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Clonal distribution of superantigen genes in clinical S. aureus isolates 1
running title: Clonal distribution of S. aureus superantigen genes 2
3
S. Holtfreter1, D. Grumann
1, M. Schmudde
1, H.T.T. Nguyen
1, P. Eichler
1, B. Strommenger
4,
4
K. Kopron2, J. Kolata
1, S. Giedrys-Kalemba
2, I. Steinmetz
3, W. Witte
4, B.M. Bröker
1 5
1Institute for Immunology and Transfusion Medicine, University of Greifswald, Germany 6
2Dept. Microbiology & Immunology, Pomeranian Medical University, Sczcecin, Poland 7
3Friedrich-Loeffler Institute for Medical Microbiology, University of Greifswald, Germany 8
4National Reference Center for Staphylococci, Wernigerode, Germany 9
10
word count: 5098 11
12
Corresponding author: 13
Barbara M. Bröker 14
Institut für Immunologie 15
Universität Greifswald 16
F.- Sauerbruch-Strasse / Diagnostikzentrum 17
17487 Greifswald 18
Germany 19
20
phone: 0 38 34 – 86 55 95 / 96 21
FAX: 0 38 34 – 86 54 90 22
e-mail: [email protected] 23
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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00204-07 JCM Accepts, published online ahead of print on 30 May 2007
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Abstract (218) 24
Staphylococcus aureus is both a successful human commensal and a major pathogen. The 25
elucidation of the molecular determinants of virulence, in particular the assessment of the 26
contributions of the genetic background versus those of mobile genetic elements (MGEs), has 27
proved difficult in this variable species. To address this, we have simultaneously determined 28
the genetic background (spa-typing) and the distribution of all 19 known superantigens and 29
the exfoliative toxins A and D (multiplex PCR) as markers for MGEs. Methicillin sensitive S. 30
aureus strains from Pomerania, 107 nasal and 88 blood culture isolates, were investigated. All 31
superantigen-encoding MGEs were linked more or less tightly to the genetic background. 32
Thus, each S. aureus clonal complex was characterised by a typical repertoire of superantigen 33
and exfoliative toxin genes. However, within each S. aureus clonal complex and even within 34
the same spa-type, virulence gene profiles varied remarkably. Therefore, virulence genes of 35
nasal and blood culture isolates were separately compared in each clonal complex. The results 36
indicated a role in infection for the MGE harbouring the exfoliative toxin D gene. In contrast, 37
there was no association of superantigen genes with blood stream invasion. In summary, we 38
show here that the simultaneous assessment of virulence gene profiles and the genetic 39
background increases the discriminatory power of genetic investigations into the mechanisms 40
of S. aureus pathogenesis. 41
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Introduction 42
Staphylococcus aureus is a major human pathogen capable of causing a wide range of 43
infections, such as skin and tissue infections, toxin-mediated diseases, pneumonia and 44
bacteraemia. At the same time, S. aureus is a persistent coloniser of the human nose in 20% of 45
the population and intermittently carried by another 30% (62). Colonisation with S. aureus is 46
a major risk factor for staphylococcal infections (47, 62). In carriers, 80% of nosocomial S. 47
aureus bacteraemia cases have an endogenous origin, which underlines the importance of host 48
factors (60, 64). On the other hand, there is plenty of evidence that S. aureus clones differ in 49
their disease-evoking potential, but it has been difficult to explain these differences at the 50
molecular level (39, 41, 51). 51
The species S. aureus has a highly clonal population structure with ten predominant clonal 52
lineages as demonstrated by various genotyping analyses, such as pulsed field gel 53
electrophoresis (PFGE), multilocus sequence typing (MLST) and spa genotyping (9, 11, 41). 54
Spa genotyping is based on variations of the polymorphic region within the protein A gene 55
(spa), which belongs to the staphylococcal core genome (22). It has a high discriminatory 56
power similar to PFGE but results can be more easily compared between laboratories (2, 30, 57
56). Moreover, spa typing, MLST and PFGE are highly concordant, and spa typing data can 58
be easily mapped onto the MLST S. aureus database (www.spaserver.ridom.de) (30, 56). 59
Whole genome microarrays recently revealed that the S. aureus genome consists of a core 60
genome (~75%), a core variable genome (~10%) and mobile genetic elements (MGE, ~15%) 61
(39). MGEs, such as plasmids, phages, pathogenicity islands and genomic islands, carry a 62
variety of staphylococcal resistance and virulence genes. MGEs can be distributed by two 63
distinct mechanisms. Firstly, MGEs are passed on to daughter cells by vertical transmission 64
and are, therefore, strongly associated with lineages (38). Secondly, MGEs can be 65
horizontally transferred and, therefore, spread between lineages (38, 40). Sometimes such 66
MGEs are conspicuously absent from certain clonal complexes, presumably due to 67
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restrictions on horizontal transmission (31). Consequently, the distribution patterns of MGE 68
among clonal lineages reflect their mobility (39). For example, several research groups have 69
reported that certain staphylococcal superantigens (SAg) genes are associated with particular 70
clonal lineages (6, 27, 44, 51, 58). 71
SAgs are secreted toxins, which induce a strong activation of large T cell subpopulations. 72
This can result in toxic shock (25). 80% of all S. aureus strains harbour SAg genes, on 73
average 5 to 6, among which the egc SAgs are the most prevalent (5, 24, 26, 48). Most of the 74
19 described S. aureus SAgs, SEA-SEE, SEG-SER, SEU and TSST-1, are encoded on 75
phages and pathogenicity islands (38). Staphylococcal phage Φ3 encodes either sea (strain 76
Mu50), sep (N315) or sea-sek-seq (MW2) (3, 31). A family of related pathogenicity islands 77
carry seb-sek-seq (SaPI1, COL), tst-sec3-sel (SaPI2; N315 and Mu50) or sec-sel (SaPI3; 78
MW2) (3, 17, 31). The enterotoxin gene cluster, egc, encoding seg-sei-sem-sen-seo and 79
sometimes seu, is located on the genomic island νSAβ (31, 38). Other SAg genes are found on 80
plasmids (sed-sej-ser) or on SCCmec (seh) (3, 49, 68). 81
It remains elusive how staphylococcal virulence is determined on a molecular level. 82
Regarding the S. aureus core genome, nasal and invasive strains probably do not differ 83
fundamentally, because they fall into the same main clusters (11, 41). Similarly, analysis of 84
the core variable genome, which comprises lineage-specific genes for surface proteins and 85
regulatory factors, did not identify factors clearly related to virulence (39). This suggests that 86
staphylococcal virulence might primarily depend on MGE-encoded toxin or resistance genes 87
(27) as has been shown for the antibiotic resistance cassette SCCmec and the gene of the 88
Panton-Valentine-leukocidin, pvl (10, 41). However, except for some toxins, it has been 89
difficult to assess the contribution of individual virulence determinants to S. aureus 90
pathogenicity (32, 36, 39, 51). 91
Since many MGE-encoded virulence factors are linked to clonal complexes, analyses of their 92
association with invasiveness could be biased by the underlying clonal population structure 93
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(51). Consequently, we propose that the simultaneous determination of the genetic 94
background (clonal lineage) and virulence genes will increase the discriminatory power of 95
investigations into the mechanisms of S. aureus pathogenesis. However, to date such studies 96
are rare (7, 27, 50). Because of their extraordinary variability in the species S. aureus, we 97
have chosen S. aureus superantigens as a model to test this approach. 98
Here we show the results a comprehensive analysis of the diversity of staphylococcal SAgs in 99
correlation with the genetic background in a large collection of S. aureus strains including 100
107 nasal and 88 blood culture isolates from Western Pomerania in the North-East of 101
Germany. Our aims were to investigate to which degree the distribution of the known SAg 102
genes is linked to the underlying clonality of the population, and to test whether the analysis 103
of SAg-carrying MGEs within defined clonal complexes would reveal differences between 104
nasal and invasive isolates. 105
106
Materials and Methods 107
Study population and bacterial isolates. Nasal-carriage isolates: Two nose swabs were 108
obtained from 121 healthy blood donors at the Institute of Immunology and Transfusion 109
Medicine, University of Greifswald, over a course of at least 10 weeks from April to October 110
2002 (T strains) (table 1). In a follow-up study (SH strains) nose swabs were obtained from 111
114 healthy blood donors between February 2005 and February 2006 (table 1). A strain shift 112
as defined by different SAg and agr gene profiles of the first and second nasal isolate 113
occurred in 5/51 persistent carriers (T009, T098, T166, T169; SH24). If the SAg and agr 114
genes in both isolates were identical, only the first isolate was analysed further. All 115
participants gave informed consent, and both studies were approved by the Ethics Board of 116
the University of Greifswald. 117
Blood culture isolates: Blood culture isolates (BK, n=88) were obtained by the Friedrich-118
Loeffler-Institute of Medical Microbiology, University of Greifswald, from May to December 119
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2002 (n=32) and from January 2005 to February 2006 (n=56) (table 1). Most isolates were 120
obtained from patients from different wards of the University Hospital of Greifswald (40 121
internal medicine, 10 surgery, 9 intensive care unit, 7 neurology, 3 neurosurgery, 2 122
neonatology, 2 urology, 1 gynaecology, 4 paediatrics). Six isolates were from a general 123
hospital near Greifswald (Wolgast); 4 strains were isolated from patients from a 124
neurorehabilitation centre, Greifswald. Only one isolate was included from each patient. We 125
observed no spatial or temporal clustering of S. aureus genotypes. 126
Nasal-carriage isolates from Sczcecin: 108 nasal S. aureus isolates (SZ) were obtained from 127
362 blood donors at the Department of Microbiology and Immunology, Pomeranian Medical 128
University, Sczcecin, Eastern Pomerania, Poland, in March 2006. All participants gave 129
informed consent, and the study was approved by the Ethics Board of the University of 130
Sczcecin. 131
Control strains for the polymerase chain reaction (PCR)-based assays included A920210 132
(egc, eta, agr4) (28), CCM5757 (seb, sek, seq, agr1), Col (seb, sek, seq, mecA, agr1) (3), 133
FRI1151m (sed, sej, ser, agr1) (27), FRI137 (sec, seh, sel, egc+seu, agr2), FRI913 (sea, sec, 134
see, sek, sel, seq, tst, agr1), N315 (sep, sec, sel, tst, egc, mecA, agr2) (31), TY114 (etd, agr3) 135
and 8325-4 (no SAg genes). 136
S. aureus identification and DNA isolation. S. aureus was identified using standard 137
diagnostic procedures and a gyrase PCR (see below). Total DNA of S. aureus was isolated 138
with the Promega Wizzard® DNA purification kit (Promega, Mannheim, Germany) according 139
to the manufacturer’s instructions. 140
spa genotyping. PCR for amplification of the S. aureus protein A (spa) repeat region was 141
performed according to the published protocol (2, 22). PCR products were purified with the 142
QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and sequenced using both 143
amplification primers from a commercial supplier (SeqLab, Goettingen, Germany). The 144
forward and reverse sequence chromatograms were analysed with the Ridom StaphType 145
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software (Ridom GmbH, Würzburg, Germany). A spa type is deduced from the sequence and 146
number of spa repeats, which are generated by point mutations and intrachromosomal 147
recombination events. Mutation of a single base pair results in a different spa type. With the 148
BURP algorithm (Ridom GmbH) spa types were clustered into different groups, the 149
calculated cost between members of a group being less than or equal to five. Spa types shorter 150
than five repeats were excluded from the analysis, because they do not allow the reliable 151
deduction of ancestries. 152
MLST genotyping. MLST genotyping was performed on selected S. aureus isolates 153
(indicated with an asterisk in Table 3 and 4) according to published protocols (9). Otherwise, 154
MLST-CCs were deduced from BURP grouping of spa types using the Ridom SpaServer 155
database (www.spaserver.ridom.de) (56). 156
SAg multiplex PCR Polymerase chain reactions. 16SrRNA PCR was performed with each 157
DNA-preparation to control DNA quality and absence of PCR inhibitors (24). Gyrase primers 158
allowed the identification of S. aureus (37). Methicillin resistance was detected with mecA-159
specific primers (45). All strains were negative for the Panton-Valentine-leukocidin (pvl) 160
genes as tested by PCR for lukS-lukF (27). Six sets of multiplex PCRs were established, 161
partially based on published protocols, to amplify I) sea, seh, sec and tst, II) sed, etd, eta and 162
sek, III) see, seb, sem, sel and seo, IV) sen, seg, seq and sej, V) sei, ser, seu and sep and VI) 163
agr group I-IV (suppl. table 1). Primer pairs for detection of sea-see, seh, sem, seo, ser, seu, 164
tst, eta, etd, pvl and agr group I-IV were previously described (27, 28, 35, 55, 67). Primers for 165
seg, sei and sej were modified from published primer sequences (42), and primers for sel, sek, 166
sen, sep, and seq were designed for this study (suppl. table 1). 167
Single and multiplex PCRs were performed with the GoTaq Flexi DNA polymerase system 168
(Promega). Each reaction mix (25 µl) contained 5 µl 5x GoTaq reaction buffer, 100 µM 169
deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP; Roche Diagnostics, 170
Mannheim, Germany), 5 mM MgCl2, 150-400 nM of each primer, 1.0 U GoTaq Flexi DNA 171
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polymerase and 10-20 ng of template DNA. An initial denaturation of DNA at 95 °C for 5 172
min was followed by 30 cycles of amplification (95 °C for 30 s, 55 °C for 30 s and 72 °C for 173
60 s), ending with a final extension phase at 72 °C for 10 min. All PCR products were 174
resolved by electrophoresis in 1.5% agarose gels (1x TBE buffer), stained with Etbr and 175
visualised under UV light. Positive controls included DNA from SAg gene-positive S. aureus 176
reference strains. In addition to standard PCR controls for contamination events, S. aureus 177
strain 8325-4 served as SAg gene-negative control. 178
sem gene sequencing. The PCR for amplification of the sem gene variant was performed with 179
sequencing primers (sems-1,-2) flanking the sem open reading frame using the HotGoldStar 180
Polymerase system (Eurogentec, Seraign, Belgium). Each reaction mix (50 µl) contained 5 µl 181
10x HotGoldStar reaction buffer, 200 µM deoxynucleoside triphosphates, 4 mM MgCl2, 1 182
µM of primers (sems-1, sems-2), 1.0 U HotGoldstar DNA polymerase and 10-20 ng of 183
template DNA. An initial denaturation of DNA at 95 °C for 10 min was followed by 30 cycles 184
of amplification (95 °C for 30 s, 56.8 °C for 40 s and 72 °C for 60 s), ending with a final 185
extension phase at 72 °C for 10 min. Sequencing was performed as described above. 186
Statistical analysis. Differences between groups were assessed using the chi-square test. P 187
values of <0.05 were considered statistically significant. Contingency tables were used to 188
compare the prevalence of a particular SAg gene or agr type between clonal complexes. 189
Nucleotide sequence of the sem gene variant found in CC30 (sem_CC30) is deposited at 190
Genbank (accession number EF551341). 191
Results 192
In this study we investigated the spa genotypes, agr group and SAg gene patterns of nasal and 193
blood culture S. aureus isolates from Western Pomerania, in the North-East of Germany. The 194
demographic data are summarised in table 1. A total of 107 nasal isolates were obtained from 195
healthy blood donors during two studies on S. aureus nasal colonisation (T strains, SH 196
strains). Similar colonisation patterns were observed in both study groups (Table 1). Only one 197
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nasal S. aureus strain was methicillin-resistant (T184-2). To investigate, whether the spa-198
defined core genomes or SAg-carrying MGEs differed between non-invasive and invasive 199
isolates, we additionally screened 88 MSSA blood culture isolates (table 1). The MSSA 200
isolates were obtained from the University Hospital of Greifswald or other close-by medical 201
facilities over the same time periods as the nasal isolates to avoid the potential confounding 202
effects of differences in geographical location or time periods. Most isolates were collected at 203
different wards of the University Hospital of Greifswald, most frequently at the internal 204
medicine ward (n=40), surgical ward (n=10) and intensive care unit (n=9), 4 strains were 205
isolated from outpatients (for details see materials and methods section). The patients had an 206
average age of 58.3 years and 61.4% were male. 207
208
Identification of clonal lineages by spa typing 209
Spa typing of nasal and blood culture isolates from Western Pomerania revealed 93 different 210
spa types, varying in length from 1 (t779) to 13 (t1660, t379) repeats. 75 spa types were 211
present in single isolates, whereas 10 spa-types were represented by at least five isolates. The 212
largest clone was t008, which comprised 24 isolates (12.3% of all isolates). Moreover, we 213
identified 30 new spa types not included in the Ridom SpaServer database. BURP clustering 214
assigned the 93 different spa types to five major and five minor clonal complexes (fig. 1). The 215
major complexes (containing >5% of the isolates) included MLST-CC8, CC15, CC25, CC30 216
and CC45, which together incorporated 73.3% of all isolates. In contrast, the minor 217
complexes CC5, CC12, CC22, CC121 and CC395 accounted for 13.3% of all strains. (11, 41) 218
(1, 39, 63) Singletons, that could not be assigned to a major CC by spa typing, occurred 219
among the nasal (n=14; 13.1%) and blood culture strains (n=5; 5.7%). Since clustering 220
parameters excluded spa types shorter than five spa repeats, three nasal and five blood culture 221
isolates were excluded from BURP grouping. Moreover, one isolate (BK067) was non-222
typable, because we were not able to amplify the spa gene by PCR. Overall, our results 223
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confirm the predominance of major clonal lineages as reported previously in other studies (11, 224
41). However, the frequencies of clonal lineages varies considerably between the different S. 225
aureus strain collections (1, 39, 63), suggesting large geographical variations. 226
227
228
Distribution of nasal and blood culture isolates between clonal complexes 229
As expected, both nasal and blood culture isolates were present in most clonal complexes (11, 230
41). CC5 (n=7) was exceptional, because it contained only nasal isolates, and all of them 231
belonged to the same spa type t002 (6.5%, P ≤ 0.05). Moreover, two clonal lineages were 232
clearly represented in different proportions (figure 1) between nasal and invasive S. aureus 233
isolates. CC8 was overrepresented among blood culture isolates compared to nasal isolates 234
(21.6% vs. 10.3%, P ≤ 0.05) while, CC30 was underrepresented among blood culture strains 235
(11.4% vs. 27.1%, P ≤ 0.01). In CC30, spa-type t012 was predominant among nasal strains, 236
whereas t021 was the most frequent spa-type in blood culture isolates (4/10 vs. 1/29, P≤0.01, 237
figure 2). 238
To assess the spreading of CC30 within the healthy population, we additionally screened 362 239
healthy blood donors from Sczcecin, Eastern Pomerania, Poland, in a cross-sectional 240
approach. Similar to the Western Pomeranian population, 26/108 (24.1%) of the nasal isolates 241
from Sczcecin belonged to CC30 (suppl. table 2), which is thus the dominating S. aureus 242
lineage in the Pomeranian community. A high prevalence of CC30 has also been reported 243
from other geographical areas worldwide (1, 39, 63). Spa type t012 was the dominant 244
colonising clone and probably the evolutionary founder of the CC30 cluster in both Western 245
(10/29 CC30 isolates) and Eastern Pomerania (7/26, figure 2). However, there were also 246
remarkable differences in the spa type composition between the two CC30 collections 247
suggesting a divergent evolution, which was probably enforced by the former East German-248
Polish border. 249
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A subanalysis of strains isolated in 2002 versus 2005/06 (data not shown) showed that CC395 250
was not yet detected in 2002 but a total of 7 isolates representing different, closely related spa 251
types were discovered in 2005/06. While a sampling bias cannot be excluded, it appears more 252
likely this S. aureus clone was recently introduced into the area, increased in frequency in the 253
population and rapidly diversified into the observed cluster of closely related spa types. 254
Moreover, within the blood culture isolates, we observed an expansion of the lineages CC15, 255
CC45 and CC8, which together accounted for 34.4% of the strains in 2002 compared to 256
60.7% in 2005/06. Even though these differences are not significant due to small case 257
numbers, they suggest that the S. aureus population structure is highly dynamic. 258
259
Distribution of agr types 260
The accessory gene regulator (agr) is a global regulator of virulence gene expression, and four 261
different agr subgroups, agr I-IV, are known. The agr locus belongs to the core variable 262
genome and is strongly linked with clonal lineages (27, 39). In agreement with others (27, 43, 263
51, 65), we observed that the clonal lineages CC8, CC22, CC25, CC45, and CC395 264
harboured agr I, and all CC5, CC12, and CC15 isolates were characterised by agr II (figure 265
3A, table 2, 3). Moreover, CC30 isolates carried agr III, while agr IV occurred only in the 266
CC121 lineage. 267
We also noted a single agr I isolate within the CC30 cluster (BK085) and one agr IV isolate 268
in CC45 (BK004); this was occasionally observed before (51, 65). Interstrain recombination 269
events could account for these exceptional strains, which needs further investigation (19, 54, 270
57). Five blood culture isolates could not be typed with our agr multiplex PCR system, 271
presumably due to a deletion of the agr locus (S. Holtfreter, unpublished data) (18). 272
273
Distribution of SAg genes 274
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We then determined the presence of the 19 known SAg genes, as well as of the eta and etd 275
genes using a system of five multiplex PCRs. A comprehensive overview on the spa-defined 276
clonal lineages, their respective agr types and SAg gene patterns is provided in table 2 for the 277
nasal isolates and table 3 for the blood culture isolates from Western Pomerania. 278
None of the SAg genes were randomly distributed between the clonal complexes (P ≤ 0.001; 279
contingency table analysis), but rather strongly associated with the clonal lineages. This 280
suggests that most MGEs are predominantly transferred vertically, while horizontal 281
transmission between different lineages is limited. However, MGEs were also found in strains 282
of divergent clonal lineages that do not share a recent ancestor. Here, the exchange of MGEs 283
appears to be favoured between some CCs. The distribution of selected SAg genes as well as 284
etd within the major CCs is depicted in figure 3B, C. 285
Egc SAgs, that cluster on the S. aureus genomic island vSAβ, were strictly linked to the 286
clonal background which is in agreement with previous studies(8, 38, 39). The egc cluster 287
was present in all CC5, CC22 and CC45 isolates but completely absent from CC8, CC12, 288
CC15 and CC395. Egc genes also characterised one subcluster of the CC25 lineage (t078 and 289
relatives), whereas they were missing from the other (t056 and relatives, fig. 3B, table 2, 3). 290
Moreover, we observed an egc variant, which was almost exclusively linked to the CC30 291
background. This egc variant is characterised by an sem allelic variant that escapes detection 292
with our standard PCR due to three point mutations within the binding site of the sem forward 293
primer (EF551341), and an additional seu gene, and probably corresponds to the reported 294
egc2 variant (fig. 3, table 2, 3) (4, 34). Our data clearly show that the egc-encoding genomic 295
island is transferred only vertically. 296
Other SAgs with very strong linkage to certain CCs were tst, sec-sel and sed-sej-ser (fig. 3B, 297
table 2, 3). The tst gene, which is located on a family of related islands, was strongly linked to 298
the CC30 background as reported in previous studies (39, 51). Sec-sel are co-localised on the 299
pathogenicity island SaPI3 and were detected mainly in CC45 isolates. Finally, the plasmid-300
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encoded SAg genes sed-sej-ser were usually found in CC8. These results suggest that 301
horizontal transfer of the respective islands and plasmids between clonal lineages is rare. 302
SAg genes with a broader distribution were the phage-encoded sea, which was occasionally 303
detected in CC8, 30, 45 and 395, and the SaPI-encoded seb, which was infrequently found in 304
CC5, 8, 12, 25 and 45 (figure 3B, table 2, 3). 305
Furthermore, we observed some new SAg gene combinations indicating the existence of so 306
far undescribed MGE variants. For example, tst and seb are usually located on two different 307
related SaPIs, either of which integrates into the same genomic locus. The rare observation of 308
seb in a tst-positive isolate, as detected in this study and by others (33) can be explained by 309
the mosaic structure of MGEs, where short mosaic fragments can spread to other MGEs of the 310
same type by homologous recombination (39). Similarly, seb-sek-seq are usually clustered on 311
SaPI1, but we and others found seb without seq-sek in several strains (16, 48), suggesting a 312
new SaPI variant. This is intriguing and needs more investigation. 313
314
agr/SAg gene profiles of S. aureus clonal complexes 315
As a consequence of their linkage to the genetic background, each clonal complexe is 316
characterised by typical SAg gene patterns. However, within clonal complexes and even 317
within the same spa types, we observed considerable variation in the prevalence of the SAgs 318
that constitute these lineage-specific patterns. There were between 1 (CC15) and 8 (CC8) 319
different SAg genotypes within the major clonal lineages. This indicates frequent acquisition 320
and loss of SAg-carrying MGEs within lineages (tables 2 and 3). 321
The characteristic SAg gene profiles and agr groups of the clonal lineages are summarised in 322
table 4. Within CC25, spa sequencing discriminated two sublineages, egc-positive t078 323
strains (and relatives) and egc-negative t056 isolates (and relatives). The former were more 324
frequent among the invasive strains (9/11 vs. 4/14, P ≤ 0.01). Within this t078 cluster, eight 325
strains additionally harboured the etd gene. Importantly, these were found exclusively in the 326
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blood culture isolates (P ≤ 0.001), so that CC25 isolates harbouring the etd-encoding 327
pathogenicity island appear to be more virulent than those without. Others have reported the 328
etd locus to be associated with the MRSA ST80 lineage as well as with a lineage carrying agr 329
I, egc and sometimes seb (67); the latter likely represents the CC25 t078 subcluster described 330
here. 331
As shown above, CC30 isolates were more prevalent among nasal strains, while CC8 was 332
significantly overrepresented among blood culture isolates. However, there were no 333
differences in the SAg gene profiles between nasal and invasive isolates, neither in CC30 nor 334
in CC8 (table 2, 3). CC15 isolates never harboured SAg genes, presumably due to restrictions 335
on horizontal transmission (61). The SAg gene patterns of the minor lineages CC12, 22, 121 336
and 395 are based on small case numbers and need to be confirmed. The SAg and agr profiles 337
reported for MRSA strains largely correspond to our findings with MSSA isolates, which 338
demonstrates that the SCCmec cassettes show a greater horizontal mobility than the MGEs 339
encoding SAgs (table 4). 340
341
Discussion 342
Recent analyses showed that all S. aureus genotypes that efficiently colonise humans have 343
given rise to life-threatening pathogens, but that some clonal lineages appear to be more 344
virulent than others (41). Analysis of the core variable genome could not clearly attribute 345
virulence to any of the factors examined (39). In fact, each of the ten dominant S. aureus 346
lineages has a unique combination of core variable genes, such as surface-associated and 347
regulatory genes (27, 39). This suggests that associated resistance and virulence genes 348
encoded on MGEs could determine staphylococcal virulence, in which case their horizontal 349
mobility has to be taken into account. Accordingly, we performed a comprehensive survey of 350
the distribution of the 19 staphylococcal SAg genes, the exfoliative toxin genes, and the agr 351
type in the known S. aureus clonal lineages to answer the following question: Are there 352
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differences in the core genome and/or the SAg-carrying MGEs between nasal and blood 353
culture isolates? 354
Our results clearly showed that MGEs carrying SAg genes were strongly associated with the 355
clonal background. They either did not spread between different genetic lineages at all, such 356
as egc-carrying pathogenicity island vSAβ (8, 38, 39, 58), or the efficiency of such genetic 357
exchange was low, as in the case of tst, which is encoded by a family of related pathogenicity 358
islands (39, 40), and also in the case of the plasmid-encoded SAg genes sed-sej-ser (14, 15). 359
The sea-carrying phage Φ3 and the SaPI-encoded seb were distributed more broadly (39, 51). 360
This shows that the genetic distribution of SAg-carrying MGEs occurs mainly by vertical 361
transmission. The degree of horizontal mobility varies considerably between different MGEs. 362
Barriers for horizontal transfer could be the incompatibility of related bacteriophages 363
(bacteriophage immunity), SaPIs and plasmids, varying susceptibility to transduction or 364
conjugation, or the Sau1 restriction modification system, as has recently been proposed by 365
Lindsay and co-workers (38, 61). Notably, the lineage CC15 completely lacks SAg genes. It 366
can be assumed that the restriction modification system of this lineage prevents acquisition of 367
any SAg gene encoding MGE by horizontal transfer. 368
The group of Jarraud have reported associations of agr type with diseases, in particular with 369
the toxin-mediated TSS and exfoliative diseases (27). Since the agr locus belongs to the core 370
variable genes and was strictly associated with clonal lineages, this observation may reflect 371
the links of these clonal lineages and their associated virulence gene patterns with disease. For 372
example, most cases of menstrual toxic shock syndrome are caused by S. aureus lineage 373
CC30, which is characterized by tst and agr III (6, 13, 27, 46). 374
As a result of the described restrictions of horizontal gene transfer each clonal complex was 375
characterised by a typical SAg and exfoliative toxin gene profile and agr type, as is described 376
in detail in the results section (table 2). These typical MGE profiles explain many of the 377
differences of virulence and disease symptoms that are oberserved between S. aureus 378
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lineages. However, in case of tight linkeage, the relative contributions of MGEs and core or 379
core variable genome cannot be resolved using the tools of molecular epidemiology. In other 380
words, a preponderance of virulence-associated genes among invasive S. aureus isolates 381
could be caused or, on the contrary, masked by an uneven distribution of clonal complexes 382
between nasal and invasive strains (51). A striking example is the egc-encoding genomic 383
island vSAβ which we found always and exclusively in members of CC5, CC22, CC30, 384
CC45 and a subcluster of CC25. On the CC25 background, egc was associated with 385
invasiveness, but on the CC5 background it characterised nasal isolates. Such associations 386
should therefore be interpreted with caution. 387
Within each lineage and even within the same spa type we observed considerable variation of 388
SAg genes. The transfer of bacteriophages appears to be quite frequent both during 389
colonisation and infection (20, 21, 44). The colonising strain T098 even lost the sea-carrying 390
phage between the first and second sampling, since the PCRs for sea and the phage-specific 391
integrase gene became negative, while spa type, PFGE pattern and antibiogram remained 392
identical (unpublished observations). This illustrates the high degree of horizontal mobility of 393
phages between strains of similar genetic background (39, 51). In such cases, the impact of 394
MGEs can be readily assessed by comparing invasive and non-invasive S. aureus isolates of 395
similar genetic background. In our study, the strict association of etd with invasiveness on the 396
CC25 genetic background strongly suggests that etd – or associated virulence genes on that 397
island – contributes to disease. The exfoliative toxin D induces intradermal blister formation 398
by cleavage of desmoglein 1 and is associated with cutaneous abscesses and furuncles (66). 399
This shows that virulence gene analysis can increase the discriminatory power of other 400
genotyping methods, as has also been suggested by others (27, 51, 59). In contrast, in the CC8 401
and CC30 the SAg gene profiles did not differ between nasal and invasive strains rendering 402
an important contribution of SAgs to the invasion process unlikely. We conclude that 403
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restricting a comparative analysis of virulence factors to those CCs that harbour the respective 404
virulence genes will increase its sensitivity and specificity. 405
In agreement with our results, similar consensus repertoires of virulence genes (e.g. SAg 406
genes, agr group, hemolysins) have also been reported for MRSA clones of CC5, 8 and 30 407
(7). The fact that staphylococcal lineages of different geographical regions show similar MGE 408
profiles suggests that these lineages are evolutionary old and share a conserved genomic 409
structure. On this conserved genetic background the mecA gene shows a highly dynamic 410
behaviour, which illustrates the extraordinary selective pressure exerted on the species 411
S. aureus by therapeutic intervention. Interestingly, the reported SAg profiles within clonal 412
complexes are less variable in MRSA strains than in our MSSA collection (7). Likely reasons 413
are the relatively recent acquisition of SCCmec and the shaping of the population structure of 414
MRSA by local outbreaks in hospital and community. 415
In Pomerania, CC30 was significantly more common among nasal strains than among blood 416
culture isolates. It appears that the local CC30 population is optimised for symptom-free 417
colonisation and probably causes systemic infections only under very accommodating 418
conditions. Intriguingly, Wertheim et al. reported that in the Netherlands CC30 isolates tend 419
to be more prevalent among endogenous invasive strains compared to non-invasive strains 420
(63). Though there are some differences in the ways the Dutch and the Pomeranian strains 421
were collected, this means that the diagnosis “CC30” alone is of limited information. In 422
support, two CC30 MRSA clones, the HA-MRSA ST36:USA200 and the CA-MRSA 423
ST30:USA1100, induce very different disease types, which has been attributed to differences 424
in their virulence gene repertoire (7). A detailed comparison of the Dutch and the Pomeranian 425
CC30 populations will, hopefully, reveal more factors that predispose to invasiveness. 426
In addition to virulence gene assessment, the analysis of individual spa types or MLST types 427
within CCs can be informative. In CC30 the spa-type t012 was most prevalent among nasal 428
strains, whereas t021 dominated the blood culture isolates. Similarly, within the CC25 429
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lineage, t078 isolates were overrepresented among invasive strains. However, while high 430
resolution typing methods based on sequence variations of the core genome may help to 431
identify aggressive S. aureus clones, virulence gene typing is much more likely to provide 432
clues for the underlying molecular mechanisms. 433
To conclude, we have shown here that S. aureus clonal complexes are characterised by 434
consensus repertoires of SAg genes. However, within each lineage and even within the same 435
spa-type, there was remarkable variation of SAg gene profiles. For etd our data indicate a role 436
in blood stream invasion, while rendering it unlikely for SAgs. Using SAgs as an example for 437
highly variable virulence genes, we have shown here that the simultaneous assessment of 438
virulence gene profiles and genetic background can provide new insights into S. aureus 439
virulence. 440
441
Legends 442
Table 1: Characteristics of the study population. 443
1T, SH study: Two nose swabs were obtained from healthy blood donors over a course of at 444
least ten weeks. Among the blood donors from both studies were on average 56.5% 445
noncarriers, 21.0% persistent carriers (2 positive nose swabs) and 22.5% intermittent carriers 446
(one positive nose swab). 447
448
Figure 1: Distribution of nasal and blood culture isolates within clonal complexes. Spa 449
types were clustered into 10 CCs by BURP analysis using a cost of 5 as threshold for 450
clustering. MLST-CC nomenclature was deduced from spa-CCs using the Ridom SpaServer 451
database. CC30 was overrepresented among nasal strains (P=0.01), CC8 was overrepresented 452
among blood culture isolates (P=0.05) and CC5 included only nasal isolates (P=0.05). 453
454
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Figure 2: Clonal relationship of Pomeranian CC30 isolates of different clinical origin. A) 455
nasal isolates from Western Pomerania, B) nasal isolates from Sczcecin and C) blood 456
culture isolates from Western Pomerania. Clusters were created with the BURP algorithm 457
of the Ridom SpaType Software. The size of each spa circle reflects the number of isolates 458
belonging to this spa type. The thickness of the connecting line reflects the calculated costs, 459
which express the evolutionary distance. The founder of a cluster, i.e. the spa type with the 460
highest number of direct relatives, is shaded in grey. Blood culture isolates from one spa type 461
were not outbreak-related. Spa type t037 isolates were excluded from the CC30 cluster, 462
because they are known to belong to MLST ST239 (CC8). Spa t037 have arisen from a single 463
recombination event that involved the exchange of a DNA fragment, encoding the spa t037 464
gene, between MLST30 and MLST 239 (52, 53). 465
Figure 3: Distribution of A) agr subgroups I to IV, B) SAg genes or SAg gene 466
combinations and C) etd within the five major clonal complexes. The overall height of the 467
bars denotes the total number of isolates within the complex. The height of the shaded area 468
represents the number of isolates positive for the respective gene. The minor lineages CC5, 469
12, 22, 121, 395 and singletons were excluded from this analysis. egc variant = egc cluster 470
with seu and an sem allelic variant. * one strain harboured only seb-seq (T198-1, CC8). 471
472
Table 2: Distribution of SAg genes, agr type and eta and etd genes within spa-defined 473
clonal complexes among nasal isolates (n=107). Spa types were clustered into 10 clonal 474
complexes by BURP analysis, which are colour-coded according to figure 1. For construction 475
of the consensus tree, several reference strains with unknown SAg gene pattern were included 476
in the BURP clustering (shaded in grey). MLST-CCs were deduced from BURP grouping of 477
spa types (56). MLST-CCs labelled with an asterisk were MLST-sequenced. SAg genes, agr 478
type, eta and etd genes were determined by multiplex PCR. All strains tested negative for the 479
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pvl gene (not shown). Staphylococcal enterotoxins (SEs) are indicated by single letters (a = 480
sea, …). 481
1Spa type t037 isolates were grouped into spa-CC012, but are known to belong to MLST 482
ST239 (CC8). Spa t037 have arisen from a single recombination event that involved the 483
exchange of a >200 kb DNA fragment, encoding the spa gene, between MLST30 and 484
MLST239 (52, 53). 485
2Spa-CC1655 isolates were clustered into MLST-CC395 after MLST-sequencing of two 486
representative strains. 487
3T184-2 was tested mecA positive. 488
4Spa type t091 were grouped into spa-CC084, but belonged to ST7 (singleton) according to 489
MLST sequencing. 490
491
Table 3: Distribution of SAg genes, agr type and eta and etd genes within spa-defined 492
clonal complexes among blood culture isolates (n=87). For background information please 493
refer to table 2. S. aureus clinical isolate BK067 was not spa-typable and, therefore, excluded 494
from this analysis. 495
1Spa type t037 isolates were grouped into spa-CC012, but are known to belong to MLST 496
ST239 (CC8) (52, 53). 497
2Spa type t1662 is a singleton according to spa typing, but was grouped into MLST-CC30 498
after MLST sequencing (ST30). 499
3Spa-CC1655 isolates were clustered into MLST-CC395 after MLST-sequencing of two 500
representative strains. 501
4Spa type t091 were grouped into spa-CC084, but belonged to ST7 (singleton) according to 502
MLST sequencing. 503
504
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Table 4: SAg gene and agr signatures of S. aureus clonal complexes. For each 505
staphylococcal clonal cluster the characteristic agr subgroup, SAg genes as well as eta and 506
etd genes are indicated. The number of isolates tested positive for a virulence gene is provided 507
in brackets. SAg genes that occurred in more than 50% of isolates of one CC are shown in 508
bold letters. SAg genes which are clustered on MGEs are linked by a hyphen. An addition, the 509
SAg/agr signatures of MRSA outbreak clones and whole-genome sequenced strains are 510
provided. 511
*Sej and ser, which cluster with sed on a plasmid, were not determined in this study (12). 512
**Egc genes were present in one subcluster of the CC25 lineage (t078 and relatives, n=13), 513
whereas they were missing in the other one (t056 and relatives, n=12). The etd gene occurred 514
only in blood culture isolates of CC25 (n=8/11, P ≤ 0.001). 515
516
Suppl. Table 1: Oligonucleotide primers and reference strains used for SAg gene 517
detection. 518
519
Suppl. Table 2: Distribution of SAg genes, agr type and eta and etd genes of CC30 nasal 520
isolates from Sczcecin, Poland. 30 nasal isolates from healthy blood donors from Sczcecin, 521
Poland, showed the agr/SAg gene profile characteristic for CC30 isolates (agr III, egc with 522
seu and sem variant). spa typing demonstrated that 28 of these isolates belong to CC30. 523
1Spa type t037 isolates were grouped into spa-CC012, but are known to belong to MLST 524
ST239 (CC8) (52, 53). 525
526
Suppl. Table 3: Epidemiological background information on blood culture isolates. 527
Wards belong to the University Hospital Greifswald, if not further indicated. 528
529
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Table 1 : Characteristics of the study populations.
nasal isolates
(T) nasal isolates
(SH) blood culture isolates (BK)
nasal isolates (SZ)
study area Western
Pomerania Western
Pomerania Western
Pomerania Sczcecin
study population healthy blood
donors healthy blood
donors hospital patients
healthy blood donors
subjects, no. 121 114 88 362
time period apr – oct 2002 feb 2005 – feb
2006
apr 2002 – oct 2002; n=32
feb 2005 – feb 2006; n=56
mar 2006
mean age (±SD), years 33.8 (±11.4) 33.3 (±11.5) 58.3 (±21.3) 28.9 (±9.2)
male sex, % 64.5 62.3 61.4 52.8
S. aureus isolates, no. 55 52 88 108
colonisation status (2 nose swabs)
1
noncarrier, no. (%) 70 (57.8 %) 63 (55.3 %) - -
1x positive, no. (%) 29 (24.0 %) 24 (21.0 %) - -
2x positive, no. (%) 22 (18.2 %) 27 (23.7 %) - -
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t1674
t012
t019
t021
t1653
t046
t012
t700
t019
t018
t021t122
t486
t1649
t253
t338
t483
t1654t1657
t1658
t1675
t1667
t012
t964
t017
t018
t019
t021t122
t840
t1504
t275
t318
t342
t1641 t1642
t1650
t1672
t1660
costs: 5
costs: 4
costs: 3
costs: 2
costs: 1
A) nasal CC30 isolates, Western Pomerania
C) blood culture CC30 isolates,
Western Pomerania
B) nasal CC30 isolates, Sczcecin
Figure 2: Clonal relationship of Pomeranian CC30 isolates of different clinical origin. A) nasal isolates
from Western Pomerania, B) nasal isolates from Sczcecin and C) blood culture isolates from Western
Pomerania.
t1662
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Table 4: SAg gene and agr signatures of S. aureus clonal complexes.
CC number agr type egc other SAg genes eta, etd
MSSA 7 agr II egc+ seb-seq-sek (1), sed-sej-ser (1), sep (3) CC5
N315 (MRSA) agr II egc+ tst-sec-sel (ref: 31)
MSSA 30 agr I egc- sed-sej-ser (23), sea (5), seb-sek-seq (3), seb-seq (1), tst (1), sec-sel (1)
COL (MRSA), spa type t008 agr I egc- seb-sek-seq (ref: 17) CC8
Lyon-MRSA, spa type t008 agr I egc- sea (13/13), seb (1/13), sed (3/13)* (ref. 12)
CC12 MSSA 6 agr II egc- sec-sel (5), sep (5), seb (1)
CC15 MSSA 20 agr II egc- -
MSSA 5 agr I (4), agr- (1) egc+ sec-sel (1) CC22
EMRSA-15, spa type t032 agr I egc+ sec-sel (ref. 29)
CC25 MSSA 25 agr I (23), agr- (2) egc+ (13 **) seb (3), seu (1) etd (8**, all BK)
MSSA 39 agr III (38), agr I (1) egc-variant+ tst (27), sea (17), sep (1) CC30
EMRSA-16, spa type t018 agr III egc-variant+ tst, sea (ref. 12, 23)
MSSA 26 agr I (24), agr IV (1), agr- (1)
egc+ sec-sel (19), tst (1), sea (1), seb (1), sed-sej-ser (1), seu (1) CC45
Berlin MRSA agr I egc+ - (ref. 12)
CC121 MSSA 2 agr IV (1), agr- (1) egc+ seu (2) eta (1)
CC395 MSSA 7 agr I egc- tst-sec-sel (5), sea (6), sek-seq (2), see (1) etd (1)
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