Background: The antigenic heterogeneity of GAS poses a challenge for vaccine design [...]

Streptococcus pyogenes is a leading cause of infection-related mortality in humans globally. The characteristic cell wall-anchored group A carbohydrate (GAC) is expressed by all S. pyogenes strains and consists of a polyrhamnose backbone with alternating N-acetylglucosamine (GlcNAc) side chains, of which 25% are decorated with glycerol phosphate (GroP). The genes in the gacA-L cluster are critical for GAC biosynthesis, with gacH-L being responsible for the characteristic GlcNAc-GroP decoration, which confers the agglutination in rapid test diagnostic assays and contributes to S. pyogenes pathogenicity. Historical research papers described S. pyogenes isolates, so-called A-variant strains, that lost the characteristic GlcNAc side chain following serial animal passage. Genomic analysis of a single viable historic parent/A-variant strain pair revealed a premature inactivating stop codon in gacI, explaining the described loss of the GlcNAc side chain. Subsequently, we analysed the genetic variation of the 12 gacA-L genes in a collection of 2021 S. pyogenes genome sequences. Although all gac genes (gacA-L) displayed genetic variation, we only identified 26 isolates (1.3%) with a premature stop codon in one of the gac genes. Twelve out of 26 (46%) isolates contained a premature stop codon in gacH, which encodes the enzyme responsible for the GroP modification. To study the functional consequences of the different premature stop codons for GacH function, we plasmid-expressed three gacH variants in a S. pyogenes gacH-deficient strain. Cell wall analysis confirmed GacH loss of function for the studied gacH variants through the significant reduction of GAC GroP, complete resistance to killing by the human bactericidal enzyme group IIA-secreted phospholipase and susceptibility to zinc toxicity. Overall, our data provide a comprehensive overview of the genetic variation of the gacA-L cluster in a global population of S. pyogenes strains and the functional consequences of rare inactivating mutations in gacH for host interaction.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes . Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes . More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

ABSTRACT Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as “pilin types.” Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools ( pubmlst.org ) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies. IMPORTANCE Precision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

AbstractStreptococcus dysgalactiaesubsp.equisimilis(SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species,Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE andS. pyogenes.More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were found across species. Assessing potential cross-protection from leadingS. pyogenesvaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

The clinico-epidemiological features of diseases caused by group A streptococci (GAS) is presented through the lens of the ecology, population genetics, and evolution of the organism. The serological targets of three typing schemes (M, T, SOF) are themselves GAS cell surface proteins that have a myriad of virulence functions and a diverse array of structural forms. Horizontal gene transfer expands the GAS antigenic cell surface repertoire by generating numerous combinations of M, T, and SOF antigens. However, horizontal gene transfer of the serotype determinant genes is not unconstrained, and therein lies a genetic organization that may signify adaptations to a narrow ecological niche, such as the primary tissue reservoirs of the human host. Adaptations may be further shaped by selection pressures such as herd immunity. Understanding the molecular evolution of GAS on multiple levels-short, intermediate, and long term-sheds insight on mechanisms of host-pathogen interactions, the emergence and spread of new clones, rational vaccine design, and public health interventions.

ABSTRACT Group A streptococci (GAS) are highly prevalent human pathogens whose primary ecological niche is the superficial epithelial layers of the throat and/or skin. Many GAS strains with a strong tendency to cause pharyngitis are distinct from strains that tend to cause impetigo; thus, genetic differences between them may confer host tissue-specific virulence. In this study, the FbaA surface protein gene was found to be present in most skin specialist strains but largely absent from a genetically related subset of pharyngitis isolates. In an Δ fbaA mutant constructed in the impetigo strain Alab49, loss of FbaA resulted in a slight but significant decrease in GAS fitness in a humanized mouse model of impetigo; the Δ fbaA mutant also exhibited decreased survival in whole human blood due to phagocytosis. In assays with highly sensitive outcome measures, Alab49ΔfbaA was compared to other isogenic mutants lacking virulence genes known to be disproportionately associated with classical skin strains. FbaA and PAM (i.e., the M53 protein) had additive effects in promoting GAS survival in whole blood. The pilus adhesin tip protein Cpa promoted Alab49 survival in whole blood and appears to fully account for the antiphagocytic effect attributable to pili. The finding that numerous skin strain-associated virulence factors make slight but significant contributions to virulence underscores the incremental contributions to fitness of individual surface protein genes and the multifactorial nature of GAS-host interactions.

The secreted cysteine proteinase SpeB is an important virulence factor of group A streptococci (GAS), whereby SpeB activity varies widely among strains. To establish the degree to which SpeB activity correlates with disease, GAS organisms were recovered from patients with pharyngitis, impetigo, invasive disease or acute rheumatic fever (ARF), and selected for analysis using rigorous sampling criteria; > 300 GAS isolates were tested for SpeB activity by casein digestion assays, and each GAS isolate was scored as a SpeB-producer or non-producer. Highly significant statistical differences (p < 0.01) in SpeB production are observed between GAS recovered from patients with ARF (41.5% SpeB-non-producers) compared to pharyngitis (20.5%), invasive disease (16.7%), and impetigo (5.5%). SpeB activity differences between pharyngitis and impetigo isolates are also significant, whereas pharyngitis versus invasive isolates show no significant difference. The disproportionately greater number of SpeB-non-producers among ARF-associated isolates may indicate an altered transcriptional program for many rheumatogenic strains and/or a protective role for SpeB in GAS-triggered autoimmunity.

PURPOSE OF REVIEW:Group A streptococci (GAS) are a common cause of pharyngitis and impetigo, and distinct throat strains and skin strains have been long recognized. This review aims to describe recent advances in molecular differences between throat and skin strains, and the pathogenic mechanisms used by virulence factors that may distinguish between these two groups.RECENT FINDINGS:Recent findings include a new typing scheme for GAS strains based on sequence clusters of genes encoding the entire surface-exposed portion of M protein; correlations between emm-based typing schemes, clinical disease and surface adhesins; covalent bond formation mediated by GAS pili and other adhesins in binding to host ligands; a key role for superantigens in oropharyngeal infection via binding major histocompatibility complex class II antigen; and migration of GAS-specific Th17 cells from the upper respiratory tract to the brain, which may be relevant to autoimmune sequelae.SUMMARY:The gap between molecular markers of disease (correlation) and virulence mechanisms (causation) in the establishment of tissue tropisms for GAS infection currently remains wide, but the gap also continues to narrow. Whole genome sequencing combined with mutant construction and improvements in animal models for oropharyngeal infection by GAS may help pave the way for new discoveries.

Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development.

Streptococcus pyogenes (group A Streptococcus; GAS) is a strict human pathogen with a very high prevalence worldwide. This review highlights the genetic organization of the species and the important ecological considerations that impact its evolution. Recent advances are presented on the topics of molecular epidemiology, population biology, molecular basis for genetic change, genome structure and genetic flux, phylogenomics and closely related streptococcal species, and the long- and short-term evolution of GAS. The application of whole genome sequence data to addressing key biological questions is discussed.

Group A Streptococcus (GAS) M protein is an important virulence factor and potential vaccine antigen, and constitutes the basis for strain typing (emm-typing). Although >200 emm-types are characterized, structural data were obtained from only a limited number of emm-types. We aim to evaluate the sequence diversity of near-full-length M proteins from worldwide sources and analyse their structure, sequence conservation and classification. GAS isolates recovered from throughout the world during the last two decades underwent emm-typing and complete emm gene sequencing. Predicted amino acid sequence analyses, secondary structure predictions and vaccine epitope mapping were performed using MUSCLE and Geneious software. A total of 1086 isolates from 31 countries were analysed, representing 175 emm-types. emm-type is predictive of the whole protein structure, independent of geographical origin or clinical association. Findings of an emm-type paired with multiple, highly divergent central regions were not observed. M protein sequence length, the presence or absence of sequence repeats and predicted secondary structure were assessed in the context of the latest vaccine developments. Based on these global data, the M6 protein model is updated to a three representative M protein (M5, M80 and M77) model, to aid in epidemiological analysis, vaccine development and M protein-related pathogenesis studies.

See related article on page 1522Group A streptococcus (GAS) is a highly prevalent bacterial pathogen that has been recovered from its human hosts throughout the world. Most epidemiological studies characterize GAS isolates by their emm type, a molecular marker that has ∼200 distinct forms. The emm gene encodes M protein, a surface fibril that is a major virulence factor and target of protective immunity. Collection of GAS, along with the molecular typing of isolates, has been highly active throughout much of the world for the past several decades. Within any one community over a 1- or 2-year surveillance period, roughly 25 to 40 distinct emm types can be detected among the GAS isolates.1Shulman S.T. Tanz R.R. Dale J.B. Beall B. Kabat W. Kabat K. Cederlund E. Patel D. Rippe J. Li Z. Sakota V. North American Streptococcal Pharyngitis Surveillance GroupSeven-year surveillance of North American pediatric group A streptococcal pharyngitis isolates.Clin Infect Dis. 2009; 49: 78-84Crossref PubMed Scopus (84) Google Scholar, 2McGregor K. Bilek N. Bennett A. Kalia A. Beall B. Carapetis J. Currie B. Sriprakash K. Spratt B. Bessen D. Group A streptococci from a remote community have novel multilocus genotypes but share emm types and housekeeping alleles with isolates from worldwide sources.J Infect Dis. 2004; 189: 717-723Crossref PubMed Scopus (40) Google Scholar, 3Sakota V. Fry A.M. Lietman T.M. Facklam R.R. Li Z.Y. Beall B. Genetically diverse group A streptococci from children in far-western Nepal share high genetic relatedness with isolates from other countries.J Clin Microbiol. 2006; 44: 2160-2166Crossref PubMed Scopus (43) Google Scholar Each host community has a unique array of emm types, and the degree of overlap in the spectrum of emm types decreases for host populations in rough proportion to their geographical distance. Even within the same community, the predominating emm types can shift from year to year.1Shulman S.T. Tanz R.R. Dale J.B. Beall B. Kabat W. Kabat K. Cederlund E. Patel D. Rippe J. Li Z. Sakota V. North American Streptococcal Pharyngitis Surveillance GroupSeven-year surveillance of North American pediatric group A streptococcal pharyngitis isolates.Clin Infect Dis. 2009; 49: 78-84Crossref PubMed Scopus (84) Google ScholarIn this issue of The American Journal of Pathology, Fittipaldi et al4Fittipaldi N. Beres S.B. Olsen R.J. Kapur V. Shea P.R. Watkins M.E. Cantu C.C. Laucirica D.R. Jenkins L. Flores A.R. Lovgren M. Ardanuy C. Liñares J. Low D.E. Tyrrell G.J. Musser J.M. Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.Am J Pathol. 2012; 180: 1522-1534Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar present a magnified view of the unfolding emergence and epidemic spread of an emm59 strain of GAS. Before 2004, when the epidemic had its beginnings in western Canada, recovery of emm59 isolates was rare. Steer at al5Steer A.C. Law I. Matatolu L. Beall B.W. Carapetis J.R. Global emm type distribution of group A streptococci: systematic review and implications for vaccine development.Lancet Infect Dis. 2009; 9: 611-616Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar recently provided a meta-analysis of >100 reports on GAS molecular epidemiology for >38,000 isolates collected from 37 countries since 1980: in every region of the world, emm59 strains are absent from the list of the 25 most prevalent emm types, even when those lists are further stratified by disease. GAS isolates of emm type 59 were largely below the radar before 2003, but have since accounted for >540 cases of invasive disease in Canada.6Tyrrell G.J. Lovgren M. St Jean T. Hoang L. Patrick D.M. Horsman G. Van Caeseele P. Sieswerda L.E. McGeer A. Laurence R.A. Bourgault A.M. Low D.E. Epidemic of group A Streptococcus M/emm59 causing invasive disease in Canada.Clin Infect Dis. 2010; 51: 1290-1297Crossref PubMed Scopus (39) Google ScholarGeneration of Epidemic emm59 GASHow does an epidemic begin? One idea is that a pre-existing strain finds itself in a new environment or in a new host population, whereupon the conditions encountered are favorable for its reproductive growth and sustained transmission. Either the organism migrates to a new community, or the conditions within a community undergo a drastic change. An alternative hypothesis states that a genetic change in a bacterium leads to an increase in fitness, which in turn leads to rapid spread of the new genetic variant. The genetic change may have resulted in acquisition of a new virulence factor or an antigenic shift, leading to increased disease severity and/or enhanced transmission. The fitness of a microorganism often reflects the host and/or environmental conditions, and each genetic variant can experience a different level of fitness within the same community. For example, if the new bacterium undergoes a change in antigenic structure and is present within an immunologically naïve host population, the organism can experience a leap in fitness whereby the conditions are now ripe for its clonal expansion through enhanced transmission. This occurred with serotype 19A pneumococcus after widespread vaccination of the host population with the heptavalent conjugate vaccine.7Brueggemann A.B. Pai R. Crook D.W. Beall B. Vaccine escape recombinants emerge after pneumococcal vaccination in the United States.PLoS Pathog. 2007; 3: e168Crossref PubMed Scopus (289) Google Scholar For another example, if the organism acquires an antibiotic resistance gene and is present in an environment where antibiotic usage is high, an outbreak with the new strain might occur. Positive selection on the new genetic variant is the driving force behind its expansion to epidemic proportions.Fittipaldi et al4Fittipaldi N. Beres S.B. Olsen R.J. Kapur V. Shea P.R. Watkins M.E. Cantu C.C. Laucirica D.R. Jenkins L. Flores A.R. Lovgren M. Ardanuy C. Liñares J. Low D.E. Tyrrell G.J. Musser J.M. Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.Am J Pathol. 2012; 180: 1522-1534Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar present a reasonable argument that a change in host and/or environmental conditions is unlikely to have been a primary cause of the emm59 epidemic, because none of the other coexisting GAS strains experienced a similar surge in prevalence within the same communities. Thus, a genetic mechanism likely produced the epidemic emm59 organism. The authors examined the genetic differences that distinguish historic emm59 isolates from the contemporary epidemic emm59 isolates. By mapping such genetic differences, they sought to identify gene candidates that may be responsible for the shift in virulence or transmissibility that led to the epidemic. To do this, the authors exploited advances in next-generation sequencing technology and determined the nucleotide sequence of the genomes of ∼600 isolates of emm59 GAS. Initially using one representative historic and one epidemic isolate for comparison, ∼150 single-nucleotide polymorphisms (SNPs) or indels (insertions or deletions) were uncovered within the core genome. In addition, the epidemic isolate lost one prophage (which harbored two virulence genes, a superantigen and phospholipase) and gained a prophage remnant at another locus, relative to the historic isolate. Might the loss of a virulence gene have led to the epidemic? Do any of the SNPs or indels lie within or adjacent to genes having an established role in GAS virulence? That analysis is currently ongoing at the authors' laboratory.Is it possible that the genetic change hypothesis is incorrect? It is possible, but another strong argument weighs heavily in favor of this hypothesis. Fittipaldi et al4Fittipaldi N. Beres S.B. Olsen R.J. Kapur V. Shea P.R. Watkins M.E. Cantu C.C. Laucirica D.R. Jenkins L. Flores A.R. Lovgren M. Ardanuy C. Liñares J. Low D.E. Tyrrell G.J. Musser J.M. Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.Am J Pathol. 2012; 180: 1522-1534Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar used animal models to test the virulence of representative historic and contemporary emm59 isolates. Under three different models for infection of normally sterile soft tissue after injection of bacteria at a subcutaneous or intramuscular site, the epidemic emm59 isolates displayed significantly greater virulence than the historic isolate. The animal findings showing differences in virulence provide supporting evidence that changes in virulence capacity evolved only recently. Thus, it appears that there may be a genetic change (yet to be defined) underlying the emm59 epidemic.Tissue-Specific Properties of the Epidemic emm59 StrainInvasive disease accounts for a large measure of morbidity and mortality due to GAS; however, infection at superficial epithelial tissue sites is far more prevalent.8Carapetis J.R. Steer A.C. Mulholland E.K. Weber M. The global burden of group A streptococcal diseases.Lancet Infect Dis. 2005; 5: 685-694Abstract Full Text Full Text PDF PubMed Scopus (1904) Google Scholar It is from the superficial tissue sites that most person-to-person transmission initiates. The most highly prevalent infections caused by GAS are pharyngitis and nonbullous impetigo. GAS is also associated with asymptomatic carriage at the throat; among school-aged children, the carriage rate often exceeds 20%. Compared with understanding of oropharyngeal carriage, there is less of an understanding of GAS colonization of normal skin. However, in one seminal prospective surveillance study involving children living on the Red Lake Indian Reservation (in northern Minnesota), where GAS pyoderma was endemic, GAS was frequently isolated from normal skin before the development of impetigo lesions.9Ferrieri P. Dajani A.S. Wannamaker L.W. Chapman S.S. Natural history of impetigo I. Site sequence of acquisition and familial patterns of spread of cutaneous streptococci.J Clin Invest. 1972; 51: 2851-2862Crossref PubMed Scopus (101) Google Scholar Thus, the primary reservoirs for GAS are the oropharyngeal mucosa and skin, and these tissues accommodate both carriage and infectious processes.Within just a few years, emm59 GAS in Canada rose from being rare to becoming the most common cause of invasive GAS infections, accounting for 13% of all invasive isolates collected over a 4-year period.6Tyrrell G.J. Lovgren M. St Jean T. Hoang L. Patrick D.M. Horsman G. Van Caeseele P. Sieswerda L.E. McGeer A. Laurence R.A. Bourgault A.M. Low D.E. Epidemic of group A Streptococcus M/emm59 causing invasive disease in Canada.Clin Infect Dis. 2010; 51: 1290-1297Crossref PubMed Scopus (39) Google Scholar However, no parallel rise in emm59 GAS isolates associated with pharyngitis occurred, with emm59 accounting for <0.5% of total pharyngitis isolates.4Fittipaldi N. Beres S.B. Olsen R.J. Kapur V. Shea P.R. Watkins M.E. Cantu C.C. Laucirica D.R. Jenkins L. Flores A.R. Lovgren M. Ardanuy C. Liñares J. Low D.E. Tyrrell G.J. Musser J.M. Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.Am J Pathol. 2012; 180: 1522-1534Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Thus, if the emm59 strain is transmitted primarily through a respiratory route, either it is extraordinarily virulent and has a very high invasive index, or there is a very strong tendency toward an asymptomatic course when colonizing the oropharynx. The far more likely explanation is that the primary mode of transmission for emm59 strains is via skin contact.Fittipaldi et al4Fittipaldi N. Beres S.B. Olsen R.J. Kapur V. Shea P.R. Watkins M.E. Cantu C.C. Laucirica D.R. Jenkins L. Flores A.R. Lovgren M. Ardanuy C. Liñares J. Low D.E. Tyrrell G.J. Musser J.M. Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.Am J Pathol. 2012; 180: 1522-1534Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar have begun to investigate the tissue-specific properties of the epidemic emm59 strain by comparison with an emm1 GAS strain (MGAS5005); emm1 is among the most prevalent emm types recovered from cases of pharyngitis in the United States and Canada in recent years.1Shulman S.T. Tanz R.R. Dale J.B. Beall B. Kabat W. Kabat K. Cederlund E. Patel D. Rippe J. Li Z. Sakota V. North American Streptococcal Pharyngitis Surveillance GroupSeven-year surveillance of North American pediatric group A streptococcal pharyngitis isolates.Clin Infect Dis. 2009; 49: 78-84Crossref PubMed Scopus (84) Google Scholar The emm1 and emm59 strains were compared for survival and growth in human saliva,10Shelburne 3rd, S.A. Granville C. Tokuyama M. Sitkiewicz I. Patel P. Musser J.M. Growth characteristics of and virulence factor production by group A Streptococcus during cultivation in human saliva.Infect Immun. 2005; 73: 4723-4731Crossref PubMed Scopus (63) Google Scholar where essential nutrients may be limiting and antimicrobial peptides may be active. The emm1 strain exhibited significantly greater survival in human saliva than the epidemic emm59 strain. The emm1 strain also showed significantly higher levels of colonization in a mouse oropharyngeal model. The findings support the notion that the MGAS5005 strain has a significantly higher tropism for the throat, compared with the epidemic emm59 strain.In contrast to the experimental models for throat infection, in a newly developed model for mouse skin lesions the emm1 and emm59 isolates showed equivalent levels of skin-to-skin transmission.4Fittipaldi N. Beres S.B. Olsen R.J. Kapur V. Shea P.R. Watkins M.E. Cantu C.C. Laucirica D.R. Jenkins L. Flores A.R. Lovgren M. Ardanuy C. Liñares J. Low D.E. Tyrrell G.J. Musser J.M. Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.Am J Pathol. 2012; 180: 1522-1534Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Thus, the collective data suggest that the emm1 strain is highly effective at infecting both throat and skin; the latter finding has strong epidemiological support in the high association of emm1 strains with soft-tissue infection. However, the MGAS5005 strain (emm1) under study is a sterile-site isolate with a mutation in the global transcriptional regulator gene covS and, consequently, exhibits the invasive transcriptome profile (TP), as opposed to the pharyngitis TP.11Sumby P. Whitney A.R. Graviss E.A. DeLeo F.R. Musser J.M. Genome-wide analysis of group a streptococci reveals a mutation that modulates global phenotype and disease specificity.PLoS Pathog. 2006; 2: e5Crossref PubMed Scopus (325) Google Scholar Perhaps the invasive TP of MGAS5005 provides an explanation for why this isolate effectively transmits in the mouse skin lesion model (a full-thickness incision), particularly if infection is established by introduction of bacteria into subepithelial tissue.12Cole J.N. Barnett T.C. Nizet V. Walker M.J. Molecular insight into invasive group A streptococcal disease.Nat Rev Microbiol. 2011; 9: 724-736Crossref PubMed Scopus (286) Google Scholar However, why an invasive TP strain such as MGAS5005 has a high capacity for oropharyngeal colonization is not entirely clear. Both the invasive TP and the pharyngitis TP are associated with invasive disease, but mounting evidence suggests that the pharyngitis TP is essential for infection of the throat.11Sumby P. Whitney A.R. Graviss E.A. DeLeo F.R. Musser J.M. Genome-wide analysis of group a streptococci reveals a mutation that modulates global phenotype and disease specificity.PLoS Pathog. 2006; 2: e5Crossref PubMed Scopus (325) Google Scholar, 12Cole J.N. Barnett T.C. Nizet V. Walker M.J. Molecular insight into invasive group A streptococcal disease.Nat Rev Microbiol. 2011; 9: 724-736Crossref PubMed Scopus (286) Google Scholar, 13Ikebe T. Ato M. Matsumura T. Hasegawa H. Sata T. Kobayashi K. Watanabe H. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates.PLoS Pathog. 2010; 6: e1000832Crossref PubMed Scopus (130) Google Scholar In some ways, the findings of Fittipaldi et al4Fittipaldi N. Beres S.B. Olsen R.J. Kapur V. Shea P.R. Watkins M.E. Cantu C.C. Laucirica D.R. Jenkins L. Flores A.R. Lovgren M. Ardanuy C. Liñares J. Low D.E. Tyrrell G.J. Musser J.M. Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.Am J Pathol. 2012; 180: 1522-1534Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar raise more questions than they provide answers, and the streptococcal field as a whole may need to reconcile these data in the future. The transcriptome profiles of the epidemic and historic emm59 strains were not reported,4Fittipaldi N. Beres S.B. Olsen R.J. Kapur V. Shea P.R. Watkins M.E. Cantu C.C. Laucirica D.R. Jenkins L. Flores A.R. Lovgren M. Ardanuy C. Liñares J. Low D.E. Tyrrell G.J. Musser J.M. Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.Am J Pathol. 2012; 180: 1522-1534Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar and this points to another ripe avenue for future investigations.Experimental data suggest that the emm1 strain has a higher affinity for the throat than does the emm59 strain. This finding gains additional support from epidemiological data showing that the emm59 strain is recovered only rarely from cases of pharyngitis. Superficial tissue site preferences for infection (ie, throat versus skin) for individual GAS strains have long been noted.14Wannamaker L.W. Differences between streptococcal infections of the throat and of the skin I.N Engl J Med. 1970; 282: 23-31Crossref PubMed Scopus (184) Google Scholar A useful biomarker for preferred tissue sites of infection is found in the emm pattern genotype, which is based on the 3′ ends of emm genes. There are three distinct genotype-defined groups: throat specialists causing pharyngitis (patterns A, B, and C), skin specialists causing impetigo (pattern D), and generalists, which as a group have no overriding preference (pattern E).15Bessen D.E. Kumar N. Hall G.S. Riley D.R. Luo F. Sabharwal H. Tengra F.K. Lizano S. Ford C.N. McShan W.M. Nyugen S.V. Dunning Hotopp J.C. Tettelin H. Genetic linkage mapping of determinants of tissue tropisms in group A Streptococcus.J Bacteriol. 2011; 193: 6651-6663Crossref PubMed Scopus (39) Google ScholarThe emm pattern was determined for three non-Canadian emm59/ST172 isolates examined by other investigators in earlier studies; all were pattern D (skin specialists).16McGregor K.F. Spratt B.G. Kalia A. Bennett A. Bilek N. Beall B. Bessen D.E. Multi-locus sequence typing of Streptococcus pyogenes representing most known emm types and distinctions among subpopulation genetic structures.J Bacteriol. 2004; 186: 4285-4294Crossref PubMed Scopus (101) Google Scholar, 17Bessen D.E. McGregor K.F. Whatmore A.M. Relationships between emm and multilocus sequence types within a global collection of Streptococcus pyogenes.BMC Microbiol. 2008; 8: 59Crossref PubMed Scopus (39) Google Scholar In a recent study comparing the accessory gene regions of GAS strains of >90 different emm types,15Bessen D.E. Kumar N. Hall G.S. Riley D.R. Luo F. Sabharwal H. Tengra F.K. Lizano S. Ford C.N. McShan W.M. Nyugen S.V. Dunning Hotopp J.C. Tettelin H. Genetic linkage mapping of determinants of tissue tropisms in group A Streptococcus.J Bacteriol. 2011; 193: 6651-6663Crossref PubMed Scopus (39) Google Scholar all but a few pattern D strains are associated with a single tight, genetically related cluster. The atypical pattern D exceptions include emm59, emm81, and emm85 strains, each of which have the sof gene, which is otherwise a distinguishing feature of pattern E strains. In fact, the emm59, emm81, and emm85 pattern D strains tend to group with the majority of pattern E strains and harbor pilus-encoding fibronectin-collagen-T (FCT) genetic regions that are also more typical of those of pattern E strains. Recently, emm85 isolates have been recovered from impetigo lesions in a tropical Australian community.2McGregor K. Bilek N. Bennett A. Kalia A. Beall B. Carapetis J. Currie B. Sriprakash K. Spratt B. Bessen D. Group A streptococci from a remote community have novel multilocus genotypes but share emm types and housekeeping alleles with isolates from worldwide sources.J Infect Dis. 2004; 189: 717-723Crossref PubMed Scopus (40) Google Scholar In a London (UK) hospital, emm81 isolates obtained from a variety of wound and soft-tissue infections in patients had the highest prevalence among all emm types.18McGregor K.F. Spratt B.G. Identity and prevalence of multilocus sequence typing-defined clones of group A streptococci within a hospital setting.J Clin Microbiol. 2005; 43: 1963-1967Crossref PubMed Scopus (26) Google Scholar An emm81 strain was recently associated with an outbreak of ulcerated skin lesions (ecthyma) among Israeli soldiers.19Wasserzug O. Valinsky L. Klement E. Bar-Zeev Y. Davidovitch N. Orr N. Korenman Z. Kayouf R. Sela T. Ambar R. Derazne E. Dagan R. Zarka S. A cluster of ecthyma outbreaks caused by a single clone of invasive and highly infective Streptococcus pyogenes.Clin Infect Dis. 2009; 48: 1213-1219Crossref PubMed Scopus (32) Google Scholar The atypical pattern D strains having a pattern E-like genotype (ie, emm59, emm81, and emm85) may possess factors that are required to initiate infection at the skin. However, unlike the typical pattern D strains, which tend to be associated with impetigo and are seemingly uncommon causes of invasive disease, the atypical pattern D strains may have an enhanced capacity for gaining access to deeper tissue. Future studies that examine the genetic similarities and differences between atypical pattern D strains and the typical pattern D and E strains may uncover the virulence factors critical for the initial skin infection and those necessary for subsequent invasion into deeper tissue.Most of the Canadian cases of invasive disease caused by the emm59 strain involved bacteremia or cellulitis, although there were also several instances of necrotizing fasciitis.6Tyrrell G.J. Lovgren M. St Jean T. Hoang L. Patrick D.M. Horsman G. Van Caeseele P. Sieswerda L.E. McGeer A. Laurence R.A. Bourgault A.M. Low D.E. Epidemic of group A Streptococcus M/emm59 causing invasive disease in Canada.Clin Infect Dis. 2010; 51: 1290-1297Crossref PubMed Scopus (39) Google Scholar The age distribution of invasive disease due to emm59 was strikingly different from nonbullous impetigo, which affects primarily children (although adults also can have impetigo).8Carapetis J.R. Steer A.C. Mulholland E.K. Weber M. The global burden of group A streptococcal diseases.Lancet Infect Dis. 2005; 5: 685-694Abstract Full Text Full Text PDF PubMed Scopus (1904) Google Scholar In the Canadian epidemic, there was a peak incidence of invasive disease involving infants, which dropped sharply by 2 years of age and rose again to a high level for patients 25 to 60 years of age.6Tyrrell G.J. Lovgren M. St Jean T. Hoang L. Patrick D.M. Horsman G. Van Caeseele P. Sieswerda L.E. McGeer A. Laurence R.A. Bourgault A.M. Low D.E. Epidemic of group A Streptococcus M/emm59 causing invasive disease in Canada.Clin Infect Dis. 2010; 51: 1290-1297Crossref PubMed Scopus (39) Google Scholar In addition, there were several notable risk factors for invasive disease caused by the emm59 strain, which were significant compared with all other GAS strains causing invasive disease within the very same communities. The risk factors for emm59 invasive disease included alcohol abuse and illicit drug use, homelessness, and hepatitis C virus infection.6Tyrrell G.J. Lovgren M. St Jean T. Hoang L. Patrick D.M. Horsman G. Van Caeseele P. Sieswerda L.E. McGeer A. Laurence R.A. Bourgault A.M. Low D.E. Epidemic of group A Streptococcus M/emm59 causing invasive disease in Canada.Clin Infect Dis. 2010; 51: 1290-1297Crossref PubMed Scopus (39) Google Scholar Thus, the emm59 strain epidemic may have originated and/or been amplified within a disadvantaged host population.Additional Genetic Data on emm59 EmergenceData on multilocus sequencing typing (MLST) of GAS are available in a valuable resource compiled by investigators from throughout the world. Five emm59/ST172 isolates are listed in the MLST database (http://spyogenes.mlst.net; last accessed January 2, 2012). Of these, the oldest emm59 isolate was recovered from an impetigo lesion in the U.S. in 1969. Three other emm59 isolates originated from normally sterile tissue sites. The fifth emm59/ST172 isolate (strain 149405, a blood isolate from the Czech Republic collected in 2004) was tested for resistance to antibiotics; it is reported to be susceptible to erythromycin and clindamycin, but resistant to tetracycline. Fittipaldi et al4Fittipaldi N. Beres S.B. Olsen R.J. Kapur V. Shea P.R. Watkins M.E. Cantu C.C. Laucirica D.R. Jenkins L. Flores A.R. Lovgren M. Ardanuy C. Liñares J. Low D.E. Tyrrell G.J. Musser J.M. Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.Am J Pathol. 2012; 180: 1522-1534Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar also report historic emm59/ST172 strains from Japan and Spain.Another plausible hypothesis for the emm59 epidemic is migration of an established clone into a new community, whereby the new host population and/or the new environment have risk factors that, in turn, enable the new migrant to grab a foothold and quickly spread. Tetracycline resistance in GAS is widespread among numerous emm types and typically arises via newly acquired resistance genes (as opposed to mutation of core genes). Nonetheless, no such genomic distinctions in antibiotic resistance genes were reported for the historic versus Canadian epidemic emm59 strains,4Fittipaldi N. Beres S.B. Olsen R.J. Kapur V. Shea P.R. Watkins M.E. Cantu C.C. Laucirica D.R. Jenkins L. Flores A.R. Lovgren M. Ardanuy C. Liñares J. Low D.E. Tyrrell G.J. Musser J.M. Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.Am J Pathol. 2012; 180: 1522-1534Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar raising the reasonable possibility that all of the emm59 strains are tetracycline-resistant, like the sole emm59 isolate reportedly tested after its recovery in the Czech Republic. Because tetracycline is not a recommended treatment for GAS infections, there are few recognized treatment failures, and resistance to this antibiotic often goes unnoticed. However, if a tetracycline-resistant strain migrates into a community having unusually high tetracycline usage, there may be enough positive selection pressure for the strain to reproduce and increase in numbers to exceed a critical threshold, thereby yielding sufficient penetration in the community to support its continual transmission to new hosts. Macrolide consumption is a key factor leading to increased prevalence of macrolide-resistant GAS,20SeppälÄ H. Klaukka T. Vuopio-Varkila J. Muotiala A. Helenius H. Lager K. Huovinen P. The effect of changes in the consumption of macrolide antibiotics on erythromycin resistance in group A streptococci in Finland Finnish Study Group for Antimicrobial Resistance.N Engl J Med. 1997; 337: 441-446Crossref PubMed Scopus (1059) Google Scholar which is often rooted in a heterogeneous set of genetically distinct clones. However, a sharp increase in prevalence of macrolide-resistant GAS can also be due to a single dominant clone.21Freeman A.F. Shulman S.T. Macrolide resistance in group A Streptococcus.Pediatr Infect Dis J. 2002; 21: 1158-1160Crossref PubMed Scopus (21) Google Scholar Knowledge of the antibiotic resistance profiles of the Canadian emm59 strains is critical information that can aid in a more thorough consideration of the possible mechanisms by which the epidemic arose, because the selective pressure exerted by antibiotics is among the strongest to which bacteria are subject.It remains possible that the epidemic emm59 clone existed elsewhere in the world for a long period of time, emerging to cause an epidemic in Canada because of unknown host or environmental risk factors that are uniquely specific to the emm59 clone. Alternatively, the initial rise in prevalence of a migrant may be due to entirely random factors. It is also plausible that the contemporary emm59 clone is not rare but, rather, is abundant in an undersampled region. Although surveillance for GAS is extensive, in many pockets of the world, particularly much of Africa and parts of Asia, there is a dearth of surveillance reports. Of the >38,000 isolates evaluated in a meta-analysis of GAS surveillance studies,5Steer A.C. Law I. Matatolu L. Beall B.W. Carapetis J.R. Global emm type distribution of group A streptococci: systematic review and implications for vaccine development.Lancet Infect Dis. 2009; 9: 611-616Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar the vast majority of organisms (>32,000) originate from countries with market economies. Another possible scenario is that the emm59 clone persisted in a region that does indeed undergo frequent surveillance, but that it tended not to cause an extensive degree of severe disease because the host and/or environmental risk factors did not align. If the emm59 c

ABSTRACT Group A Streptococcus (GAS) has a rich evolutionary history of horizontal transfer among its core genes. Yet, despite extensive genetic mixing, GAS strains have discrete ecological phenotypes. To further our understanding of the molecular basis for ecological phenotypes, comparative genomic hybridization of a set of 97 diverse strains to a GAS pangenome microarray was undertaken, and the association of accessory genes with emm genotypes that define tissue tropisms for infection was determined. Of the 22 nonprophage accessory gene regions (AGRs) identified, only 3 account for all statistically significant linkage disequilibrium among strains having the genotypic biomarkers for throat versus skin infection specialists. Networked evolution and population structure analyses of loci representing each of the AGRs reveal that most strains with the skin specialist and generalist biomarkers form discrete clusters, whereas strains with the throat specialist biomarker are highly diverse. To identify coinherited and coselected accessory genes, the strength of genetic associations was determined for all possible pairwise combinations of accessory genes among the 97 GAS strains. Accessory genes showing very strong associations provide the basis for an evolutionary model, which reveals that a major transition between many throat and skin specialist haplotypes correlates with the gain or loss of genes encoding fibronectin-binding proteins. This study employs a novel synthesis of tools to help delineate the major genetic changes associated with key adaptive shifts in an extensively recombined bacterial species.

Infections caused by multiresistant Gram-positive bacteria represent a major health burden in the community as well as in hospitalized patients. Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium are well-known pathogens of hospitalized patients, frequently linked with resistance against multiple antibiotics, compromising effective therapy. Streptococcus pneumoniae and Streptococcus pyogenes are important pathogens in the community and S. aureus has recently emerged as an important community-acquired pathogen. Population genetic studies reveal that recombination prevails as a driving force of genetic diversity in E. faecium, E. faecalis, S. pneumoniae and S. pyogenes, and thus, these species are weakly clonal. Although recombination has a relatively modest role driving the genetic variation of the core genome of S. aureus, the horizontal acquisition of resistance and virulence genes plays a key role in the emergence of new clinically relevant clones in this species. In this review, we discuss the population genetics of E. faecium, E. faecalis, S. pneumoniae, S. pyogenes and S. aureus. Knowledge of the population structure of these pathogens is not only highly relevant for (molecular) epidemiological research but also for identifying the genetic variation that underlies changes in clinical behaviour, to improve our understanding of the pathogenic behaviour of particular clones and to identify novel targets for vaccines or immunotherapy.

ABSTRACT Streptococcus pyogenes is an important pathogen that causes a variety of diseases. The most common infections involve the throat (pharyngitis) or skin (impetigo); however, the factors that determine tissue tropism and severity are incompletely understood. The S. pyogenes NAD+ glycohydrolase (SPN) is a virulence factor that has been implicated in contributing to the pathogenesis of severe infections. However, the role of SPN in determining the bacterium's tissue tropism has not been evaluated. In this report, we examine the sequences of spn and its endogenous inhibitor ifs from a worldwide collection of S. pyogenes strains. Analysis of average pairwise nucleotide diversity, average number of nucleotide differences, and ratio of nonsynonymous to synonymous substitutions revealed significant diversity in spn and ifs. Application of established models of molecular evolution shows that SPN is evolving under positive selection and diverging into NAD+ glycohydrolase (NADase)-active and -inactive subtypes. Additionally, the NADase-inactive SPN subtypes maintain the characteristics of a functional gene while ifs becomes a pseudogene. Thus, NADase-inactive SPN continues to evolve under functional constraint. Furthermore, NADase activity did not correlate with invasive disease in our collection but was associated with tissue tropism. The ability to cause infection at both the pharynx and the skin (“generalist” strains) is correlated with NADase-active SPN, while the preference for causing infection at either the throat or the skin (“specialist” strains) is associated with NADase-inactive SPN. These findings suggest that SPN has a NADase-independent function and prompt a reevaluation of the role of SPN in streptococcal pathogenesis.

This chapter contains sections titled: Habitats, Transmission, and Disease Classical Strain Typing Multilocus Sequence Typing (MLST) Based on Housekeeping Genes Species Boundaries and Gene Flow Niche-driving Genes Bacterial Population Dynamics and Selection Machinery of Genetic Change, Revisited References

Background Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging global pathogen that can colonize and infect humans. Although most SDSE isolates possess the Lancefield group G carbohydrate, a significant minority have the group C carbohydrate. Isolates are further sub-typed on the basis of differences within the emm gene. To gain a better understanding of their molecular epidemiology and evolutionary relationships, multilocus sequence typing (MLST) analysis was performed on SDSE isolates collected from Australia, Europe and North America. Methodology/Principal Findings The 178 SDSE isolates, representing 37 emm types, segregate into 80 distinct sequence types (STs) that form 17 clonal complexes (CCs). Eight STs recovered from all three continents account for >50% of the isolates. Thus, a small number of STs are highly prevalent and have a wide geographic distribution. Both ST and CC strongly correlate with group carbohydrate. In contrast, eleven STs were associated with >1 emm type, suggestive of recombinational replacements involving the emm gene; furthermore, 35% of the emm types are associated with genetically distant STs. Data also reveal a history of extensive inter- and intra-species recombination involving the housekeeping genes used for MLST. Sequence analysis of single locus variants identified through goeBURST indicates that genetic change mediated by recombination occurred ∼4.4 times more frequently than by point mutation. Conclusions/Significance A few genetic lineages with an intercontinental distribution dominate among SDSE causing infections in humans. The distinction between group C and G isolates reflects recent evolution, and no long-term genetic isolation between them was found. Lateral gene transfer and recombination involving housekeeping genes and the emm gene are important mechanisms driving genetic variability in the SDSE population.