. Only 4 of 15 (27%) of the bacitracin-susceptible strains but 10 of 12 (83%) of the bacitracinresistant strains of S. milleri with group C antigen produced acid from trehalose. Two isolates of S. milleri with group G antigen were unique among the isolates in this study in that they produced acid from raffinose and melibiose. The biochemical patterns for S. equisimilis (group C) and the large-colony group G streptococci were virtually identical. With few exceptions, the strains in both groups were characterized by acid production from ribose, hydrolysis of both 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 Lancefield group antigen. Approximately one-third each of S. equisimilis and the large-colony group G streptococci (33 and 27%, respectively) was susceptible to bacitracin, but none hydrolyzed PYR. In contrast, all strains of S. pyogenes were susceptible to bacitracin and hydrolyzed PYR. Although S. pyogenes, S. equisimilis, and the large-colony group G streptococci generally produced larger colonies than did S. milleri, colony size was not a definitive characteristic. Several isolates of group C streptococci produced colonies of only moderate size, yet they were identified as S. equisimilis because they produced acid from ribose, hydrolyzed,b-gur, and were negative in the VP test. Three strains of group G streptococci which were biochemically most consistent with the large-colony form appeared morphologically similar to S. milleri. Three isolates remained unidentified by the methods used in this study. All three were nongroupable in the Streptex system. One strain, isolated from a throat specimen, produced a biochemical pattern similar to that described by Facklam (11) for Streptococcus sanguis II. S. sanguis II, however, is described by Facklam (11) as not being beta hemolytic. The remaining two isolates, one from a hand abscess and the other from a throat specimen, produced unusual but identical biochemical patterns. These two strains do not fit any of the 28 phenons of streptococci described by Bridge and Sneath (6) and may represent a new species of streptococcus. In terms of serological identification, the incidence of beta-hemolytic S. milleri among the clinical isolates of beta-hemolytic streptococci in our study was as follows: S. milleri represented 27 of 48 (56%) isolates of group C streptococci, all isolates of group F streptococci, and 15 of 18 (83%) isolates of nongroupable streptococci. The precise incidence of beta-hemolytic S. milleri among clinical isolates of group A and group G streptococci was not determined in this study because not all isolates with group A or G antigen were included. However, this incidence is estimated to be low, since only three isolates of S. milleri with group A antigen and five isolates of S. milleri with group G antigen were found as a result of examining all clinical isolates of group A and group G streptococci for colony morphology. DISCUSSION The results of this study indicate that Lancefield serological grouping does not provide sufficient information for

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5 776 LAWRENCE ET AL. TABLE 3. Differentiation of beta-hemolytic S. milleri from other beta-hemolytic streptococci Reaction" for: Hydrolysis of: Acid Streptococci VP Bacitracin production test PYR,B-GUR susceptibility from PYRp-GUR ~~~~~~~ribose Group A S. milleri S. pyogenes + v - + Group C S. milleri v S. equisimilis v + Group G S. milleri Large-colony v + Nongroupable S. milleri - Unidentified Group F S. milleri a -, Negative; +, positive; v, variable. species identification of beta-hemolytic streptococci of groups A, C, and G isolated from humans. It also shows that the conventional biochemical tests used for identifying species of viridans streptococci do not distinguish betahemolytic S. milleri strains from other beta-hemolytic species of streptococci. The isolates identified in this study as S. milleri conform in most aspects to the description of this species given by Colman and Williams (8) and by Ball and Parker (2). These investigators noted that esculin hydrolysis and acid production from lactose, raffinose, and melibiose were less common among beta-hemolytic strains than among non-betahemolytic strains of S. milleri. However, our data and those of Poole and Wilson (29) suggest that these reactions may vary among beta-hemolytic S. milleri in relation to their Lancefield group antigens (Table 2). We found that a majority of beta-hemolytic S. milleri strains with group C or G antigen were positive for esculin hydrolysis and acid production from lactose. In addition, most of these strains hydrolyzed NAG. The opposite was true for beta-hemolytic S. milleri strains with group A or F antigen or with no group antigen. Poole and Wilson (29) found a similar pattern for esculin hydrolysis and acid production from lactose among the beta-hemolytic streptococci which they identified as S. milleri. In both studies, acid production from trehalose was less common among beta-hemolytic S. milleri strains with group C antigen than among the majority of beta-hemolytic S. milleri. We also found that over half (56%) of our S. milleri isolates with group C antigen were susceptible to bacitracin, whereas all of our other S. milleri isolates were resistant. Acid production from raffinose was found only among S. milleri isolates with group G antigen. Two of our five (40%) and 46% of Poole and Wilson's (29) minutecolony-forming beta-hemolytic group G isolates produced acid from raffinose. These strains are biochemically similar to a subset of S. milleri described by Ball and Parker (2) as producing acid from raffinose, melibiose, and less often mannitol. J. CLIN. MICROBIOL. The serological and biochemical heterogeneity observed among the S. milleri strains in this and previous investigations (2, 8) suggests the possibility that S. milleri may represent more than one species. DNA hybridization studies (15, 37) support the inclusion of S. intermedius, S. constellatus, 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 additional inclusion of minute beta-hemolytic strains with Lancefield group A, C, or G antigen or with no group antigen under S. milleri. Hybridization studies may also determine whether there is any taxonomic significance associated with bacitracin susceptibility among minute beta-hemolytic group C streptococci or with acid prodution from raffinose and melibiose among minute beta-hemolytic group G streptococci and among the strains described by Ball and Parker (2). In the absence of further genetic data, the inclusion of these strains under S. milleri is based on their serological and physiological similarities to accepted members of this species. All of our S. milleri isolates were positive in the VP test (4 to 6 h) and were differentiated from S. pyogenes, S. equisimilis, and the large-colony group G streptococci on this basis. Our data agree with the findings of Bucher and von Graevenitz (7). In studies by Ball and Parker (2) and Poole and Wilson (29), although the vast majority of S. milleri strains were positive in the VP test, a few were negative when a method requiring 5 days of incubation was used. In addition, Ball and Parker (2) found that 11% of the S. pyogenes strains thus tested produced a positive VP reaction. This suggests that the value of the VP reaction as a differential test may depend on the method used and that additional tests may be necessary to differentiate betahemolytic S. milleri strains from other species of betahemolytic streptococci. Our data indicate that several tests can be used as alternatives or supplements to the VP test for this purpose. Although only three isolates of beta-hemolytic S. milleri with group A antigen were found, all three strains were negative in the PYR test. In contrast, all S. pyogenes strains were positive. Thus, the PYR test may serve as a rapid method for differentiating these organisms. Our limited data suggest that the bacitracin test may also be used for this purpose. However, the PYR test has the advantage of differentiating between S. pyogenes and the bacitracinsusceptible strains of either S. equisimilis or the large-colony group G streptococci, since all strains of the latter are PYR negative (14; J. Lawrence, D. M. Yajko, and W. K. Hadley, Program Abstr. 24th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 19, 1984). We found that serological testing is necessary to differentiate between S. equisimilis and the large-colony group G streptococci. As evident from this study and from previous descriptions of these organisms (9, 27), S. equisimilis and the large-colony group G streptococci have virtually identical biochemical patterns. Recent DNA-DNA hybridization tests have shown that S. equisimilis, large-colony group G streptococci, and group L streptococci actually represent a single species (15). The clinical significance of S. milleri has been examined by several investigators (25, 28-31, 33). However, since in most clinical laboratories beta-hemolytic isolates of S. milleri are not distinguished from other species of streptococci, the incidence and pathogenicity of S. milleri may be underestimated. In the present study, a high incidence of S. milleri was observed among the clinical isolates examined. The

6 VOL. 22, 1985 CHARACTERIZATION OF BETA-HEMOLYTIC S. MILLERI 777 occurrence of streptococcal group A, C, and G antigens among beta-hemolytic strains of S. milleri demonstrates that correct identification of beta-hemolytic streptococci to the species level requires a combination of serological and biochemical tests. The rapid biochemical tests described here 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 similar findings (J. Clin. Microbiol. 22: , 1985). They found that S. milleri accounted for 75% of group C, 15% of group G, 100% of group F, and 75% of nongroupable betahemolytic streptococci of clinical origin. No S. milleri with group A antigen were found in their study. LITERATURE CITED 1. Andrewes, F. W., and T. J. JIorder A study of the streptococci pathogenic for man. Lancet ii: , , and Ball, L. C., and M. T. 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J. Syst. Bacteriol. 30: Slifkin, M., and G. M. Gil Rapid biochemical tests for the identification of group A, B, C, F, and G streptococci from throat cultures. J. Clin. Microbiol. 18: Smith, F. R., and J. M. Sherman The hemolytic streptococci of human feces. J. Infect. Dis. 62: Welborn, P. P., W. K. Hadley, E. Newbrun, and D. M. Yajko Characterization of strains of viridans streptococci by deoxyribonucleic acid hybridization and physiological tests. Int. J. Syst. Bacteriol. 33: