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Antimicrobial Agents and Chemotherapy, January 2004, p. 285-296, Vol. 48, No. 1
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.1.285-296.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Local Variants of Staphylococcal Cassette Chromosome mec in Sporadic Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Coagulase-Negative Staphylococci: Evidence of Horizontal Gene Transfer?

Anne-Merethe Hanssen,* Gry Kjeldsen, and Johanna U. Ericson Sollid*

Department of Microbiology and Virology, Institute of Medical Biology, University of Tromsø, Tromsø, Norway

Received 8 April 2003/ Returned for modification 7 July 2003/ Accepted 23 September 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mecA gene in Staphylococcus aureus is located on the genetic element staphylococcal cassette chromosome (SCC). Different SCCmecs have been classified according to their putative recombinase genes (ccrA and ccrB) and overall genetic composition. Clinical isolates of coagulase-negative staphylococci (CoNS; n = 39) and S. aureus (n = 20) from Norway, India, Italy, Finland, the United States, and the United Kingdom were analyzed by pulsed-field gel electrophoresis, which showed that most isolates were genetically unrelated. Cluster analyses of 16S rRNA gene and pta sequences confirmed the traditional biochemical species identification. The mecI, mecR1, mecA, and ccrAB genes were detected by PCRs, identifying 19 out of 20 S. aureus and 17 out of 39 CoNS isolates as carriers of one of the three published ccrAB pairs. New variants of SCCmec were identified, as well as CoNS isolates containing ccrAB genes without the mec locus. ccrAB and mec PCRs were verified by hybridization. Sequence alignments of ccrAB genes showed a high level of diversity between the ccrAB alleles from different isolates, i.e., 94 to 100% and 95 to 100% homology for ccrAB1 and ccrAB2, respectively. All of the ccrAB3 genes identified were identical. Genetically unique and sporadic methicillin-resistant S. aureus (MRSA) contained local variants of ccrAB gene pairs identical to those found in MR-CoNS but different from those in MRSA from other regions. Allelic variants of ccrAB in isolates from the same geographic region showed sequence conservation independent of species. The species-independent sequence conservation found suggests that there is a closer genetic relationship between ccrAB2 in Norwegian staphylococci than between ccrAB2 sequences in international MRSA and Norwegian MRSA. This might indicate that different staphylococcal species acquire these genes locally by horizontal gene transfer.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Methicillin resistance in staphylococci is caused by expression of PBP2a (PBP') encoded by the mecA gene (9, 16, 31, 39, 43). mecA is located on a genetic element called the staphylococcal cassette chromosome (SCC) in Staphylococcus aureus (17, 24). SCCmec is a group of mobile DNA elements of 21 to 67 kb that is integrated into the chromosome of methicillin-resistant S. aureus (MRSA) at a unique site (attBscc) located near the S. aureus origin of replication (18). For movement, SCCmec carries one of three described cassette chromosome recombinase A and B gene pairs (ccrA and ccrB), which encode recombinases of the invertase-resolvase family (21, 24). Different SCCmecs in S. aureus have been characterized and classified according to their putative recombinase genes (ccrAB) and overall genetic composition (24). SCCmec types I to IV are defined by the particular combination of type 1, 2, or 3 ccrAB gene pairs and class A, B, C, or D mec complexes. The latter classification is defined by the genetic organization of the mec regulatory genes (mecR1 and mecI) (18, 25, 30, 36).

The origin of SCCmec is unknown. There has been no report of SCCmec in bacteria other than staphylococci (18). The mechanism(s) responsible for mecA transfer is not known, but evidence supports horizontal transfer of mec DNA between staphylococcal species and of the mecA gene between different gram-positive genera (4). One assumes that the ccr and mec genes were brought together in coagulase-negative staphylococci (CoNS) from an unknown source (5, 18, 40, 45), where deletion in the mec regulatory genes occurred, before the genes were transferred into S. aureus to generate MRSA (5, 19, 34, 40).

The overall prevalence of MRSA is still very low in Norway. Only 0.2% of the S. aureus isolates from blood cultures and wound specimens were identified as MRSA in 2001 (NORM/NORM-VET 2001, consumption of antimicrobial agents and occurrence of antimicrobial resistance in Norway; report available at http://www.zoonose.no). Some MRSA isolates have their origin in other countries, while others are sporadic and epidemiologically unrelated (2, 3; Surveillance of communicable diseases in Norway 1999, available at http://www.fhi.no/filer/pdf/smittevern4.pdf). Methicillin-resistant CoNS (MR-CoNS) strains, on the contrary, are abundant. As many as 80% of blood culture CoNS isolates are MR-CoNS (35), and this is similar to the prevalence of 25 to 75% reported from Scandinavia (22) and from the rest of the world (11, 46).

Our hypothesis is that Norwegian sporadic MRSA strains are genetically unique and different from MRSA strains from other regions and contain SCCmec genes obtained from MR-CoNS from the same geographic region. This was explored by comparing the molecular compositions of two basic structures in the resistance cassette, i.e., the recombinase genes ccrAB and the mec gene complex, in genetically unrelated isolates of MRSA, S. epidermidis, S. haemolyticus, S. hominis, and S. warneri from Norway, Finland, India, Italy, the United Kingdom, and the United States.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains. Thirty-nine clinical isolates of CoNS from different hospitals in Norway and 20 clinical isolates of MRSA from Norway, India, Italy, Finland, the United States, and the United Kingdom were included (Table 1). For reference in all analyses, mecA-negative strain S. aureus NCTC 8325 and SCCmec-positive strains S. aureus NCTC 10442, N315, and 85/2082 (21) were included (Table 1). In addition, S. epidermidis ATCC 12228 (methicillin susceptible, mecA negative) and S. epidermidis ATCC 27626 (methicillin resistant, mecA positive) were used (Table 1). All clinical isolates were selected because of differences in the mec gene complex and variations in the level of oxacillin resistance. Other criteria were that the isolates should be genetically different by pulsed-field gel electrophoresis (PFGE) analysis, and they were isolated between 1990 and 2001.


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  		TABLE 1. Characteristics of the 20 S. aureus isolates, 39 CoNS isolates, and six reference strains used in this study

 
Bacterial identification and susceptibility testing. All clinical isolates were identified by a positive Gram stain, the presence of catalase, and the presence or absence of clumping factor (bound coagulase) by Staphaurex Plus* (Murex Biotech, Dartford, England). All clinical isolates were identified to the species level by ID32 Staph (bioMérieux, Marcy l'Etoile, France) in accordance with the manufacturer's instructions. MICs of oxacillin were determined by Etest (AB Biodisk) on Mueller-Hinton agar (Difco) supplemented with 2% NaCl. In accordance with the NCCLS (34a) interpretive standards (micrograms per milliliter), CoNS strains for which the MIC was <=0.25 µg/ml were considered oxacillin susceptible, while CoNS strains for which the MIC was >=0.5 µg/ml were considered oxacillin resistant. S. aureus strains for which the MIC was <=2 µg/ml were considered oxacillin susceptible, whereas S. aureus strains for which the MIC was >=4 µg/ml were considered oxacillin resistant.

PFGE. Preparation of chromosomal DNA was performed as described earlier (8), with a few modifications. Lysozyme (Sigma) was added to the EC buffer (0.1 M EDTA [pH 8.0], 0.06 M Tris-HCl [pH 8.0], 1 M NaCl, 0.5% Brij 58, 0.2% deoxycholate [Sigma], 0.5% N-lauroylsarcosine [Sigma]) to a final concentration of 0.25 mg/ml. Four- and 10-µl volumes of a 1-mg/ml solution of lysostaphin (Sigma) were added to the cell suspensions of CoNS and MRSA, respectively. The cell suspension was mixed with 300 µl of 2% low-melting-point agarose (LMP, preparative grade for large fragments; Promega). Solid agarose plugs were lysed in 3 ml of EC buffer at 37°C without shaking for 1 h (MRSA) or overnight (CoNS). The plugs were incubated at 55°C for 1 h in 1 ml of TE buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA) and washed three times with 3 ml of TE buffer at room temperature before macrorestriction with 20 U of SmaI (Promega) for 2 h (MRSA) or overnight (CoNS) at room temperature. Restriction fragments of DNA were separated by PFGE with a CHEF-DRIII device (Bio-Rad Laboratories, Inc., Richmond, Calif.) through 1% agarose gel (LE, analytical grade agarose; Promega) in 45 mM Tris-borate-1 mM EDTA running buffer. The pulse times were 1 to 35 s for 24 h at 200 V and 14°C for CoNS and 5 to 60 s for 23 h at 200 V and 14°C for MRSA. After electrophoresis, gels were stained with ethidium bromide, rinsed in water, and photographed under UV light with the GelDoc 1000 system (Bio-Rad). The PFGE types were defined on the basis of the DNA banding patterns in accordance with the criteria of Tenover et al. (42) for bacterial strain typing and analyzed both visually and by GelCompar software version 2.5 (Applied Maths, Sint-Martens-Latem, Belgium).

PCR amplification. Template DNA was prepared by dissolving a 1-µl loopful of bacteria in 1 ml of TE buffer and centrifuging it at 3,000 x g for 5 min, and the pellet was resuspended in 100 µl of TE buffer. The suspension was boiled for 10 min before centrifugation at 3,000 x g for 5 min. Alternatively, InstaGene Matrix (Bio-Rad) was used to isolate DNA. The supernatant served as the PCR template. PCRs were performed in a GeneAmp PCR system (models 2400 and 9700; Applied Biosystems). The PCRs were carried out with the Applied Biosystems standard PCR mixture with GeneAmp PCR buffer (with 15 mM MgCl2) and 1 U of Taq DNA polymerase in a final volume of 55 µl containing 5 µl of template DNA, 0.24 pmol of each primer per µl, and 80 µM each deoxynucleoside triphosphate. Different parts of the mec complex were amplified, including the genes mecA, mecI, and mecR1 (both the membrane-spanning and penicillin-binding parts of mecR1; Table 2). A 1,370-bp fragment of the 16S rRNA gene (rDNA) and the housekeeping gene pta (encoding phosphate acetyltransferase) were amplified (Table 2). Selected PCRs for detection of different regions of the cassette chromosome recombinase (ccr) genes were established. ccrAB primers were designed on the basis of the nucleotide sequences of S. aureus NCTC 10442 SCCmec type I (GenBank accession no. AB033763), S. aureus N315 SCCmec type II (GenBank accession no. D86934), and S. aureus 85/2082 SCCmec type III (GenBank accession no. AB037671). PCR elongation times were adjusted in accordance with the expected sizes of the PCR amplicons, and the alignment temperatures were adjusted in accordance with the specific nucleotide sequences of the primers and hence their melting temperatures. Alternative primer sets for ccrAB were tested on all strains by using primers and PCRs described by Ito et al. (21). All strains were also tested for the presence of IS1272 and SCCmec type IV with the primers and PCR described by Okuma et al. (36). The oligonucleotide primers and PCR amplicons used in this study are listed in Table 2.


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  		TABLE 2. Oligonucleotide primers and PCR amplicons used in this study

 
Southern blot hybridization. Southern blot transfer of PFGE SmaI-digested genomic DNA to a positively charged nitrocellulose membrane (Roche) was carried out by vacuum blotting (Vacugene XL system; Pharmacia Biotech) in accordance with the manufacturer's instructions. Total DNAs from the following bacteria were used as templates for probe synthesis: S. aureus NCTC 10442 (ccrA1 and ccrB1 probes), S. aureus strain N315 (ccrA2 and ccrB2 probes), and S. aureus strain 85/2082 (ccrA3 and ccrB3 probes). PCR amplicons were used as probes (Table 2) and labeled with the PCR DIG probe synthesis kit (Roche) and purified by agarose gel electrophoresis, followed by extraction with a QIAquick gel extraction kit (QIAGEN). Hybridization was carried out at 68°C, and detection was performed with the DIG luminescence detection kit (Roche) in accordance with the manufacturer's instructions. Dot blotting was performed by standard techniques (7). All CoNS and MRSA strains listed in Table 1 were analyzed by Southern blot hybridization.

DNA sequencing. PCR products were purified with the QIAquick PCR purification kit (QIAGEN). Heterogeneity in the ccrAB, mecA, pta, and 16S rDNAs was identified by bidirectional DNA sequencing of the PCR products with BigDye Terminator v3.0 cycle sequencing ready reactions (Applied Biosystems, Warrington, United Kingdom). All cycle sequencing reactions were performed at 96°C for 1 min, followed by 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min for 25 cycles. PCR sequencing products were precipitated in a total volume of 100 µl of 96% ethanol-3 M Na acetate-70% ethanol, dried, and dissolved in formamide and loading buffer in accordance with the manufacturer's instructions before application to an ABI Prism 377 sequence analyzer (Applied Biosystems).

Computer analyses and sequence accession numbers. The nucleotide sequences and deduced amino acid sequences were edited by using the Chromas software (version 2.21) and aligned with the BioEdit sequence alignment editor, v5.0.9 (15). Nucleotide sequences were compared to sequences in the GenBank, EMBL, DDBJ, and PDB databases, and protein sequences were compared to nonredundant GenBank CDS translations, by using the BLASTN, BLASTP, and BLASTX local alignment search tools (1).

Phylogenetic trees. Phylogenetic trees (rooted) were generated from aligned sequences by using the parsimony, distance, and maximum-likelihood methods contained in the PAUP* 4.0b1 software (41). The topologies of the phylogenetic trees were evaluated by bootstrap analyses with 1,000 replicates to give the degree of confidence intervals for each node on the phylogenetic trees. The following reference sequences were used in the phylogenetic analyses: ccrA1 and ccrB1 from S. aureus NCTC 10442 (GenBank accession no. AB033763), ccrA2 and ccrB2 from S. aureus N315 (GenBank accession no. D86934), ccrA3 and ccrB3 from S. aureus 85/2082 (GenBank accession no. AB037671), ccrA2 from S. aureus strain CA05 (GenBank accession no. AB063172), ccrB2 from S. aureus (GenBank accession no. AB063172.), ccrA and ccrB from S. epidermidis ATCC 12228 (GenBank accession no. AE015929), pta from Haemophilus influenzae (GenBank accession no. U32799), pta1 and pta40 from S. aureus (http://www.mlst.net), and ccrA1 and ccrB1 from S. hominis ATCC 27844 (GenBank accession no. AB063171).

Nucleotide sequence accession numbers. The sequences determined in this study were submitted to the EMBL/GenBank database and assigned the accession numbers listed in Table 1.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotypic and genotypic methicillin resistance. The Etest MICs of oxacillin are presented in Table 1. Twenty S. aureus strains (MIC, >=4 µg/ml) and 36 CoNS strains (MIC, >=0.5 µg/ml) were considered oxacillin resistant. For three CoNS strains, the oxacillin MICs were <=0.25 µg/ml, and they were considered oxacillin susceptible. S. aureus NCTC 8325 and S. epidermidis ATCC 12228 were considered methicillin susceptible. S. aureus NCTC 10442, N315, and 85/2082 and S. epidermidis ATCC 27626 were considered methicillin resistant. All methicillin-resistant isolates contained mecA, and 14 out of 20 MRSA isolates and 17 out of 39 CoNS isolates showed genetic rearrangements in the mec regulatory genes (mecR1 and mecI; Table 3).


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  		TABLE 3. Types of ccrAB and mec genes observed in MRSA and CoNS isolates by PCR

 
Genetic relationships. All of the strains in this study (S. aureus, n = 20; CoNS, n = 39), including the 6 reference strains, were typed by PFGE. Twenty-four MRSA strains (reference strains included) were classified into 22 different PFGE types (Fig. 1a). Isolates 9-11 (Norway) and 9-13 (Norway) showed one band difference in the PFGE patterns and were considered closely related. According to demographic data, no known connections between these patients were reported. MRSA isolates 9-05 (Norway) and 39-37 (Norway) showed no band differences and were considered indistinguishable. Thirty-eight different PFGE types were assigned among the 41 CoNS isolates (reference strains included) (Fig. 1b). MR-CoNS isolates 8-33 and 8-71 (both from the University Hospital of North Norway) were considered genotypically indistinguishable, while isolates 13-48 and 13-53 (both from the University Hospital of North Norway) were closely related, showing only one band difference. CoNS isolates 6-30 and 6-37 were also considered indistinguishable. We included all of the isolates in the ccrAB gene analyses, keeping in mind that the above-mentioned strains showed clonal relationships.




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  		FIG. 1. PFGE dendrograms representing the genetic relatedness of 20 S. aureus strains and 4 reference strains (a) and that of 39 CoNS strains and 2 reference strains (b). Strain designations and species are specified on the right end of the dendrogram. A low-range PFGE ladder (New England Biolabs, Beverly, Mass.) was used as the molecular size marker. PFGE patterns were analyzed with GelCompar II version 2.5 (Applied Maths) with a 1.1% molecular weight position tolerance. Cluster analyses were performed with Dice comparison settings and an unweighted pair group method with arithmetic mean dendrogram type. The scale bar at the top of the dendrogram represents similarity.

 
Identification of ccrAB genes. Nineteen of the 20 MRSA isolates and 17 of the 39 CoNS isolates contained one of the three published ccrAB genotypes, and all three types were identified in both MRSA and CoNS isolates (Table 3). Sequence analysis of these amplicons revealed that they were ccrAB genes since the corresponding amino acid sequences were homologous. Of the 20 MRSA isolates tested, ccrAB type 2 dominated (n = 11), while ccrAB type 3 dominated (n = 10) among CoNS isolates. The majority of the CoNS isolates (22 of 39) did not have any of the three known ccrAB types. Of the 20 MRSA strains tested, 1 was untypeable. The untypeable group of CoNS were mostly S. epidermidis isolates (n = 14), but also a large group of S. haemolyticus isolates (n = 8) and 1 S. hominis strain were represented here. Our ccrAB PCR results were confirmed with alternative primer sets described by Ito et al. (21). Southern blot hybridization and dot blot hybridization performed with all three known ccrAB types as probes confirmed the negative PCR results. The ccrAB and mec probes hybridized to the same PFGE SmaI fragment, confirming that the mec and ccrAB genes were on the same SCCmec fragment (data not shown).

Verification of IS1272 insertion. Nineteen strains (S. aureus, n = 12; CoNS, n = 7) tested positive for an IS1272 insertion upstream of mecA, indicating that they were carriers of SCCmec type I or IV (Table 3). Four strains had ccrAB1 and were considered carriers of SCCmec type I, while 11 strains had ccrAB2 and were considered carriers of SCCmec type IV.

Combinations of ccrAB types and mec gene complexes. PCR amplification results for the mec regulators and mecA genes are shown in Table 3. Previously reported combinations of ccrAB types and classes of mec gene complexes are indicated in the table by their respective SCCmec types. The three known ccrAB types were combined with the class of mec gene complexes reported earlier in 18 out of 20 MRSA strains. S. aureus isolate 9-07 and three CoNS isolates (8-55, 13-27, and 13-33) contained new combinations of ccrAB types and mec gene complexes not reported earlier. S. epidermidis strain 8-55 contained ccrAB2 combined with all three of the mec regulator regions tested, as well as an IS1272 insertion in the mec complex. S. hominis strains 13-27 and 13-33 contained ccrAB1 in combination with a complete mec gene complex. MRSA strain 9-07 contained ccrAB3 but lacked mecI and mecR1B (Table 3). PCR results were confirmed by Southern blot and dot blot hybridization (data not shown). There was some discrepancy between PCR and hybridization results when mecI was tested for in CoNS isolates 8-23 and 8-80; i.e., they were PCR negative for mecI but hybridization positive for mecI. These isolates were included in further studies and considered to have rearrangements in the mec regulatory genes. The mecR1A-specific PCR was negative in MRSA strains 9-39 and 9-40, but a homologous region could be identified by hybridization. Alternative primers, amplifying the region between the mecA promoter and mecR1B (data not shown), resulted in PCR products of the same size in isolates 9-30 and 9-40 as for SCCmec type III control strain 85/2082.

mecA-negative CoNS isolates. All known types of ccrAB genes were found in three CoNS isolates lacking the mecA gene (Table 3). Two S. epidermidis strains, 6-42 and 8-39, tested positive for ccrAB1, and S. epidermidis strain 13-63 tested positive for ccrAB3. All of the mecA-negative isolates also tested negative for the mecR1 and mecI genes. The negative mecA, mecI, mecR1B, and mecR1A PCR results were confirmed by dot blot and Southern blot hybridization of chromosomal DNA (data not shown).

ccrAB gene sequence alignments. DNA sequences obtained from positive ccrA and ccrB PCRs in 17 CoNS strains and 19 MRSA strains were aligned. DNA sequence comparisons showed 94 to 100% homology for ccrAB1 and 95 to 100% homology for ccrAB2 genes. In the ccrA1 and ccrA2 alleles, 44 to 52% of the nucleotide substitutions resulted in amino acid shifts, while 3 to 16% of the nucleotide substitutions in the ccrB1 and ccrB2 alleles resulted in amino acid shifts. The ccrAB3 gene pairs were all 100% identical (data not shown). The genetic relationship between allelic ccr sequences was also analyzed and displayed as phylogenetic trees (Fig. 2a to d).




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  		FIG. 2. Parsimony tree showing phylogenetic relationships among ccrA1 genes (a), ccrB1 genes (b), ccrA2 genes (c), ccrB2 genes (d), and pta genes (e) in MRSA and CoNS isolates. Outgroups are S. aureus ccrA2 (GenBank accession no. AB063172), S. aureus ccrB2 (GenBank accession no. AB063172), S. aureus ccrA3 (GenBank accession no. AB037671), S. aureus ccrB3 (GenBank accession no. AB037671), and H. influenzae pta (GenBank accession no. U32799).

 
Molecular typing of staphylococcal species. The 16S rDNA and the housekeeping gene pta were included as reference sequences for molecular typing of the different staphylococcal species. Only the MRSA (n = 19; and reference strains N315, NCTC 10442, and 85/2082) and CoNS (n = 17; and reference strain ATCC 12228) isolates that gave positive amplification of ccr genes were investigated. The pta and 16S rDNAs from 19 MRSA isolates, 14 CoNS isolates, and reference strains ATCC 12228, N315, NCTC 10442, and 85/2082 were sequenced, aligned, and compared to each other (data not shown). S. hominis strains 8-39, 13-27, and 13-33 gave no positive PCR amplification product with the pta primers used, but Southern blot hybridization with a pta probe (based on NCTC 10442) gave positive hybridization signals with these strains. These strains were therefore not included in the phylogenetic pta analysis. The phylogenetic tree for the pta gene (Fig. 2e) clustered S. aureus, S. epidermidis, and S. warneri in different groups. 16S rDNA sequence comparisons showed only minor sequence variation (99% homology) between staphylococcal species (data not shown), but the S. hominis strains were separated from S. aureus and other CoNS species by 16S rDNA analyses. These results confirmed the biochemical species identification results obtained by ID32 Staph.

Phylogenetic relationship between ccrA and ccrB alleles from individual isolates. The phylogenetic relationships among the ccrA1, ccrB1, ccrA2, and ccrB2 genes are displayed in Fig. 2a to d. Isolates containing ccrAB1 alleles were clustered in two main groups, both containing alleles obtained from different staphylococcal species; i.e., one cluster contained alleles from S. hominis, while the other cluster contained identical genes from S. aureus, S. hominis, and S. epidermidis (Fig. 2a and b). Three CoNS isolates (6-42, 8-39, and ATCC 27844) appeared in different clusters in the phylogenetic trees for the ccrA1 and ccrB1 genes. Comparisons of the ccrA2 and ccrB2 alleles generated phylogenetic trees with three main clusters (Fig. 2c and d). The largest group comprised alleles from sporadic MRSA and MR-CoNS isolates from Norway, while the two other groups contained alleles from isolates from the United States and Japan. The Japanese strain S. aureus N315 showed a close phylogenetic relationship with MRSA strains 9-37 and 9-38 from the United States, whereas S. epidermidis ATCC 12228 and MRSA strain CA05 clustered closely. Norwegian isolates S. epidermidis 8-55 and S. warneri 8-80 and MRSA isolates 9-05, 9-11, 9-13, 9-17, 39-06, 39-37, 39-61, and 40-22 showed a close relationship. The parsimony, maximum-likelihood, and distance methods gave similar tree topologies for ccrA1, ccrB1, ccrA2, ccrB2, and pta (not shown). The robustness of the trees was supported by bootstrap values of more than 70% for all trees, except in the ccrB1 and ccrB2 trees, where one of the nodes had bootstrap values of 59 and 60%, respectively.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study aimed to explain the appearance of genetically unique and sporadic MRSA isolates by identifying a possible genetic relationship between SCCmecs in methicillin-resistant staphylococci from one geographic region.

Our data indicate that there might exist new or rearranged types of SCCmec that have not yet been characterized in MRSA or CoNS isolates. We identified all three known ccrAB gene types among the CoNS and MRSA isolates tested. The ccrAB gene pair types were combined with the class of mec gene complexes reported earlier (25) in 18 out of 20 MRSA strains, while all three types of ccrAB genes were found in combination with both complete and rearranged mec regulatory genes in CoNS.

All three types of ccrAB genes also exist in the absence of mec genes in CoNS. Three isolates, which were mec negative and methicillin sensitive, contained one of the three ccrAB gene pairs tested. This is not surprising since SCCmec is described as not being dependent on mecA, and it can contain different sets of antibiotic resistance determinants (21). This was also observed by Luong et al. (29), who characterized an SCCcap1 element in a mec-negative S. aureus strain, and by Katayama et al. (26), who described a type I SCC in a methicillin-susceptible S. hominis strain. Thus, different SCCs most likely have a function(s), independently of resistance genes, as mobile genetic elements in staphylococcal chromosomes. This is in accordance with the conclusion of Ito and coworkers (21), who suggested that SCC is not confined to antibiotic resistance alone but may serve as a general genetic information exchange system in staphylococci.

ccrAB gene pairs occur not only in the absence of mecA in CoNS but also in multiple copies. A BLAST search of the GenBank database for the whole genome of mecA-negative and methicillin-susceptible strain S. epidermidis ATCC 12228 (GenBank accession no. AE015929) revealed that it contains two ccrAB gene pair homologues and one truncated ccrA copy of only 120 bp. The first gene pair shows homology to SCCmec type IV ccrAB2 (subtype a) (93 to 99%), while the second ccrAB gene pair is very different from all known ccrAB types. Thus, chromosomal cassette recombinase genes might be rather common in staphylococci, contributing to genetic exchange by their recombinases with invertase-resolvase activity (24).

The mec locus was observed in the absence of the three known types of ccrAB genes, both in MRSA and in CoNS. One MRSA isolate and 22 out of 39 CoNS isolates lacked ccrAB gene homologues but carried mec genes and were methicillin resistant. Enright et al. (13) reported that not all MRSA isolates can be typed by using the published ccr and mec PCR methods, indicating the existence of novel SCCmec types. Another possibility is that the ccrAB gene pair has been deleted, as Ito et al. (21) suggest for methicillin-susceptible S. aureus strain ATCC 25923 carrying a DNA fragment inserted in orfX, with structural similarities to the end sequences (i.e., attL and attR) in SCCmec. Alternatively, mecA could have been chromosomally inserted independently of any ccrAB gene homologue. The ccr-untypeable strains are under further investigation to identify possible novel ccrAB gene pairs.

We observed nucleotide sequence conservation among ccrAB alleles obtained from one geographic region, i.e., Norway, while distantly related ccrAB alleles show differences of up to 4%, e.g., in ccrAB2. This is clearly visualized in the cluster analyses performed on the ccrAB sequences shown in Fig. 2a to d. Even more remarkable is the strong sequence conservation observed independently of species; e.g., Norwegian isolates of S. warneri (strain 8-80) and S. epidermidis (strain 8-55) contain ccrAB2 homologues that are identical to the ccrAB2 alleles in sporadic MRSA isolates from the same region. Interestingly, these isolates are obtained from different time periods, i.e., 1994 through 2001. The observed sequence conservation suggests that there is a closer genetic relationship between ccrAB2 in Norwegian staphylococci, independently of species, than between ccrAB2 in international MRSA and Norwegian MRSA isolates.

It is noteworthy that the phylogenetic analysis performed on the housekeeping gene pta clearly separated all S. aureus isolates from S. epidermidis and S. warneri, as illustrated in Fig. 2e. The cluster analyses of 16S rDNA and pta sequences separated staphylococci according to species, while the corresponding analyses of ccrAB genes clustered staphylococcal isolates according to the geographic regions where they were isolated.

In general, very little is known about S. epidermidis SCCmec since most data are obtained from studies of MRSA. CoNS strains have been suggested to serve as a reservoir for antibiotic resistance genes that can be transferred to strains of S. aureus, as well as other gram-positive organisms (23, 27, 32, 33). Neither the mechanism(s) responsible nor the organisms involved in mecA transfer are known, but it has been suggested that the flow of mec is from CoNS to S. aureus (6). Wielders and coworkers (44) reported a possible horizontal in vivo transfer of mecA into S. aureus in the presence of MR-CoNS during antibiotic treatment, and a sporadic MRSA emerged de novo. The SCCmec type IV frequently found in community-acquired S. epidermidis strains among healthy individuals has been suggested to be responsible for the conversion of commensal S. aureus to MRSA (18).

Our results, showing the conservation in the ccrAB alleles obtained from different staphylococcal species from the same geographic region, support the idea of horizontal gene transfer even though the direction of transfer cannot be identified. The appearance of MRSA with similar ccrAB gene pair variants could be explained by clonal spread of one single MRSA with a specific ccrAB allele, but that would give rise to MRSA isolates with indistinguishable or related genetic prints generated by PFGE. Only two genetically related pairs of MRSA were identified in our study, and only one of these pairs (i.e., strains 9-05 and 39-37) could be considered indistinguishable according to PFGE. Furthermore, clonal spread cannot explain the appearance of one specific ccrAB allele in different species. On the other hand, most of the isolates that have identical ccrAB alleles show differences in the mec complex, hence disfavoring the suggestion of horizontal transfer of SCCmec. In contrast to the recombinase genes, mecA is very well conserved, with only 1 to 2 bp differences out of 2,007 bp among S. aureus, S. epidermidis, and S. sciuri (14, 21). This might imply that there is a difference in selective pressure on the ccrAB and mecA gene products or that these genes have different evolutionary histories. A high mutational rate in the ccrAB genes could explain the large sequence variations observed (up to 4% for ccrB2) within one species, but the occurrence of independent mutational events resulting in identical ccrAB genes in different species is a highly unlikely explanation. This illustrates the complexity of the origin and transfer of SCCmec, and questions about horizontal gene transfer remain unanswered.

Three isolates interrupt the clustering of ccrAB1 alleles in the phylogenetic analyses, and they are all mecA negative. S. epidermidis 6-42 and S. hominis ATCC 27844 containing ccrA1 cluster together, apart from S. hominis 8-39, while strain 8-39 ccrB1 clusters together with ATCC 27844, apart from strain 6-42. One isolate interrupts the ccrAB2 clusters, i.e., MRSA isolate 9-61 from Finland. The ccrA2 sequence shows 97% identity to that of N315, while the ccrB2 sequence forms a separate clade closely related to the "Norwegian sequences." ccrAB alleles from all other isolates are clustered in the same groups when both the ccrA and ccrB genes are studied. The difference in clustering observed cannot be explained from our analyses but could be due to recombination between homologous gene pairs, and this adds to the complexity of the issue.

In conclusion, we have found that genetically unique and sporadic MRSA isolates contain local variants of ccrAB gene pairs that are identical to those found in MR-CoNS but different from those in MRSA isolates from other regions. New combinations of mec complexes and ccrAB types suggest that there are new or rearranged types of SCCmec in both MRSA and MR-CoNS. The existence of ccrAB without mec in CoNS is not surprising, but the existence of a mec complex in the absence of the known ccrAB types in both MRSA and MR-CoNS isolates indicates that the recombinase genes in our study are very distantly related to the known ccrAB types or that they are just deleted from the chromosome. It may also indicate that mecA can be transferred between staphylococcal cells independently of ccrAB. The local variants of ccrAB genes found in both MRSA and MR-CoNS might indicate that these genes have been transmitted horizontally among staphylococci and that the isolates probably are feeding from the same gene pool. However, we cannot conclude anything about the introduction of mecA since the combination of ccrAB alleles and mec complex vary between species. Either MRSA and CoNS isolates with identical ccrAB genes may contain derivatives of the same SCCmec with rearrangements in the mec complex, or types of SCC are spread among staphylococci before the mec complex, in any configuration, is introduced. Since there is no doubt that S. aureus and CoNS share the same gene pool containing resistance genes and recombinases, there is a need to continue the search for the SCCmec origin, as well as the mechanism(s) and route(s) of transfer among staphylococci.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the University of Tromsø, Tromsø, Norway, and the Norwegian National Health Association (Nasjonalforeningen for Folkehelsen).

We thank K. Hiramatsu of Juntendo University, Tokyo, Japan; E. Myhre of Lund University Hospital, Lund, Sweden; J. Vuopio of the National Public Health Institute, Helsinki, Finland; Y. Tveten of Telelab, Skien, Norway; P. Gaustad of Rikshospitalet, University Hospital, Oslo, Norway; and the Department of Microbiology, University Hospital of North Norway, for kindly providing strains. We thank Liselotte Buarø, Mette S. Wesmajervi, and Bianca Nygård for excellent technical assistance. Thanks to Kaare M. Nielsen for making the phylogenetic trees. We also thank Kristin H. Dahl, Arnfinn Sundsfjord, Claus Klingenberg, Trond Flægstad, and Ørjan Olsvik for critical reading of the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address for Anne-Merethe Hanssen: Department of Microbiology and Virology, Faculty of Medicine, Institute for Medical Biology, University of Tromsø, N-9037 Tromsø, Norway. Phone: 47 77 64 57 52. Fax: 47 77 64 53 50. E-mail: annemh{at}fagmed.uit.no. Present address for Johanna U. E. Sollid: Institute of Medicine, University of Bergen, Haukeland Hospital, N-5021 Bergen, Norway. Phone: 47 55 97 30 75. Fax: 47 55 97 29 50. E-mail: johanna{at}fagmed.uit.no. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Antimicrobial Agents and Chemotherapy, January 2004, p. 285-296, Vol. 48, No. 1
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.1.285-296.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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