Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) strains carrying the Panton-Valentine leucocidin (PVL) genes have been reported worldwide and are a serious threat to public health. The PVL genes encode a highly potent toxin which is involved in severe skin infections and necrotizing pneumonia, even in previously healthy individuals. We assessed the prevalence of PVL-positive MRSA in The Netherlands for two periods of time: (i) 1987 through 1995 and (ii) 2000 and 2002, and determined their characteristics by using multilocus sequence typing and staphylococcal chromosome cassette (SCCmec) typing. It was found that up to 15% of all MRSA isolates detected in The Netherlands harbored the PVL genes. Most PVL-positive MRSA isolates were obtained from severe soft tissue infections in relatively young individuals. The first PVL-positive MRSA described in The Netherlands, isolated in 1988, was a single-locus variant of the “Berlin” epidemic MRSA clone. The 20 PVL-positive MRSA isolates studied in 2000 and 2002 consisted of five different sequence types (STs) that belonged to four clonal complexes. One of the STs, ST80, is considered to be a widespread European clone and was the most predominant ST (60%) in this study, while ST37 had never been found to be associated with PVL-positive MRSA. Most isolates harbored SCCmec type IV, a supposed marker for community-acquired MRSA. The number and type of virulence-associated genes varied among the different STs.
Methicillin-resistant Staphylococcus aureus (MRSA) infections have been recognized for decades as hospital acquired or healthcare associated (2, 6, 20). Nowadays, MRSA is also recognized as a worldwide emerging community-acquired pathogen (CA-MRSA) (5, 30, 32). CA-MRSA generally causes primary skin and soft tissue infections, such as furunculosis and abscesses, but necrotizing tissue infections and lethal hemorrhagic pneumonia, even in previously healthy individuals, have recently been described as well (5, 21). Most CA-MRSA strains have a common virulence factor: Panton-Valentine leucocidin (PVL) (8, 9, 13, 27). CA-MRSA infections spread easily by direct skin-to-skin contact. Outbreaks in closed living communities, as with jail inmates, military recruits, and gay men, have been reported in the United States (16, 26, 28). The spread of PVL-positive S. aureus has also been described within families (25) and among healthcare staff (31).
In 2003, Vandenesch et al. reported that all PVL-positive CA-MRSA isolates shared a type IV staphylococcal chromosome cassette mec (SCCmec) and the presence of clones specific to the United States, Europe, or Oceania (32). Hence, the PVL-positive CA-MRSA clone isolated in France and Switzerland was of sequence type 80 (ST80), determined by multilocus sequence typing (MLST). CA-MRSA isolates displaying ST80 have also been observed in other European countries (2, 13, 32; B. Liljequist and D. Morrison, personal communication).
Due to the implementation of successful search-and-destroy guidelines, the incidence of MRSA in The Netherlands is very low (<1% in Dutch hospitals). Nevertheless, analysis of MRSA strains obtained from the Dutch MRSA surveillance program showed the presence of the PVL genes in MRSA with diverse STs (34). In this study, we have characterized 197 MRSA isolates randomly chosen from the national collection in The Netherlands. The PVL genes were detected in 5 of the 99 isolates (5%) collected in 2000 and in 15 of the 98 isolates (15%) collected in 2002. The genetic characteristics of the PVL-positive MRSA isolates were compared with those observed in other countries and continents.
MATERIALS AND METHODS
General setting and organization of the survey.
The National Institute of Public Health and the Environment (RIVM) serves as the national reference center for surveillance of MRSA in The Netherlands. On behalf of the Dutch Health Inspectorate, each hospital is requested to send all first clinical MRSA isolates from sporadic infections and from nosocomial outbreaks to the RIVM for confirmation and further typing.
Bacterial isolates.
To estimate the prevalence of PVL-positive MRSA in The Netherlands, MRSA isolates were randomly chosen from the RIVM national MRSA surveillance collection for two periods of time: (i) for the period 1987 through 1995, 216 isolates were selected from a collection of 1,768 MRSA isolates; (ii) for the years 2000 and 2002, 99 and 98 MRSA isolates, respectively, were selected from a collection of 1,590 MRSA isolates. Identification of MRSA was confirmed by mecA PCR assay (12), Martineau PCR assay for species identification (22), and oxacillin susceptibility testing by E-test on Mueller-Hinton agar plus 2% NaCl with 24 h of incubation at 35°C (interpretation criteria were used according to the guidelines of the CLSI [formerly NCCLS]) (23).
Seventeen epidemic MRSA strains (EMRSA 1 to 17) from the United Kingdom, 6 epidemic German MRSA strains (Northern German MRSA, Southern German MRSA, Hannover area MRSA, South Eastern German/Western Austrian MRSA, Vienna MRSA, and Berlin MRSA), and strains representing the Brazilian (HSJ216/ATCC BAA-43), Iberian (HPV107/ATCC BAA-44), Greek (GRE14/413), and pediatric (New York/Japan) MRSA clones were included in this study for comparison with Dutch MRSA clones (1, 3, 7).
Typing of MRSA isolates. (i) MLST.
MLST was carried out according to the method of Enright et al. (15) by sequencing an internal fragment of seven unlinked housekeeping genes to determine the following allelic profiles: carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glp), guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), and acetyl coenzyme A acetyltransferase (yqiL). A clonal group was defined as a group of isolates having a strictly identical sequence of all seven genes (11, 15). Clustering of the allelic profiles of the PVL-positive MRSA and comparison with allelic profiles of all S. aureus isolates present in the MLST database (www.mlst.net) was performed using the recently developed algorithm, called eBURST (Based Upon Related Sequence Types, enhanced version) (17). This algorithm divides an MLST data set into groups of related isolates and clonal complexes and predicts the founding (ancestral) genotype of each clonal complex (17).
(ii) SCCmec multiplex PCR. The SCCmec multiplex PCR assay was performed according to the method of Oliveira and de Lencastre (24). Briefly, the multiplex PCR includes eight loci selected on the basis of previously described mec element sequences. The resulting amplicon patterns represent the four major SCCmec types (I through IV) or derivatives of these. PCR products were visualized with ethidium bromide on 3% agarose gels.
(iii) PVL genes. The PVL genes (lukS-PV/lukF-PV) were detected by PCR according to the method of Lina et al. (21). PCR products were resolved by electrophoresis and visualized with ethidium bromide on 1.5% agarose gels.
(iv) Detection of virulence-associated genes.
The detection of virulence-associated genes by PCR was performed as described by Vandenesch et al. (32). Briefly, the presence of the accessory gene regulator (agr) allele group and 22 specific staphylococcal virulence genes (16 superantigenic toxins [sea, seb, sec, sed, see, seg, seh, sei, sej, sen, seo, sem, tst, eta, etb, and edin], 3 hemolysins [hla, hlb, and hlg], and 3 leucocidins [lukS-lukF, lukE-lukD, and lukM]) were determined as described previously (19). The amplification of gyrA was used as a quality control for each DNA extract.
RESULTS
The PVL genes were detected in 2 of the 216 MRSA isolates (1%) selected from the first study period (1987 through 1995). One isolate was detected in 1988 (ST508, SCCmec type IV; ST508 is a single-locus variant [SLV] of the Berlin epidemic clone ST45) and the second one in 1995 (ST80, SCCmec type IV). Furthermore, the PVL genes were detected in 5 of the 99 MRSA isolates (5%) selected from the year 2000 and in 15 of the 98 MRSA isolates (15%) selected from 2002. The majority of these 20 Dutch PVL-positive MRSA isolates (70%) were community acquired, since in these cases, soft tissue infections were already present at hospital admission. The remaining PVL-positive MRSA isolates (30%) might indicate transmission within the hospital.
The 20 PVL-positive MRSA isolates detected in 2000 and 2002 were fully characterized according to their ST, SCCmec and agr type, and toxin gene profiles (Table 1). PVL-positive MRSA ST508 was not further analyzed, as it was not detected recently. Twelve of the 20 isolates (60%) were assigned ST80 based on MLST. This major clone displayed agr type 3. Four other isolates had agr allele type 3 but were either ST30 (three isolates) or ST37 (one isolate). Four MRSA isolates had the agr type 1 allele and were either ST59 (three isolates) or ST8 (one isolate).
TABLE 1.
Characterization of 20 PVL-positive MRSA isolates recovered in The Netherlands in 2000 and 2002
| ST (determined by MLST) | No. of isolates | agr typea | SCCmec type (n) | Detection of toxin geneg
|
||||
|---|---|---|---|---|---|---|---|---|
| secb | egcc | edind | hlge | etdf | ||||
| 8 | 1 | 1 | IV (1) | − | − | − | − | − |
| 30 | 3 | 3 | I (1) | − | + | − | − | − |
| IV (2) | − | + | − | − | − | |||
| 37 | 1 | 3 | I (1) | + | + | − | − | − |
| 59 | 3 | 1 | III (2) | − | − | − | + | − |
| IV (1) | − | − | − | + | − | |||
| 80 | 12 | 3 | IV (12) | − | − | + | − | + |
Accessory gene regulator.
Staphylococcal enterotoxin c gene.
Staphylococcal enterotoxin genes (seg, sei, sem, sen, and seo genes are located on the same gene cluster).
Epidermal differentiation inhibitor gene.
Gamma hemolysin gene.
Exfoliatin D gene.
+, present; −, absent.
SCCmec type IV was detected in 16 of the 20 PVL-positive MRSA isolates. Unexpectedly, SCCmec type I was detected in two isolates (one of ST30 and the other of ST37) and SCCmec type III was detected in two isolates of ST59. Hence, isolates of the same ST (either ST30 or ST59) harbored different types of SCCmec.
The toxin genes detected in the isolates differed considerably according to the STs. Isolates of ST8 contained none of the toxin genes listed in Table 1, whereas isolates of other STs displayed various combinations of toxin genes. Overall, the genetic characteristics of the PVL-positive MRSA isolates recovered in The Netherlands reflect a diversity of the genetic background associated with these strains.
The different STs of the PVL-positive MRSA isolates were compared with the entire S. aureus MLST database (Fig. 1). The 5 STs detected in the PVL-positive S. aureus isolates differed from each other by more than two alleles and were not related to the same clonal complex (CC), except for ST30 and ST37. The predominant clone, ST80, is part of a minor clonal complex, which is comprised of only two STs, ST80 and ST153. ST153, which is an SLV of ST80, originated also from a European country, namely Denmark. Two of the five PVL-positive MRSA STs, ST8 and ST30, are known epidemic MRSA types (32) and are the presumed primary founders of two of the five major CCs harboring multiple epidemic and pandemic MRSA clones (Fig. 1) (29). So is ST8 thought to be the founder of CC-8, containing the epidemic clones EMRSA-2 and -6 (ST8); EMRSA-10 (ST254; Hannover clone), which is an SLV of ST8; EMRSA-8 and the archaic clone (ST250), which is also an SLV of ST8; EMRSA-1, -4, -7, and -11 (ST239; Brazilian and Portuguese clone), also an SLV of ST8; EMRSA-9 (ST240), a double-locus variant of ST8; and finally, EMRSA-5 (ST247; Iberian clone), which is also a double-locus variant of ST8. ST30 is thought to be the primary founder of CC-30, which contains EMRSA-16 (ST36), an SLV of ST30. ST59 is the presumed founder of CC-59, which consists of, in addition to ST59, three SLVs of ST59, named ST87, ST338, and ST359 (Fig. 1).
FIG. 1.
Population snapshot of S. aureus. The genetic relationships of the entire S. aureus database, including the five PVL-positive MRSA STs described in this study, are presented using a single eBURST diagram. The numbers represent STs and are indicated by black solid circles. The area of each circle corresponds to the abundance of the isolates of the ST in the input data. Presumed primary and secondary founders of clonal complexes are indicated in blue and yellow, respectively, while solid lines represent radial links from primary founders to each of its SLVs, which share identical alleles at 6 of the 7 loci, e.g., ST37 is an SLV of the primary founder ST30. The STs representing the PVL-positive MRSA isolates are marked by a red circle. Additional information about the eBURST algorithm can be found at www.mlst.net.
DISCUSSION
We have demonstrated the recent simultaneous coemergence of CA-MRSA of different pandemic clones in The Netherlands. Five different STs were found among the 20 PVL-positive MRSA strains, obtained from the random MRSA selections from 2000 and 2002, that belonged to 4 different clonal complexes.
Despite the low incidence of MRSA in The Netherlands (<1% in Dutch hospitals [35] and 0.03% at hospital admission [36]), MRSA strains harboring PVL genes as stable genetic markers pose a new threat to public health. In general, patients with PVL-positive S. aureus are often children or adolescents. In our study, the median age of the patients was somewhat higher (31 years in the 2000 study and 25 years in the 2002 study) (data not shown). PVL-positive S. aureus is mainly associated with skin and soft tissue infections and sometimes with necrotizing pneumonia with poor prognosis (13, 18, 33). Recently, van der Flier et al. described a fatal case of necrotizing pneumonia caused by PVL-positive CA-MRSA in a 15-year-old girl in The Netherlands (33). It remains unknown why younger (and previously healthy) individuals have such high lethality and mortality rates when infected with PVL-producing S. aureus. Possibly, a preceding influenza virus infection might pave the way for a secondary infection with (PVL-positive) S. aureus (18, 33).
The first PVL-positive MRSA isolate described in The Netherlands, ST508 from 1988, is an SLV of the ST45 (Berlin epidemic) clone. MRSA clone ST45 emerged in The Netherlands in 2000 and reached epidemic proportions in 2002, but since 2003, the outbreak is under control. None of these outbreak ST45 MRSA isolates were PVL-positive (35).
The 20 Dutch PVL-positive S. aureus isolates from 2000 and 2002 were shown, by determination of the ST, agr type, SCCmec type, and toxin gene profile, to belong to various clones. Most isolates harbored SCCmec type IV, a supposed marker for CA-MRSA. Nevertheless, type IV SCCmec can in some cases also be found in hospital-acquired MRSA (10, 24). In general, it is assumed that type IV SCCmec can be transferred relatively easy and is therefore present in a wide range of S. aureus backgrounds (4, 14, 37). However, this does not rule out that MRSA isolates harboring other SCCmec elements, like types I and III found in our study, can be PVL positive as well and might predominantly represent hospital-associated strains (10, 24, 34). It is emphasized that PVL-positive MRSA isolates might not only emerge in the community but also in the hospital environment (34).
PVL-positive MRSA clone ST80 with an agr type 3 allele and a SCCmec type IV was the predominant clone detected in The Netherlands. This clone has also been observed in other European countries, such as France, Germany, Greece, Scotland, Sweden, and Switzerland. However, to our knowledge, MRSA clone ST80 (with or without PVL genes) has not been observed on other continents, except Africa (in Algeria) (F. Tenover and J. Etienne, personal communication). This confirms that this MRSA clone is, at least until now, a predominantly European clone. PVL-positive MRSA clone ST80 was observed for the first time in The Netherlands in 1995 (no further epidemiological data available). Until 1999, this MRSA type was observed only sporadically in our country. Since 2000, PVL-positive MRSA clone ST80 has become endemic to The Netherlands.
PVL-positive MRSA isolates with the agr 3 allele were also detected in the isolates ST30 and ST37, which belong to the same clonal complex. This is the first report of ST37 MRSA carrying the PVL genes. The PVL-negative counterpart of ST37 represents an epidemic MRSA strain in The Netherlands, comprising almost 10% of all isolates sent to the RIVM in 2002. This suggests that the PVL bacteriophage could have been transferred into that recipient MRSA strain. ST30 has been observed before in Oceania (Southwest Pacific clone) and probably originated from Europe, based on data from the S. aureus MLST website reported by Enright et al. (15).
One PVL-positive MRSA strain in our study displayed ST8 with an agr 1 allele. Recently, this ST8 PVL-positive clone was found in an outbreak among jail inmates in the United States, where 91% of the isolates were of SCCmec type IV (26). PVL-positive S. aureus isolates with ST8 are also linked to recent infections observed in the gay community in several major U.S. cities, such as Boston, San Francisco, and Washington, D.C. (16, 28). Given its apparent epidemic potential, infections with this type of PVL-positive MRSA might increase in the near future. Preliminary data from 2003 and 2004 already seem to substantiate this hypothesis for The Netherlands (data not shown).
Three Dutch PVL-positive isolates were of ST59 with an agr1 allele. ST59 has been detected previously in, and probably originated from, the United States (32). In the case of the recent PVL-positive MRSA outbreak in the Los Angeles county jail, 89% of all isolates comprised ST8, ST30, and ST59 (26).
In conclusion, our data suggest a relatively recent emergence of PVL-positive MRSA in The Netherlands, the simultaneous coemergence of CA-MRSA of various lineages, and the spread of CA-MRSA in The Netherlands that initially has been described for other countries and continents, such as France for ST80, the United States for ST8 and ST59, and Oceania for ST30. The results of the present study are worrisome because up to 15% of all MRSA isolates encountered in The Netherlands carried the PVL genes. The precise incidence of infections in The Netherlands with PVL-positive S. aureus is unknown because community-acquired infections, especially superficial skin infections, are rarely characterized. Our data support the urgent need for further studies to monitor and prevent the spread of PVL-positive S. aureus in both the community and the hospital environment before additional resistance and virulence markers are acquired.
Acknowledgments
We thank M. Enright (University of Bath), H. Grundmann (RIVM), H. de Lencastre and A. Tomasz (University of Lisbon), and W. Witte (RKI) for kindly providing the EMRSA, German, and global epidemic MRSA strains. We also thank M. Enright and E. Feil (University of Bath) for the use of the S. aureus MLST database and the eBURST algorithm, respectively.
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