Annotated Whole-Genome Multilocus Sequence Typing Schema for Scalable High-Resolution Typing of Streptococcus pyogenes.

Streptococcus pyogenes is a major human pathogen with high genetic diversity, largely created by recombination and horizontal gene transfer, making it difficult to use single nucleotide polymorphism (SNP)-based genome-wide analyses for surveillance. Using a gene-by-gene approach on 208 complete genomes of S. pyogenes, a novel whole-genome multilocus sequence typing (wgMLST) schema was developed, comprising 3,044 target loci. The schema was used for core-genome MLST (cgMLST) analyses of previously published data sets and 265 newly sequenced draft genomes with other molecular and phenotypic typing data. Clustering based on cgMLST data supported the genetic heterogeneity of many emm types and correlated poorly with pulsed-field gel electrophoresis macrorestriction profiling, superantigen gene profiling, and MLST sequence type, highlighting the limitations of older typing methods. While 763 loci were present in all isolates of a data set representative of S. pyogenes genetic diversity, the proposed schema allows scalable cgMLST analysis, which can include more loci for an increased resolution when typing closely related isolates. The cgMLST and PopPUNK clusters were broadly consistent in this diverse population. The cgMLST analyses presented results comparable to those of SNP-based methods in the identification of two recently emerged sublineages of emm1 and emm89 and the clarification of the genetic relatedness among isolates recovered in outbreak contexts. The schema was thoroughly annotated and made publicly available on the chewie-NS online platform (https://chewbbaca.online/species/1/schemas/1), providing a framework for high-resolution typing and analyzing the genetic variability of loci of particular biological interest.

Infecciones por Chlamydia trachomatis en un hospital portugués de atención terciaria: estudio retrospectivo de 11 años / Chlamydia trachomatis Infections in a Tertiary Care Portuguese Hospital: An 11-Year Retrospective Study

Antecedentes Las infecciones por Chlamydia trachomatis siguen siendo un problema de salud mundial. Los objetivos de este estudio fueron determinar el número de infecciones por C.trachomatis por año, caracterizar los rasgos demográficos de la población infectada e identificar las infecciones de transmisión sexual (ITS) asociadas. Con dicha finalidad se recogieron los casos diagnosticados en los últimos 11años en el principal centro hospitalario de Lisboa. Materiales y métodos Se incluyeron todas las infecciones causadas por C.trachomatis y confirmadas por el laboratorio entre el 1 de enero de 2009 y el 31 de diciembre de 2019. Resultados Se recogieron un total de 6.404 muestras, de las cuales 602 fueron positivas para C.trachomatis y correspondían a un total de 550 pacientes. El 60% de los casos fueron diagnosticados por dermatólogos-venereólogos. La mayoría de los casos se observaron en pacientes de sexo masculino (n=371; 67,5%), con una mediana de edad de 26,1años (desviación estándar: 7,98) y con un rango de edad que oscilaba entre los 13 y los 68años. Más de la mitad de los casos (51,3%) se diagnosticaron en pacientes menores de 25años. En 87 pacientes (15,8%) se hallaron antecedentes de otra ITS. En 198 pacientes (36%) se diagnosticaron además otras ITS concomitantes, y 43 pacientes (7,8%) presentaron una ITS posteriormente. En cuanto al seguimiento, 196 pacientes (correspondientes al 35,6% del total de pacientes) no acudieron a una reevaluación tras el diagnóstico. Conclusión El número de casos confirmados de C.trachomatis ha demostrado un incremento con el transcurso de los años, afectando principalmente a pacientes menores de 25años. La infección por C.trachomatis se asociará especialmente a la infección por Neisseria gonorrhoeae; sin embargo, las infecciones concomitantes por otras ITS serán también muy prevalentes. Teniendo en cuenta las posibles complicaciones de la infección por C.trachomatis (AU)

Background Chlamydia trachomatis infections remain a burden worldwide. The goals of this study were to determine the number of C.trachomatis infections per year, characterize the demographic traits of the infected population and identify the associated sexually transmitted infections (STI), over the last 11years, in Lisbon main Hospital Centre. Materials and methods Laboratorial confirmed C.trachomatis infections diagnosed between January 1st, 2009 and December 31st, 2019 were evaluated. Results A total of 6404 samples were collected, with 602 being positive for C.trachomatis, corresponding to 550 patients. 60% of diagnoses were made by dermatovenereologists and most cases occurred in males (n=371; 67.5%), with a median age of 26.1years (standard deviation 7.98), ranging from 13 to 68years. More than half the cases (51.3%) occurred below the age of 25years. In 87 patients (15.8%) there was previous history of STI, in 198 patients (36%) a concomitant STI was present and 43 patients (7.8%) developed a posterior STI. Concerning follow-up, 196 patients (corresponding to 35.6% of total patients) did not show for a re-evaluation after the diagnosis was made. Conclusion Confirmed cases of C.trachomatis infection have risen along the years, affecting mainly people younger than 25 years. C.trachomatis infection is especially associated with Neisseriagonorrhoeae infection, but other STI are also very prevalent. Considering the potential complications of C.trachomatis infection, screening and treatment are necessary. The high percentage of diagnoses made by dermatovenereologists support the pivotal role of dermatovenereology in diagnosing C.trachomatis infections (AU)

Estudio retrospectivo acerca de la evolución y el desarrollo de resistencias antimicrobianas en casos diagnosticados de gonorrea en un hospital de nivel terciario en Portugal durante 10 años / Gonorrhea in a Tertiary Care Portuguese Hospital: A 10-year Retrospective Study of the Evolution of Cases and Drug Resistance of the Isolates

INTRODUCCIÓN: Las infecciones de transmisión sexual siguen siendo un problema mundial de salud pública. Aunque tradicionalmente se considera una infección tratable, la aparición de resistencias de la Neisseria gonorrhoeae (N. gonorrhoeae) a los agentes antimicrobianos se ha convertido en un tema de vital importancia. El objetivo del presente estudio fue valorar tanto la incidencia como la resistencia frente a los agentes antimicrobianos desarrollada por las cepas de N. gonorrhoeae en un hospital portugués durante 10 años. MÉTODOS: Se incluyeron aquellos casos con confirmación en laboratorio de infecciones por N. gonorrhoeae diagnosticados entre los años 2009 y 2018. Se estudió además la sensibilidad frente a la penicilina, las tetraciclinas, el ciprofloxacino, la azitromicina y la cefotaxima, así como las características clínicas y demográficas relacionadas. RESULTADOS: Desde el año 2009 al 2018, en nuestro centro fueron detectados un total de 440 casos de infecciones por N. gonorrhoeae, evidenciándose además un incremento anual significativo en el número de casos (p < 0,05). La mayoría de los casos se observaron en pacientes del sexo masculino (97,9%), con una media de edad de 25 años. En el 88,7% de los casos el tratamiento utilizado fue la asociación de ceftriaxona con azitromicina. La resistencia objetivada frente a la penicilina, las tetraciclinas y el ciprofloxacino permaneció elevada durante todo el período del estudio. CONCLUSIONES: La resistencia antimicrobiana de la infección por N. gonorrhoeae apareció al poco tiempo después de la introducción del tratamiento antimicrobiano. Para poder combatir esta situación es necesario implantar una mayor vigilancia, así como realizar más estudios que busquen combinar tanto datos epidemiológicos como de sensibilidad. En nuestra población, la N. gonorrhoeae sigue siendo muy sensible a los tratamientos antibióticos que se recomiendan actualmente. Sin embargo, el tratamiento con ciprofloxacino, azitromicina (en monoterapia) y la penicilina se deben de evitar como tratamiento empírico

BACKGROUND: Sexually Transmitted Infections remain a major public health concern worldwide. Although traditionally considered treatable, the emergence of Neisseria gonorrhoeae resistance to antimicrobials is currently a serious problem. The goal of this study was to evaluate the incidence and trends of antimicrobial resistance over the last 10 years in N. gonorrhoeae isolates from a Portuguese Centre. METHODS: Laboratorial confirmed N. gonorrhoeae infections diagnosed between 2009 and 2018 were evaluated. Susceptibilities to penicillin, tetracycline, ciprofloxacin, azithromycin and cefotaxime were studied, along with demographic and clinical characteristics. RESULTS: From 2009 to 2018, 440 cases of N. gonorrhoeae infection were diagnosed in our center, with a significant yearly increase (p < 0.05). Most cases occurred in males (97.9%), with a median age of 25 years. In 88.7% of the cases, treatment with ceftriaxone plus azithromycin was used. Resistances to penicillin, tetracycline and ciprofloxacin remained high throughout the study period. CONCLUSIONS: Antimicrobial resistance of N. gonorrhoeae appeared shortly after the introduction of antimicrobials. To combat this problem, improved surveillance and more studies combining susceptibility and epidemiological data are needed. In our population, N. gonorrhoeae remains highly susceptible to the antibiotics currently recommended for its treatment, whereas ciprofloxacin, azithromycin (in monotherapy) and penicillin should be avoided as empirical treatment

Gonorrhea in a Tertiary Care Portuguese Hospital: A 10-year Retrospective Study of the Evolution of Cases and Drug Resistance of the Isolates. / Estudio retrospectivo acerca de la evolución y el desarrollo de resistencias antimicrobianas en casos diagnosticados de gonorrea en un hospital de nivel terciario en Portugal durante 10 años.

BACKGROUND: Sexually Transmitted Infections remain a major public health concern worldwide. Although traditionally considered treatable, the emergence of Neisseria gonorrhoeae resistance to antimicrobials is currently a serious problem. The goal of this study was to evaluate the incidence and trends of antimicrobial resistance over the last 10 years in N. gonorrhoeae isolates from a Portuguese Centre. METHODS: Laboratorial confirmed N. gonorrhoeae infections diagnosed between 2009 and 2018 were evaluated. Susceptibilities to penicillin, tetracycline, ciprofloxacin, azithromycin and cefotaxime were studied, along with demographic and clinical characteristics. RESULTS: From 2009 to 2018, 440 cases of N. gonorrhoeae infection were diagnosed in our center, with a significant yearly increase (p<0.05). Most cases occurred in males (97.9%), with a median age of 25 years. In 88.7% of the cases, treatment with ceftriaxone plus azithromycin was used. Resistances to penicillin, tetracycline and ciprofloxacin remained high throughout the study period. CONCLUSIONS: Antimicrobial resistance of N. gonorrhoeae appeared shortly after the introduction of antimicrobials. To combat this problem, improved surveillance and more studies combining susceptibility and epidemiological data are needed. In our population, N. gonorrhoeae remains highly susceptible to the antibiotics currently recommended for its treatment, whereas ciprofloxacin, azithromycin (in monotherapy) and penicillin should be avoided as empirical treatment.

Changes in emm types and superantigen gene content of Streptococcus pyogenes causing invasive infections in Portugal.

Fluctuations in the clonal composition of Group A Streptococcus (GAS) have been associated with the emergence of successful lineages and with upsurges of invasive infections (iGAS). This study aimed at identifying changes in the clones causing iGAS in Portugal. Antimicrobial susceptibility testing, emm typing and superantigen (SAg) gene profiling were performed for 381 iGAS isolates from 2010-2015. Macrolide resistance decreased to 4%, accompanied by the disappearance of the M phenotype and an increase of the iMLSB phenotype. The dominant emm types were: emm1 (28%), emm89 (11%), emm3 (9%), emm12 (8%), and emm6 (7%). There were no significant changes in the prevalence of individual emm types, emm clusters, or SAg profiles when comparing to 2006-2009, although an overall increasing trend was recorded during 2000-2015 for emm1, emm75, and emm87. Short-term increases in the prevalence of emm3, emm6, and emm75 may have been driven by concomitant SAg profile changes observed within these emm types, or reflect the emergence of novel genomic variants of the same emm types carrying different SAgs.

Capsule impairs efficient adherence of Streptococcus agalactiae to intestinal epithelium in tilapias Oreochromis sp.

Streptococcosis caused by Streptococcus agalactiae is one of the most important diseases in the tilapia aquaculture industry. The role of the capsule of Streptococcus agalactiae in adherence to fish surfaces has not been evaluated and the mechanism of capsular regulation during adhesion has not been described. The aim of this study was to evaluate the role of the capsule of S. agalactiae during adhesion to intestinal epithelium of tilapia (Oreochromis sp.) in an ex vivo infection model. We show that the capsule impairs the adhesion of bacteria to host intestinal epithelium. Wild type (WT) strain SaTiBe08-18 (S. agalactiae recovered from tilapia) had reduced adhesion (P < 0.0001) in comparison with its unencapsulated mutant of SaTiBe08-18 (Δcps). When WT was treated with sterile saline solution (pH 5) before infection of intestine explants, the adhesion was reached. The results suggest that the capsule impairs the adhesion of S. agalactiae to tilapia intestine and that the acidic milieu could regulate adherence of encapsulated strains. We found GlcNAc on the surface of adherent Δcps but not over the capsule in WT. This difference could be explained by the GlcNAc composition of Lancefield group B antigen and the peptidoglycan in GBS (Group B Streptococcus) and also may be related with better exposure of glycosylated adhesins in unencapsulated fish GBS. Understanding capsular regulation during adhesion of S. agalactiae may provide new leads to find a successful anti-adherence therapy to prevent streptococcosis in tilapia.

Changing epidemiology of group B streptococcal infections among adults in Iceland: 1975-2014.

We studied the bacterial characteristics and incidence of invasive infections caused by group B streptococci (GBS) in adults in Iceland in 1975-2014. A total of 145 isolates were characterized by serotyping, antimicrobial susceptibility, multilocus sequence typing and surface protein gene profiling. Disease incidence increased during the studied period (p <0.001), reaching 2.17 cases/100 000 person-years in 2013-14. Overall, serotype Ia was the most frequently found (23%), but serotypes Ib, II, III and V showed similar prevalence (14%-17%). Although there were notable changes in the proportion of most serotypes during the study period, only the decline of serotype III was statistically supported (p = 0.003) and was reflected in a decrease of clonal complexes CC17 and CC19 that included most serotype III isolates (p <0.04). On the other hand, the increase in frequency of CC1 was caused by two lineages expressing distinct serotypes: ST1/V/alp3 and ST196/IV/eps. Underlying the relative stability of serotype Ia were major changes in the lineages expressing this serotype, with an increase in the relative importance of CC23, including both ST23/Ia/eps and ST24/Ia/bca lineages, and a decrease in CC7. Nine cases of invasive GBS disease were caused by ST7, of possible zoonotic origin. All isolates were susceptible to penicillin. Rates of erythromycin and clindamycin resistance were 8.3% and 9.7%, respectively. An over-representation of resistance solely to clindamycin was associated with the unusual lsaC gene and serotype III ST19/rib lineage (p <0.001).

Evolving epidemiology of carbapenemase-producing Enterobacteriaceae in Portugal: 2012 retrospective cohort at a tertiary hospital in Lisbon.

Despite great efforts to enhance European epidemiological surveillance on carbapenemase-producing Enterobacteriaceae (CPE), information from several countries remains scarce. To address CPE epidemiology in Portugal, we have undertaken a retrospective cohort study of adults with CPE cultures identified in the microbiology laboratory of a tertiary hospital, in 2012. Sixty patients from 25 wards or intensive care units were identified. This is, to the best of our knowledge, the first report of clinical data on CPE in Portugal. It shows a hospital-wide CPE dissemination and alerts us to an evolving epidemiological situation not previously described.

Declining macrolide resistance in Streptococcus pyogenes in Portugal (2007-13) was accompanied by continuous clonal changes.

OBJECTIVES: Macrolide resistance among Streptococcus pyogenes [group A streptococci (GAS)] in Portugal decreased between 1999 and 2006 and this decrease was accompanied by alterations in the prevalence of macrolide resistance phenotypes and clonal composition of the population. The aims of this study were to determine the macrolide resistance rate, resistance phenotypes and clones of GAS recovered from pharyngitis in 2007-13 in Portugal. METHODS: Antimicrobial susceptibility was tested by disc diffusion. Macrolide-resistant isolates were characterized by emm typing, T typing, PFGE profiling and MLST, and the presence of macrolide resistance determinants was determined by PCR. RESULTS: We found continuing changes in macrolide resistance phenotypes and a persistent decline in overall erythromycin resistance, from 10% in 2007 to 1% in 2013. During this period there was a marked increase in emm11-ST403 cMLSB isolates, the disappearance of the emm3-ST315 M lineage and changes in the prevalence of previously identified GAS clones. Unexpectedly, the decline in erythromycin resistance and the decreasing prevalence of the MLSB phenotype were accompanied by a high consumption of long-acting and intermediate-acting macrolides, known to select for resistance and particularly for the erm(B) gene. CONCLUSIONS: The continuous decline in macrolide resistance detected since 2000, accompanied by a high clonal instability, emphasizes the importance of considering factors other than antibiotic consumption in explaining the prevalence of macrolide-resistant GAS.

Epidemiological survey of the first case of vancomycin-resistant Staphylococcus aureus infection in Europe.

We report on the follow-up and epidemiological study triggered by the isolation of the first vancomycin-resistant Staphylococcus aureus (VRSA) detected in Europe. The patient and 53 close contacts were screened for S. aureus colonization and all isolates recovered were characterized by multiple molecular typing methods. The VRSA remained confined to the infected foot of the patient and was not detected in any of the close contacts. Nasal colonization with S. aureus was detected in 20 subjects, of whom 15 carried methicilin-susceptible isolates with the remaining five harbouring methicilin-resistant S. aureus (MRSA). The majority of the isolates belonged to clones that have been previously shown to be prevalent in Portugal, both in the hospital setting and in the community. Only one isolate, an MRSA, was closely related to the VRSA. Like most of the characterized VRSA isolates from other countries, the VRSA isolated in Portugal belonged to clonal complex (CC) 5. Despite the absence of VRSA dissemination, the recent increase in the incidence of lineages belonging to CC5 in some European countries, including Portugal, may result in more frequent opportunities for the emergence of VRSA.

PCR based differentiation between Streptococcus dysgalactiae subsp. equisimilis strains isolated from humans and horses.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) can be severely pathogenic in humans and is increasingly isolated from horses with respiratory, reproductive or other diseases, although it is often considered a commensal bacterium. Here a PCR protocol is described for identifying SDSE recovered from humans. A multiplex PCR targeting the 16S rRNA and the streptokinase precursor gene has been optimized for differentiating between SDSE strains isolated from humans and those isolated from horses. Previously, the sequence of the streptokinase precursor gene of SDSE recovered from horses has been found in two human cases of pneumonia in Japan. Although further evaluation is required, the findings of this study suggest that SDSE strains are host-specific and this multiplex PCR protocol can be useful in further epidemiological studies and for investigating the zoonotic potential of SDSE.

Invasive infections due to Streptococcus pyogenes: seasonal variation of severity and clinical characteristics, Iceland, 1975 to 2012.

Epidemiology and clinical characteristics of invasive Group A streptococcal infections (IGASI) are highly variable. Long-term studies are needed to understand the interplay between epidemiology and virulence. In a population-based study of IGASI in Iceland from 1975 to 2012, 288 cases were identified by positive cultures from normally sterile body sites. Charts were reviewed retrospectively and emm-types of viable Streptococcus pyogenes isolates (n=226) determined. Comparing the first and last decade of the study period, IGASI incidence increased from 1.09 to 3.96 cases per 100,000 inhabitants per year. The most common were emm types 1 (25%), 28 (11%) and 89 (11%); emm1 strains were most likely to cause severe infections. Infections in adults were significantly more likely to be severe during the seasonal peak from January to April (risk ratio: 2.36, 95% confidence interval: 1.34­4.15). Significant seasonal variability in severity was noted among patients with diagnosis of sepsis, respiratory infection and cellulitis, with 38% of severe infections in January to April compared with 16% in other months (p<0.01). A seasonal increase in severity of IGASI suggested that generalised seasonal increase in host susceptibility, rather than introduction of more virulent strains may play a role in the pathogenesis of these potentially fatal infections.

Decreasing incidence and changes in serotype distribution of invasive pneumococcal disease in persons aged under 18 years since introduction of 10-valent and 13-valent conjugate vaccines in Portugal, July 2008 to June 2012.

The 10-valent pneumococcal conjugate vaccine (PCV10) became available in Portugal in mid-2009 and the 13-valent vaccine (PCV13) in early 2010. The incidence of invasive pneumococcal disease (IPD) in patients aged under 18 years decreased from 8.19 cases per 100,000 in 2008­09 to 4.52/100,000 in 2011­12. However, IPD incidence due to the serotypes included in the 7-valent conjugate vaccine (PCV7) in children aged under two years remained constant. This fall resulted from significant decreases in the number of cases due to: (i) the additional serotypes included in PCV10 and PCV13 (1, 5, 7F; from 37.6% to 20.6%), particularly serotype 1 in older children; and (ii) the additional serotypes included in PCV13 (3, 6A, 19A; from 31.6% to 16.2%), particularly serotype 19A in younger children. The decrease in serotype 19A before vaccination indicates that it was not triggered by PCV13 administration. The decrease of serotype 1 in all groups, concomitant with the introduction of PCV10, is also unlikely to have been triggered by vaccination, although PCVs may have intensified and supported these trends. PCV13 serotypes remain major causes of IPD, accounting for 63.2% of isolates recovered in Portugal in 2011­12, highlighting the potential role of enhanced vaccination in reducing paediatric IPD in Portugal.

Serotype discrimination of encapsulated Streptococcus pneumoniae strains by Fourier-transform infrared spectroscopy and chemometrics.

Fourier-transform infrared (FTIR) spectroscopy was evaluated as an alternative method for serotyping encapsulated Streptococcus pneumonia strains. Sixty-nine invasive strains from the Microbiology Laboratory of the Medicine Faculty of University of Lisbon were selected for the analysis. The encapsulated strains used in this work (9N, 9V, 14, 19A, 19F, 23A, 23B and 23F) were serotyped by the standard capsular reaction test using the chessboard method and specific sera. FTIR spectra were analysed with chemometric methods. Results showed that the best spectral region unveiling serotype difference is located at 1185-900 cm(-1). A partial least squares discriminant analysis model was calibrated using this spectral region against the chessboard method serotyping. FTIR spectroscopy proved to be able to correctly predict 100% of strains according to the serogroups (9, 14, 19, 23). A segregated analysis showed that it was also possible to differentiate serotypes belonging to the same serogroup although spectral differences are less pronounced as was initially expected.

Multilocus sequence analysis of Streptococcus canis confirms the zoonotic origin of human infections and reveals genetic exchange with Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus canis is an animal pathogen that occasionally causes human infections. Isolates recovered from infections of animals (n = 78, recovered from 2000 to 2010 in three European countries, mainly from house pets) and humans (n = 7, recovered from 2006 to 2010 in Portugal) were identified by phenotypic and genotypic methods and characterized by antimicrobial susceptibility testing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and emm typing. S. canis isolates presented considerable variability in biochemical profiles and 16S rRNA. Resistance to antimicrobial agents was low, with the most significant being tet(M)- and tet(O)-mediated tetracycline resistance. MLST analysis revealed a polyclonal structure of the S. canis population causing infections, where the same genetic lineages were found infecting house pets and humans and were disseminated in distinct geographic locations. Phylogenetic analysis indicated that S. canis was a divergent taxon of the sister species Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis and found evidence of acquisition of genetic material by S. canis from S. dysgalactiae subsp. equisimilis. PFGE confirmed the MLST findings, further strengthening the similarity between animal and human isolates. The presence of emm-like genes was restricted to a few isolates and correlated with some MLST-based genetic lineages, but none of the human isolates could be emm typed. Our data show that S. canis isolates recovered from house pets and humans constitute a single population and demonstrate that isolates belonging to the main genetic lineages identified have the ability to infect the human host, providing strong evidence for the zoonotic nature of S. canis infection.

Distribution of pilus islands in Streptococcus agalactiae that cause human infections: insights into evolution and implication for vaccine development.

At least one pilus island, PI-1 (70%), PI-2a (79%), or PI-2b (21%), was found among 898 Streptococcus agalactiae (group B streptococcus [GBS]) isolates recovered from humans, supporting the use of pilus proteins in vaccines. The stability and dominance of PI-1 and PI-2a in multiple serotypes and founder multilocus sequence types disseminated worldwide suggest it could be the PI combination present in ancestral GBS human pathogens.

Superantigen gene complement of Streptococcus pyogenes--relationship with other typing methods and short-term stability.

The profiling of the superantigen (SAg) encoding genes has been frequently used as a complementary typing method for group A streptococci (GAS), but a confusing gene nomenclature and a large diversity of primers used in screening has led to some conflicting results. The aim of this work was to develop a polymerase chain reaction (PCR) method capable of efficiently amplifying all the known allelic variants of these genes, and to evaluate the congruence of this methodology with other commonly used molecular typing methods. The presence of the 11 known SAg genes and two other exotoxin-encoding genes (speB and speF) was tested in a collection of 480 clinical GAS isolates, using two multiplex PCR reactions. The SAg gene profile was compared with other typing methods. Four naturally occurring deletions involving the genes speB, speF, and rgg were characterized, two of which were found among invasive isolates. The absence of the chromosomally encoded genes speG and smeZ was supported by Southern blot hybridization and associated with specific GAS lineages, while the presence of phage-encoded genes was more variable. Positive associations between SAg genes or between SAg profiles and emm types or pulsed-field gel electrophoresis (PFGE) clusters were observed. The results suggest that the SAg profile diversifies faster than other properties commonly used for molecular typing, such as emm type and multilocus sequence typing (MLST) sequence types (STs), and can be a useful complement in GAS molecular epidemiology. Still, the short-term stability of the SAg gene profile among prevalent genetic lineages may largely explain the observed associations between SAg genes.