JAC Advance Access published online on November 11, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn435
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Original research |
Genetic diversity of methicillin-resistant Staphylococcus aureus carrying type IV SCCmec in Örebro County and the western region of Sweden
1 Department of Clinical Microbiology, Örebro University Hospital, Örebro, Sweden 2 Department of Infection Control Science, Juntendo University, Postgraduate School, Tokyo, Japan 3 Department of Bacteriology, Juntendo University, School of Medicine, Tokyo, Japan 4 Department of Medical Microbiology and Parasitology, China Medical University, Shenyang, China 5 Tuberculosis Research Section, Laboratory of Clinical Infectious Disease, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, USA 6 Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden
* Corresponding author. Present address: Department of Microbiology, Aleris MEDILAB, SE 183 15 Täby, Sweden. E-mail: carolina.berglund{at}aleris.se
Received 13 June 2008; returned 7 August 2008; revised 30 August 2008; accepted 22 September 2008
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Background: Recent studies have shown a predominance of type IV SCCmec among the methicillin-resistant Staphylococcus aureus (MRSA) isolated in the low endemic areas of Örebro County and the western region of Sweden. However, many of these isolates were not possible to classify as existing subtypes IVa, IVb, IVc or IVd.
Methods: We analysed 16 such MRSA isolates by multilocus sequence typing, spa typing, staphylocoagulase (SC) typing and detection of type IVg and IVh SCCmec. MRSA that remained as unknown type IV SCCmec were investigated by long-range PCR covering the J1 region; however, only two isolates were possible to amplify by PCR. The nucleotide sequences of the entire SCCmec of these two MRSA were determined. In addition, isolates that had unknown SC types were investigated by nucleotide sequencing of the coa genes.
Results: Five of 16 isolates were classified as type IVg SCCmec, and four isolates had type IVh SCCmec. Two subtypes of type IV SCCmec shared J1 regions previously identified in other types of SCCmec, types I.2 and II.2. The novel elements were designated as type IVi and IVj SCCmec. In addition, the genetic backgrounds of these Swedish MRSA were diverse and constituted at least nine sequence types and eight SC types, including four new types of SC.
Conclusions: Type IV SCCmec is occurring in heterogeneous clones of MRSA in Sweden, and the majority of the type IV SCCmec were identified in community-acquired MRSA. We describe two novel subtypes of type IV SCCmec with common J1 regions shared by other types of SCCmec, which indicate that J1 regions occurred as primordial SCC.
Key Words: MRSA , community-acquired MRSA , staphylococcal cassette chromosome mec , staphylocoagulase
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The staphylococcal cassette chromosome mec (SCCmec) is the genetic element that carries the methicillin resistance determinant, mecA, that makes it possible for staphylococci to continue cell wall assembly and growth in the presence of β-lactam antibiotics. SCCmec is integrated at the 3' end of the open reading frame X (orfX) at the specific site attBSCC located close to the origin of replication in the staphylococcal chromosome. SCCmec contains a characteristic combination of two essential genetic components; the mec gene complex with mecA and its regulator genes, and the cassette chromosome recombinase (ccr) gene complex that provides mobility. The SCCmec allotypes are classified as I, II, III, IV, V or VI, depending on the combination of mec gene complex (class A, B or C) and ccr gene complex (type 1, 2, 3, 4 or 5) contained.1–3 Subtyping of SCCmec is based on nucleotide differences in the three non-essential junkyard (J) regions.1 The J1 region located between the ccr genes and the downstream chromosomal region is the most important region for further subtyping of type IV SCCmec. There are five major subtypes, IVa (IV.1), IVb (IV.2), IVc (IV.3), IVd (IV.4) and IVg (IV.5), of the type IV SCCmec, described so far.4–8 A sixth subtype was recently described in EMRSA-15 and was designated as type IVh (IV.6).9
Characterization of methicillin-resistant Staphylococcus aureus (MRSA) in a low endemic area—Örebro County, Sweden—has revealed a significant diversity and the presence of 19 sequence types (STs) among a collection consisting of 57 isolates, indicating that the MRSA have arisen on several different occasions by independent acquisition of the SCCmec.10 In addition, the majority of these MRSA were isolated from individuals who had not recently been in contact with the healthcare system and were therefore classified as community-acquired MRSA (CA-MRSA).10 Similar findings have been made when investigating a collection of MRSA from the western region of Sweden.11 The most common genotypes in Örebro County were ST45, ST80 and ST5, but also representatives from the five major pandemic clones have been sporadically identified.10 MRSA genotypes related to those found in Örebro County have also been described in, for example, Ireland and Denmark, an indication that common MRSA clones are circulating in northern Europe.12,13
Characterization of CA-MRSA from different countries shows that they usually carry type IV SCCmec, a small element that generally does not contain any additional resistance genes.4,14 SCCmec type IV is also found in various genetic backgrounds, a finding that suggests that this element is more mobile than the other types of SCCmec.15 Recent studies have shown a predominance of type IV SCCmec among the MRSA isolated both in Örebro and in the western region of Sweden; however, many of these isolates are not possible to classify as subtype IVa, IVb, IVc or IVd.11,16
The aim of this study was to characterize type IV SCCmec that carried subtypes other than IVa, IVb, IVc or IVd, from MRSA isolated in Örebro County and the western region of Sweden by molecular methods and nucleotide sequencing of the SCCmec.
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MRSA (n = 16) that had previously been characterized as type IV SCCmec by detection of the class B mec complex and the ccr type 2 genes using the scheme developed by Ito et al. and adjusted for real-time LightCycler PCR (Roche Diagnostics, Mannheim, Germany) and could not be further subtyped as IVa, IVb, IVc or IVd were further analysed.4,17 The MRSA were isolated during 1983–2005, and outbreak isolates or isolates representing intrafamilial spread were excluded. Thirteen of the isolates were classified as community-acquired. Community-acquired infection was defined as identification of MRSA in the outpatient setting or a culture positive for MRSA within 48 h after hospital admission. In addition, the patient had no medical history of MRSA infection or colonization and no medical history in the last year of hospitalization, admission to a nursing home, dialysis or surgery. The patient had no permanent indwelling catheters or medical devices that pass through the skin.
Seven of the Swedish MRSA were isolated in Örebro County and were stored at Örebro University Hospital, Örebro, Sweden, and nine originated from the western region of Sweden, Sahlgrenska University Hospital, Gothenburg.
The MICs of different antimicrobial agents were determined by the agar dilution method according to CLSI guidelines. The following agents were tested: ampicillin, cefazolin, erythromycin, gentamicin, tetracycline and vancomycin (all from Sigma Chemical Co., USA), teicoplanin (Aventis Pharma, France), ceftizoxime (Astellas Pharma Inc., Japan), ciprofloxacin (Bayer Yakuhin Co., Ltd, Japan) and imipenem (Banyu Pharmaceutical Co., Ltd, Japan). S. aureus ATCC 29213 was used as a reference strain.
The SC type (I–VIII) was determined by multiplex PCR to detect types III, IV, VII and VIII (MPCR:A) and types I, II, V and VI (MPCR:B) separately, as described previously.18
Nucleotide sequencing of novel SC genes
Determination of the SC genes (coa) was performed by PCR amplification and subsequently nucleotide sequencing the genes as described previously.19
Multiple alignments were performed with GENETYX-MAC Version 13.0.3 (GENETYX Corporation). The phylogenetic tree was created using the neighbour-joining method in GENETYX-MAC. Support for individual nodes on the tree was determined by bootstrap analysis.
Multilocus sequence typing (MLST)
MLST was performed with primer sequences developed by Enright et al.20 and Crisostomo et al.21 using real-time LightCycler System PCR and amplification of the seven housekeeping genes in the same PCR program.10 The STs were grouped into clonal complexes (CCs) and shared a common ancestor as predicted by the based upon related STs (eBURST) software v3.
spa typing was carried out according to Harmsen et al.22 and was adapted to LightCycler System PCR with the SYBR Green I dye. Samples contained 0.5 µM of forward primer, 0.3 µM of reverse primer and 3 mM MgCl2 and were pre-incubated for 10 min at 95°C and then subjected to 35 cycles of amplification run according to the following schedule: denaturation at 95°C for 0 s, annealing at 55°C for 5 s and extension at 72°C for 18 s. Melting curve analysis was conducted by continuously registering the fluorescence while slowly raising the temperature (0.1°C/s) from 65°C to 95°C. Prior to sequencing, the products amplified by LightCycler System PCR were purified using a MultiScreen PCRµ96 Plate (Millipore AB, Solna, Sweden) and subsequently sequenced on both strands using the PCR primers and an ABI PRISM BigDye Terminator version 3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Stockholm, Sweden), and separation was performed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). spa types were determined with the Ridom StaphType software (Ridom GmbH, Würzburg, Germany) and grouped using the BURP (‘Based Upon Repeat Pattern’) algorithm.
Detection of lukS-PV and lukF-PV
A previously described protocol was used to detect the Panton–Valentine leucocidin (PVL) genes employing LightCycler® FastStart DNA MasterPLUS SYBR Green I and modified regarding the PCR conditions.23,24
SCCmec typing was initially performed by using real-time LightCycler® System PCR to detect the essential genetic components mecA, mecR1, IS1272, ccrA and ccrB according to Berglund et al.16 and was confirmed by the multiplex scheme described by Kondo et al.25 In the latter scheme, six multiplex PCRs identified the ccr gene complex, the mec gene complex and specific structures in the J regions.
Detection of type IVg and IVh SCCmec
Primers designed in the J1 region of the isolate M03-68 (DQ106887 [GenBank] ), IV g Left 5'-GCAAGCTGTTATCGGCATTT-3' and IV g Right 5'-GATCGTTCGTGTTTGTGTGC-3', were used to detect type IVg SCCmec and yielded a product of a size of 378 bp.7 The PCR was run at 94°C for 2 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 1 min and extension at 72°C for 1 min, followed by a final extension at 72°C for 2 min before cooling at 4°C.
Type IVh SCCmec was detected by employing primers J IVh F 5'-TTCCTCGTTTTTTCTGAACG-3' and J IVh R 5'-CAAACACTGATATTGTGTCG-3'.9 The PCR was run at 94°C for 1 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 1 min and extension at 72°C for 1 min, followed by a final extension at 72°C for 1 min before cooling at 4°C.
Nucleotide sequencing of SCCmec
Type IV SCCmec was initially identified by detection of a class B mec complex and the type 2 ccr, and isolates carrying subtypes other than IVa, IVb, IVc, IVd, IVg or IVh were further investigated by nucleotide determination of the J1 region. Two isolates, JCSC6668 and JCSC6670, were possible to analyse by PCR amplification from the ccr genes to the right chromosomal region. Other isolates were not possible to amplify due to unknown chromosomal regions or were designated as type IVg or IVh SCCmec. The entire nucleotide sequence was determined of two SCCmec from isolates JCSC6668 and JCSC6670. The SCCmec of isolates JCSC6668 and JCSC6670 were amplified by using overlapping primers and several long-range PCRs carried out with the Expand High Fidelity PCR System (Roche Diagnostics) to cover the entire cassettes. The region from orfX to the hypervariable region (HVR) was amplified using primers cR1 and mD2 and from HVR to mecA using primers mD3 and mA9.26 The mec complex was covered using primer mA2 located in mecA and IS7 located in IS1272, and the region from the mec complex to the ccr genes was amplified using primers IS8 and 
7.17,25 The region from the ccr genes to the chromosomal region was amplified using primer 5 located in ccr and in the right chromosomal region the primer cLm1 was used for isolate JCSC6668 while primer cL5 was used for JCSC6670.27 Additional primers (cR2 and mR3) were used to cover the entire orfX.26 Oligonucleotide primers used for long-range PCR amplification of SCCmec are listed in Table 1, and the locations are indicated in Figure 1. The PCR products were purified using the High Pure PCR Purification Kit (Roche Diagnostics GmbH) and subsequently sequenced on both strands using the ABI PRISM BigDye Terminator version 3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Warrington, UK), and separation was performed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The sequences were analysed and assembled using the BioEdit Sequence Alignment Editor.
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orfs of more than 100 bp were identified with the GenomeGambler v.1.5 software and were compared with sequence databases at the National Center for Biotechnology Information with the basic local alignment search tool (BLAST; National Library of Medicine, Bethesda, MD, USA) for annotation and prediction of functions. The ccr genes were investigated using the clustal W (1.83) multiple alignment, and amino acid identities were calculated using the EMBOSS pairwise alignment algorithms.
Nucleotide sequence accession numbers
The sequence of the SCCmec elements of strains JCSC6668 (CCUG41764) and JCSC6670 (CCUG27050) have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers AB425823 and AB425824, respectively. The sequences of coa of S. aureus JCSC6074, JCSC6666, JCSC6075, JCSC6667, JCSC6671, JCSC6674 and JCSC6669 strains have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers AB436965, AB436966, AB436967, AB436968, AB436969, AB436970 and AB436971, respectively.
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Characterization of Swedish MRSA with type IV SCCmec
In order to define the collection of MRSA isolated in Sweden carrying type IV SCCmec, we determined the MLST, spa and SC types of these strains. As shown in Table 2, the selection of Swedish MRSA carrying type IV SCCmec demonstrated a high diversity of genotypes. A heterogeneous pattern of MICs was observed, and several isolates showed notable low values of carbapenems and cephalosporins. Sixteen Swedish MRSA strains were distributed into nine STs (as shown in Table 2), and these were classified into seven CCs and two singletons.
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spa typing revealed that there were 13 different spa types among these 16 Swedish MRSA strains. Most of the spa types (11 of 13) were represented by single isolates, whereas two spa types (t015 and t032) were represented by at least two strains.
SC types were determined using multiplex PCRs.18 Eight of 16 strains were classified into four SC types (II, III, IV and VII), whereas the other eight strains could not be characterized according to SC types I–VIII. SC type VII was the major SC group in this study, which included five strains of MRSA ST1, ST45 and ST59. The eight SC non-typeable strains were represented by four genotypes according to MLST (ST22, ST45, ST140 or ST182) and carried either type IVg SCCmec, IVh SCCmec or unknown subtypes of type IV SCCmec.
Five of the 16 MRSA were classified as subtype IVg SCCmec, and these represented five different genotypes as depicted by spa and MLST (ST1, ST30, ST45, ST59 or ST140). In contrast, four of the MRSA were classified as subtype IVh SCCmec, and these were all ST22. However, one of the MRSA carrying type IVh SCCmec had a different, yet closely related, spa type (t020) and carried the PVL locus, in comparison with the other MRSA with type IVh SCCmec that represented t032 and were PVL-negative. In addition, isolate JCSC6668 was positive in SCCmec multiplex PCR for identifying S01 located at the J1 region of type II.2 SCCmec, indicating the presence of the J1 region similar to type II.2 SCCmec (data not shown).
Identification of novel type of SC
The structures of coa genes of the isolates that had unknown SC types were investigated by nucleotide sequencing of the genes and comparison with existing type I–X coa genes. We determined the nucleotide sequences of coa of the eight MRSA: JCSC6674, JCSC6665, JCSC6666, JCSC6667, JCSC6669, JCSC6671, JCSC6074 and JCSC6075. The coa genes of these eight isolates were classified into four groups based on their nucleotide sequences. The coa genes of four MRSA, JCSC6075, JCSC6667, JCSC6671 and JCSC6674, were identical to each other. We designated the coa type of these isolates as type XIa. The nucleotide sequence of type XIa showed >99% identity to the partial sequence of coa of HinfI coagulase type D strain RS54.28 Type D was one of the major clones of MRSA isolated in Scotland in the 1990s, and the PFGE banding patterns of the strains were related to EMRSA-15.28 The coa of JCSC6669 showed 98% identity to the partial sequence of coa of HinfI coagulase type I* strain a48 isolated in Aberdeen, and it was designated as type XIb. The coa of two isolates, JCSC6074 and JCSC6666, were identical to each other. The nucleotide identity between coa sequences except repeat sequences of these two isolates and that of type X coa was 92.8%, and therefore, the coa were designated as type Xb. The other non-typeable coa of JCSC6665 showed 92.1% identity to the coa sequence without repeat sequence of type VII coa. We designated it as type VIIb.
We investigated the phylogenetic relation among the new coa of Swedish MRSA and the type I–X coa by using those nucleotide sequences, except repeat sequences, which is shown in Figure 2. The phylogenetic tree of coa also showed that Swedish MRSA of type IV SCCmec demonstrated a high diversity.
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Identification of novel SCCmec type IV
Seven MRSA remained as unknown type IV SCCmec as five isolates were carrying type IVg SCCmec and four isolates were classified as carrying type IVh SCCmec. Five of the seven MRSA carrying novel subtypes of type IV SCCmec were not possible to amplify by long-range PCR covering the J1 region. In two isolates, JCSC6668 and JCSC6670, 
12 kb PCR products were covering the J1 regions. These isolates were initially characterized as type IV SCCmec by the presence of type 2 ccr and class B mec complex and were further investigated by long-range PCR and determination of the nucleotide sequence of the J1 region. The J1 regions of JCSC6668 and JCSC6670 had high homology to corresponding regions previously described in type II.2 and I.2 SCCmec, respectively, and the entire nucleotide sequences of the two elements were therefore determined in order to properly describe the SCCmec.
Characteristics of the two SCCmec
The entire SCCmec elements were amplified in isolates JCSC6668 and JCSC6670 by long-range PCR and were subsequently sequenced. The SCCmec of JCSC6668 was 22 534 bp long and the SCCmec of JCSC6670 was 22 323 bp, and both SCCmec were demarcated by integration site sequences (ISSs) of SCCmec. Also, two characteristic direct repeated (DR) regions were located at the upstream extremity of the two SCCmec and one DR at the downstream region.
The orfs of the two type IV SCCmec are summarized in Table 3. Both SCCmec investigated carried the class B mec complex as determined by the presence of a truncated copy of IS1272 located downstream of mecR1. However, the SCCmec of JCSC6668 had an altered class B mec complex with an integration of a 1014 bp ISSep1-like transposase located upstream of a 12 bp truncated IS1272. This novel mec complex consisting of IS431/mecA/
mecR1/ISSep1/IS1272 was regarded as a variant class B mec complex designated as class B.3 (Figure 1).
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The two SCCmec identified in JCSC6668 and JCSC6670 carried the type 2 ccr and surrounding orfs. The amino acid homology of ccrA in JCSC6668 was 98.9% identical to ccrA described in a type IVa SCCmec (accession number AB063172 [GenBank] ) and in a Staphylococcus epidermidis (accession number DQ514334 [GenBank] ). The ccrB was 1629 bp and had 99.6% identity to the ccrB described in an isolate representing USA300 (accession number CP000730 [GenBank] ), in a type IVc SCCmec (accession number AB266532 [GenBank] ), among others. The ccrA of JCSC6670 had 100% amino acid identity to ccrA described in S. aureus isolate ZH47 and the type IVE SCCmec (accession numbers AM292304 [GenBank] and AJ810121 [GenBank] ), as well as in Staphylococcus warneri and S. epidermidis (accession numbers DQ225180 [GenBank] and DQ196433 [GenBank] ). The ccrB of JCSC6670 was 21 bp longer than ccrB in JCSC6668 and had 100% identity to ccrB described in type IVa and IVd SCCmec (accession numbers AB266531 [GenBank] and AB097677 [GenBank] ) as well as in isolate ZH47.
The J1 region of isolate JCSC6668 was 3.3 kb and showed 99.2% to 99.4% identity to the corresponding orfs described in the type II.2 SCCmec of strain JCSC3063 (accession number AB127982 [GenBank] ). The length was equal in the type II.2 SCCmec and consisted of two orfs representing hypothetical proteins with unknown functions. In addition, the J2 region of JCSC6668 had high homology to a corresponding region in type II.2 SCCmec of strain JCSC3063, although the latter consisted of a much larger region, due to insertion of, for example, Tn554.
The isolate JCSC6670 highly corresponded (99.4% to 100% identity) to the J1 region described in the SCCmec type I.2 identified in strain PL72 (accession number AB433542) (H. Xiao, T. Ito, F. Takeuchi, X. X. Ma, M. Takasu, Y. Uehara, D. Oliveira, H. de Lencastre and K. Hiramatsu, unpublished results) and was 4.7 kb. The J1 regions of both elements consisted of four orfs of equal length, which were considered as hypothetical proteins. Although the SCCmec of JCSC6670 and PL72 had different ccr, the J2 region was also well conserved in these different cassettes.
In contrast to the J1 region, the two investigated type IV SCCmec were well conserved in the J3 and J2 regions. The J3 regions located from orfX to mecA were 99.9% identical in JCSC6668 and JCSC6670 and contained both the downstream constant region and the HVR commonly identified among type IV SCCmec. In addition, this region was 99.9% similar to the existing type IVa SCCmec in isolate CA05 (accession number AB063172 [GenBank] ).27 The J2 regions were 99.6% identical among the two SCCmec investigated and 99.5% or 99.9% identical to the type IVa SCCmec of CA05.
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We describe two new variants of type IV SCCmec in a collection of Swedish MRSA constituting type IV SCCmec with unknown subtypes, i.e. not type IVa, IVb, IVc or IVd. The type IV SCCmec of JCSC6668 and JCSC6670 were investigated by nucleotide sequencing and were found to possess J1 regions with high identity to J1 regions previously identified in type II.2 and I.2 SCCmec, respectively. High amino acid identities were also observed in the J2 regions. The finding of equal J1 regions explains why the isolate JCSC6668 was positive in the multiplex PCR for S01, which would have indicated the presence of a type II SCCmec according to the multiplex PCR described by Kondo et al.25 Although this specific finding of equal J1 regions is unique, difficulties in assigning SCCmec due to similarities in J regions have been reported previously.25 Furthermore, variation has recently also been observed in type II SCCmec, which carries type 2 ccr genes as does type IV SCCmec.12,25,29,30
The occurrence of diverse J1 regions among type IV SCCmec from Sweden indicated independent insertions of mec complex into the J1 regions that may have served as the original SCC. The SCCmec is suggested to initially have originated by integration of a mec gene complex into the J1 region located between the ccr genes and the downstream chromosomal region.25 In this study, we identified nearly identical J1 regions in different types of SCCmec, a finding that provides further support for this hypothesis. However, no ISSs were identified inside the SCCmec that would suggest an organization of two preceding SCC elements. It is possible that the type IV SCCmec described in this study shared a common origin with the type II.2 and I.2 SCCmec, respectively, and that these elements may have arisen by independent integration of the mec complex.
No type IV SCCmec have yet been described to carry these compositions of the J1 region and, for that reason, we designate the two new subtypes as types IVi SCCmec (JCSC6668) and IVj SCCmec (JCSC6670), or IV.7 and IV.8 according to the nomenclature recently suggested by Chongtrakool et al.8 MRSA from the whole world are continuously being investigated and, consequently, more SCCmec variants are being identified. This new information gives us an idea of the origin and relatedness of SCCmec.
The size of the type IV SCCmec of MRSA is more variable and the composition more diverse than among the other SCCmec types. There are until today six known subtypes of type IV SCCmec based on structural differences in the J1 region located between the ccr complex and the right chromosomal region of SCCmec. Type IVa was described and frequently found in CA-MRSA from the USA and Australia and has then been identified all over the world, including Sweden.4,16,27,31,32 Type IVb has been scarcely found since it was first described in the USA, which is in contrast to subtype IVc that is present in MRSA spread all over Europe as one of the dominating clones.4,5,16,33,34 Types IVc and IVd were frequently found in MRSA isolated in Japan in the early 1980s.6 Subtype IVg was found in MRSA from bovine milk in Korea.7 In the present study, we describe MRSA with type IVg SCCmec isolated from humans, and they all displayed different genetic backgrounds as depicted by MLST, indicating independent introduction of the SCCmec into the chromosome of S. aureus. The most recently described subtype, IVh, was specific for EMRSA-15 and reported to be highly homologous to the strain PL72, although the entire SCCmec was not investigated.9 Four of the Swedish MRSA carried type IVh SCCmec with the same genetic background, ST22, as the Portuguese MRSA with subtype IVh. Interestingly, one of the MRSA with type IVh SCCmec carried the PVL locus, which may indicate the introduction of the PVL genes subsequent to the integration of the SCCmec.
The collection of 16 isolates further analysed in the present study represented highly diversified MRSA designated as type IV SCCmec, which consisted of at least nine different genetic backgrounds as depicted by MLST, SC typing and spa typing. The concordance between these three methods was high, although the SCCmec showed a more complex picture. The occurrence of subtype IVg, new subtypes and unknown subtypes in the same genotype indicated a high level of recombination or represented highly mobile type IV SCCmec. Type IV SCCmec is smaller in size than the other SCCmec types; it is found among S. aureus with several different genetic backgrounds and generally does not contain any additional resistance genes, which may facilitate mobility of the cassette. The larger size of type II SCCmec is thought to be the explanation for the observation that this element is not excised by the ccr recombinases and not readily spread among several genetic backgrounds.35 In addition, the presence of four new SC types in the collection of Swedish MRSA further indicated a diverse nature. The similarity of XIa and XIb to MRSA strains described in Scotland probably represents the clonal spread of S. aureus in Europe.28
This study provides new information about the variation of type IV SCCmec and how these elements may have arisen and spread by site-specific excision driven by the type 2 ccr genes. In order to more easily designate the increasing number of SCCmec variants, we recommend the use of the alternative nomenclature based on ccr type and mec class as well as also including the description of J regions indicated by numbers representing differences in the J1, J2 and J3 regions.8
In conclusion, type IV SCCmec is occurring in heterogeneous clones of MRSA in Sweden, and it seems that type IV SCCmec itself is a highly diversified genetic element. We describe two novel subtypes of type IV SCCmec with common J1 regions shared by other types of SCCmec, which indicate that J1 regions occurred as primordial SCC.
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This study was supported by grants from the Sweden–Japan Foundation, the Swedish Society for Medical Research, the Swedish Institute of Biomedical Laboratory Science, the Örebro County Council Research Committee, Sweden, and a Grant-in-Aid for 21st Century COE Research, Ministry of Education and Science, Japan.
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None to declare.
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We thank Christina Åhrén, Christina Welinder-Olsson and Leif Larsson, at the Sahlgrenska University Hospital, Gothenburg, Sweden, for providing MRSA isolates. Tadashi Baba at the Department of Microbiology and Infection Control Science, Juntendo University is appreciated for professional advice and assistance during the orf analysis and Jin Jingxun at the Department of Infection Control Science, Juntendo University for conducting the PCR for type IVh SCCmec.
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1 . Ito T, Okuma K, Ma XX, et al. Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC. Drug Resist Updat (2003) 6:41–52.[CrossRef][Web of Science][Medline]
2
.
Ito T, Ma XX, Takeuchi F, et al. Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC. Antimicrob Agents Chemother (2004) 48:2637–51.
3 . Oliveira DC, Tomasz A, de Lencastre H. The evolution of pandemic clones of methicillin-resistant Staphylococcus aureus: identification of two ancestral genetic backgrounds and the associated mec elements. Microb Drug Resist (2001) 7:349–61.[CrossRef][Web of Science][Medline]
4
.
Okuma K, Iwakawa K, Turnidge JD, et al. Dissemination of new methicillin-resistant Staphylococcus aureus clones in the community. J Clin Microbiol (2002) 40:4289–94.
5 . Dufour P, Gillet Y, Bes M, et al. Community-acquired methicillin-resistant Staphylococcus aureus infections in France: emergence of a single clone that produces Panton–Valentine leukocidin. Clin Infect Dis (2002) 35:819–24.[CrossRef][Web of Science][Medline]
6
.
Ma XX, Ito T, Chongtrakool P, et al. Predominance of clones carrying Panton–Valentine leukocidin genes among methicillin-resistant Staphylococcus aureus strains isolated in Japanese hospitals from 1979 to 1985. J Clin Microbiol (2006) 44:4515–27.
7
.
Kwon NH, Park KT, Moon JS, et al. Staphylococcal cassette chromosome mec (SCCmec) characterization and molecular analysis for methicillin-resistant Staphylococcus aureus and novel SCCmec subtype IVg isolated from bovine milk in Korea. J Antimicrob Chemother (2005) 56:624–32.
8
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Chongtrakool P, Ito T, Ma XX, et al. Staphylococcal cassette chromosome mec (SCCmec) typing of methicillin-resistant Staphylococcus aureus strains isolated in 11 Asian countries: a proposal for a new nomenclature for SCCmec elements. Antimicrob Agents Chemother (2006) 50:1001–12.
9
.
Milheirico C, Oliveira DC, de Lencastre H. Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: ‘SCCmec IV multiplex. J Antimicrob Chemother (2007) 60:42–8.
10
.
Berglund C, Molling P, Sjoberg L, et al. Multilocus sequence typing of methicillin-resistant Staphylococcus aureus from an area of low endemicity by real-time PCR. J Clin Microbiol (2005) 43:4448–54.
11 . Welinder Olsson C, Larsson L, Berglund C, et al. Emergence of community-associated MRSA in the western part of Sweden over a 20 year period. In: Abstracts of the Twelfth International Symposium on Staphylococci and Staphylococcal Infections, Maastricht, The Netherlands, 2006. Poster no. P150.
12
.
Shore A, Rossney AS, Keane CT, et al. Seven novel variants of the staphylococcal chromosomal cassette mec in methicillin-resistant Staphylococcus aureus isolates from Ireland. Antimicrob Agents Chemother (2005) 49:2070–83.
13
.
Faria NA, Oliveira DC, Westh H, et al. Epidemiology of emerging methicillin-resistant Staphylococcus aureus (MRSA) in Denmark: a nationwide study in a country with low prevalence of MRSA infection. J Clin Microbiol (2005) 43:1836–42.
14 . Robinson DA, Enright MC. Multilocus sequence typing and the evolution of methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect (2004) 10:92–7.[CrossRef][Web of Science][Medline]
15
.
Enright MC, Robinson DA, Randle G, et al. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci USA (2002) 99:7687–92.
16 . Berglund C, Molling P, Sjoberg L, et al. Predominance of staphylococcal cassette chromosome mec (SCCmec) type IV among methicillin-resistant Staphylococcus aureus (MRSA) in a Swedish county and presence of unknown SCCmec types with Panton–Valentine leukocidin genes. Clin Microbiol Infect (2005) 11:447–56.[CrossRef][Web of Science][Medline]
17
.
Ito T, Katayama Y, Asada K, et al. Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother (2001) 45:1323–36.
18 . Sakai F, Takemoto A, Watanabe S, et al. Multiplex PCRs for assignment of staphylocoagulase types and subtypes of type VI staphylocoagulase. J Microbiol Methods (2008) 75:312–7.[CrossRef][Web of Science][Medline]
19
.
Watanabe S, Ito T, Takeuchi F, et al. Structural comparison of ten serotypes of staphylocoagulases in Staphylococcus aureus. J Bacteriol (2005) 187:3698–707.
20
.
Enright MC, Day NP, Davies CE, et al. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol (2000) 38:1008–15.
21
.
Crisostomo MI, Westh H, Tomasz A, et al. The evolution of methicillin resistance in Staphylococcus aureus: similarity of genetic backgrounds in historically early methicillin-susceptible and -resistant isolates and contemporary epidemic clones. Proc Natl Acad Sci USA (2001) 98:9865–70.
22
.
Harmsen D, Claus H, Witte W, et al. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol (2003) 41:5442–8.
23 . Berglund C, Prevost G, Laventie BJ, et al. The genes for Panton–Valentine leukocidin (PVL) are conserved in diverse lines of methicillin-resistant and methicillin-susceptible Staphylococcus aureus. Microbes Infect (2008) 10:878–84.[CrossRef][Web of Science][Medline]
24 . Johnsson D, Molling P, Stralin K, et al. Detection of Panton–Valentine leukocidin gene in Staphylococcus aureus by LightCycler PCR: clinical and epidemiological aspects. Clin Microbiol Infect (2004) 10:884–9.[CrossRef][Web of Science][Medline]
25
.
Kondo Y, Ito T, Ma XX, et al. Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob Agents Chemother (2007) 51:264–74.
26
.
Ito T, Katayama Y, Hiramatsu K. Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315. Antimicrob Agents Chemother (1999) 43:1449–58.
27
.
Ma XX, Ito T, Tiensasitorn C, et al. Novel type of staphylococcal cassette chromosome mec identified in community-acquired methicillin-resistant Staphylococcus aureus strains. Antimicrob Agents Chemother (2002) 46:1147–52.
28 . Carter PE, Begbie K, Thomson-Carter FM. Coagulase gene variants associated with distinct populations of Staphylococcus aureus. Epidemiol Infect (2003) 130:207–19.[CrossRef][Medline]
29
.
Hisata K, Kuwahara-Arai K, Yamanoto M, et al. Dissemination of methicillin-resistant staphylococci among healthy Japanese children. J Clin Microbiol (2005) 43:3364–72.
30
.
Cha HY, Moon DC, Choi CH, et al. Prevalence of the ST239 clone of methicillin-resistant Staphylococcus aureus and differences in antimicrobial susceptibilities of ST239 and ST5 clones identified in a Korean hospital. J Clin Microbiol (2005) 43:3610–4.
31 . Larsen A, Stegger M, Goering R, et al. Emergence and dissemination of the methicillin resistant Staphylococcus aureus USA300 clone in Denmark (2000–2005). Euro Surveill (2007) 12:22–4.
32
.
Ip M, Yung RW, Ng TK, et al. Contemporary methicillin-resistant Staphylococcus aureus clones in Hong Kong. J Clin Microbiol (2005) 43:5069–73.
33
.
Wannet WJ, Spalburg E, Heck ME, et al. Emergence of virulent methicillin-resistant Staphylococcus aureus strains carrying Panton–Valentine leucocidin genes in the Netherlands. J Clin Microbiol (2005) 43:3341–5.
34 . Vandenesch F, Naimi T, Enright MC, et al. Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton–Valentine leukocidin genes: worldwide emergence. Emerg Infect Dis (2003) 9:978–84.[Web of Science][Medline]
35
.
Jansen WT, Beitsma MM, Koeman CJ, et al. Novel mobile variants of staphylococcal cassette chromosome mec in Staphylococcus aureus. Antimicrob Agents Chemother (2006) 50:2072–8.
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