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Journal of Clinical Microbiology, March 2006, p. 841-846, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.841-846.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Clonal Relationships between Invasive and Noninvasive Lancefield Group C and G Streptococci and emm-Specific Differences in Invasiveness

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

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

Received 10 August 2005/ Returned for modification 26 October 2005/ Accepted 13 December 2005


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ABSTRACT
 
Lancefield group G and group C streptococci (GGS and GCS, respectively) are pathogens responsible for a number of life-threatening infections. A collection of 116 recent (1998 to 2004) invasive (n = 28) and noninvasive (n = 88) GGS and GCS clinical isolates from Portugal were characterized. All isolates were identified as Streptococcus dysgalactiae subsp. equisimilis and characterized by emm typing and DNA macrorestriction profiling using pulsed-field gel electrophoresis (PFGE). emm typing revealed the presence of 22 distinct types, including 3 novel types. PFGE identified 14 clones with more than two isolates, but over half of the isolates were concentrated in 3 large clones. Individual clones and emm types showed a low level of association, since the majority of the clones included more than one emm type and the same emm type was found among diverse genetic backgrounds. Two emm types, stg2078 and stg10, were significantly more frequent among invasive isolates, and another two, stg6792 and stg166b, were present only in noninvasive isolates, suggesting a correlation between emm type and invasive disease potential.


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INTRODUCTION
 
Beta-hemolytic, large-colony-forming (diameter, >0.5 mm) Lancefield group G and group C streptococci (GGS and GCS, respectively) are increasingly recognized as important human pathogens (6, 16, 23), but the study of these infections has been hampered by the heterogeneity of the species presenting Lancefield group C and G polysaccharides. The taxonomic classification of both GGS and GCS is in a state of flux, but at least three species have been found in human infections: Streptococcus dysgalactiae subsp. equisimilis, Streptococcus equi subsp. zooepidemicus, and Streptococcus canis (12). Characterization of the isolates recovered from human infections to the species level is uncommon, and most of the available data consider GGS and GCS independently, with more information available on GGS bacteremia (6, 27) and much less for GCS (5). Invasive GGS infections were mostly reported to be caused by S. dysgalactiae subsp. equisimilis (6, 27). This species may also present group C polysaccharide, and such isolates have been recovered from human infections (26). However, few studies present species identification of GCS, and a study from Hong Kong reported that most group C beta-hemolytic streptococcal bacteremias are caused by S. equi subsp. zooepidemicus (28), while the contributions of the different species to GCS infections in other geographic locations remain mostly unknown.

Although both GGS and GCS are part of the normal human flora (9), they have also been identified as causative agents of infections of the respiratory tract (29), infections of the skin and soft tissue, and life threatening infections such as endocarditis, bacteremia, and meningitis, frequently with a poor prognosis (3, 9). More recently, an increasing number of reports described the association with streptococcal syndromes typically caused by Streptococcus pyogenes (Lancefield group A streptococcus [GAS]), such as streptococcal toxic shock syndrome (STSS) (16, 19) and acute rheumatic fever (15). Despite differences in the disease burden attributable to each of these organisms, GGS, GCS, and GAS are closely related genetically and share several virulence factors (9, 12). Proteins similar to the M protein (encoded by the emm gene), a key virulence factor in GAS, have long been identified in GGS and GCS from human infections, and the emm genes of both species share structural features such as substantial polymorphisms at the 5' end (21). The M protein contributes considerably to the invasive capacity of GAS by mediating the antiphagocytic, adherence, and internalization processes (7). For GAS, different emm genes have been suggested as genotypic markers for tissue site preference (20), and a recent study reevaluated an older observation that isolates expressing certain M proteins were more frequently associated with invasive disease (11). It is therefore conceivable that the M protein also plays an important part in GGS and GCS pathogenesis, although its role remains largely unexplored and most of the work has concentrated on the presence of superantigens in these bacteria (16, 19).

The M protein has also been used to differentiate strains, and the sequence-based typing scheme developed for GAS, relying on the hypervariable region of the emm gene, was successfully applied to GGS and GCS (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). Other molecular typing techniques, such as macrorestriction profiling using pulsed-field gel electrophoresis (PFGE), have also been used successfully with pyogenic streptococci, including GGS and GCS (2).

The higher rate of isolation of these organisms in recent studies may be due to improved detection methods, an increase in virulence, or an expanding population of compromised hosts. In this study we have used emm typing and PFGE to characterize a collection of invasive (i.e., recovered from normally sterile sites) and noninvasive GGS and GCS isolated in Portugal during the years 1998 to 2004. The molecular characterization of these isolates allowed us to test whether PFGE-defined clones associate specifically with certain emm types and if there was a higher invasive disease potential of particular emm types and PFGE-defined clones.


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MATERIALS AND METHODS
 
Bacterial isolates. A total of 116 beta-hemolytic, large-colony-forming (diameter, >0.5 mm) GGS and GCS isolates from clinical infections were collected from June 1998 to December 2004. The distribution of isolates over the study period was as follows: 5 in 1998, 14 in 1999, 12 in 2000, 14 in 2001, 14 in 2002, 35 in 2003, and 22 in 2004. The Lancefield groups and sources of the isolates are given in Table 1. The strains were recovered in nine laboratories, located in Portugal, that were asked to submit all nonduplicate GGS and GCS isolates associated with human infections.


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  		TABLE 1. GGS and GCS isolates recovered from invasive and noninvasive infections during 1998 to 2004 in Portugal

Species identification. Strains were identified to the genus level by the submitting laboratory. Upon receipt, the Lancefield group was confirmed by a commercial latex agglutination technique, the Slidex Strepto-Kit (bioMérieux, Marcy-l'Etoile, France). All further studies were carried out at the Laboratory of Microbiology of the Lisbon School of Medicine. Beta-hemolysis and colony size were confirmed in tryptic soy agar (Oxoid, Hampshire, England) supplemented with 5% (vol/vol) defibrinated sheep blood, after overnight incubation at 37°C. Further identification to the species level was done using the API 20 Strep system (bioMérieux, Marcy-l'Etoile, France). Additionally, all the invasive isolates were subjected to 16S rRNA sequencing. Two generic primers for gram-positive bacteria (24) were used to amplify and sequence a 1,423-bp fragment of the 16S rRNA coding region. Template DNA for the PCR was prepared according to the method recommended for emm typing (http://www.cdc.gov/ncidod/biotech/strep/protocols.htm). One microliter of DNA template lysate was added to the PCR mixture containing 1x PCR buffer (100 mM Tris-HCl, 500 mM KCl, pH 9, 0.1% gelatin, 1% [vol/vol] Triton X-100), 200 µM deoxynucleoside triphosphates (MBI Fermentas, Vilnius, Lithuania), 0.5 µM primers, 1.5 mM MgCl2, and 1 U of SUPER Tth DNA polymerase (HT Biotech, Cambridge, United Kingdom) in a final volume of 50 µl. PCR conditions for amplification were as follows: 35 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min, and a final extension at 72°C for 10 min in a Biometra T gradient thermocycler (Goettingen, Germany). Amplification products were purified using a High Pure PCR product purification kit (Roche, Mannheim, Germany) and sequenced with the primers used for amplification. The sequences were compared to those deposited in the Ribosomal Database Project II (http://rdp.cme.msu.edu/) and GenBank.

emm typing. Isolates were emm typed by following the protocols and recommendations for GAS of the Centers for Disease Control and Prevention (CDC) (http://www.cdc.gov/ncidod/biotech/strep/protocols.htm). Briefly, emm genes from GGS and GCS were amplified with primer 1 and primer 2 and sequenced with emmseq2. The first 240 bases of sequence were used to query the CDC streptococcal emm sequence database (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). When a type assignment was not obtained, the full sequence was used to query GenBank. An emm type was defined as ≥95% identical over the first 160 bases of sequence obtained with primer emmseq2 (1). Sequences that did not show an identity to previously described sequences sufficient for emm type assignment were submitted to Bernard Beall at the CDC and were assigned an emm type designation.

PFGE macrorestriction profiling. Agarose plugs of total DNA were prepared according to methods described elsewhere (13), with some modifications. Briefly, cells were embedded in agarose plugs and lysed with a solution containing 5 U/ml mutanolysin, 1 mg/ml lysozyme, and 30 µg/ml RNase A (Sigma-Aldrich, Steinheim, Germany) for 5 h at 37°C and with 0.5 mg/ml proteinase K (Roche, Mannheim, Germany) for 17 h at 50°C. After digestion with SmaI (Fermentas, Vilnius, Lithuania), the fragments were resolved by PFGE as described previously (13).

PFGE patterns were compared by using Bionumerics software (Applied Maths, Sint-Martens-Latem, Belgium) to create dendrograms by the unweighted-pair group method with arithmetic means (UPGMA). The Dice similarity coefficient was used with optimization and position tolerance settings of 1.0 and 1.5, respectively. Clones were defined as groups of isolates (n ≥ 2) presenting profiles ≥80% related on the dendrogram, as previously described for Streptococcus pneumoniae (22).

Estimation of the invasiveness of emm types and PFGE-defined clones. In order to compare the probability of invasive disease due to individual emm types and clones, an empirical odds ratio (OR) and 95% confidence intervals (CI) (8) were calculated by reference to all other emm types and clones as previously described (4). The OR was calculated as (ad)/(bc), where a is the number of invasive A emm types or clones, b is the number of noninvasive A emm types or clones, c is the number of non-A emm types or clones, and d is the number of noninvasive non-A emm types or clones. It follows from the formula presented that it is not possible to calculate an OR when no isolates of a given emm type or clone were recovered from invasive infections.

The choice of using all other emm types and clones to measure the reference OR was supported by prior studies (reference 4 and references therein) that also provide a discussion of the strong points of this method. The ORs calculated in this way will be comparable between studies, although the set of all other strains that is used to measure the reference OR will be different. In contrast, the alternative hypotheses, such as choosing an arbitrary emm type or clone or using the pooled minor emm types or clones as a reference, suffer from more-severe pitfalls. The choice of a particular emm type or clone will cause the ORs to change significantly depending on which emm type or clone is chosen and will prevent comparison of the ORs between studies when that particular emm type or clone is not found. The pooling of minor types suffers from similar limitations, and both alternatives reduce statistical efficiency relative to that obtained by using all other emm types or clones, justifying the use of all other emm types and clones to measure the reference OR. An OR of 1 indicates that the emm type or clone was equally likely to be invasive or noninvasive, whereas an OR of >1 or <1 indicated an increased or reduced probability to cause invasive disease, respectively.


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RESULTS
 
Isolate identification. All 116 isolates were identified with the API 20 Strep system alone or together with 16S rRNA gene sequencing and were shown to be S. dysgalactiae subsp. equisimilis independently of their Lancefield group polysaccharide. All but five strains could be identified to the species level by using the API 20 Strep system with a confidence in the identification above 95%, according to the manufacturer's literature. Each biochemical profile obtained with a confidence in the identification below 99% had a matching 16S rRNA gene sequence available, confirming the species identification. Only one isolate had an inconclusive identification with the API 20 Strep system (a group G blood isolate that fermented mannitol and sorbitol); however, the sequence of the 16S rRNA gene clearly identified it as S. dysgalactiae subsp. equisimilis. Overall, 16 distinct biochemical profiles were obtained. Almost half of the strains (44%) fermented lactose, and 41% hydrolyzed esculin. Other tests for which results differed occasionally included pyrrolidone arylamidase and beta-galactosidase production.

The sequence of the 16S rRNA gene was obtained for all 28 invasive isolates; they differed at only 1 base in a 1,419-bp fragment of the gene and were all 99.9% identical to S. dysgalactiae subsp. equisimilis ATCC 35666 (GenBank accession number AB096755).

emm type distribution and clonal relationships among isolates. emm genes were successfully amplified and sequenced for all the 116 S. dysgalactiae subsp. equisimilis isolates analyzed in our study. We found 22 distinct emm types (Table 2), including 3 with novel emm sequences: stgLP1, stgLP2, and stgLP3. Overall, the eight most prevalent emm types accounted for approximately 75% of the isolates (Table 2). Most emm types were found exclusively among either group G or group C isolates. Exceptions occurred with stc839 (nine group C isolates and one group G isolate) and stg485 (six group C and three group G isolates).


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  		TABLE 2. emm types and distribution of invasive isolates among PFGE clones

Analysis of the PFGE patterns generated after digestion with SmaI revealed 14 clones differing greatly in size (Fig. 1). Over half of the isolates were distributed in three large clones, D33, J19, and F16 (the subscript number in each clone designation is the number of isolates) (Table 2). Clone D33 stands out, because it comprised almost one-third of the isolates analyzed. During the study period there were no clear trends regarding the prevalences of the various emm types, and isolates representing the three major clones were recovered during all the years of the study and in most laboratories.


Figure 1
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  		FIG. 1. PFGE SmaI macrorestriction profile analysis of GGS and GCS isolates from invasive and noninvasive infections. (A) Dendrogram showing UPGMA cluster analysis of the PFGE profiles of the 116 isolates studied. Dice coefficients (percentages) are indicated in the scale above the dendrogram. Each clone (defined as a group of ≥2 isolates with a Dice coefficient of ≥80%) is represented by a triangle proportional to the number of isolates included in the clone. Clones are designated by capital letters and a subscript number indicating the number of isolates included in the clone. (B) PFGE profiles of representatives of each clone. Capital letters above the lanes correspond to clone designations; m, lambda ladder PFGE marker (New England Biolabs, Beverly, MA).

Half of the clones included both group G and C isolates; however, the largest clone (D33) included no group C isolates, and more than half of GCS (n = 14) were concentrated in two clones—G7 and J19—of which G7 included only GCS isolates. The majority of the clones included more than one emm type (Table 2). In D33 nine distinct emm types were found; however, stg10 (n = 16) and stg166b (n = 6) together accounted for two-thirds of the isolates. Only clone G7, already noted for including only GCS isolates, was associated with a single emm type (stc839). Within clones, subgroups of the same emm type could be defined; still, there was no similarity cutoff that allowed the separation of all isolates presenting distinct emm types.

With the exception of stg10, all the emm types had representatives in more than one clone, although the majority of the isolates of each emm type were represented in a single clone. In contrast, stg480 and stg2078 were highly dispersed among distinct genetic backgrounds.

Invasive isolates. None of the clones identified was significantly associated with invasive infection. However, it is noteworthy that a significant number of invasive isolates (stg10 [n = 6] and stg2078 [n = 3]) were tightly clustered within clone D33 at a similarity of 97.5% in the dendrogram.

ORs calculated to evaluate the invasiveness of the various emm types are presented in Table 2. The emm types stg10 and stg2078 were significantly more associated with invasive disease. While all the stg10 isolates were present in a single clone, D33, stg2078 isolates were found to correspond to diverse genetic backgrounds (represented in three different clones). The emm types stg6792 (n= 14) and stg166b (n = 8), each distributed in two clones, were found only among noninvasive isolates.


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DISCUSSION
 
This study aimed to characterize human infections caused by GGS and GCS. Identification to the species level revealed that all isolates were S. dysgalactiae subsp. equisimilis independently of their Lancefield polysaccharide group. This finding is not unique, since this species has been shown to express both C and G polysaccharides (26) and a recent study of GGS and GCS pharyngitis in the United States identified S. dysgalactiae subsp. equisimilis as the sole species (29), but it was in sharp contrast with the situation in Hong Kong. In this region, S. dysgalactiae subsp. equisimilis was the only species associated with GGS (27) but S. equi subsp. zooepidemicus was the only species identified in GCS bacteremia (28). In the latter study, the same strains were found in pigs and humans, suggesting a zoonotic origin of the infections. The data presented here support a different epidemiology of invasive GCS infections in Portugal, possibly not involving animal exposure, similar to that proposed in a report from the United States (5). In spite of the high phenotypic heterogeneity observed, resulting in a diversity of profiles with the API 20 Strep system, all dubious identifications were confirmed using 16S rRNA sequencing as the "gold standard," substantiating previous suggestions of the usefulness of this system for species identification of GGS and GCS (26, 27).

More than 150 emm types are currently recognized for GAS, and a smaller number were found exclusively in GGS or GCS (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). We identified 22 distinct emm types, including 3 novel types, among the 116 isolates tested; however, none of the emm types identified had also been found in GAS, in spite of previous reports documenting the occurrence of typical GAS emm types among GGS (21).

PFGE analysis revealed considerable genetic diversity among S. dysgalactiae subsp. equisimilis isolates, indicating that there are several distinct clones causing human infection. Nevertheless, the prevalences of the different clones among this streptococcal population differed considerably, and three large clones accounted for over half of the isolates. Since none of the clones appeared to result from a specific outbreak, it is conceivable that some particular characteristics in the genomes of these strains are responsible for their predominance.

Isolates bearing either C or G group antigen were not clearly separated from each other by PFGE, which is in agreement with the classification of these isolates into a single taxon and confirms previous data based on whole-cell protein profiles (25). An interesting observation was that PFGE-defined clones did not include a single emm type but, in contrast to GAS (10), predominantly comprised isolates with distinct emm types. Many isolates with diverse emm types clustered with higher Dice coefficients than those presenting the same emm type, suggesting that it is unlikely that the observed clustering of isolates with distinct emm types is due to a lack of discriminatory power of PFGE. This observation supports previous suggestions of higher rates of genetic exchange among GGS and GCS than among GAS (18). The only exception to the heterogeneity of genetic backgrounds associated with each emm type was the most prevalent type in our study, stg10, which was found associated with the largest clone (D33) only.

Numerous studies have identified a number of host factors, including malignancy, predisposing to invasive GGS or GCS infection (6). Studies on bacterial factors promoting invasiveness have focused on STSS and its association with the expression of superantigens (16, 19). Although none of the isolates in our collection was associated with a diagnosis of STSS, 24% (n = 28) of our isolates were from invasive disease. These isolates were recovered from normally sterile sites of patients that were hospitalized at the time of isolation, attesting to the severity of the infections and the aggressiveness of the strains. We investigated if either individual emm types or clones were more associated with invasive disease than would be expected by chance by calculating ORs comparing the prevalences of individual emm types and clones in invasive and noninvasive infections. While no clone was significantly associated with invasiveness, two emm types (stg10 and stg2078) had significant ORs.

The presence of stg10 in a single clone precludes the evaluation of whether the overrepresentation among invasive isolates is due to this emm type or to the clonal expansion of a specific strain bearing this emm type. However, the nonrandom distribution of invasive isolates within this clone points to the existence of a specific genetic background responsible for the increased virulence, where factors other than the M protein may also play a role.

In contrast to stg10, stg2078 isolates had a polyclonal origin. Therefore, the observed overrepresentation among invasive isolates cannot be attributable to clonal expansion of a given strain. In this case, our observations suggest that isolates with stg2078 have a higher probability of causing invasive disease independently of their genetic background, suggesting a role for the product of the emm gene or that of a closely linked gene. Heterogeneity in the genomic region flanking the emm gene has already been described among GGS and GCS (14). Although this variability was not correlated with invasiveness, a number of virulence-related genes are known to cluster in this region in GAS, and it is conceivable that alterations of the same region in GGS and GCS lead to changes in virulence. A further indication that strains with stg2078 have increased invasive potential is the fact that this emm type was not more frequently detected globally than other emm types. Interestingly, stg2078 has previously been reported as one of the dominant emm types (present in 3 of 21 isolates) among isolates causing invasive disease (17) and was the only emm type expressed by two isolates among a collection of GGS and GCS (n= 12) associated with STSS (16). In contrast, a study conducted in Israel failed to identify any stg2078 isolate among 94 GGS from cases of bacteremia (6).

Although the potential of isolates with specific M proteins to cause certain disease manifestations remains to be established, numerous studies point to a nonrandom M type distribution among invasive isolates of GAS (7). Our observations suggest that specific emm types are associated with increased invasiveness of GGS and GCS strains. Nevertheless, host factors as well as bacterial traits other than the M protein may contribute to the frequency of invasive disease. This is supported by the finding of emm type variability among invasive isolates (10 emm types among 22 invasive isolates) reported in this paper and elsewhere (6, 16). Geographic differences in the prevalences of particular clones or emm types may also play an important role, since a study of GGS bacteremia in Israel failed to identify any stg2078 isolates and type stg10 was represented by a small number of isolates (3 of 94) (6). This study also identified stg6792 (n = 3) and stg166b (n = 6) among isolates causing bacteremia, whereas in our collection neither of these emm types was identified in invasive isolates. However, the Israeli study does not provide information on the frequency of these emm types in noninvasive disease, precluding a direct comparison, but these differences highlight the importance of conducting studies similar to that presented here in diverse geographic locations.

The data presented highlight the importance of studying both GGS and GCS to fully evaluate the prevalence and severity of infections by S. dysgalactiae subsp. equisimilis and suggest an association between emm type and invasive disease potential in this bacterium.


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ACKNOWLEDGMENTS
 
This work was partly supported by Fundação Calouste Gulbenkian, Portugal.

Members of the Portuguese Group for the Study of Streptococcal Infections are as follows: Luís Lito and Maria José Salgado, Hospital de Santa Maria, Lisbon; Filomena Martins and Maria Ana Pessanha, Hospital de São Francisco Xavier, Lisbon; José Diogo, Ana Rodrigues, and Isabel Nascimento, Hospital Garcia de Orta, Almada; Fernanda Cotta and J. Correia da Fonseca, Hospital de São João, Porto; Paulo Lopes, Ismália Calheiros, Luísa Felício, Cacilda Magalhães, and Lourdes Sobral, Centro Hospitalar de Vila Nova de Gaia; Valquíria Alves and Antónia Read, Hospital Pedro Hispano, Matosinhos; Ana Paula Castro, Hospital de Vila Real; and Ana Paula M. Vieira and Francisco B. Moniz, Hospital Senhora da Oliveira, Guimarães.


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


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Journal of Clinical Microbiology, March 2006, p. 841-846, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.841-846.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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