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Journal of Clinical Microbiology, June 2007, p. 2044-2047, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00496-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Nonoutbreak Surveillance of Group A Streptococci Causing Invasive Disease in Portugal Identified Internationally Disseminated Clones among Members of a Genetically Heterogeneous Population{triangledown}

A. Friães, M. Ramirez,* J. Melo-Cristino, and the Portuguese Group for the Study of Streptococcal Infections

Instituto de Microbiologia, Instituto de Medicina Molecular, Faculdade Medicina, Universidade de Lisboa, Lisboa, Portugal

Received 6 March 2007/ Accepted 12 April 2007


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ABSTRACT
 
The typing of 160 invasive Streptococcus pyogenes isolates confirmed the importance of pulsed-field gel electrophoresis and multilocus sequence typing for defining clones. The results identified an extremely diverse population and highlighted the importance of both internationally disseminated and local clones not previously associated with invasive disease.


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TEXT
 
A reemergence of invasive disease caused by Streptococcus pyogenes (a member of the group A streptococci [GAS]) has been noted since the late 1980s, both in North America and in Europe (3). This increase in the incidence of GAS infections has frequently been associated with specific clones, suggesting the possibility that the rise of particularly virulent clones is responsible for this reemergence. The identification of GAS clones in surveillance and epidemiological studies has frequently relied on serotyping using two variable surface antigens, the T antigen (T typing) and the M protein (M typing, or emm typing, as the protein is encoded by the emm gene) (3). Recent work suggests that emm typing alone is not sufficient to unambiguously identify GAS clones and that this method must be complemented with an analysis by either pulsed-field gel electrophoresis (PFGE) or multilocus sequence typing (MLST) (1). A total of 160 nonduplicate GAS isolates recovered from normally sterile sites (150 from blood, 7 from pleural fluid, and 3 from cerebrospinal fluid) were collected in 19 laboratories distributed throughout Portugal that were asked to submit all isolates between 2000 and 2005. The number of participating laboratories was not constant, and this variation was reflected in the number of isolates available in each of the study years: 6 in 2000 (3 laboratories), 15 in 2001 (8 laboratories), 17 in 2002 (10 laboratories), 27 in 2003 (12 laboratories), 39 in 2004 (15 laboratories), and 56 in 2005 (19 laboratories).

Twelve different T serotypes (20) were identified, and 23 isolates (14%) were nontypeable (Simpson's index of diversity [SID] ± 95% confidence interval [CI], 88.2% ± 2.3%) (1), whereas 30 different emm types were identified (SID ± 95% CI, 92.0% ± 2.0%). The presence of genes encoding GAS pyrogenic toxins was studied by PCR (2, 9, 16, 18). As expected, the results confirmed the presence of the chromosomal genes speB and speF in all strains except one. Eleven different exotoxin gene profiles were identified (SID ± 95% CI, 82.2% ± 3.0%), but a significant fraction of the isolates (16%) were negative for all but the chromosomal genes (Table 1).


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  		TABLE 1. Properties of the PFGE clones of invasive S. pyogenes isolates from Portugal

The dendrogram based on the PFGE profiles of SmaI- or Cfr9I-digested total DNA (1, 19) from the isolates identified 13 major clusters accounting for 86% of all isolates (SID ± 95% CI, 92.0% ± 2.1%) (Fig. 1). Among the 37 isolates characterized by MLST (5), four new alleles were identified and submitted to the S. pyogenes MLST database, namely, recP82, gki98, gtr73, and murI68, as well as seven new sequence types (STs; ST258 and ST406 to ST411) that are all single-locus variants of preexisting STs, except for ST258, which is a double-locus variant of three preexisting STs. The overall level of correspondence between PFGE clusters and emm types was high, with a Wallace coefficient of 0.890, meaning that only 1 out of every 10 pairs of isolates grouped into the same PFGE cluster will not share the same emm type (1). In most cases, the presence of more than one emm type in the same lineage is associated with the presence of more than one ST. In two instances, although the isolates grouped by PFGE shared the same emm type, they presented different STs (PFGE clusters B and D) (Fig. 1 and Table 1). However, the two pairs of STs found among isolates expressing each of these emm types (ST15-ST406 and ST382-ST411) are single-locus variants of each other, supporting a close genetic relationship between the isolates as indicated by PFGE.


Figure 1
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  		FIG. 1. PFGE SmaI/Cfr9I macrorestriction profile analysis of S. pyogenes isolates from invasive infections in Portugal. (A) Dendrogram showing a cluster analysis of the PFGE profiles of the 160 isolates studied as determined by the unweighted-pair group method using average linkages. Dice coefficients (percentages) are indicated in the scale above the dendrogram. Each major clone (defined as a group of ≥5 isolates with a Dice coefficient of ≥80%) is represented by a triangle with a size proportional to the number of isolates included in the cluster, followed by a capital letter designating the clone and a subscript number indicating the number of isolates it comprises. (B) PFGE profiles of representatives of each clone. Capital letters above the lanes correspond to the clone designations; m, lambda PFGE ladder marker (New England Biolabs, Beverly, MA).

Globally, both emm type and PFGE cluster assignment were excellent predictors of the exotoxin gene profile, with Wallace coefficients of 0.906 and 0.871, respectively, such that any two isolates grouped together by these characteristics had a high probability (close to 90%) of sharing the same exotoxin gene profile.

In most countries, emm types 1, 3, and 28 have traditionally been associated with invasive GAS disease (4). In this study, emm types 1 and 3 were also those expressed by isolates in the main PFGE clusters, A and B, which accounted for 30% of all isolates and were associated with ST28 and ST15-ST406, respectively. Several studies report a correlation between the presence of the speA gene and invasive GAS isolates, especially emm1 isolates causing streptococcal toxic shock syndrome (13, 22). In the present study, speA was also detected in all isolates of the two major PFGE clusters, but all other isolates lacked speA, and the most frequent exotoxin gene found was speC. The exotoxin gene profile of the main invasive clone in Portugal is in agreement with previous reports for isolates of the same emm type (emm1) in other countries (2, 18, 23). On the other hand, though ssa has recently been reported as being more prevalent among M1/emm1 invasive isolates than among M1/emm1 noninvasive isolates (4, 17), it was not found among any of the emm1 isolates in this study. Taken together with the high Wallace coefficient found for the PFGE clusters and the exotoxin gene profiles, this result suggests that there are two distinct lineages carrying the emm1 allele responsible for invasive infections and emphasizes the importance of using PFGE or MLST to fully characterize GAS clones (1).

When we examined the association of the major PFGE clusters with the age groups of the patients, only the distribution of PFGE cluster E among isolates from the three age groups considered (≤18 years, 18 to 60 years, and >60 years) was significantly different from the expected distribution, with this cluster assignment being more frequent among isolates from pediatric patients than among those from adults (P = 0.003; one-tailed Fisher's exact test) (10). Although emm12, the dominant type in this PFGE cluster, corresponded to the most abundant M type found among healthy children in Tokyo during an extensive longitudinal study (8), an association between M12 and pediatric invasive disease was not previously noted. The fact that the isolates described here were not geographically or temporally clustered suggests that a clone defined by a characteristic PFGE profile, ST36, and carrying the emm12 allele is associated with children and pediatric invasive disease in particular.

To evaluate the geographic spread of the clones identified, we resorted to the data available in the S. pyogenes MLST database (http://spyogenes.mlst.net). Representatives of ST28 are widely dispersed, being found in several countries worldwide, while ST15, associated with the B PFGE cluster, has been found only in Europe and North America. The isolates exhibiting emm28, the third-most-frequent emm type in this and most studies of invasive GAS disease, were dispersed among several PFGE clusters, with macrolide-resistant isolates grouping together and apart from susceptible isolates (data not shown), suggesting that there may be other genetic differences between these isolates. However, the PFGE distinction was not supported by the other methods used to characterize them, with all isolates presenting ST52 and identical exotoxin profiles (Table 1). ST52 is also widely geographically dispersed, being found in several countries worldwide.

In contrast, the STs found in the other two largest PFGE clusters, C (ST164 and ST11) and D (ST382, ST411, and ST402), have a much more limited geographic distribution. ST164 has been found in Israel, and ST11 has been found in Trinidad and the United States, while ST382 has been found in Spain, Russia, and Austria, and ST411 and ST402 have been limited to Portugal. Moreover, none of these STs were previously found to cause invasive infections outside of Portugal. Interestingly, the ST11 isolates found in the MLST database were associated exclusively with impetiginous lesions, while the ST11 isolates reported here were recovered from blood. Several lines of evidence suggest that invasive isolates in industrialized countries reside mainly in a throat reservoir (7). Although the identification in this study of macrolide-resistant isolates representing the major clones found among isolates causing pharyngitis (19) is in agreement with this suggestion (data not shown), the data available regarding ST11 isolates recovered in other countries suggest that strains with this ST may reside in another important GAS reservoir, the impetiginous lesion.

While most isolates presented emm alleles associated exclusively with S. pyogenes, one isolate had an emm allele (stG1750) that has been identified only among group G streptococci (GGS), according to the Centers for Disease Control and Prevention database. The MLST analysis of the stG1750 isolate revealed a novel allelic profile which was assigned ST258, but all alleles at each of the genes had already been found in other STs present in the S. pyogenes MLST database. An eBURST analysis (6) identified this ST as a singleton, but there are three double-locus variants in the S. pyogenes MLST database. Taken together, the results of molecular typing support the identification of this isolate as S. pyogenes, raising the possibility that it may have acquired the emm gene from GGS by lateral gene transfer. Evidence for the horizontal transfer of emm genes between GAS and GGS has been reported previously (21), but this emm type was not found among a large collection of group G and C streptococci responsible for human infections in Portugal during the same time period as the isolates analyzed in this report (14).

Previous studies have addressed specific questions regarding GAS infections in Portugal (12, 15, 19), but this is the largest and most detailed study of GAS invasive isolates from Portugal, allowing the characterization of the main lineages of S. pyogenes causing invasive disease. This study identified extensive diversity among GAS isolates, as attested by the high SIDs of all typing methodologies, that results in a lower potential coverage of GAS invasive infections in Portugal by a future 26-valent vaccine (69%) than that in the United States (11).


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ACKNOWLEDGMENTS
 
This work was supported partly by Fundação para a Ciência e a Tecnologia (PTDC/SAUESA/72321), Portugal.

The members of the Portuguese Group for the Study of Streptococcal Infections are as follows: Centro Hospitalar de Cascais, Ana Fonseca and Adriana Coutinho; Centro Hospitalar de Coimbra, Ana Florinda Alves and Luís Albuquerque; Centro Hospitalar de Vila Nova de Gaia, Paulo Lopes, Ismália Calheiros, Luísa Felício, and Lourdes Sobral; Hospital Central do Funchal, Teresa Afonso; Hospital Infante D. Pedro, Aveiro, Elmano Ramalheira and Ana Margarida Paradela; Hospital D. Estefânia, Lisboa, Rosa M. Barros and Maria Isabel Peres; Hospital Garcia de Orta, Almada, José Diogo, Ana Rodrigues, and Isabel Nascimento; Hospital Pedro Hispano, Matosinhos, Valquíria Alves, Antónia Read, and Margarida Monteiro; Hospital de Santa Luzia, Elvas, Ilse Fontes; Hospital de Santa Maria, Lisboa, Luís Lito, Maria Luís Fernandes, and Maria José Salgado; Hospital de Santa Marta, Lisboa, Margarida Pinto and Hermínia Choon; Hospital de Santo António, Porto, Ana Paula Castro, Maria Helena Ramos, and José M. Amorim; Hospital de São Francisco Xavier, Lisboa, Filomena Martins, Maria Ana Pessanha, and Elsa Gonçalves; Hospital de São João, Porto, Fernanda Cotta, Maria José Machado Vaz, and Cidália Pina-Vaz; Hospital de São Marcos Braga, Maria Alberta Faustino and Adelaide Alves; Hospital Senhora da Oliveira, Guimarães, Ana Paula M. Vieira; Hospitais da Universidade de Coimbra, Rosa Velho, Rui Tomé, Celeste Pontes, and Graça Ribeiro; Hospital de Vila Real, Ana Paula Castro; and Hospital dos SAMS, Lisboa, Luísa Cabral and Olga Neto.


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FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Microbiologia, Faculdade Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, PT 1649-028 Lisboa, Portugal. Phone: 351-21 799 9460. Fax: 351-21 799 9459. E-mail: ramirez{at}fm.ul.pt Back

{triangledown} Published ahead of print on 25 April 2007. Back


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Journal of Clinical Microbiology, June 2007, p. 2044-2047, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00496-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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