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Journal of Clinical Microbiology, August 2007, p. 2669-2680, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00204-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Clonal Distribution of Superantigen Genes in Clinical Staphylococcus aureus Isolates{triangledown} ,{dagger}

S. Holtfreter,1 D. Grumann,1 M. Schmudde,1 H. T. T. Nguyen,1 P. Eichler,1 B. Strommenger,4 K. Kopron,2 J. Kolata,1 S. Giedrys-Kalemba,2 I. Steinmetz,3 W. Witte,4 and B. M. Bröker1*

Institute for Immunology and Transfusion Medicine, University of Greifswald, Greifswald, Germany,1 Department of Microbiology and Immunology, Pomeranian Medical University, Sczcecin, Poland,2 Friedrich-Loeffler Institute for Medical Microbiology, University of Greifswald, Greifswald, Germany,3 National Reference Center for Staphylococci, Wernigerode, Germany4

Received 26 January 2007/ Returned for modification 30 March 2007/ Accepted 17 May 2007


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ABSTRACT
 
Staphylococcus aureus is both a successful human commensal and a major pathogen. The elucidation of the molecular determinants of virulence, in particular assessment of the contributions of the genetic background versus those of mobile genetic elements (MGEs), has proved difficult in this variable species. To address this, we simultaneously determined the genetic backgrounds (spa typing) and the distributions of all 19 known superantigens and the exfoliative toxins A and D (multiplex PCR) as markers for MGEs. Methicillin- sensitive S. aureus strains from Pomerania, 107 nasal and 88 blood culture isolates, were investigated. All superantigen-encoding MGEs were linked more or less tightly to the genetic background. Thus, each S. aureus clonal complex was characterized by a typical repertoire of superantigen and exfoliative toxin genes. However, within each S. aureus clonal complex and even within the same spa type, virulence gene profiles varied remarkably. Therefore, virulence genes of nasal and blood culture isolates were separately compared in each clonal complex. The results indicated a role in infection for the MGE harboring the exfoliative toxin D gene. In contrast, there was no association of superantigen genes with bloodstream invasion. In summary, we show here that the simultaneous assessment of virulence gene profiles and the genetic background increases the discriminatory power of genetic investigations into the mechanisms of S. aureus pathogenesis.


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INTRODUCTION
 
Staphylococcus aureus is a major human pathogen capable of causing a wide range of infections, such as skin and tissue infections, toxin-mediated diseases, pneumonia, and bacteremia. At the same time, S. aureus is a persistent colonizer of the human nose in 20% of the population and is intermittently carried by another 30% (61). Colonization with S. aureus is a major risk factor for staphylococcal infections (47, 61). In carriers, 80% of nosocomial S. aureus bacteremia cases have an endogenous origin, which underlines the importance of host factors (59, 63). On the other hand, there is plenty of evidence that S. aureus clones differ in their disease-evoking potentials, but it has been difficult to explain these differences at the molecular level (39, 41, 51).

The species S. aureus has a highly clonal population structure with 10 predominant clonal lineages, as demonstrated by various genotyping analyses, such as pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and spa genotyping (9, 11, 41). spa genotyping is based on variations of the polymorphic region within the protein A gene (spa), which belongs to the staphylococcal core genome (22). It has a high discriminatory power similar to that of PFGE, but results can be more easily compared between laboratories (2, 30, 55). Moreover, spa typing, MLST, and PFGE are highly concordant, and spa-typing data can be easily mapped onto the MLST S. aureus database (http://www.spaserver.ridom.de) (30, 55).

Whole-genome microarrays recently revealed that the S. aureus genome consists of a core genome (~75%), a core variable genome (~10%), and mobile genetic elements (MGEs) (~15%) (39). MGEs, such as plasmids, phages, pathogenicity islands, and genomic islands, carry a variety of staphylococcal resistance and virulence genes. MGEs can be distributed by two distinct mechanisms. First, MGEs are passed on to daughter cells by vertical transmission and are, therefore, strongly associated with lineages (38). Secondly, MGEs can be horizontally transferred and thus spread between lineages (38, 40). Sometimes such MGEs are conspicuously absent from certain clonal complexes, presumably due to restrictions on horizontal transmission (31). Consequently, the distribution patterns of MGEs among clonal lineages reflect their mobility (39). For example, several research groups have reported that certain staphylococcal superantigen (SAg) genes are associated with particular clonal lineages (6, 27, 44, 51, 57).

SAgs are secreted toxins that induce a strong activation of large T-cell subpopulations. This can result in toxic shock (25). Eighty percent of all S. aureus strains harbor SAg genes, on average five or six, among which the egc SAgs are the most prevalent (5, 24, 26, 48). Most of the 19 described S. aureus SAgs, SEA to SEE, SEG to SER, SEU, and toxic shock syndrome toxin 1 (TSST-1), are encoded on phages and pathogenicity islands (38). Staphylococcal phage {Phi}3 carries either sea (strain Mu50), sep (N315), or sea-sek-seq (MW2) (3, 31). A family of related pathogenicity islands carry seb-sek-seq (SaPI1 in strain COL), tst-sec3-sel (SaPI2 in strains N315 and Mu50), or sec-sel (SaPI3 in strain MW2) (3, 17, 31). The enterotoxin gene cluster, egc, including seg-sei-sem-sen-seo and sometimes seu, is located on the genomic island vSAß (31, 38). Other SAg genes are found on plasmids (sed-sej-ser) or on the antibiotic resistance cassette SCCmec (seh) (3, 49, 67).

The way in which staphylococcal virulence is determined on a molecular level remains elusive. Regarding the S. aureus core genome, nasal and invasive strains probably do not differ fundamentally, because they fall into the same main clusters (11, 41). Similarly, analysis of the core variable genome, which comprises lineage-specific genes for surface proteins and regulatory factors, did not identify factors clearly related to virulence (39). This suggests that staphylococcal virulence might primarily depend on MGE-encoded toxin or resistance genes (27), as has been shown for SCCmec and the gene of the Panton-Valentine leukocidin, the PVL locus (10, 41). However, except for some toxins, it has been difficult to assess the contributions of individual virulence determinants to S. aureus pathogenicity (32, 36, 39, 51).

Since many MGE-encoded virulence factors are linked to clonal complexes, analyses of their association with invasiveness could be biased by the underlying clonal population structure (51). Consequently, we propose that the simultaneous determination of the genetic background (clonal lineage) and virulence genes will increase the discriminatory power of investigations into the mechanisms of S. aureus pathogenesis. However, to date, such studies are rare (7, 27, 50). Because of their extraordinary variability in the species S. aureus, we have chosen S. aureus SAgs as a model to test this approach.

Here, we show the results of a comprehensive analysis of the diversity of staphylococcal SAgs in correlation with the genetic background in a large collection of S. aureus strains, including 107 nasal and 88 blood culture isolates from Western Pomerania in northeastern Germany. Our aims were to investigate to what degree the distribution of the known SAg genes is linked to the underlying clonality of the population and to test whether the analysis of SAg-carrying MGEs within defined clonal complexes would reveal differences between nasal and invasive isolates.


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MATERIALS AND METHODS
 
Study population and bacterial isolates. (i) Nasal-carriage isolates. Two nose swabs were obtained from 121 healthy blood donors at the Institute of Immunology and Transfusion Medicine, University of Greifswald, over a course of at least 10 weeks from April to October 2002 (T strains) (Table 1). In a follow-up study (SH strains), nose swabs were obtained from 114 healthy blood donors between February 2005 and February 2006 (Table 1). A strain shift, as defined by different SAg and accessory gene regulator (agr) gene profiles, of the first and second nasal isolates occurred in 5/51 persistent carriers (T009, T098, T166, T169, and SH24). If the SAg and agr genes in both isolates were identical, only the first isolate was analyzed further. All participants gave informed consent, and both studies were approved by the Ethics Board of the University of Greifswald.


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  		TABLE 1. Characteristics of the study population

(ii) Blood culture isolates. Blood culture isolates (BK; n = 88) were obtained by the Friedrich-Loeffler-Institute of Medical Microbiology, University of Greifswald, from May to December 2002 (n = 32) and from January 2005 to February 2006 (n = 56) (Table 1). Most isolates were obtained from patients from different wards of the University Hospital of Greifswald (40 internal medicine, 10 surgery, 9 intensive-care unit, 7 neurology, 3 neurosurgery, 2 neonatology, 2 urology, 1 gynecology, and 4 pediatrics). Six isolates were from a general hospital near Greifswald (Wolgast); four strains were isolated from patients from a neurorehabilitation center in Greifswald. Only one isolate from each patient was included. We observed no spatial or temporal clustering of S. aureus genotypes.

(iii) Nasal-carriage isolates from Sczcecin. One hundred eight nasal S. aureus isolates (SZ) were obtained from 362 blood donors at the Department of Microbiology and Immunology, Pomeranian Medical University, Sczcecin, Eastern Pomerania, Poland, in March 2006. All participants gave informed consent, and the study was approved by the Ethics Board of the University of Sczcecin.

(iv) Control strains. Control strains for the PCR-based assays included A920210 (egc, eta, and agr-4) (28), CCM5757 (seb, sek, seq, and agr-1), Col (seb, sek, seq, mecA, and agr-1) (3), FRI1151m (sed, sej, ser, and agr-1) (27), FRI137 (sec, seh, sel, egc plus seu, and agr-2), FRI913 (sea, sec, see, sek, sel, seq, tst, and agr-1), N315 (sep, sec, sel, tst, egc, mecA, and agr-2) (31), TY114 (etd and agr-3), and 8325-4 (no SAg genes).

S. aureus identification and DNA isolation. S. aureus was identified using standard diagnostic procedures and a gyrase PCR (see below). Total DNA of S. aureus was isolated with the Promega Wizard DNA purification kit (Promega, Mannheim, Germany) according to the manufacturer's instructions.

spa genotyping. PCR for amplification of the S. aureus protein A (spa) repeat region was performed according to the published protocol (2, 22). PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and sequenced using two amplification primers from a commercial supplier (SeqLab, Goettingen, Germany). The forward and reverse sequence chromatograms were analyzed with the Ridom StaphType software (Ridom GmbH, Würzburg, Germany). A spa type is deduced from the sequence and number of spa repeats, which are generated by point mutations and intrachromosomal recombination events. Mutation of a single base pair results in a different spa type. With the BURP algorithm (Ridom GmbH), spa types were clustered into different groups, the calculated cost between members of a group being ≤5. The calculated cost reflects the evolutionary distance between two isolates. spa types shorter than five repeats were excluded from the analysis, because they did not allow the reliable deduction of ancestries.

MLST genotyping. MLST genotyping was performed on selected S. aureus isolates (also see Fig. 4 and 5) according to published protocols (9). Otherwise, MLST clonal complexes (CCs) were deduced from BURP grouping of spa types using the Ridom SpaServer database (http://www.spaserver.ridom.de) (55).


Figure 4
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  		FIG. 4. Distribution of SAg genes, agr types, and eta and etd genes within spa-defined clonal complexes among nasal isolates (n = 107). spa types were clustered by BURP analysis into 10 clonal complexes, which are color coded according to the scheme in Fig. 1. For construction of the consensus tree, several reference strains with unknown SAg gene patterns were included in the BURP clustering (shaded in grey). MLST CCs were deduced from BURP grouping of spa types (55). MLST CCs labeled with an asterisk were MLST sequenced. SAg genes, agr types, and eta and etd genes were determined by multiplex PCR. All strains tested negative for the PVL locus (not shown). Staphylococcal enterotoxins (SEs) are indicated by single letters (a = sea, etc.). Footnotes are as follows. 1, spa type t037 isolates were grouped into spa CC012 but are known to belong to MLST ST239 (CC8). spa t037 isolates have arisen from a single recombination event that involved the exchange of a >200-kb DNA fragment including the spa gene between MLST30 and MLST239 (52, 53). 2, spa CC1655 isolates were clustered into MLST CC395 after MLST sequencing of two representative strains. 3, T184-2 tested mecA positive. 4, spa type t091 isolates were grouped into spa CC084 but belonged to ST7 (singleton) according to MLST sequencing.


Figure 5
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  		FIG. 5. Distribution of SAg genes, agr types, and eta and etd genes within spa-defined clonal complexes among blood culture isolates (n = 87). For background information, see the legend to Fig. 4. S. aureus clinical isolate BK067 was not spa typeable and therefore was excluded from this analysis. Footnotes are as follows. 1, spa type t037 isolates were grouped into spa CC012 but are known to belong to MLST ST239 (CC8) (52, 53). 2, spa type t1662 is a singleton according to spa typing but was grouped into MLST CC30 after MLST sequencing (ST30). 3, spa CC1655 isolates were clustered into MLST CC395 after MLST sequencing of two representative strains. 4, spa type t091 isolates were grouped into spa CC084 but belonged to ST7 (singleton) according to MLST sequencing.

SAg multiplex PCRs. 16S rRNA PCR was performed with each DNA preparation to control the DNA quality and absence of PCR inhibitors (24). Gyrase primers allowed the identification of S. aureus (37). Methicillin resistance was detected with mecA-specific primers (45). All strains were negative for the PVL locus, as tested by PCR for lukS-lukF (27). Six sets of multiplex PCRs were established, partially based on published protocols, to amplify (i) sea, seh, sec, and tst; (ii) sed, etd, eta, and sek; (iii) see, seb, sem, sel, and seo; (iv) sen, seg, seq, and sej; (v) sei, ser, seu, and sep; and (vi) agr-1 to -4 (see Table S1 in the supplemental material). Primer pairs for detection of sea to see, seh, sem, seo, tst, eta, etd, the PVL locus, and agr-1 to -4 were previously described (27, 28, 35, 66). Primers for seg, sei, and sej were modified from published primer sequences (42), and primers for sel, sek, sen, sep, seq, ser, and seu were designed for this study (see Table S1 in the supplemental material).

Single and multiplex PCRs were performed with the GoTaq Flexi DNA polymerase system (Promega). Each reaction mixture (25 µl) contained 5 µl 5x GoTaq reaction buffer, 100 µM deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP; Roche Diagnostics, Mannheim, Germany), 5 mM MgCl2, 150 to 400 nM of each primer, 1.0 U GoTaq Flexi DNA polymerase, and 10 to 20 ng of template DNA. An initial denaturation of DNA at 95°C for 5 min was followed by 30 cycles of amplification (95°C for 30 s, 55°C for 30 s, and 72°C for 60 s), ending with a final extension phase at 72°C for 10 min. All PCR products were resolved by electrophoresis in 1.5% agarose gels (1x Tris-borate-EDTA buffer), stained with ethidium bromide, and visualized under UV light. Positive controls included DNA from SAg gene-positive S. aureus reference strains. In addition to standard PCR controls for contamination events, S. aureus strain 8325-4 served as a SAg gene-negative control.

sem gene sequencing. The PCR for amplification of the sem gene variant was performed with sequencing primers (sems-1 and -2) flanking the sem open reading frame using the HotGoldStar Polymerase system (Eurogentec, Seraign, Belgium). Each reaction mixture (50 µl) contained 5 µl 10x HotGoldStar reaction buffer, 200 µM deoxynucleoside triphosphates, 4 mM MgCl2, 1 µM of primers (sems-1 and sems-2), 1.0 U HotGoldstar DNA polymerase, and 10 to 20 ng of template DNA. An initial denaturation of DNA at 95°C for 10 min was followed by 30 cycles of amplification (95°C for 30 s, 56.8°C for 40 s, and 72°C for 60 s), ending with a final extension phase at 72°C for 10 min. Sequencing was performed as described above.

Statistical analysis. Differences between groups were assessed using the chi-square test. P values of <0.05 were considered statistically significant. Contingency tables were used to compare the prevalences of a particular SAg gene or agr type between clonal complexes.

Nucleotide sequence accession number. The nucleotide sequence of the sem gene variant found in CC30 (sem_CC30) has been deposited at GenBank (accession number EF551341).


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RESULTS
 
In this study, we investigated the spa genotypes, agr groups, and SAg gene patterns of nasal and blood culture S. aureus isolates from Western Pomerania in northeastern Germany. The demographic data are summarized in Table 1. A total of 107 nasal isolates were obtained from healthy blood donors during two studies on S. aureus nasal colonization (T strains and SH strains). Similar colonization patterns were observed in both study groups (Table 1). Only one nasal S. aureus strain was methicillin resistant (T184-2). To investigate whether the spa-defined core genomes or SAg-carrying MGEs differed between noninvasive and invasive isolates, we additionally screened 88 methicillin-susceptible S. aureus (MSSA) blood culture isolates (Table 1). The MSSA isolates were obtained from the University Hospital of Greifswald or other nearby medical facilities over the same time periods as the nasal isolates to avoid the potential confounding effects of differences in geographical locations or time periods. Most isolates were collected in different wards of the University Hospital of Greifswald, most frequently in the internal medicine ward (n = 40), surgical ward (n = 10), and intensive-care unit (n = 9); four strains were isolated from outpatients (for details, see Materials and Methods). The patients were on average 58.3 years old, and 61.4% were male.

Identification of clonal lineages by spa typing. spa typing of nasal and blood culture isolates from Western Pomerania revealed 93 different spa types, varying in length from 1 (t779) to 13 (t1660 and t379) repeats. Seventy-five spa types were present in single isolates, whereas 10 spa types were represented by at least five isolates. The largest clone was t008, which comprised 24 isolates (12.3% of all isolates). Moreover, we identified 30 new spa types not included in the Ridom SpaServer database. BURP clustering assigned the 93 different spa types to five major and five minor CCs (Fig. 1). The major complexes (containing >5% of the isolates) included MLST CC8, CC15, CC25, CC30, and CC45, which together incorporated 73.3% of all isolates. In contrast, the minor complexes CC5, CC12, CC22, CC121, and CC395 accounted for 13.3% of all strains. Singletons that could not be assigned to a major CC by spa typing occurred among the nasal (n = 14; 13.1%) and blood culture (n = 5; 5.7%) strains. Since clustering parameters excluded spa types shorter than five spa repeats, three nasal and five blood culture isolates were excluded from BURP grouping. Moreover, one isolate (BK067) was nontypeable, because we were not able to amplify the spa gene by PCR. Overall, our results confirm the predominance of major clonal lineages as reported previously in other studies (11, 41). However, the frequencies of clonal lineages varied considerably between the different S. aureus strain collections (1, 39, 62), suggesting large geographical variations.


Figure 1
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  		FIG. 1. Distribution of nasal and blood culture isolates within clonal complexes. spa types were clustered into 10 CCs by BURP analysis using a cost of 5 as the threshold for clustering. MLST CC nomenclature was deduced from spa CCs using the Ridom SpaServer database. CC30 was overrepresented among nasal strains (P = 0.01), CC8 was overrepresented among blood culture isolates (P = 0.05), and CC5 included only nasal isolates (P = 0.05).

Distribution of nasal and blood culture isolates between clonal complexes. As expected, both nasal and blood culture isolates were present in most clonal complexes (11, 41). CC5 (n = 7) was exceptional, because it contained only nasal isolates, and all of them belonged to the same spa type, t002 (6.5%; P ≤ 0.05). Moreover, two clonal lineages were clearly represented in different proportions between nasal and invasive S. aureus isolates (Fig. 1). CC8 was overrepresented among blood culture isolates compared to nasal isolates (21.6% versus 10.3%; P ≤ 0.05), while CC30 was underrepresented among blood culture strains (11.4% versus 27.1%; P ≤ 0.01). In CC30, spa type t012 was predominant among nasal strains, whereas t021 was the most frequent spa type in blood culture isolates (4/10 versus 1/29; P ≤ 0.01) (Fig. 2).


Figure 2
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  		FIG. 2. Clonal relationships of Pomeranian CC30 isolates of different clinical origins. (A) Nasal isolates from Western Pomerania. (B) Nasal isolates from Sczcecin. (C) Blood culture isolates from Western Pomerania. Clusters were created with the BURP algorithm of the Ridom SpaType Software. The size of each spa circle reflects the number of isolates belonging to the spa type. The thickness of the connecting lines reflects the calculated costs, which express the evolutionary distances. The founder of a cluster, i.e., the spa type with the highest number of direct relatives, is shaded in gray. Blood culture isolates from one spa type were not outbreak related. spa type t037 isolates were excluded from the CC30 cluster because they are known to belong to MLST ST239 (CC8). spa t037 isolates have arisen from a single recombination event that involved the exchange of a DNA fragment, including the spa t037 gene, between MLST30 and MLST 239 (52, 53).

To assess the spreading of CC30 within the healthy population, we additionally screened 362 healthy blood donors from Sczcecin, Eastern Pomerania, Poland, in a cross-sectional approach. Similar to the Western Pomeranian population, 26/108 (24.1%) of the nasal isolates from Sczcecin belonged to CC30 (data not shown), which was thus the dominating S. aureus lineage in the Pomeranian community. High prevalences of CC30 have also been reported from other geographical areas worldwide (1, 39, 62). spa type t012 was the dominant colonizing clone and probably the evolutionary founder of the CC30 cluster in both Western (10/29 CC30 isolates) and Eastern (7/26) Pomerania (Fig. 2). However, there were also remarkable differences in the spa type compositions between the two CC30 collections, suggesting a divergent evolution, which was probably enforced by the former East German-Polish border.

A subanalysis of strains isolated in 2002 versus 2005-2006 (data not shown) showed that CC395 was not yet detected in 2002 but a total of seven isolates representing different, closely related spa types were discovered in 2005-2006. While a sampling bias cannot be excluded, it appears more likely that this S. aureus clone was recently introduced into the area, increased in frequency in the population, and rapidly diversified into the observed cluster of closely related spa types. Moreover, within the blood culture isolates, we observed an expansion of the lineages CC15, CC45, and CC8, which together accounted for 34.4% of the strains in 2002 compared to 60.7% in 2005-2006. Even though these differences are not significant due to small case numbers, they suggest that the S. aureus population structure is highly dynamic.

Distribution of agr types. agr is a global regulator of virulence gene expression, and four different agr subgroups, agr-1 to -4, are known. The agr locus belongs to the core variable genome and is strongly linked with clonal lineages (27, 39). In agreement with others (27, 43, 51, 64), we observed that the clonal lineages CC8, CC22, CC25, CC45, and CC395 harbored agr-1 and all CC5, CC12, and CC15 isolates were characterized by agr-2 (Fig. 3A, 4, and 5). Moreover, CC30 isolates carried agr-3, while agr-4 occurred only in the CC121 lineage.


Figure 3
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  		FIG. 3. Distribution of (A) agr-1 to -4, (B) SAg genes or SAg gene combinations, and (C) etd within the five major clonal complexes. The overall heights of the bars denote the total number of isolates within the complex. The height of the shaded area represents the number of isolates positive for the respective gene. The minor lineages CC5, -12, -22, -121, and -395 and singletons were excluded from this analysis. An egc variant is an egc cluster with seu and an sem allelic variant. *, one strain harbored only seb-seq (T198-1; CC8).

We also noted a single agr-1 isolate within the CC30 cluster (BK085) and one agr-4 isolate in CC45 (BK004); this had occasionally been observed before (51, 64). Interstrain recombination events could account for these exceptional strains, and this needs further investigation (19, 54, 56). Five blood culture isolates could not be typed with our agr multiplex PCR system, presumably due to a deletion of the agr locus (18; S. Holtfreter, unpublished data).

Distribution of SAg genes. We then determined the presence of the 19 known SAg genes, as well as of the eta and etd genes, using a system of five multiplex PCRs. Comprehensive overviews of the spa-defined clonal lineages and their respective agr types and SAg gene patterns are provided in Fig. 4 for the nasal isolates and Fig. 5 for the blood culture isolates from Western Pomerania.

None of the SAg genes were randomly distributed between the clonal complexes (P ≤ 0.001; contingency table analysis) but rather were strongly associated with the clonal lineages. This suggests that most MGEs are predominantly transferred vertically while horizontal transmission between different lineages is limited. However, MGEs were also found in strains of divergent clonal lineages that do not share a recent ancestor. Here, the exchange of MGEs appears to be favored between some CCs. The distribution of selected SAg genes, as well as etd, within the major CCs is depicted in Fig. 3B and C.

egc SAgs, which cluster on the S. aureus genomic island vSAß, were strictly linked to the clonal background, which is in agreement with previous studies (8, 38, 39). The egc cluster was present in all CC5, CC22, and CC45 isolates but completely absent from CC8, CC12, CC15, and CC395. egc genes also characterized one subcluster of the CC25 lineage (t078 and relatives), whereas they were missing from the other (t056 and relatives) (Fig. 3B, 4, and 5). Moreover, we observed an egc variant that was almost exclusively linked to the CC30 background. This egc variant was characterized by an sem allelic variant that escaped detection with our standard PCR due to three point mutations within the binding site of the sem forward primer (EF551341) and an additional seu gene, and it probably corresponds to the reported egc2 variant (Fig. 3, 4, and 5) (4, 34). Our data clearly show that the egc-containing genomic island is transferred only vertically.

Other SAgs with very strong linkage to certain CCs were tst, sec-sel, and sed-sej-ser (Fig. 3B, 4, and 5). The tst gene, which is located on a family of related islands, was strongly linked to the CC30 background, as reported in previous studies (39, 51). sec-sel are colocalized on the pathogenicity island SaPI3 and were detected mainly in CC45 isolates. Finally, the plasmid-borne SAg genes sed-sej-ser were usually found in CC8. These results suggest that horizontal transfer of the respective islands and plasmids between clonal lineages is rare.

SAg genes with a broader distribution were the phage-borne sea, which was occasionally detected in CC8, -30, -45, and -395, and seb, on SaPI, which was infrequently found in CC5, -8, -12, -25, and -45 (Fig. 3B, 4, and 5).

Furthermore, we observed some new SAg gene combinations, indicating the existence of yet-undescribed MGE variants. For example, tst and seb are usually located on two different related SaPIs, either of which integrates into the same genomic locus. The rare observation of seb in a tst-positive isolate, as detected in this study and by others (33), can be explained by the mosaic structure of MGEs, where short mosaic fragments can spread to other MGEs of the same type by homologous recombination (39). Similarly, seb-sek-seq are usually clustered on SaPI1, but we and others found seb without seq-sek in several strains (16, 48), suggesting a new SaPI variant. This is intriguing and needs more investigation.

agr and SAg gene profiles of S. aureus clonal complexes. As a consequence of their linkage to the genetic background, each clonal complex is characterized by typical SAg gene patterns. However, within clonal complexes and even within the same spa types, we observed considerable variation in the prevalences of the SAgs that constitute these lineage-specific patterns. There were between one (CC15) and eight (CC8) different SAg genotypes within the major clonal lineages. This indicates frequent acquisition and loss of SAg-carrying MGEs within lineages (Fig. 4 and 5).

The characteristic SAg gene profiles and agr groups of the clonal lineages are summarized in Table 2. Within CC25, spa sequencing discriminated two sublineages, egc-positive t078 strains (and relatives) and egc-negative t056 isolates (and relatives). The former were more frequent among the invasive strains (9/11 versus 4/14; P ≤ 0.01). Within this t078 cluster, eight strains additionally harbored the etd gene. Importantly, they were found exclusively in the blood culture isolates (P ≤ 0.001), so that CC25 isolates harboring the pathogenicity island containing etd appear to be more virulent than those without it. Others have reported the etd locus to be associated with the methicillin-resistant S. aureus (MRSA) ST80 lineage, as well as with a lineage carrying agr-1, egc, and sometimes seb (66); the latter likely represents the CC25 t078 subcluster described here.


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  		TABLE 2. SAg gene and agr signatures of S. aureus clonal complexesa

As shown above, CC30 isolates were more prevalent among nasal strains, while CC8 was significantly overrepresented among blood culture isolates. However, there were no differences in the SAg gene profiles between nasal and invasive isolates, either in CC30 or in CC8 (Fig. 4 and 5). CC15 isolates never harbored SAg genes, presumably due to restrictions on horizontal transmission (60). The SAg gene patterns of the minor lineages CC12, -22, -121, and -395 are based on small case numbers and need to be confirmed. The SAg and agr profiles reported for MRSA strains largely correspond to our findings with MSSA isolates, which demonstrates that the SCCmec cassettes show a greater horizontal mobility than the MGEs encoding SAgs (Table 2).


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DISCUSSION
 
Recent analyses have shown that all S. aureus genotypes that efficiently colonize humans have given rise to life-threatening pathogens but that some clonal lineages appear to be more virulent than others (41). Analysis of the core variable genome could not clearly attribute virulence to any of the factors examined (39). In fact, each of the 10 dominant S. aureus lineages has a unique combination of core variable genes, such as surface-associated and regulatory genes (27, 39). This suggests that associated resistance and virulence genes carried on MGEs could determine staphylococcal virulence, in which case their horizontal mobility has to be taken into account. Accordingly, we performed a comprehensive survey of the distribution of the 19 staphylococcal SAg genes, the exfoliative toxin genes, and the agr types in the known S. aureus clonal lineages to answer the following question: are there differences in the core genome and/or the SAg-carrying MGEs between nasal and blood culture isolates?

Our results clearly showed that MGEs carrying SAg genes were strongly associated with the clonal background. Either they did not spread between different genetic lineages at all, such as the egc-carrying pathogenicity island vSAß (8, 38, 39, 57), or the efficiency of such genetic exchange was low, as in the case of tst, which is carried by a family of related pathogenicity islands (39, 40), and also in the case of the plasmid-borne SAg genes sed-sej-ser (14, 15). The sea-carrying phage {Phi}3 and seb, located on SaPI, were distributed more broadly (39, 51). This shows that the genetic distribution of SAg-carrying MGEs occurs mainly by vertical transmission. The degrees of horizontal mobility vary considerably between different MGEs.

Barriers to horizontal transfer could be the incompatibility of related bacteriophages (bacteriophage immunity), SaPIs, and plasmids; varying susceptibilities to transduction or conjugation; or the Sau1 restriction modification system (38, 60). Notably, the lineage CC15 completely lacks SAg genes. It can be assumed that the restriction modification system of this lineage prevents the acquisition of any SAg gene-carrying MGE by horizontal transfer.

Jarraud et al. have reported associations of agr types with diseases, in particular with the toxin-mediated toxic shock syndrome and exfoliative diseases (27). Since the agr locus belongs to the core variable genes and was strictly associated with clonal lineages, this observation may reflect the links of these clonal lineages and their associated virulence gene patterns with disease. For example, most cases of menstrual toxic shock syndrome are caused by S. aureus lineage CC30, which is characterized by tst and agr-3 (6, 13, 27, 46).

As a result of the described restrictions on horizontal gene transfer, each clonal complex was characterized by a typical SAg and exfoliative toxin gene profile and agr type, as is described in detail in Results above (Table 2). These typical MGE profiles explain many of the differences in virulence and disease symptoms that are observed between S. aureus lineages. However, in cases of tight linkage, the relative contributions of MGEs and the core or core variable genome cannot be resolved using the tools of molecular epidemiology. In other words, a preponderance of virulence-associated genes among invasive S. aureus isolates could be caused or, on the contrary, masked by an uneven distribution of clonal complexes between nasal and invasive strains (51). A striking example is the egc-carrying genomic island vSAß, which we found always and exclusively in members of CC5, CC22, CC30, CC45, and a subcluster of CC25. On the CC25 background, egc was associated with invasiveness, but on the CC5 background, it characterized nasal isolates. Such associations should therefore be interpreted with caution.

Within each lineage and even within the same spa type we observed considerable variation of SAg genes. The transfer of bacteriophages appears to be quite frequent during both colonization and infection (20, 21, 44). The colonizing strain T098 even lost the sea-carrying phage between the first and second samplings, since the PCRs for sea and the phage-specific integrase gene became negative while the spa types, PFGE patterns, and antibiograms remained identical (unpublished observations). This illustrates the high degree of horizontal mobility of phages between strains of similar genetic backgrounds (39, 51). In such cases, the impact of MGEs can be readily assessed by comparing invasive and noninvasive S. aureus isolates with similar genetic backgrounds. In our study, the strict association of etd with invasiveness on the CC25 genetic background strongly suggests that etd—or associated virulence genes on that island—contributes to disease. The exfoliative toxin D induces intradermal blister formation by cleavage of desmoglein 1 and is associated with cutaneous abscesses and furuncles (65). This shows that virulence gene analysis can increase the discriminatory power of other genotyping methods, as has also been suggested by others (27, 51, 58). In contrast, in CC8 and CC30, the SAg gene profiles did not differ between nasal and invasive strains, rendering an important contribution of SAgs to the invasion process unlikely. We conclude that restricting a comparative analysis of virulence factors to those CCs that harbor the respective virulence genes will increase its sensitivity and specificity.

In agreement with our results, similar consensus repertoires of virulence genes (e.g., SAg genes, agr groups, and hemolysins) have also been reported for MRSA clones of CC5, -8, and -30 (7). The fact that staphylococcal lineages from different geographical regions show similar MGE profiles suggests that these lineages are evolutionarily old and share a conserved genomic structure. On this conserved genetic background, the mecA gene shows highly dynamic behavior, which illustrates the extraordinary selective pressure exerted on the species S. aureus by therapeutic intervention. Interestingly, the reported SAg profiles within clonal complexes are less variable in MRSA strains than in our MSSA collection (7). Likely reasons are the relatively recent acquisition of SCCmec and the shaping of the population structure of MRSA by local outbreaks in hospitals and communities.

In Pomerania, CC30 was significantly more common among nasal strains than among blood culture isolates. It appears that the local CC30 population is optimized for symptom-free colonization and probably causes systemic infections only under very accommodating conditions. Intriguingly, Wertheim et al. reported that in The Netherlands CC30 isolates tend to be more prevalent among endogenous invasive strains than noninvasive strains (62). Though there are some differences in the ways the Dutch and the Pomeranian strains were collected, this means that the diagnosis "CC30" alone conveys limited information. In support of this, two CC30 MRSA clones, the hospital-acquired MRSA ST36:USA200 and the community-acquired MRSA ST30:USA1100, induce very different disease types, which has been attributed to differences in their virulence gene repertoires (7). A detailed comparison of the Dutch and the Pomeranian CC30 populations will hopefully reveal more factors that predispose to invasiveness.

In addition to virulence gene assessment, the analysis of individual spa types or MLST types within CCs can be informative. In CC30, the spa type t012 was most prevalent among nasal strains, whereas t021 dominated the blood culture isolates. Similarly, within the CC25 lineage, t078 isolates were overrepresented among invasive strains. However, while high-resolution typing methods based on sequence variations of the core genome may help to identify aggressive S. aureus clones, virulence gene typing is much more likely to provide clues to the underlying molecular mechanisms.

In conclusion, we have shown here that S. aureus clonal complexes are characterized by consensus repertoires of SAg genes. However, within each lineage, and even within the same spa type, there was remarkable variation of SAg gene profiles. For etd, our data indicate a role in bloodstream invasion while rendering it unlikely for SAgs. Using SAgs as an example of highly variable virulence genes, we have shown here that the simultaneous assessment of virulence gene profiles and genetic background can provide new insights into S. aureus virulence.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Immunologie, Universität Greifswald, F.-Sauerbruch-Strasse/Diagnostikzentrum, 17487 Greifswald, Germany. Phone: 0 38 34-86 55 95. Fax: 0 38 34-86 54 90. E-mail: broeker{at}uni-greifswald.de Back

{triangledown} Published ahead of print on 30 May 2007. Back

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/. Back


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Journal of Clinical Microbiology, August 2007, p. 2669-2680, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00204-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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