Laboratory Diagnosis of Streptococcus pyogenes

(group A streptococci)
Barbara Spellerberg, PhD 1 and Claudia Brandt, PhD2
Created: February 10, 2016.

Introduction
Historically, Streptococcus pyogenes (group A streptococci) was first cultured and
identified as the cause of erysipelas by Friedrich Fehleisen in 1883, and it received its
species designation from Rosenbach in 1884. Today, laboratory diagnosis of group A
streptococcal infections still largely relies on culturing bacteria from clinical specimens.
To detect streptococci in clinical samples (and especially S. pyogenes), the material is most
often cultured on blood agar plates, which facilitates an easy preliminary screen for β-
hemolytic colonies. Subsequent confirmation of suspicious colonies as S. pyogenes can be
achieved by several easy, rapidly performed laboratory tests that are still widely applied in
clinical microbiology, despite the increasing use of automated identification systems. In
contrast to the diagnosis of acute S. pyogenes infections, the diagnosis of poststreptococcal
diseases, such as glomerulonephritis, acute rheumatic fever, and cerebral disorders relies
on the determination of specific antibodies. For epidemiological studies and outbreak
investigations, different typing methods have been developed. In addition to classical
serology based typing methods, well-established molecular typing systems are available,
which provide large databases of already characterized strains. This chapter will try to give
a comprehensive overview of classical microbiology and serology tests, molecular
methods, automated systems, as well as both molecular and conventional typing methods
that are used for the identification and characterization of S. pyogenes.

1 Institute of Medical Microbiology and Hygiene, University of Ulm, Albert Einstein Allee 11,
89081 Ulm, Germany; Email: barbara.spellerberg@uniklinik-ulm.de. 2 Institute of Medical
Microbiology, Johann Wolfgang Goethe University, Paul Ehrlich Str. 40, 60596 Frankfurt,
Germany; Email: claudia.brandt@em.uni-frankfurt.de.

Corresponding author.

NLM Citation: Spellerberg B, Brandt C. Laboratory Diagnosis of Streptococcus pyogenes (group

A streptococci). 2016 Feb 10. In: Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus
pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of
Oklahoma Health Sciences Center; 2016-.

The authors appreciate using pre-published information in this chapter with permission from
ASM Press ©2015 American Society for Microbiology. No further reproduction or distribution is
permitted without the prior written permission of American Society for Microbiology.
2 Streptococcus pyogenes

Culturing techniques
Streptococci are generally grown on agar media supplemented with blood. This technique
allows the detection of β-hemolysis, which is important for subsequent identification
steps, and enhances the growth of streptococci by the addition of an external source of
catalase. Selective media for culturing Gram-positive bacteria (such as agar media that
contains phenylethyl alcohol, or Columbia agar with colistin and nalidixic acid) also
provide adequate culturing conditions for S. pyogenes. Optimal incubation conditions for
the vast majority of streptococcal strains include a temperature range of 35°C to 37°C in
the presence of 5% CO2 or under anaerobic conditions. These conditions are optimized
for culturing streptococcal species that belong to the viridans group, but they may not be
ideal for growing S. pyogenes.
Special procedures have been developed to optimize the identification of S. pyogenes in
throat cultures. When properly performed and interpreted, culturing throat swabs on a
5% sheep blood agar with trypticase soy base incubated in air remains the gold standard
and reference method for the diagnosis of S. pyogenes acute pharyngitis (Murray, Wold,
Schreck, & Washington, 1976; Shulman, et al., 2012). These conditions represent reliable
and well-accepted methods with a sensitivity of 90% or higher, as shown with studies
using duplicate throat cultures (Bisno, 2001; Murray, Wold, Schreck, & Washington,
1976). In most cases of acute streptococcal pharyngitis, ample growth of typical colonies
can be observed after 24 hours of incubation at 35-37°C. If only a few colonies of S.
pyogenes appear after incubation under these conditions, interpretation becomes more
difficult, and these patients are most likely to be streptococcal carriers, rather than acutely
infected individuals (Bisno, 2001). Differentiation between a carrier and an acutely
infected individual could also reflect inadequate specimen collection, a lack of optimal
conditions for incubation, and inaccurate reading of plates. False negative results with
small numbers of organisms most likely occur because of overgrowth of upper respiratory
tract microorganisms that produce toxic materials to mask hemolysis. To increase
detection rates after the initial 18 to 24 hours of incubation, negative cultures should be
re-examined after an additional 24 hours of incubation. For presumptive identification of
S. pyogenes, cultures should be tested for bacitracin susceptibility and PYR activity (as
described below). A definitive diagnosis should include a positive Lancefield group A
antigen test. Negative results can be confirmed after a total culture time of 48 hours.
A number of studies have been performed to enhance S. pyogenes isolation, including
analysis of incubation conditions in anaerobic or CO2 enriched atmospheres, as well as
the use of various media selective for β-hemolytic streptococci (Kellogg, 1990; Kurzynski
& Meise, 1979; Welch, Hensel, Pickett, & Johnson, 1991; Milatović, 1981). In view of
existing cost limitations and uncertain benefits, these additional efforts are not generally
recommended. Incubation in anaerobic or a CO2-enriched atmosphere more frequently
leads to the isolation of non-S. pyogenes β-hemolytic streptococci (Altun, Almuhayawi,
Ullberg, & Özenci, 2013). To suppress the growth of commensal respiratory microbiota,
including other β-hemolytic streptococci, Streptococcus-selective media may be used,
Laboratory Diagnosis of Streptococcus pyogenes (group A streptococci) 3

which is highly sensitive for the isolation of S. pyogenes (Welch, Hensel, Pickett, &
Johnson, 1991). Another option is the addition of a blood agar plate that contains
sulfamethoxazole-trimethoprim to inhibit the normal respiratory microbiota (Kurzynski
& Meise, 1979). Details of these study results have been summarized in several
publications (Bisno, Gerber, Gwaltney, Kaplan, & Schwartz, 1997; Kellogg, 1990).
A concern rarely addressed when culturing pharyngeal specimens for S. pyogenes on
blood agar plates is the role of nonhemolytic S. pyogenes isolates. Culture based screening
relies on the detection of β-hemolytic colonies and subsequent identification steps.
However, clinical nonhemolytic S. pyogenes isolates that carry deletions of SLS genes have
been published (Yoshino, et al., 2010). Moreover, nonhemolytic S. pyogenes strains have
repeatedly been implicated as causing pharyngitis, as well as invasive infections (James &
McFarland, 1971; Cimolai, Trombley, & Bhanju, 2002; Dierksen & Tagg, 2000; Jantsch, et
al., 2013). Standard throat cultures will not detect these strains and it is currently
unknown if there is a true burden of disease caused by nonhemolytic S. pyogenes strains.

Morphology
To identify S. pyogenes in clinical samples, blood agar plates are screened for the presence
of β-hemolytic colonies. The typical appearance of S. pyogenes colonies after 24 hours of
incubation at 35-37°C is dome-shaped with a smooth or moist surface and clear margins.
They display a white-greyish color and have a diameter of > 0.5 mm, and are surrounded
by a zone of β-hemolysis that is often two to four times as large as the colony diameter.
Microscopically, S. pyogenes appears as Gram-positive cocci, arranged in chains (Figure
1).
4 Streptococcus pyogenes

Figure 1: Typical appearance of S. pyogenes on sheep-blood agar plates, following 24 hour incubation under
aerobic conditions.

Conventional identification tests

After the detection of β-hemolytic colonies displaying a typical S. pyogenes morphology,
catalase testing confirms that the isolates represent streptococci. A few easy, rapidly
performed laboratory tests can then be applied for definite species identification. Since
each of the tests, which are detailed below, has some limitations, the most reliable
identification results can be achieved by combining two of the following methods.

Lancefield antigen determination

Rebecca Lancefield was the first to develop a method for distinguishing β-hemolytic
streptococci into different species by determining the presence of Lancefield antigens on
streptococcal surfaces through antibodies (Lancefield, 1933). Currently, commercially
available Lancefield antigen grouping sera, obtained from many different suppliers, are
still widely applied in microbiology laboratories for the differentiation of β-hemolytic
streptococci. The commercial kits provide substrates for rapid antigen extraction and
subsequent agglutination by specific antibodies, and are typically directed towards
Lancefield antigens A, B, C, F, and G. While there is a good correlation between the
Laboratory Diagnosis of Streptococcus pyogenes (group A streptococci) 5

presence of certain Lancefield antigens with specific streptococcal species, this correlation
is not 100% in the cases of Lancefield group A, C, or G antigens. Except for rare
mutations, all S. pyogenes strains harbor the Lancefield group A antigen on their surface,
but the presence of the group A antigen is not limited to S. pyogenes. It has also been
found in species from the Streptococcus anginosus group (Facklam, 2002), as well as in rare
Streptococcus dysgalactiae subsp. equisimilis isolates (Brandt, Haase, Schnitzler, Zbinden,
& Lütticken, 1999). Therefore the detection of Lancefield group A necessitates further
testing for a reliable species diagnosis of S. pyogenes, which can be achieved by bacitracin
susceptibility discs or a PYR determination test.

PYR test
The PYR test is a rapid colorimetric method often used to distinguish S. pyogenes from
other β-hemolytic streptococci with a similar morphology (such as S. dysgalactiae subsp.
equismilis) and tests for the presence of the enzyme pyrrolidonyl aminopeptidase. This
enzyme hydrolyzes L-pyrrolidonyl-β-naphthylamide (PYR) to β-naphthylamide, which
produces a red color when a cinnamaldehyde reagent is added. The test can be performed
on paper strips that contain dried chromogenic substrates for the pyrrolidonyl
aminopeptidase within a few minutes (Kaufhold, Lütticken, & Schwien, 1989). PYR spot
tests are available from a number of commercial vendors. For standard laboratory
identification procedures, PYR positive β-hemolytic streptococci that display the typical
morphology of S. pyogenes can be presumptively identified as S. pyogenes. Other PYR
positive β-hemolytic streptococcal species, such as Streptococcus iniae and Streptococcu
porcinus, are primarily animal-associated species and are rarely identified in human
clinical specimens. To avoid potential misidentification, it is important to distinguish
Streptococcus from Enterococcus prior to PYR testing, and to keep in mind that species
and strains from other closely related genera may be PYR-positive (including the genera
Abiotrophia, Aerococcus, Enterococcus, Gemella, Staphylococcus, and Lactococcus).
Enterococci presenting with β-hemolysis are occasionally found on blood agar plates;
however, PYR-positive β-hemolytic enterococcal isolates display a different colonial
morphology, and when combined with other phenotypic characteristics, are easily
distinguished from streptococci. To avoid false positive reactions caused by other PYR-
positive bacterial species, which may be present in mixed cultures, this test should only be
performed on pure cultures.

Bacitracin susceptibility
Streptococcus pyogenes can be differentiated from other non-group A β-hemolytic
streptococci by their increased sensitivity to bacitracin. The bacitracin test, along with the
Lancefield antigen A test, is used for greater specificity in the identification of S. pyogenes,
since other β-hemolytic strains of streptococci that may contain the group A antigen are
resistant to bacitracin. The bacitracin test is also used to distinguish S. pyogenes from
other β-hemolytic streptococci that are PYR-positive, such as S. iniae and S. porcinus. To
perform a bacitracin susceptibility test, it is important to make a subculture of the strain
to be tested on a sheep blood agar plate (SBA), since placing the bacitracin disc on a
6 Streptococcus pyogenes

primary plate could give variable results. The strain being tested is streaked with several
individual colonies of a pure culture on an SBA agar plate and a disk containing 0.04 U of
bacitracin is placed on the SBA plate. After overnight incubation at 35°C in 5% CO2, a
zone of inhibition surrounding the disc indicates the susceptibility of the strain. It is
noteworthy that bacitracin-resistant strains of S. pyogenes have been observed in a
number of European countries (Malhotra-Kumar, Wang, Lammens, Chapelle, &
Goossens, 2003; Mihaila-Amrouche, Bouvet, & Loubinoux, 2004; James & McFarland,
1971); however, bacitracin resistance has not yet been reported in the US to date.

Species determination of S. pyogenes in automated identification systems

During the last decade, automated bacterial identification systems have gained more and
more importance in the clinical laboratory. Currently, a variety of products that
incorporate batteries of physiologic tests are commercially available for species
identification of streptococci. These products generally perform well with commonly
isolated pathogenic streptococci, such as S. pyogenes. Although automated bacterial
identification by MALDI-TOF (Bruker Corporation, 2015; bioMérieux, Inc., 2015) has
limitations in identifying several streptococcal species (including S. dysgalactiae subsp.
equisimilis, which may be misidentified as S. pyogenes), the results for S. pyogenes
correspond well to species identification by conventional tests (Schulthess, et al., 2013).
New commercial systems for the identification of streptococci include the FDA approved
Verigene Gram-positive blood culture (BC-GP) nucleic acid test (Nanosphere, Inc., 2014)
and the FilmArray platform (BioFire Diagnostics, LLC, 2015) for the direct identification
of bacterial pathogens from blood culture bottles (Altun, Almuhayawi, Ullberg, & Özenci,
2013; Wojewoda, et al., 2013). Highly favorable results from the application of these
systems were obtained for major bacterial pathogens, including S. pyogenes and S.
agalactiae. The direct identification of S. pyogenes from blood culture bottles enables the
rapid administration of a suitable antibiotic treatment, which offers a considerable
advantage for patients suffering from life-threatening invasive diseases. However, for the
majority of S. pyogenes isolates encountered in the clinical laboratory, serologic tests, in
conjunction with presumptive physiologic tests (as described above) still offer an
acceptable and cost-effective alternative to commercially available identification systems.

Antibiotic resistance testing

Penicillin remains the drug of choice for the empirical treatment of S. pyogenes infections,
despite over sixty years of use. S. pyogenes has also remained uniformly susceptible to
penicillin and resistance testing for penicillins or other β-lactams approved for treatment
of S. pyogenes is not necessary for clinical purposes, in accordance with CSLI
recommendations. In contrast to Streptococcus agalactiae, for which rare isolates with
reduced susceptibility to penicillin have been reported and mutations in the cell wall
synthesis enzyme Pbp2x were found, S. pyogenes isolates with altered penicillin
susceptibilities that could be confirmed by a reference laboratory have not yet been
encountered. Nevertheless, more than 10% of patients report suspected or confirmed
allergies to penicillins, which frequently leads to the use of macrolides as an alternative
Laboratory Diagnosis of Streptococcus pyogenes (group A streptococci) 7

treatment. Since macrolide resistance rates among S. pyogenes isolates have been
increasing in North America as well as in Europe (Desjardins, Delgaty, Ramotar,
Seetaram, & Toye, 2004), resistance testing is mandatory for these substances. S. pyogenes
macrolide resistance rates correlate with the use of macrolides in clinical practice, and
geographic differences in resistance rates are often due to differences in macrolide use.
Susceptibility testing for macrolides should be performed using erythromycin, since
resistance and susceptibility of azithromycin, clarithromycin, and dirithromycin can be
predicted by testing erythromycin. To detect inducible clindamycin resistance in S.
pyogenes, CLSI recommends a double-disc diffusion assay. Similar to the resistance
situation for penicillins, a reduced susceptibility to glycopeptides has not yet been found
in S. pyogenes. Currently, resistance testing will most often be performed by automated
systems that provide ready-made panels of antibiotics for different bacterial species.
Further discussion of antibiotic resistance can be found in a subsequent chapter.

Direct antigen detection of S. pyogenes from throat specimens

S. pyogenes infections represent the most common cause of acute pharyngitis, and account
for up to 37% of pediatric cases (Shaikh, Leonard, & Martin, 2010) and 5 to 15% of cases
in adults (Shulman, et al., 2012). If a diagnosis can be provided rapidly, prompt initiation
of antibiotic therapy will relieve symptoms, avoid sequelae, and reduce transmission rates.
This is most often achieved through the application of so-called “rapid antigen tests”.
Numerous assays for direct detection of the group A-specific carbohydrate antigen in
throat swabs by agglutination methods or immunoassays (enzyme, liposome, or optical)
have become commercially available during the past two decades. A list of FDA-cleared
tests is accessible online (U.S. Department of Health and Human Services, 2015).
Although these tests provide rapid results to allow early treatment decisions, culturing
throat swabs for S. pyogenes remains the gold standard. The sensitivities of rapid antigen
tests range from 58% to 96%, but have never equaled that of culture tests (Facklam, 1987;
Uhl, et al., 2003). Therefore, national advisory committees continue to recommend
confirmation of negative rapid test results with a throat culture in children and
adolescents (Shulman, et al., 2012). However, a routine back-up throat culture is
dispensable in adult patients, due to the low incidence of streptococcal pharyngitis and
rheumatic fever in this age group. The specificity of rapid antigen tests is generally high,
even though false-positive antigen results can be seen from patients previously diagnosed
and/or treated for S. pyogenes (Chapin, Blake, & Wilson, 2002), or patients colonized with
non-S. pyogenes streptococcal species that carry the Lancefield group A antigen. Despite
the obvious advantages of a rapid diagnosis, it must be noted that the positive predictive
value of rapid group A antigen tests is currently low in the adult population, and can
frequently result in unnecessary antimicrobial therapy (Peterson & Thomson, 1999).

Nucleic acid detection techniques

Several years ago, a method based on nucleic acid detection was first introduced for direct
S. pyogenes diagnosis from clinical throat swabs. The GASDirect test identifies specific
rRNA sequences of S. pyogenes in pharyngeal specimens by a single-stranded
8 Streptococcus pyogenes

chemiluminescent nucleic acid probe (Hologic, Inc., 2015; Steed, Korgenski, & Daly, 1993;
Pokorski, Vetter, Wollan, & Cockerill, 1994). The test has performed well in comparison to
standard streptococcal culture methods and has received FDA clearance. Sensitivity and
specificity ranged from 89%–95% and 98%–100%, respectively, as compared to culture
results, which reached a sensitivity of 98%–99% (Chapin, Blake, & Wilson, 2002; Steed,
Korgenski, & Daly, 1993; Pokorski, Vetter, Wollan, & Cockerill, 1994). The GASDirect test
can be applied for primary testing, has also been used as a backup test to negative antigen
tests (Nakhoul & Hickner, 2013), and is suitable for batch screening of throat cultures.
A commercial polymerase chain reaction (PCR) method for the direct detection of S.
pyogenes using the illumigene system (Meridian Bioscience, Inc., 2015) has recently
received FDA clearance. Excellent sensitivity (99%) and specificity (99.6%) were
demonstrated for the illumigene test in a multicenter evaluation study (Anderson, et al.,
2013; Buchan & Ledeboer, 2014). This test relies on loop-mediated isothermal
amplification (LAMP) technology with S. pyogenes specific primers. In 2015, two point of
care tests for the detection of S. pyogenes in throat swabs using rapid automated PCR
technology received FDA clearance. Both the cobas Strep A test, running on the cobas
Liat platform (F. Hoffmann-La Roche Ltd., 2015) and the Simplexa Group A Strep Direct
Test (Focus Diagnostics, Inc., 2013) provide PCR results for individual samples within 20
minutes. Apart from FDA-released documents, however, scientific publications on the
performance of these tests concerning S. pyogenes detection, are not yet publicly available.

Serologic tests
The diagnosis of poststreptococcal diseases, such as rheumatic fever or
glomerulonephritis, can be aided by the detection of certain streptococcal antibodies.
Such a diagnosis is rarely useful in acute infections, since antibody development takes
about one to two weeks after the onset of acute infection to be detectable in serum
samples. Rising antibody levels only occur in patients suffering from S. pyogenes
infections and streptococcal carriers do not experience an increase of antibody titers (Shet
& Kaplan, 2002), when acute and convalescent sera are compared. A fourfold rise in
antibody titers is regarded as a definitive proof of antecedent streptococcal infection.
While the measurement of a definite antibody rise is the more reliable detection method,
serum of patients suffering from glomerulonephritis or rheumatic fever may have reached
peak antibody levels at the onset of symptoms and thus will not experience any further
rises in these levels. Multiple variables influence antibody levels: these include the site of
infection, time since the onset of symptoms, age of the patient, the background prevalence
of streptococcal infections in a particular region, seasonal changes, and patient
comorbidities (Ayoub, et al., 2003). Age is an especially important determinant of
streptococcal antibody levels. Due to the frequent exposure to S. pyogenes, children
between 6 and 15 years display the highest antibody titers, as compared with very young
infants and adults: thus, “normal levels” in children may considerably exceed regular
background titers of adults. Prompt antibiotic treatment of acute infections can reduce the
magnitude of, but will not abolish the immune response to streptococcal antigens. The
Laboratory Diagnosis of Streptococcus pyogenes (group A streptococci) 9

most widely used antibodies for the diagnosis of poststreptococcal diseases are anti-
streptolysin O and anti-DNase B.
Streptolysin O is a cholesterol-dependent hemolysin of S. pyogenes that belongs to the
group of thiol-activated cytolysins. Antibody levels against streptolysin O (ASO) start
rising after one week of infection and reach maximum levels at about three to six weeks of
infection. The upper limits of normal (ULN) of ASO are 240–320 in the pediatric age
group 6–15 years (Shet & Kaplan, 2002). While the ASO response following streptococcal
upper respiratory tract infection is usually high, pyoderma caused by S. pyogenes does not
elicit a strong ASO response. Streptococcus dysgalactiae subsp. equisimilis can also produce
streptolysin O; thus, elevated ASO titers are not specific to S. pyogenes infections. The
classical test for measuring ASO titers is a neutralization assay, where hemolysis through
streptolysin O is inhibited by patient serum that harbors antistreptolysin O antibodies.
Results are expressed as Todd units, which is the reciprocal of the highest titer not
showing any hemolysis. Newer tests based on latex agglutination and nephelometric
measurements are also available.
S. pyogenes produces several nucleases that are important for the escape of bacteria from
neutrophil extracellular traps (Sumby, et al., 2005). Among the four streptococcal
deoxyribonucleases (DNase), the immunologic host response is most consistent against
DNase B. DNase B titers start appearing at two weeks after the onset of infection, and may
not reach maximum titers for six to eight weeks. Similar to ASO titers, the upper limits of
normal for pediatric patients (6–15 years) is much higher, and the ULN for Anti DNase B
in this age group is 640 (Shet & Kaplan, 2002). Anti-DNase B titers tend to remain
elevated longer than the ASO titers and are more reliable than ASO for the confirmation
of a preceding streptococcal skin infection. Moreover, since only 80–85% of patients with
rheumatic fever will present with elevated ASO titers, additional DNase titers may be
helpful. Since DNase B is specific for S. pyogenes and not present in Streptococcus
dysgalactiae subsp. equisimilis, increased ASO levels without changes in the anti-DNase B
titers may indicate Streptococcus dysgalactiae subsp. equisimilis infections. The classic anti-
DNase B assay is a neutralization assay, and is based on the inhibition of nuclease activity
through antibodies present in patient serum. Other less standardized assay techniques
that are available include a latex agglutination test.
A more historical test is the hemagglutination-based streptozyme test that was developed
to detect antibodies against multiple extracellular streptococcal products as a
supplementary test in the clinical laboratory. However, variabilities in the standardization
of the test and an inconsistent specificity have been reported (Gerber, Wright, &
Randolph, 1987). Tests for the detection of antibodies against the group A carbohydrate,
as well as serotype-specific antibodies, are measured for research purposes only and
usually have no clinical use in the diagnosis of streptococcal infection.
10 Streptococcus pyogenes

Figure 2. Common antigenic proteins of S. pyogenes used for diagnostic and typing purposes.

Typing of Streptococcus pyogenes

In most clinical cases of acute infections, subtyping of group A streptococcal strains has
no immediate diagnostic or therapeutic consequences. Such typing is typically performed
by reference laboratories for epidemiologic surveys or in outbreak situations, and may
provide important information about the evolutionary relatedness of various strains.
Although classical antibody-dependent typing systems of surface proteins have been used
for many years, molecular methods have become more and more prevalent, since they do
not require the maintenance of rarely used large antibody panels or the establishment of
specialized techniques. As an additional advantage, the determination of DNA sequences
is independent from culture conditions and gene expression.
Conventional typing of S. pyogenes is based upon the antigenic specificity of the surface-
expressed T and M proteins. (Johnson & Kaplan, 1993). The trypsin-resistant T protein is
part of the pilus structures (Mora, et al., 2005). T type identification can be achieved by
Laboratory Diagnosis of Streptococcus pyogenes (group A streptococci) 11

commercially available assays that use approximately 20 recognized anti-T sera.

Molecular analysis has successfully established an association between pilus genes and
recognized T-serotypes (Falugi, et al., 2008). M proteins are major antiphagocytic
virulence factors of S. pyogenes (Fischetti, 1989). The different antigenic specificities are
based on N-terminal sequence variations in the M-proteins, which are detected by
precipitation typing, using M-protein specific antisera. 83 M serotypes are currently
validated and internationally recognized as serologically unique. They are designated as
M1 to M93, in accordance with their Lancefield classifications (Facklam, et al., 2002). M
serotypes that are not included are from non-S. pyogenes organisms, such as Streptococcus
dysgalactiae subsp. equisimilis, or are duplicates of an already existing M serotype.
A molecular serotyping system has been established on the basis of the nucleotide
sequence variations that encode the amino termini of M proteins. This system is based on
the amplification and subsequent nucleotide sequencing of an emm gene fragment by a
conserved primer pair. The emm genes encode M proteins and correlate with the
Lancefield M serotypes. This methodology allows an assignment to a validated M protein
gene sequence (emm1 through emm124) and easy identification of new emm-sequence
types and subtypes. Molecular serotyping through emm sequencing has evolved into the
"gold standard" molecular methodology of S. pyogenes typing (Facklam, et al., 2002). A
large database of more than 200 distinct emm gene sequences from strains originally used
for Lancefield serotyping, including emm sequences from β-hemolytic groups C, G, and L
streptococci, is accessible at the CDC website (Centers for Disease Control and
Prevention, 2014). The current type definition is based upon the sequence of the 90
nucleotides that encode the N terminal 30 amino acid residues of the processed M
protein. This annotation is favored, as it is most consistent with the classical serology-
based typing scheme. Subtypes can be assigned by sequencing 150 nucleotides that
encode the N terminal 50 residues of the mature M protein. Due to the rapidly increasing
availability of newly sequenced S. pyogenes strains, the database is constantly growing.
However, it has not yet been determined if the newer M-proteins that are defined solely
through sequencing are functional. In most instances, the potential antiphagocytic or
opsoninogenic properties of these proteins have not been experimentally tested.
Additional molecular typing techniques applicable for S. pyogenes have been developed. In
analogy to emm typing, a nucleotide based T-typing system by determination of the tee
gene has been published (Falugi, et al., 2008). Furthermore, several years ago, an MLST
scheme was developed for S. pyogenes. In population genetic studies, a stable association
between emm type and MLST could be demonstrated for isolates obtained decades apart
and/or from different continents (Enright, Spratt, Kalia, Cross, & Bessen, 2001). In S.
pyogenes outbreaks, the restriction digestion of emm gene PCR amplicons may be a
valuable tool for the rapid identification of isolates that carry identical or highly similar
emm genes (Beall, et al., 1998). For clusters of isolates that share the same emm type,
PFGE profiles may be helpful in further distinguishing these strains (Musser, et al., 1995).
Several emm-types can be shared between different clonal groups of S. pyogenes.
12 Streptococcus pyogenes

Acknowledgments
The authors appreciate using pre-published information in this chapter with permission
from ASM Press ©2015 American Society for Microbiology. No further reproduction or
distribution is permitted without the prior written permission of American Society for
Microbiology.

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