JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1993, p. 804-807 Vol. 31, No.

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0095-1137/93/040804-04$02.00/0
Copyright © 1993, American Society for Microbiology

Role of Beta-Hemolytic Group C Streptococci in Pharyngitis:

Incidence and Biochemical Characteristics of Streptococcus
equisimilis and Streptococcus anginosus in Patients
and Healthy Controls
KAREN FOX,' JAMES TURNER,2'3 AND ALVIN FOX`*
Department of Microbiology and Immunology' and Department of Internal Medicine,2 School of Medicine,
and Thomson Student Health Center, 3 University of South Carolina, Columbia, South Carolina 29208

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Received 28 September 1992/Accepted 18 December 1992

The biochemical characteristics and the isolation rates of the two Lancefield group C streptococcal species (S.
equisimilis and S. anginosus) from patients with pharyngitis and asymptomatic controls were compared. Some
239 strains of beta-hemolytic group C streptococci were isolated from 1,480 patients (209 strains) with
pharyngitis and 227 controls (30 strains). A total of 44 strains displayed broad hemolysis, were Vogues-
Proskauer test negative, and produced glucuronidase. Some 72.7% of these strains also fermented ribose. A
second group of 159 strains was Voges-Proskauer test positive and glucuronidase negative; 98.8% also
displayed minute hemolysis and only 3.7% fermented ribose. These two groups represent typical large-colony
S. equisimilis and small-colony S. anginosus, respectively. A small number of strains (36 in total) exhibited
intermediate characteristics; morphologically, all 36 strains resembled S. anginosus, but 6 strains biochemi-
cally resembled S. equisimilis. No strains of S. equisimilis fermented sorbitol (unlike S. zooepidemicus) and all
fermented trehalose (unlike S. equi). The structural carbohydrate profiles of S. equisimilis and S. anginosus
both included galactosamine (consistent with their being group C organisms), but the profiles were not
distinguishable. In total, 78.5% of strains from controls and 83.3% of strains from patients were determined
to be the species S. anginosus. S. equisimilis was isolated from 3.0%o of patients and 2.2% of controls, and S.
anginosus was isolated from 11.1% of patients and 11.0%6 of controls. Thus, S. equisimilis and S. anginosus are
both members of the normal flora of asymptomatic individuals. The incidence and biochemical characteristics
of these two species are similar in patients who are healthy and those who have disease. However, a companion
article provides clinical evidence associating S. equisimilis (but not S. anginosus) with pharyngitis.

The roles of Streptococcus equisimilis and Streptococcus
anginosus in causing endemic pharyngitis are still controver-
sial (6, 14, 16, 19, 27), although Lancefield group C strepto-
cocci are implicated in outbreaks of pharyngitis and associ-
ated disorders (3, 4, 8, 10, 25). It is well known that S.
equisimilis and S. anginosus are often isolated from clinical
specimens. S. anginosus is the most common beta-hemolytic
group C streptococcus isolated from the human throat (5, 17,
18, 21). Other group C streptococcal species (including S.
equi and S. zooepidemicus) are generally isolated only from
patients with zoonotic infections (2, 6, 13). To this point, no
study has compared the isolation rates and biochemical
characteristics of S. equisimilis and S. anginosus in patients
with pharyngitis versus those in asymptomatic controls (7,
15, 16, 27). A significantly higher isolation rate of either
species from patients with pharyngitis than from controls
would suggest a causative role (6).
Group C streptococcal strains fall into two morphologic
categories: large and small colony types. These colony types
are distantly related genetically (22). Small-colony group C
streptococci isolated from humans are primarily S. angino-
sus, while large-colony group C streptococci are primarily S.
equisimilis. S. anginosus can also be of serological groups A,
F, and G or ungroupable and show a high degree of DNA
homology to nonhemolytic organisms (11). In the European
*
Corresponding author.
804
literature, "S. milleri" refers to S. anginosus and related
organisms. These organisms display great variabilities in
their physiological characteristics (9).
Several clinical reports have emphasized the discriminat-
ing power of the Voges-Proskauer (VP) reaction (which
detects the production of acetoin) in differentiating S. angi-
nosus from S. equisimilis (5, 17). However, a substantial
number of S. anginosus strains can be VP test negative (1,
14). S. anginosus, in contrast to S. equisimilis, generally
does not display glucuronidase activity (5). Fermentation of
ribose by S. equisimilis but not S. anginosus is also an
important physiological distinction (18). S. anginosus colo-
nies on sheep blood agar generally have minute zones of
hemolysis, while colonies of S. equisimilis have broad zones
(6). However, hemolysis characteristics have not been con-
sistently used in clinical differentiation. The ability to fer-
ment certain sugars, including trehalose, sorbitol, or ribose,
is used for differentiating the three large-colony group C
beta-hemolytic species (S. equisimilis, S. equi, and S. zooep-
idemicus [22, 24]).
The purpose of the present study was to determine the
isolation rates and physiological characteristics of S. equisi-
milis and S. anginosus from patients with pharyngitis and
healthy controls. A consecutive series of 239 human strains
of beta-hemolytic group C streptococci collected over a
1-year period was examined. The companion article (26)
correlates the clinical features of pharyngitis with the isola-
tion of S. anginosus and S. equisimilis.
VOL. 31, 1993
TABLE 1. Biochemical properties of human group C streptococci isolated from patients with pharyngitis and healthy controls
Species and patient
(total no. of Complete Acetoin
strains) hemolysis production
S. equisimilis
Sick (45) 86.7 0 100
Control (5) 100 0 100
Total (50) 88.0 0 100
S. anginosus
Sick (164) 1.2 86.0
Control (25) 0.0 72.0
Total (189) 1.0 84.1
MATERIALS AND METHODS
Strains of beta-hemolytic group C streptococci were iso-
lated on sheep blood agar plates under 5 to 7% CO2. The
strains were obtained from throat swabs of 1,480 University
of South Carolina students diagnosed clinically with pharyn-
gitis over the period 21 January 1991 to 20 January 1992.
Controls consisted of 227 students visiting the University of
South Carolina Thomson Student Health Center with non-
infectious problems (e.g., orthopedic problems, routine
physicals). The clinical evaluation of these patients and
handling of throat swab cultures to obtain pure cultures for
further microbiological characterization are described in the
companion article (26). Serogrouping was performed with a
commercial latex bead immunoagglutination test (Difco,
Detroit, Mich.). Strains which were positive for group C
antigen were identified at the Department of Microbiology
and Immunology, University of South Carolina. Colonies
were graded as small or large, and hemolysis zones around
the colonies were noted to be minute or broad. Broad
hemolysis extended from the colony as a transparent, color-
less zone resembling but not as extensive as that produced
by Staphylococcus aureus. Minute hemolysis was a zone
that was not simply limited in size but one that had no
complete clearing. All group C strains were identified by
using the battery of tests recommended previously (5).
The VP test was performed as follows. The strain was
inoculated into 0.2 ml of methyl red-VP broth and incubated
at 37°C for 4 to 6 h. One drop each of 40% KOH, 0.5%
creatine, and 5% a-naphthol were added. A red color after 30
min of incubation was considered a positive result (5). Hy-
drolysis of 4-methylumbelliferyl-D-3-glucuronide (MUMG;
glucuronidase activity) was assessed by impregnating disks
with colonies of group C streptococci and a solution of
MUMG (1 mg/ml in phosphate-buffered saline; pH 6.5). The
disks were incubated for 30 min at 37°C and fluorescence was
observed under a long-wave UV lamp (23).
Sugar fermentation reactions were also performed. The
streptococci were inoculated into phenol red broth contain-
ing trehalose, sorbitol, or ribose (10 g/liter) or a negative
control. Fermentation was monitored at 4, 24, 48, and 72 h as
indicated by a color change of the medium (from red to
yellow). The fermentation patterns distinguish S. equisimi-
lis, S. equi, and S. zooepidemicus.
For structural carbohydrate profiling, members of a se-
lected group of strains (eight in total) were each cultured in
250 ml of Todd-Hewitt broth. The bacteria were collected,
washed, heat killed, and lyophilized. Sugars were released
by hydrolysis and converted to alditol acetates and analyzed
GROUP C STREPTOCOCCI AND PHARYNGITIS 805
% of strains
Fermentation Glucuronidase
Trehalose Ribose Sorbitol activity
73.3 0 100
80.0 0 100
74.0 0 100
78.3 3.7 0 0
88.0 4.0 0 0
80.4 3.7 0 0
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by gas chromatography-mass spectrometry as described
previously (12).
RESULTS
Two hundred thirty-nine beta-hemolytic strains of group C
streptococci were isolated from 1,480 patients with pharyn-
gitis and 227 age-matched healthy controls; 209 strains were
from patients and 30 strains were from controls. These
strains were characterized by using a battery of four tests
used previously in differentiating S. equisimilis from S.
anginosus, namely, the VP reaction, glucuronidase activity,
ribose fermentation, and hemolysis characteristics. The re-
sults are summarized in Table 1.
Two groups of strains were readily discriminated. A group
of 44 strains all displayed broad hemolysis, were VP test
negative, and produced glucuronidase. Some 72.7% of this
group also fermented ribose. This group of 44 strains repre-
sents typical S. equisimilis. Of the remaining 195 strains,
only 2 strains displayed broad hemolysis. Among these 195
strains, 159 were VP test positive and glucuronidase nega-
tive. Of these 159 strains, 98.8% displayed minute hemoly-
sis, with 3.7% fermenting ribose. This group of 159 strains
represents typical S. anginosus. S. equisimilis generally had
large colonies (74%), whereas S. anginosus had small colo-
nies (90%).
In addition to typical S. equisimilis and S. anginosus
strains, 36 strains showed intermediate characteristics (14%
of strains from patients and 23% of strains from controls).
The strains of the intermediate group resembled S. angino-
sus morphologically in that they exhibited minute hemolysis
but were VP test negative. However, six strains were
glucuronidase positive, and five of those strains also fer-
mented ribose. Thus, these six strains were grouped with S.
equisimilis. Of the remaining 30 intermediate strains, none
showed glucuronidase activity and only 1 strain fermented
ribose. Thus, these 30 strains were grouped with S. angino-
sus.
Fermentation reactions are used to differentiate among VP
test-negative Lancefield group C streptococcal species (S.
equisimilis, S. zooepidemicus, and S. equis). It has been
reported that strains of S. equisimilis generally ferment
ribose and trehalose but not sorbitol. S. equis usually does
not ferment ribose, sorbitol, or trehalose, and S. zooepi-
demicus ferments sorbitol and ribose but not trehalose (22,
24). In the present study, approximately three-quarters of
the large-colony strains fermented ribose, none fermented
sorbitol, and all fermented trehalose. Thus, no S. zooepi-
demicus or S. equis strains were identified.
806 FOX ET AL.
In total, 78.5% of strains from controls and 83.3% of
strains from patients were identified as S. anginosus. S.
equisimilis was isolated from 3.0% of patients and 2.2% of
controls (Fisher exact tests P > 0.05), and S. anginosus was
isolated from 11.1% of patients and 11.0% of controls
(chi-square test, P > 0.05).
Three strains of S. equisimilis and five strains of S.
anginosus were analyzed by gas chromatography-mass spec-
trometry and were found to have similar carbohydrate
profiles typical for group C streptococci; rhamnose, ribose,
glucose, galactose, muramic acid, glucosamine, and galac-
tosamine were identified.
DISCUSSION
In the present study, two groups of strains of beta-
hemolytic group C streptococci (typical S. equisimilis and S.
anginosus strains) from human throats were delineated. In
agreement with others, it was noted that most strains of S.
equisimilis showed broad zones of beta-hemolysis and large
colonies, whereas S. anginosus showed minute zones of
hemolysis and small colonies (6). S. equisimilis, unlike S.
anginosus, did not generate acetoin but did display glucuron-
idase activity. However, a substantial number of strains
(15%) had intermediate characteristics, being VP test nega-
tive with minute zones of hemolysis. Thirty of these strains
were characterized as S. anginosus since they did not
display glucuronidase activity and (with one exception) did
not ferment ribose. Variability in the physiological charac-
teristics of S. anginosus is well documented (1, 9, 14).
The carbohydrate profiles of S. equisimilis and S. angino-
sus (as determined by gas chromatography-mass spectrom-
etry) were consistent with their being group C organisms
(20), but the profiles of the two species were not distinguish-
able.
In a classic study, Ball and Parker (1) cautioned: "Thus
although none of the single tests we employed could be
relied upon to distinguish S. milleri from the pyogenic
members of groups A, C and G, this could nearly always be
done with confidence by a complete cultural, biochemical
and serological examination." S. equisimilis and S. angino-
sus can be readily differentiated by hemolysis reaction,
acetoin generation, glucuronidase activity, and ribose fer-
mentation. However, there is a subgroup of strains which
displays intermediate characteristics, and use of all these
tests is vital for accurate discrimination of these two groups
of organisms. Sole reliance on the VP reaction would have
resulted in 30 strains of S. anginosus being misidentified as
S. equisimilis, increasing the number of strains of S. equisi-
milis from 50 to 80.
In three previous studies (17, 18, 21), 44, 27, and 19% of
group C strains, respectively, were reported to be S. equi-
similis. In our study, 20.9% were found to be S. equisimilis.
The study by Lawrence et al. (17) included strains of diverse
origins. The other two studies (18, 21), with isolation rates
similar to ours, were from patients with pharyngitis. In none
of the previous studies (17, 18, 21) was the incidence of S.
equisimilis and S. anginosus in healthy controls compared
with that in patients with pharyngitis.
In conclusion, the present study demonstrated that group
C streptococci are members of the normal flora of healthy
controls. There was no appreciable difference in the isolation
rates of S. anginosus and S. equisimilis from patients versus
controls, although it is possible that small differences in
isolation rates could have been overlooked in this study. The
physiologic characteristics of each species from patients
J. CLIN. MICROBIOL.
with pharyngitis and controls were similar. The companion
article (26), however, documents that there is a significant
correlation between the presence of S. equisimilis (but not S.
anginosus) and clinical features of pharyngitis.
ACKNOWLEDGMENTS
This work was supported by a Research and Productive Scholar-
ship award from the University of South Carolina.
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