Novel Type of Staphylococcal Cassette Chromosome mec in a Methicillin-Resistant Staphylococcus aureus Strain Isolated in Sweden

We identified a novel type of staphylococcal cassette chromosomemec (SCCmec) element carried by methicillin-resistant Staphylococcusaureus (MRSA) strain JCSC6082 isolated in Sweden. The SCCmecelement was demarcated by characteristic nucleotide sequencesat both ends and was integrated at the 3' end of orfX. The elementcarried a novel combination of a type 5 ccr gene complex andclass C1 mec gene complex. The J regions of the element werehomologous to those of the SCCmercury element of S. aureus strain85/2082, with nucleotide identity greater than 99%. However,the novel SCCmec element from JCSC6082 did not carry the meroperon nor Tn554, suggesting that evolution to SCCmec couldhave been from a common ancestor by acquisition of the classC1 mec gene complex. The novel SCCmec element from JCSC6082was flanked by a novel SCC-like chromosome cassette (CC6082),which was demarcated by two direct repeats and could be excisedfrom the chromosome independently of the SCCmec element. Ourdata suggest that novel SCCmec elements can be generated onthe staphylococcal chromosome through the recombination betweenextant SCC elements and mec gene complexes.

INTRODUCTION

Top

INTRODUCTION

Since the staphylococcal cassette chromosome mec (SCCmec) wasfirst described as the carrier of the mecA gene (9, 13), atleast six structural variants have been identified, namely typesI, II, III, IV, V, and VI SCCmec. They are typically composedof two essential components, the mec gene complex (mec) andthe cassette chromosome recombinase (ccr) gene complex (8, 10,16, 18). In addition, they are integrated at a specific integrationsite sequence (ISS) of orfX on the staphylococcal chromosome(11).

Three major classes of mec gene complex have been identifiedin methicillin-resistant Staphylococcus aureus (MRSA) strains.The prototypic mec (class A) is composed of mecA, its regulatorygenes, mecR1 and mecI, and insertion sequence IS431 downstreamof mecA. Certain variants of the mec gene complex contain insertionof either IS1272 (class B) or IS431 (class C1 and C2) at the3' portion of mecR1. The ccr genes are responsible for the chromosomalintegration and excision of the SCCmec, and there are at leastfive types of the ccr gene complex. The first four types containtwo site-specific recombinase genes, ccrA and ccrB, with surroundingopen reading frames (ORFs), while the fifth type contains onesite-specific recombinase gene, ccrC, with surrounding ORFs.The classification of SCCmec is based upon the combination ofthe mec class and the ccr type (11).

Recently, SCCmec elements with more complex structures havebeen reported, which contain multiple ccr gene complexes (7,19). In addition, mobile genetic elements that did not themselvescarry ccr gene complexes have been found integrated downstreamof orfX, such as the 31-kb arginine catabolic mobile element(ACME). Mobilization of these elements has been suggested tooccur by the activity of the recombinases encoded by the ccrABgenes located on the adjacent SCCmec (4, 5).

In Sweden, the prevalence of MRSA is still below 1%, and MRSAisolates are genetically diverse (1). At present, the community-acquiredMRSA strains are more prevalent than the nosocomial MRSA strains(Swedish Institute for Infectious Disease Control; www.smittskyddsinstitutet.se)(1). In addition, several MRSA isolates that were not carryingextant types of SCCmec elements have been identified in variousregions of Sweden (C. Berglund, unpublished data).

In this work, we describe a novel type of SCCmec element froman MRSA strain isolated in Sweden.

MATERIALS AND METHODS

Top

MATERIALS AND METHODS

MRSA strain JCSC6082 (p5747/2002) (2) was isolated in 2002 froma subcutaneous abdominal wall abscess of a previously healthy42-year-old woman. The infection was designated as communityacquired; however, the patient had undergone abdominal surgerydue to salpingitis 24 years earlier, and in the present abscesscavity the remnants of a suture were found.

The MRSA strain belonged to the epidemic and widespread sequencetype 5 (ST5) and clonal complex 5 (CC), and carried neitherthe genes for Panton-Valentine leukocidin (PVL) nor exfoliativetoxin A or B. The results of susceptibility testing using theagar dilution method according to Clinical and Laboratory StandardsInstitute guidelines showed that it was resistant to oxacillin,tetracycline, and erythromycin, with MICs of 48, 32, and 8 mg/liter,respectively (1).

Construction of fosmid libraries. Genomic DNA of JCSC6082 was prepared using ISOPLANT (NipponGene Co., Tokyo, Japan) and was subsequently used as insertDNA in the fosmid library. The fosmid libraries were createdusing the CopyControl fosmid library production kit (EpicentreBiotechnologies, Madison, WI) according to the manufacturer'srecommendation. The clones were screened by PCR with primerpairs to identify orfX, mecA, and the chromosomal region locateddownstream of SCCmec.

DNA manipulations. PCRs for screening of fosmid clones and identification of specificgenes, long-range PCR for amplification of DNA fragments coveringthe entire SCCmec, and nested PCR experiments for identificationof precise excision of the novel SCCmec, SCCmec and CC6082 together,and CC6082 alone were carried out as described previously (9).The sequences and locations of the PCR primer pairs used toamplify DNA fragments covering the entire SCCmec region areshown in Fig. 1 and Table 1.

View larger version (22K):
[in this window]
[in a new window]

FIG. 1. Structural comparisons of the novel SCCmec element and a chromosome cassette identified from JCSC6082 isolated in Sweden, SCCmercury, and type III SCCmec carried by 85/2082 and the type V SCCmec carried by WIS. The structures of these elements are illustrated based on the nucleotide sequence deposited in the DDBJ/EMBL/GenBank database under accession no. AB373032, AB037671, and AB121219. Conserved regions are indicated in blue: more than 99% homology is in dark blue and 19.1% to 93.6% homology is in light blue. Red arrowheads indicate integration site sequence of SCCmec (ISS) that comprises DR sequences, and black arrowheads indicate the positions and directions of primers used for detection of the novel type of SCCmec.

View this table:
[in this window]
[in a new window]

TABLE 1. Oligonucleotide primers employed for PCR detection of the novel type of SCCmec

Nucleotide sequencing was performed as described previously(15), and the sequences were analyzed and assembled using theBioEdit sequence alignment editor.

Analysis of ORFs. ORFs longer than 100 bp were identified with the GenomeGamblerv.1.5 software Japan (Marine Science and Technology Center andXanagen, Inc., Kawasaki, Japan) and were compared with sequencedatabases at the National Center for Biotechnology Informationwith the basic local alignment search tool BLAST (National Libraryof Medicine, Bethesda, MD) for annotation and prediction offunctions. The ccr genes were investigated using the ClustalW(1.83) multiple alignment program available at http://www.staphylococcus.net.

Nucleotide sequence accession number. The nucleotide sequence reported in this paper has been depositedin the DDBJ/EMBL/GenBank database under accession no. AB373032.

RESULTS AND DISCUSSION

Top

RESULTS AND DISCUSSION

Identification of a new member of the SCCmec family. We identified an MRSA strain, JCSC6082, which carried an unknowntype of SCCmec element according to PCR detection of the extanttypes of SCCmec (10, 14, 17). The MRSA strain JCSC6082 was consideredto be community acquired, although the patient had a historyof hospitalization 24 years previously. It may be that the patienthad acquired the MRSA strain recently and that it was a truecommunity-acquired infection. However, the specific locationof the infection in addition to a difficult-to-treat infection24 years earlier makes it tempting to consider the possibilityof a dormant infection or at least persistence of MRSA, adherentto foreign material (e.g., a suture) and embedded in biofilm,for more than 20 years until it was reidentified by the presentationof the abscess in 2002.

The structure of the SCCmec element from JCSC6082 was furtherinvestigated by construction of fosmid libraries and determinationof the entire nucleotide sequence. The overall structure ofthe 33,261-bp region encompassing the SCCmec element from JCSC6082is illustrated in Fig. 1, and the ORFs carried by this regionare listed in Table 2.

View this table:
[in this window]
[in a new window]

TABLE 2. ORFs of the novel SCCmec and chromosome cassette CC6082 in JCSC6082

The sequenced region included three ISSs comprising directlyrepeated (DR) sequences typical of SCC-like cassettes, suggestingthat JCSC6082 carried two SCC-like elements, including an SCCmecelement and an SCC-like chromosome cassette lacking ccr recombinasegenes (Fig. 1).

We then conducted nested PCR experiments in order to investigatewhether these elements could be excised from the chromosomeindependently. DNA fragments, indicating that these elementshave been excised from the chromosome, were successfully amplifiedby PCR, and nucleotide sequencing confirmed that excision hadoccurred precisely at the DR sites (data not shown).

The SCCmec element was 26,733 bp in size, i.e., as small astype IV and V SCCmec elements that are prevalent among community-acquiredMRSA (3). It carried a class C1 mec complex, which was differentfrom class C2 mec since the orientation of the IS431 inserteddownstream of mecA and disrupting the mecR1 gene was oppositeto that found in class C2 mec. The class C1 mec complex wasfirst identified in methicillin-resistant Staphylococcus haemolyticusstrain SH631 (12). To our knowledge, this is the first descriptionof a class C1 mec complex in an MRSA strain. Interestingly,in the SCCmec element from JCSC6082 the deletion of ${Delta}$ mecR1 wasnot the same as that found in the previously described C1 meccomplex (12).

The SCCmec element from JCSC6082 carried a type 5 ccr gene complex,in common with the type V SCCmec and SCCmercury elements (8,10). In this case, however, ccrC was 1,677 bp in size: i.e.,larger by 133 bp than the ccrC gene carried by SCCmercury in85/2082 (accession no. AB037671). Since the region of SCCmercurycorresponding to the entire 1,677 bp of the ccrC gene had anucleotide identity of 99.9%, it was considered that a prematurestop codon had been introduced in the ccrC of SCCmercury. TheccrC genes appear to be more diversified in comparison withccrA and ccrB, since several variants of this recombinase havebeen described (6, 19).

Consequently, we regarded the region between the first ISS andthe second ISS in JCSC6082 as a novel type of SCCmec element,since it carried a type 5 ccr gene complex (ccrC) and a classC1 mec gene complex. No additional resistance gene other thanthe mecA was identified in the element.

Interestingly, the nucleotide sequence of this novel SCCmecelement had 99% homology with the SCCmercury element of 85/2082(11) but in contrast carried the mec complex instead of themer operon and Tn554. SCCmercury has been reported to be similarto SCCcap1 carrying a type 1 capsule operon in the same manner(11). These three SCC elements might have been generated ina similar way, with incorporation of mercury resistance operonand Tn554 to generate SCCmercury, a capsule gene cluster tomake SCCcap-1, or the mec gene complex to create SCCmec. Wealso suspect that the class C1 mec complex present in this novelSCCmec may be a composite transposon itself, since it is demarcatedby two copies of IS431, which further strengthens the hypothesisthat an SCC element might evolve into an SCCmec element by acquiringa mec gene complex. However, so far, there is no evidence oftransposition ability of the mec gene complex.

Characteristics of chromosome cassette CC6082. The SCC-like chromosome cassette CC6082 was 5,617 bp in lengthand was located between the second and third ISSs, downstreamof the SCCmec element. CC6082 carried an hsdM gene encodinga type I restriction modification system DNA methylase, a geneencoding a membrane protein homologue, a gene encoding a hypotheticalprotein similar to a reverse transcriptase, and two additionalORFs of unknown function. Neither ccr genes nor a mec complexwas identified in this region. This region may be a remnantof a previously active SCC element.

Naming of novel SCC elements. The SCCmec element should be defined by a specific combinationof the class of mec and ccr gene complex, and it should be indicatedin Roman numerals. We considered that atypical elements e.g.,carrying two ccr gene complexes or non-mecA-carrying SCC, atfirst should be analyzed by determining the entire nucleotidesequences. In the case of a composite of SCC and SCCmec, theelements should be described separately and primarily be categorizedinto the type of SCCmec as determined by the ccr and mec carriedon the element demarcated by ISS and subsequently given theRoman numeral. Here, we designated the novel SCCmec elementin JCSC6082 carrying a type 5 ccr gene complex (ccrC) and aclass C1 mec gene complex as type 5C1. However, we suggest thatthis novel SCCmec should receive a Roman numeral as type VII.To avoid confusion on the naming of SCCmec elements, we awaita decision from the International Working Group on the Classificationof Staphylococcal Cassette Chromosome Elements.

ACKNOWLEDGMENTS

We thank Tadashi Baba at the Department of Bacteriology andInfection Control Science, Juntendo University, for professionaladvice and assistance during the orf analysis. We also thankLi Shan Shuang at the Department of Infection Control Science,Juntendo University, Postgraduate School, for performing thenested excision PCR.

This study was supported by grants from the Sweden-Japan Foundation,the Swedish Society for Medical Research, the Swedish Instituteof Biomedical Laboratory Science, and the Örebro CountyCouncil Research Committee, Sweden, and a Grant-in-Aid for 21stCentury COE Research from the Ministry of Education and Science,Japan.

FOOTNOTES

* Corresponding author. Mailing address: Department of Clinical Microbiology, Örebro University Hospital, SE-701 85 Örebro, Sweden. Phone: 46 19 602 15 20. Fax: 46 19 12 74 16. E-mail: carolina.berglund{at}orebroll.se

Published ahead of print on 1 August 2008.

REFERENCES

Top