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Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes: Basic Biology to Clinical Manifestations [Internet]. 2nd edition. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2022 Oct 8.
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 or automated identification systems such as MALDI TOF mass spectrometry. 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.
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 et al. 1976) (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). Sparse cultural growth in 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 prevent the detection of ß-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 could be tested for bacitracin susceptibility and PYR activity (as described below). A definitive diagnosis should include a positive Lancefield group A antigen test or species identification by automated methods. 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; Milatović, 1981; Welch, Hensel, Pickett, & Johnson, 1991). 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. To suppress the growth of commensal respiratory microbiota, including other β-hemolytic streptococci, Streptococcus-selective media may be used, 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.
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 (Figure 1). Microscopically, S. pyogenes appears as Gram-positive cocci, arranged in chains.
Typical appearance of S. pyogenes on sheep-blood agar plates, following 24-hour incubation under aerobic conditions.
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.
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 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 Streptococcus dysgalactiae subsp. equisimilis isolates (Brandt, Haase, Schnitzler, Zbinden, & Lütticken, 1999; Brandt & Spellerberg, 2009), with a recently emerging clone in the US (Chochua, et al., 2019). 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.
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 Streptococcus 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.
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 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.
During the last decades, 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, 2022; bioMérieux, Inc, 2015) initially had some limitations in identifying several beta-hemolytic streptococcal species, including the misidentification of S. dysgalactiae subsp. equisimilis as S. pyogenes. These problems have been addressed through database modifications and currently the results for S. pyogenes correspond well to species identification by conventional tests (Schulthess, et al., 2013; Jensen, Dam-Nielsen, & Arpi, 2015; Nybakken, Oppegaard, Gilhuus, Jensen, & Mylvaganam, 2021). MALDI TOF is therefore increasingly used for species identification of beta-hemolytic streptococci, often replacing the use of conventional tests. Commercial systems for the identification of streptococci include the FDA approved Verigene Gram-positive blood culture (BC-GP) nucleic acid test (Luminex Corporation, 2022) and the FilmArray platform (BioFire Diagnostics, 2022) for the direct identification of bacterial pathogens from blood culture bottles (Altun, Almuhayawi, Ullberg, & Özenci, 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 an effective antibiotic treatment, which offers a considerable advantage for patients suffering from life-threatening invasive diseases.
Penicillin remains the drug of choice for the empirical treatment of S. pyogenes infections after more than seventy 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 has not been recommended for clinical purposes, in accordance with CSLI recommendations. In 2020 however a first report was published about a mutation of the Pbp2x cell wall synthesis enzyme in two clinical S. pyogenes isolates leading to an elevation of the MIC for Ampicillin to the CLSI breakpoint value but without reaching Ampicillin resistance levels. MICs for penicillin were clearly sensitive with 0.012 mg/l in these strains (Vannice, et al., 2020). Subsequently, a large scale whole genome sequence analysis of S. pyogenes strains revealed more than 100 strains carrying Pbp2x mutations with increased MIC levels against some ß-lactam antibiotics including Penicillin, but without reaching levels of resistance (Musser, et al., 2020). As this situation may develop into a serious public health threat, increased surveillance of S. pyogenes and testing for ß-lactam resistance should be considered.
In addition, more than 10% of patients report suspected or confirmed allergies to penicillins, which frequently leads to the use of macrolides as an alternative 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) up to resistance levels of 22% in invasive isolates from the US (Fay, et al., 2021), 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 clearly 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.
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 decades. A list of FDA-cleared tests is accessible online (www.fda.gov). 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; Stewart, et al., 2014). 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. A recent systematic Cochrane review supports the use of rapid antigen test for their ability to lower antibiotic prescription rates in pharyngitis patients by about 25% (Cohen, Pauchard, Hjelm, Cohen, & Chalumeau, 2020).
Several years ago, a first nucleic acid amplification test (NAAT) was introduced for direct S. pyogenes diagnosis from clinical throat swabs. The GASDirect test (Hologic, Inc., 2022) identifies specific rRNA sequences of S. pyogenes in pharyngeal specimens by a single-stranded chemiluminescent nucleic acid probe (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.
In the meantime, multiple point of care (POC) tests for the detection of S. pyogenes in throat swabs using rapid automated PCR technology have received FDA clearance. These include the Roche cobas Strep A test, running on the cobas Liat platform (Roche Diagnostics, North America, 2022), the Abbot strep A assay (www.abbott.com), the Xpert Xpress Strep A assay (Cepheid, 2022) using the Cepheid gene expert system, the Solana GAS assay from Quidel (Quidel Corporation, 2022), the Aries Group A strep assay running on the Luminex platform (Luminex Corporation, 2022), and the Simplexa Group A Strep Direct Test (DiaSorin, Inc., 2016)(www.focusdx.com). These tests typically provide PCR results for individual samples within 20 minutes or less and CLIA clearance allows the use at the location of patient care. In general, sensitivities and specificities of the tests are comparable (Amrud, Slinger, Sant, Ramotar, & Desjardins, 2019; Thompson & McMullen, 2020; Ferrieri, Thonen-Kerr, Nelson, & Arbefeville, 2021) and surpass the sensitivity of traditional bacterial culture.
The increased sensitivity, however, is a point of discussion. While some false positive results may occur through contamination or detection of nonviable bacteria, the detection of asymptomatic S. pyogenes carriers without signs of infection is favored by the use of highly sensitive molecular tests and may results in increased prescription of antibiotics (Tanz, et al., 2019). Clear advantages are the shortened turnaround time of NAAT in comparison to bacterial culture and the detection of S. pyogenes in some patients suffering from true streptococcal infections or poststreptococcal sequelae (Ralph, et al., 2019) who present with low bacterial numbers. Overall, the use of NAAT for diagnosing streptococcal throat infections remained low (<1%) in one of the largest US surveillance studies conducted in recent years (Luo, et al., 2019), an observation that may, however, be changing (Thompson & McMullen, 2020).
The diagnosis of poststreptococcal diseases, such as rheumatic fever or glomerulonephritis, can be aided by the detection of specific streptococcal antibodies. Serologic 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 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 and used more frequently.
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 (Figure 2).
Common antigenic proteins of S. pyogenes used for diagnostic and typing purposes.
In most clinical cases of acute infections, typing and subtyping of group A streptococcal strains has no immediate diagnostic or therapeutic consequences. 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 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 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 (https://www.cdc.gov/streplab/index.html). 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.
For several years now, whole genome sequencing (WGS) has been used as a typing tool in many different streptococcal species, including S. pyogenes (Nanduri, et al., 2019; Plainvert, et al., 2018). WGS allows subtyping of strains that carry identical emm types, which enables further characterization of large S. pyogenes outbreaks of a single emm type. In addition, online tools have been developed to extract classical S. pyogenes typing characteristics from the obtained genome sequences, such as emm type and MLST data (Kapatai, Coelho, Platt, & Chalker, 2017; Chochua, et al., 2017).
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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