JAC Advance Access published online on October 23, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn430
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Original research |
Spread of invasive Spanish Staphylococcus aureus spa-type t067 associated with a high prevalence of the aminoglycoside-modifying enzyme gene ant(4')-Ia and the efflux pump genes msrA/msrB
1 Antibiotic Laboratory, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain 2 Staphylococcus Reference Laboratory, Bacteriology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain 3 Centre for Infectious Diseases Epidemiology, National Institute for Public Health and the Environment, Bilthoven, The Netherlands 4 Department of Medical Microbiology, Groningen University Medical Centre, The Netherlands 5 Consejo Superior de Investigaciones Científicas, Madrid, Spain
* Correspondence address. Antibiotic Laboratory, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. Tel: +34-91-822-3650; Fax: +34-91-509-7966; E-mail: jcampos{at}isciii.es
Received 5 February 2008; returned 29 May 2008; revised 15 September 2008; accepted 22 September 2008
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Abstract |
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Objectives: We carried out a nationwide study aimed at the determination of the molecular epidemiology and antibiotic resistance mechanisms of invasive Staphylococcus aureus in 21 Spanish hospitals.
Methods: The distributions of molecular markers, including antibiotic resistance genes, were investigated in 203 S. aureus, comprising 90 methicillin-resistant S. aureus (MRSA) and 113 methicillin-susceptible S. aureus (MSSA). Antimicrobial susceptibility was determined by standard methods. Panton–Valentine leucocidin (PVL) detection, staphylococcal cassette chromosome mec (SCCmec) types and agr types were performed/determined by PCR. All isolates were genotyped by PFGE after digestion of chromosomal DNA with SmaI. Multilocus sequence typing and spa-typing were also performed.
Results: In MRSA isolates, 74.4% were agr allotype II and were positive for SCCmec IV. Sixty-nine spa-types were identified, 18 in MRSA and 57 in MSSA. Both MRSA and MSSA variants were detected in six spa-types (8.7%). The majority of S. aureus (51.2%) were grouped into four spa-types (t067, t002, t012 and t008). The spa-type t067 was detected in 18 of the 21 (85.7%) participating hospitals, including both MRSA and MSSA in six of them; in total, 25.9% of our isolates were spa-type t067 (49% in MRSA) in comparison with 0.6% in a central spa-typing database. The prevalence of the ant(4')-Ia and msrA/msrB genes was significantly higher in the MRSA spa-type t067 than in the other MRSA spa-types. Association between spa-type t067 and ST125 is described here for the first time. A high prevalence (36.4%) of PVL-positive MSSA was detected.
Conclusions: A higher than expected prevalence of spa-type t067 isolates was found among invasive MRSA in Spain. The oxacillin, tobramycin, erythromycin and ciprofloxacin resistance profile of spa-type t067 isolates was linked to the presence of ant(4')-Ia and msrA or msrB genes.
Key Words: invasive infections , molecular epidemiology , spa-typing , resistance mechanisms
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Introduction |
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Staphylococcus aureus is an important human pathogen responsible for a wide range of nosocomial and community-acquired infections.1–4 These include invasive infections such as skin and soft tissue infections, pneumonia, bloodstream infections, osteomyelitis and endocarditis, as well as toxin-mediated syndromes such as toxic shock and food poisoning. The burden of disease imposed by S. aureus has increased over the last decade due to the spread of methicillin-resistant strains which frequently cause healthcare-associated infections, and this uncomfortable trend has been compounded by the recent emergence of community-acquired MRSA (CA-MRSA) caused by several clonal lineages that are genetically distinct from their nosocomial counterparts.5,6
For decades, various phenotypic and genotypic methods have been used for tracking the spread of methicillin-susceptible S. aureus (MSSA) and MRSA. PFGE is still the most widely used molecular typing method.7 But this method, based on the position of anonymous bands, comes with some inconveniences, such as lack of biologically meaningful grouping8 and difficulties in standardization and inter-laboratory comparison.9 Moreover, PFGE is a time- and labour-intensive technique. Typing approaches based on DNA sequencing offer some advantages over PFGE. The sequencing of the polymorphic X region of the protein A gene (spa-typing) is fast, allows for a discrete grouping and generates portable data. Combining PFGE and spa-typing provides an extremely high discrimination and thus a very fine scale for the interpretation of the results of surveillance studies.10
Staphylococcal cassette chromosome mec (SCCmec) typing is based on the molecular characterization of the mobile genetic element carrying the methicillin resistance gene (mecA).11 Six major types of SCCmec differing in sequence and size have been characterized according to the type of recombinase genes (ccr) and the class of mec complex they carry.12,13 Panton–Valentine leucocidin (PVL) is an S. aureus specific exotoxin, encoded by two co-transcribed genes designated lukF-PV and lukS-PV, and is associated with skin and soft tissue infections and severe necrotizing pneumonia.14,15 Both PVL and SCCmec type IV have been used as molecular markers for CA-MRSA infections.16 In recent years, the agr (‘accessory gene regulator’) gene has received increasing attention, as the agr locus influences the expression of many virulence traits in S. aureus and allows the classification of S. aureus into four groups.17,18
The main mechanism of aminoglycoside resistance in staphylococcal isolates is drug inactivation by cellular aminoglycoside-modifying enzymes: resistance to gentamicin and concomitant resistance to tobramycin and kanamycin in staphylococci are mediated by the bifunctional enzyme AAC(6')/APH(2''') encoded by the aac(6')-Ie-aph(2''') gene; resistance to neomycin, kanamycin, tobramycin and amikacin is mediated by an ANT(4')-I enzyme encoded by the ant(4')-Ia gene; and resistance to neomycin and kanamycin is mediated by the APH(3')-III enzyme encoded by the aph(3')-III gene.19 Ribosome modification confers MLSB resistance, principally by a single base change in the 23S rRNA by methylases encoded by erythromycin ribosomal methylase (erm) genes ermA or ermC. Resistance to macrolides and streptogramin B (MS resistance) can also occur in staphylococcal isolates with active efflux by a membrane-bound transporter protein (msrA gene).20 Quinolone resistance develops mainly as a result of chromosomal mutations in the target of quinolones, topoisomerase IV or DNA gyrase. The GrlA subunit of topoisomerase IV and the GyrA subunit of gyrase are the most common sites of resistance mutations; topoisomerase IV mutations are the most critical, since they are the primary target for many fluoroquinolones in S. aureus.21
In the winter of 2006–07, the European Antimicrobial Resistance Surveillance System (http://www.rivm.nl/earss/) promoted a study aimed at the identification of the predominant S. aureus clones in European countries by spa-typing. Previously, we have shown that new prevalent clones of S. aureus have appeared in recent years in MRSA in Spain.22,23 However, the prevalence of the genetically distinct strains defined by PFGE, spa-typing and multilocus sequence typing (MLST) and their association with other epidemiological markers such as agr, PVL and antibiotic resistance genes has never been investigated using a representative sample of invasive isolates in Spain or in other countries. We describe here the genomic diversity and antibiotic resistance mechanism of invasive isolates of S. aureus identified through a nationwide multicentre surveillance study.
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Test isolates
Twenty-one Spanish hospitals participated in this study. They were selected in order to provide a population snapshot that is demographically and geographically representative of the patient population treated in Spanish hospitals. Each hospital laboratory was asked to submit to the central reference laboratories the first five successive MSSA and the first five successive MRSA isolated from individual patients with invasive infections. If there were less than five MRSA isolates, successive MSSA isolates could be included to complete a set of 10 before submission. The duration of the sampling interval was 6 months, from 1 September 2006 to 28 February 2007. Each isolate was accompanied by epidemiological information detailing the date of isolation, origin of the specimen, demographic details (age and gender) and epidemiological context (such as healthcare-associated or outpatients). Infections were identified as community-acquired if a positive culture was obtained within the first 48 h of hospitalization.24 All microbiological and molecular studies were carried out at the reference laboratories of the Centro Nacional de Microbiología, Madrid, Spain.
Susceptibility testing and antibiotic resistance gene detection
Antimicrobial susceptibility testing was determined by the disc diffusion and microdilution methods (BD Phoenix Automated Microbiology System; Becton–Dickinson Diagnosis, Sparks, MD, USA). The antimicrobial agents tested were cefoxitin, ciprofloxacin, vancomycin, linezolid, rifampicin, erythromycin, gentamicin, tobramycin, quinupristin/dalfopristin, co-trimoxazole and clindamycin. The CLSI guidelines for susceptibility testing and qualitative interpretation were used throughout.25 Methicillin resistance was confirmed by detection of mecA using a PCR protocol as described previously.26
The macrolide–lincosamide resistance genes ermA, ermB, ermC, msrA, msrB and linA/linA' were detected by PCR as described previously;20,27,28 ermA, ermB and ermC were tested in 47 isolates resistant to erythromycin and clindamycin, msrA and msrB were tested in 32 isolates resistant to erythromycin but susceptible to clindamycin and linA/linA' were tested in two clindamycin-resistant but erythromycin-susceptible isolates.
Aminoglycoside-modifying enzyme genes aac(6')-Ie + aph(2'), ant(4')-Ia and aph(3')-III were screened by PCR as described previously.19 The enzyme gene aac(6')-Ie + aph(2') was tested in 18 isolates resistant to gentamicin, tobramycin and kanamycin; ant(4')-Ia was tested in 55 isolates that were tobramycin-resistant and gentamicin/kanamycin-susceptible and aph(3')-III was tested in 7 isolates that were kanamycin-resistant but gentamicin/tobramycin-susceptible.
The amplification and sequence analysis of the quinolone resistance determining region of grlA and gyrA was performed as described in 40 ciprofloxacin-resistant isolates.29
SCCmec types were determined using a multiplex PCR strategy that generated a specific amplification pattern for SCCmec types I, Ia, II, III, IV and IVa SCCmec structural types.11 This method does not identify the ccrAB alleles, but an excellent correlation has been demonstrated between the full characterization of SCCmec and this strategy.11 Another two PCR methods were used to determine SCCmec IV subtypes (IVb, IVc, IVd and IVh) and SCCmec V.30,31
The determination of the four agr types was performed by the multiplex PCR method previously described by Shopsin et al.18 The primers used were: a forward primer pan-agr (corresponding to conserved sequences from the agrB gene) and four reverse primers [agrI (in the agrD gene), agrII (in the agrC gene), agrIII (in the agrD gene) and agrIV (in the agrC gene)].18
All isolates were genotyped by PFGE following SmaI digestion of chromosomal DNA, prepared using a modification of the protocol described by Cookson et al.32 The resulting agarose plugs were loaded on agarose 1% w/v gels, and PFGE was performed in a CHEF-DRII apparatus (Bio-Rad, Hemel Hempstead, UK) in 0.5x Tris–borate–EDTA buffer (1x TBE is 89 mM Tris, 89 mM boric acid and 1 mM EDTA), at 6 V/cm and 12–14°C. The total run time was 23 h as follows: 5–15 s for 10 h and 15–60 s for 13 h. The gels were stained with ethidium bromide, visualized under UV illumination and photographed. Following analysis according to the criteria of Tenover et al.,33 a dendrogram was constructed with Molecular Analyst Software (Bio-Rad) using the Dice correlation coefficient34 and the unweighted pair-group method with averages, with a tolerance position of 0.8%. PFGE profiles of the MRSA isolates were named according to a previous report;23 PFGE types of MSSA isolates were assigned in this study following the Tenover criteria.33
DNA sequencing of the spa gene
To obtain a DNA preparation, a loopful of S. aureus cells were washed with distilled water and incubated with 200 µL of 6% InstaGene matrix solution (Bio-Rad, München, Germany) for 20 min at 56°C. The suspension was vortexed and heated for 8 min at 100°C and centrifuged at 8000 g for 2–3 min. DNA amplification was performed by PCR using the PuRe Taq Ready-ToGo PCR Beads (Amersham Biosciences), in a total volume of 25 µL containing LiChrosolv water (Merck), 5 µL of DNA template and 10 pmol of each primer.
The spa gene was amplified using primers spa-1113f and spa-1514r, and sequencing was performed as described previously.35 Nucleotide sequences were analysed using the software Ridom StaphTypeTM (Ridom GmbH, Würzburg, Germany) as described by Harmsen et al.36 BURP (‘based upon repeat patterns’), as implemented in the Ridom StaphType software, was used to cluster (spa-CC) spa-types in MRSA and MSSA isolates.37
The PVL genes (lukS-PV and lukF-PV) were detected by PCR as described by Lina et al.15 The PVL-positive S. aureus (PVL-SA) ATCC 49775 strain was used as a positive amplification control.
The sequence type (ST) of selected isolates of S. aureus was determined according to the method of Enright et al.38 Analysis of the results was performed using the database available at www.mlst.net.
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Characteristics of participating hospitals
The 21 participating hospitals were located in 19 Spanish provinces covering all geographic regions of the country. The estimated catchment population of these hospitals was 
6 000 000 persons, corresponding to 15% of the Spanish population. Three hospitals (14.3%) had more than 1000 beds, 8 (38.1%) had between 500 and 1000 and 10 (47.6%) had between 250 and 500.
A total of 203 unduplicated S. aureus were collected from blood cultures, of which 90 were MRSA and 113 were MSSA; all participating hospitals provided MRSA isolates (average 4.3).
One hundred and thirty-seven isolates (67.5%) were from males and 66 (32.5%) were from females. Four (2%) were from children 
14 years of age, 74 (36.5%) were from patients between 15 and 65 years of age and 125 (61.6%) were from patients >65 years.
In total, 123 cases (60.6%) were considered nosocomial infections, 67 (33%) community-acquired infections, and in 13 (6.4%) isolates, this information was unknown.
Susceptibility testing and antibiotic genes detection
Resistance to ciprofloxacin, erythromycin, clindamycin, tobramycin and gentamicin was 96.7%, 81.1%, 31.1%, 77.8% and 17.8%, respectively, in MRSA isolates and 7.1%, 16.8%, 14.1%, 2.6% and 2.6%, respectively, in MSSA isolates. Resistance to rifampicin was observed in six isolates (6.7%), all of them MRSA; no resistance to linezolid, co-trimoxazole, quinupristin/dalfopristin or vancomycin was detected.
Multiresistance (defined as resistance to three antibiotics other than oxacillin) was observed in 58 MRSA isolates (64.4%) (Table 1) and in 4 MSSA isolates (3.5%). The most frequent resistance phenotypes found in MRSA were resistance to oxacillin, ciprofloxacin, tobramycin and erythromycin (19 isolates, 21.1%), and to oxacillin, ciprofloxacin and tobramycin (17 isolates, 18.9%) (Table 1). In MSSA, the phenotype characterized by erythromycin resistance plus inducible clindamycin resistance was the most prevalent (13 isolates, 11.5%).
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The aminoglycoside-modifying enzyme gene ant(4')-Ia was detected in all 55 tobramycin-resistant but gentamicin-susceptible isolates tested; 19 isolates resistant to both gentamicin and tobramycin had the bifunctional enzyme gene aac(6')-Ie + aph(2'). The remaining seven isolates resistant to kanamycin but susceptible to gentamicin and tobramycin contained the aph(3')-IIIa gene only (Table 2).
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In all 32 erythromycin-resistant but clindamycin-susceptible isolates, the efflux genes msrA/B were detected. The 47 isolates with the erythromycin-resistant and clindamycin-resistant phenotype had the methylase genes ermA, ermB and ermC as described in Table 2.
Most of the 40 ciprofloxacin-resistant isolates tested presented the 2614 (C
T) change encoding the Ser-80Phe mutation in grlA in combination with the 2402 (CT) change encoding the Ser-84Leu mutation in gyrA. Only one isolate had the 2614 (CT) change in grlA without mutations in gyrA (Table 2).
Seventy-five (83.3%) of the 90 MRSA isolates harboured SCCmec type IV, whereas 9 (10%) and 6 (6.7%) harboured SCCmec types I and II, respectively. SCCmec type III was not detected (Table 1). Of the isolates harbouring SCCmec type IV, 55 were subtype IVa (73.3%), 18 were subtype IVc (24%) and 2 were subtype IVh (2.6%).
The great majority of MRSA isolates, 78 of 90 (86.7%), had agr type II, while only 7 (7.8%) and 5 (5.6%) isolates had agr types I and III, respectively. No agr type IV was detected in MRSA.
The distribution of agr types in MSSA was more diverse as 35 (31%), 40 (35.4%) and 35 (31%) isolates had agr types I, II and III, respectively, and 3 (2.7%) isolates had agr type IV.
Of the 90 MRSA isolates, 28 distinct PFGE profiles were identified by PFGE, of which 63 isolates (70%) were grouped into eight clonal groups. The main groups were E7 (22 isolates, 24.4% of all MRSA) and E8 (17 isolates, 18.9%). The remaining 27 isolates (defined as sporadics) were assigned to 20 different PFGE profiles with 16 of them containing a single isolate (Table 3).
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The 113 MSSA isolates studied were classified into 88 distinct PFGE profiles, 104 of which (92%) were grouped into 10 clusters (named clusters C1–C10 in this study). Four of the clusters were the most prevalent and contained 73 isolates (64.6%) distributed in clusters C2 (26 isolates), C5 (17 isolates), C6 (17 isolates) and C3 (13 isolates). Strains that were not included in any of these clusters were named as ‘not grouped’ (NG).
In 201 isolates successfully spa-typed, 18 types were detected among MRSA and 57 among MSSA. Overall, 52 (25.9%) isolates were t067, 28 (13.9%) isolates were t002 and 12 (6%) isolates were t012. The most common spa-type (t067) was detected in 18 hospitals (85.7%) with both MRSA and MSSA variants found in 6.
The 90 MRSA isolates were grouped into four clusters and two singletons consisting of: cluster MRSA-1, spa-CC002 (64 isolates, 71.1%) with 6 spa-types and t067 as the predominant spa-type (44 isolates, 68.7% of all spa-CC002 isolates and 48.8% of all MRSA isolates); cluster MRSA-2, spa-CC1154 (4 spa-types, 5 isolates, 5.6%); cluster MRSA-3, spa-CC109 (3 spa-types, 8 isolates, 8.9%); and cluster MRSA-4, with no founder identified (2 spa-types, 5 isolates, 5.5%) (Table 3).
The 111 MSSA isolates were grouped into 7 clusters and 14 singletons consisting of: cluster MSSA-1, spa-CC012 (7 spa-types, 21 isolates, 18.9%); MSSA-2, spa-CC084 (7 spa-types, 12 isolates, 10.8%); MSSA-3, spa-CC002 (5 spa-types, 23 isolates, 20.7%); MSSA-4, spa-CC078 (4 spa-types, 6 isolates, 5.4%); MSSA-5, spa-CC116 (4 spa-types, 8 isolates, 7.2%); MSSA-6, with no founder identified (2 spa-types, 2 isolates, 1.8%); MSSA-7, with no founder identified (2 spa-types, 3 isolates, 2.7%); and 14 singletons (30 isolates, 27%). Six MSSA had less than five repeats and were excluded from the analysis.
Six spa-types common to MSSA and MRSA were detected. Two of them (t002 and t067) belonged to spa-CC002 and were distributed as follows: 12 MSSA and 16 MRSA isolates were spa-type t002; and 8 MSSA and 45 MRSA isolates were spa-type t067. Other common spa-types detected were: t012, found in 11 MSSA and in 1 MRSA; t018, found in 1 MSSA and in 3 MRSA; t008, found in 4 MSSA and in 6 MRSA; and t148, found in 4 MSSA and in 1 MRSA.
Presumptive CCs/MLST lineages in all MRSA and in 72 MSSA were assigned according to published data39,40 and the data available at the Ridom StaphTypeTM database (http://spa.ridom.de/index.shtml). We found 4 clonal complexes in MRSA (CC5, CC8, CC30 and CC22; Table 3), and 11 clonal complexes in MSSA (CC5, CC8, CC30, CC45, CC15, CC25, CC1, CC7, CC12, CC22 and CC121; Table 4).
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The ciprofloxacin/tobramycin/erythromycin resistance pattern was detected in 17 of 44 (38.6%) t067 MRSA and in 2 (4.3%) MRSA isolates of the remaining 46 isolates that were not t067 (P < 0.001; OR: 13.8, 95% CI: 2.9–64.7).
Among MRSA isolates, tobramycin resistance and gentamicin susceptibility [ant(4')-Ia associated] was more prevalent in t067 isolates (33 of 44, 75%) than in isolates of other spa-types (22 of 46, 47.8%) (P = 0.008). Also, the erythromycin-resistant but clindamycin-susceptible pattern (linked to the efflux pump genes msrA or msrB) was more prevalent in spa-type t067 isolates (24 of 44, 54.5%) than in isolates with other spa-types (4 of 46, 8.7%) (P < 0.0001).
The ant(4')-Ia gene and the efflux pump genes msrA or msrB were detected in 18 of 44 (40.9%) t067 MRSA and in 2 (4.3%) of the remaining 46 MRSA isolates that were not t067 (P < 0.001; OR: 15.2, 95% CI: 3.3–71).
In MRSA, 37 of the 39 (94.9%) isolates of the PFGE clonal groups E7 and E8 belonged to spa-CC002 (Table 3). In MSSA strains, 17 of the 26 (65.4%) isolates of the PFGE cluster C2 were spa-CC012, 8 of the 13 isolates (61.5%) of the PFGE cluster C3 were spa-CC116, 13 of the 17 isolates (76.5%) of the PFGE cluster C5 were spa CC002, and, finally, 10 of the 17 isolates (58.8%) of the PFGE cluster C6 were spa-CC084. In 72 MSSA strains, it was possible to assign a presumptive CCs/MLST lineage; the molecular markers of these strains, including CCs/lineages, STs, spa-types, PFGE profiles and agr groups, are depicted in Table 4.
According to spa-typing data available at the Ridom StaphTypeTM database (http://spa.ridom.de/index.shtml; 19 July 2007, date last accessed), the most prevalent spa-types among 36 571 isolates studied worldwide were t003 (15.2%) and t032 (10.3%), but these two spa-types were very infrequent in our study (three isolates in total, 1.5%). In contrast, t002 and t067 spa-types were much more prevalent in our isolates as 28 of them (13.9%) were spa-type t002 in comparison with 1865 (5.1%) in the Ridom StaphTypeTM database (OR 2.03, 95% CI: 1.28–2.23; P = 0.0037); also, 52 of our isolates (25.9%) were spa-type t067 in comparison with 215 (0.6%) in the Ridom StaphTypeTM database (OR 58.2, 95% CI: 25.7–131.5; P < 0.0001).
PVL genes were detected in 42 (20.7%) isolates, 41 of them MSSA (36.3% of all MSSA) and 1 MRSA (1.1%) (Table 3). Eighteen of the 21 (85.7%) participating hospitals had at least one PVL-SA.
The unique PVL-positive MRSA strain (PVL-MRSA) (isolated from a 55-year-old outpatient) was resistant to oxacillin only, was ST8, carried SCCmec type IVa and belonged to agr group I and spa-type t008. This strain is similar to the CA-MRSA strain USA300 (ST8-MRSA IV) and had the same PFGE profile as CA-MRSA A1 described in Spain by Cercenado et al.41
Twenty (48.8%) of the PVL-positive MSSA (PVL-MSSA) isolates were considered to have arisen from community acquisition, while 15 (36.6%) were implicated in nosocomial infections (in 6 isolates, this information was not available).
The most prevalent agr types among PVL-MSSA were types II (20.4%) and I (13.3%). The most prevalent PFGE profiles among PVL-MSSA isolates were C5 (11 of 17; 64.7%), C6 (7 of 17; 41.2%) and C9 (5 of 6; 83.3%); cluster C2 had just one PVL-positive isolate of the 26 included in this cluster; and clusters C1 (5 isolates) and C3 (13 isolates) had no PVL-MSSA strains.
Of the 41 PVL-MSSA, 13 (31.7%) belonged to spa-CC002 (t002, 7 isolates; t067, 4 isolates; and t311 and t2225, one isolate each); 11 (26.8%) were singletons; 6 (14.6%) were spa-CC084 (t084, 3 isolates; t346, 2 isolates; and t2055, one isolate); and 4 (9.8%) were spa-CC078 (t078, 2 isolates; t258 and t1661, one isolate each). For the remaining PVL-MSSA, in three isolates (7.3%) the spa sequences were excluded because they were shorter than five repeats, two isolates (4.9%) had no founder, and in two isolates (4.9%) no spa sequences were amplified. The association between the molecular markers of invasive PVL-MSSA and presumptive CCs/MLST lineages is detailed in Table 4.
STs were determined in 52 isolates; 19 of them belonged to agr type II and spa-CC002 (t067 and t002): 12 were MSSA (9 of them PVL-MSSA) and 7 were MRSA. The remaining 33 (32 MSSA and 1 MRSA) isolates were PVL-SA. All PVL-SA isolates were typed by MLST.
The distribution of molecular markers in invasive S. aureus belonging to agrII and spa-CC002 is shown in Table 5. Of the 7 MRSA isolates, 5 (71.4%) were ST125 and 2 (28.6%) were ST5; of the 12 MSSA isolates, 1 PVL-SA isolate was ST125 and 11 (8 of them PVL-SA) were ST5 (Table 5).
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The molecular features of the PVL-MSSA isolates are shown in Table 4. The most common MLST types found were ST5 (13 isolates), ST15 (8 isolates) and ST25 (4 isolates). Of the 13 isolates belonging to spa-CC002, 7 shared all the molecular markers studied. The six isolates of spa-CC084 were ST15 (Table 4). The unique PVL-MRSA strain was ST8.
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In this study, 203 blood S. aureus isolates (90 MRSA and 113 MSSA) from 21 Spanish hospitals were typed using several molecular methods. After analysis of the data, several findings were found to be of clinical and epidemiological interest. First, spa-type t067 (ST5-MRSA-IV) was dominant in the Spanish population, a situation not described in other countries. Second, in MRSA isolates, spa-type t067 was strongly associated with multiresistance to ciprofloxacin, tobramycin and erythromycin and to the expression of the enzyme gene ant(4')-Ia and the efflux pump genes msrA/msrB. Third, the prevalence of PVL-MSSA was unexpectedly high. Fourth, an important proportion of MRSA and MSSA blood isolates had similar epidemiological markers in common, including agr, spa and MLST, suggesting that they may share common epidemic clones.
The resistance phenotypes encountered were heterogeneous (17 patterns), with a high percentage (35.6%) of MRSA isolates presenting resistance to only one or two antibiotics besides oxacillin. This finding is of clinical relevance. Most MRSA isolates exhibit multiresistance phenotypes,42 but a narrowing of resistance patterns in invasive and epidemic MRSA strains has been reported.43,44 This reduction may be the result of the emergence and dissemination of the SCCmec type IV cassette, which was also the most frequent in this study (85.5%), and in other previous studies.22,23 A decrease in MRSA isolates harbouring SCCmec type I, classically associated with most extensive multiresistance patterns, has been noticed in other European countries.45,46
In this study, the great majority of the MRSA isolates (84.4%) belonged to agr type II. In MSSA isolates, we detected similar percentages of agr types I, II or III, although 35.4% of them were also type II. These results differ from those obtained by Hallin et al.47 in 614 Belgian isolates from all body sites (8% blood isolates) in which agr type I was the most frequent among both MSSA and MRSA. In Spain, the high number of MRSA strains with agr type II could be due to the replacement of the Iberian PFGE-clone (agr type I, SCCmec I) by E7 and E8 PFGE clonal groups (agr type II, SCCmec IV).
In this study, we have detected a high prevalence of spa-CC002 (CC5/SCCmecIV) in MRSA isolates, mainly t067 (50% of all MRSA isolates), that was distributed in almost all participant hospitals. However, according to the Ridom StaphTypeTM database, the geographical distribution of the t067 spa-type is very limited and mainly reported in northern European countries such as Sweden, Norway, Denmark and Germany. The second most prevalent MRSA spa-type in our study was t002 (13.9%), which has been described in Germany,36 Austria48 and the USA.49
The predominant MRSA spa-type t067 was gentamicin-susceptible and presented the ant4' gene that confers resistance to kanamycin, tobramycin and amikacin. The spread of this clone may help to explain why gentamicin resistance has decreased in recent years in Spain.23,44
Recently, an increase in the number of circulating MRSA clones has been described,50 in comparison with the few epidemic strains described previously.51 The predominant MRSA clonal groups found in this study were ST5/125-MRSA IV which belonged to the PFGE clonal groups (E7, E8 and the closely related E20). These strains were isolated from blood infections and are very similar to the MRSA epidemic strains implicated in other clinical scenarios in this country.23 A high correlation between spa-CC002 (spa-types t067 and t002) and the previously described E7, E8 and E20 PFGE clonal groups23 has been observed in this study, as all isolates belonging to these PFGE clonal groups except two were spa-CC002; the two remaining isolates were spa-CC1154, closely related to spa-CC002 (http://spa.ridom.de/index.shtml).
In accordance with previous studies,22,23 we have shown that the E7, E8 and E20 PFGE clonal groups belonged to ST125 and harboured SCCmec IV; in addition, these same isolates mainly belonged to spa-type t067 (Table 3). This association between t067 and ST125 is described here for the first time, to our knowledge. The geographical distribution of spa-type t067 has not been reported previously. So far, ST125 has been detected in Spain (Tenerife, Canary Islands), Norway and Finland.23,52–54 Our data suggest that an endemic circulating clone is present among MRSA isolates in Spain characterized by being agr type II, very similar to the PFGE profile, ST125 and spa-type t067.
The t002 spa-type has been associated with ST5, closely related to ST125, and ST231 (http://spa.ridom.de/index.shtml). ST5 and ST125 are highly related as ST125 differs from ST5 in just one nucleotide in the amplified region of the yqiL gene, while the remaining six MLST alleles that define the ST are identical. We found that spa-type t002 and ST5 were associated in both MRSA and MSSA isolates (Tables 3 and 4). We also found that 14 MSSA and 46 MRSA isolates shared the same agr type II and the same CC002; since they also had the same MLST type ST5 or the closely related ST125, this may suggest that both MRSA and MSSA invasive isolates in Spain have common circulating clones. Moreover, nine MSSA isolates of this clone were PVL-MSSA, indicating that the eventual acquisition of the mecA gene by this clone could increase the PVL-positive prevalence in MRSA isolates.
Issartel et al.55 found that the most frequent discrepancy observed between PFGE and spa-typing methods was mainly due to MRSA isolates characterized as sporadics by PFGE but that belonged to the same spa-types, spa-CC008 and spa-CC002. This could reflect genetic events such as the insertion/deletion of different mobile genetic elements that may be involved in the epidemic behaviour of S. aureus leading to different PFGE patterns, while spa and MLST types remain indistinguishable.55
A remarkable finding from this study was that 36.4% of the MSSA isolates studied had PVL genes; since in a recent study in Spain56 this percentage was 1.6%, it seems that the prevalence of PVL-SA isolates is rapidly progressing in this country. In Argentina, 19 of 31 MSSA isolates (61.3%) were PVL-positive.57 Our PVL-MSSA isolates were distributed among different PFGE clusters, agr types, spa-types and MLST types, although most of them were associated with the prevalent spa-CC002 and the CC5/MLST lineage. Due to the predominance of CC5 and spa-CC002 among Spanish blood isolates, principally in MRSA, the potential increase in PVL prevalence in both MSSA and MRSA is a subject of concern.
The classical definition of CA-MRSA infection used in this and other studies needs a revision on the basis of the data provided by molecular markers. Morin and Hadler58 proposed a new category called ‘healthcare associated’ for a better epidemiological interpretation. In our study, only one isolate of the 90 MRSA studied had the typical characteristics of CA-MRSA (PVL-positive, single oxacillin resistance, SCCmec type IV, agr group I and a PFGE profile closely related to CA-MRSA type A141), but up to 21 (23.3%) may have been considered community-acquired according to the classical definition.
In summary, we carried out a multicentre study aimed at the molecular characterization of S. aureus causing invasive infections; 203 blood isolates (90 MRSA and 113 MSSA) from 21 Spanish hospitals were typed using several molecular methods. We found that most MRSA isolates were of agr type II and harboured SCCmec type IV; also, spa-type t067 was highly predominant in this bacterial population. A high prevalence (36.4%) of PVL-MSSA was detected. Some MRSA and MSSA blood isolates of agr type II also shared other molecular markers, including spa-CC002, CC5/MLST 5 and its closely related ST125. Moreover, in MRSA isolates, spa-type t067 was found to be associated with simultaneous resistance to ciprofloxacin, tobramycin and erythromycin, and to the detection of the enzyme gene ant(4')-Ia and the efflux pump genes msrA/msrB.
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This study was supported by research grants from Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III; Spanish Network for Research in Infectious Diseases (REIPI C03/14) and Spanish Network for Research in Infectious Diseases (REIPI RD06/0008); research grants from Fondo de Investigaciones Sanitarias (FIS PI040837); and from the Dirección General de Salud Pública, Ministry of Health, Spain (reference 1429/05-11).
The software Ridom StaphTypeTM (Ridom GmbH, Würzburg, Germany) and laboratory training were provided by EARSS. EARSS was funded by the European Commission, DG Sanco (Agreement SI2.123794).
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None to declare.
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Members of the EARSS Spain spa-typing Group are listed in the Acknowledgements section. 
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The EARSS Spain spa-typing Group: Dionisia Fontanals (Corporació Sanitària Parc Taulí, Barcelona), Rosario Moreno (Hospital General de Castellón, Castellón), Josep Lite (Hospital Mutua de Tarrassa, Barcelona), Pilar López and Gloria Royo (Hospital General Universitario de Elche, Elche), Gregoria Megías-Lobón and Eva Ojeda (Hospital General Yagüe, Burgos), Consuelo Miranda and María Dolores Rojo (Hospital Universitario Virgen de la Nieves, Granada), Ana Fleites (Hospital Central de Asturias, Oviedo), Pilar Teno (Hospital San Pedro de Alcántara, Cáceres), Pilar Berdonces (Hospital Galdakao, Vizcaya), Teresa Nebreda and Ángel Campos (Hospital General de Soria, Soria), Eugenio Garduño (Hospital Infanta Cristina, Badajoz), Carmen Raya (Hospital del Bierzo, León), Begoña Fernández (Hospital Santa María Nai, Pontevedra), María Fe Brezmes (Hospital Virgen de la Concha, Zamora), Patricia Álvarez and Marta García-Campello (Complejo Hospitalario de Pontevedra, Pontevedra), Isabel Wilhemi (Hospital Severo Ochoa, Madrid), Alberto Delgado-Iribarren (Fundación Hospital de Alcorcón, Madrid), Luisa Marco and María José Revillo (Hospital Miguel Servet, Zaragoza), Natalia Montiel (Hospital Costa del Sol, Málaga), Inocente Cuesta (Complejo Hospitalario de Jaén, Jaén), and Jorge Calvo and Luis Martínez (Hospital Marqués de Valdecilla, Santander).
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