Teruyo Ito

Structural Comparison of Three Types of Staphylococcal Cassette Chromosome mec Integrated in the Chromosome in Methicillin-Resistant Staphylococcus aureus

ABSTRACT The β-lactam resistance gene mecA ofStaphylococcus aureus is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome mec(SCCmec), identified in the chromosome of a Japanese methicillin-resistant S. aureus (MRSA) strain. We now report identification of two additional types ofmecA-carrying genetic elements found in the MRSA strains isolated in other countries of the world. There were substantial differences in the size and nucleotide sequences between the elements and the SCCmec. However, new elements shared the chromosomal integration site with the SCCmec. Structural analysis of the new elements revealed that they possessed all of the salient features of the SCCmec: conserved terminal inverted repeats and direct repeats at the integration junction points, conserved genetic organization around the mecA gene, and the presence of cassette chromosome recombinase (ccr) genes responsible for the movements of SCCmec. The elements, therefore, were considered to comprise the SCCmec family of staphylococcal mobile genetic elements together with the previously identified SCCmec. Among 38 epidemic MRSA strains isolated in 20 countries, 34 were shown to possess one of the three typical SCCmec elements on the chromosome. Our findings indicated that there are at least three distinct MRSA clones in the world with different types of SCCmec in their chromosome.

A New Class of Genetic Element, Staphylococcus Cassette Chromosome mec, Encodes Methicillin Resistance in Staphylococcus aureus

ABSTRACT We have previously shown that the methicillin-resistance genemecA of Staphylococcus aureus strain N315 is localized within a large (52-kb) DNA cassette (designated the staphylococcal cassette chromosome mec[SCCmec]) inserted in the chromosome. By sequence determination of the entire DNA, we identified two novel genes (designated cassette chromosome recombinase genes [ccrAand ccrB]) encoding polypeptides having a partial homology to recombinases of the invertase/resolvase family. The open reading frames were found to catalyze precise excision of the SCCmec from the methicillin-resistant S. aureuschromosome and site-specific as well as orientation-specific integration of the SCCmec into the S. aureuschromosome when introduced into the cells as a recombinant multicopy plasmid. We propose that SCCmec driven by a novel set of recombinases represents a new family of staphylococcal genomic elements.

Combination of Multiplex PCRs for Staphylococcal Cassette Chromosome mec Type Assignment: Rapid Identification System for mec, ccr, and Major Differences in Junkyard Regions

ABSTRACT Staphylococcal cassette chromosome mec (SCCmec) typing, in combination with genotyping of the Staphylococcus aureus chromosome, has become essential for defining methicillin-resistant S. aureus (MRSA) clones in epidemiological studies. We have developed a convenient system for SCCmec type assignment. The system consists of six multiplex PCRs (M-PCRs) for identifying the ccr gene complex (ccr), the mec gene complex (mec), and specific structures in the junkyard (J) regions: M-PCR with primer set 1 (M-PCR 1) identified five types of ccr genes; M-PCR 2 identified class A to class C mec; M-PCRs 3 and 4 identified specific open reading frames in the J1 regions of type I and IV and of type II, III, and V SCCmec elements, respectively; M-PCR 5 identified the transposons Tn554 and ΨTn554 integrated into the J2 regions of type II and III SCCmec elements; and M-PCR 6 identified plasmids pT181 and pUB110 integrated into J3 regions. The system was validated with 99 MRSA strains carrying SCCmec elements of different types. The SCCmec types of 93 out of the 99 MRSA strains could be assigned. The SCCmec type assignments were identical to those made with a PCR system that uses numerous primer pairs to identify genes or gene alleles. Our system of six M-PCRs is thus a convenient and reliable method for typing SCCmec elements.

Novel Type V Staphylococcal Cassette Chromosome mec Driven by a Novel Cassette Chromosome Recombinase, ccrC

ABSTRACT Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element composed of the mec gene complex, which encodes methicillin resistance, and the ccr gene complex, which encodes the recombinases responsible for its mobility. The mec gene complex has been classified into four classes, and the ccr gene complex has been classified into three allotypes. Different combinations of mec gene complex classes and ccr gene complex types have so far defined four types of SCCmec elements. Now we introduce the fifth allotype of SCCmec, which was found on the chromosome of a community-acquired methicillin-resistant Staphylococcus aureus strain (strain WIS [WBG8318]) isolated in Australia. The element shared the same chromosomal integration site with the four extant types of SCCmec and the characteristic nucleotide sequences at the chromosome-SCCmec junction regions. The novel SCCmec carried mecA bracketed by IS431 (IS431-mecA-ΔmecR1-IS431), which is designated the class C2 mec gene complex; and instead of ccrA and ccrB genes, it carried a single copy of a gene homologue that encoded cassette chromosome recombinase. Since the open reading frame (ORF) was found to encode an enzyme which catalyzes the precise excision as well as site- and orientation-specific integration of the element, we designated the ORF cassette chromosome recombinase C (ccrC), and we designated the element type V SCCmec. Type V SCCmec is a small SCCmec element (28 kb) and does not carry any antibiotic resistance genes besides mecA. Unlike the extant SCCmec types, it carries a set of foreign genes encoding a restriction-modification system that might play a role in the stabilization of the element on the chromosome.

Classification of staphylococcal cassette chromosome mec (SCCmec) : guidelines for reporting novel SCCmec elements.

Classification of staphylococcal cassette chromosome mec (SCCmec) : guidelines for reporting novel SCCmec elements.

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

ABSTRACT A description of staphylococcal cassette chromosome mec (SCCmec) elements carried by 615 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 11 Asian countries is reported, and a novel nomenclatural system based on their structures is proposed. The 615 strains were classified as type 3A (370 strains), type 2A (207 strains), type 2B (32 strains), type 1B (1 strain), and nontypeable (5 strains). The previously reported type III SCCmec (DDBJ/EMBL/GenBank accession no. AB037671) carried by the MRSA strain 85/2082 was ascertained to be composed of two SCC elements, type 3A SCCmec and SCCmercury. PCR analysis indicated that 310 of 370 type 3A SCCmec strains carried both SCC elements. These strains were prevalent in eight countries: Thailand, Sri Lanka, Indonesia, Vietnam, Philippines, Saudi Arabia, India, and Singapore. The remaining 60 type 3A SCCmec strains differed with respect to the left extremity polymorphism or to the presence of ccrC. Among these, two were identified as carrying only type 3A SCCmec elements, but their left extremities differed. Type 2A SCCmec strains predominated in Korea and Japan, although the frequency of the presence of ant(4′)-1 gene downstream of mecA varied (53% for Korean strains; 93% for Japanese strains). Various SCCmec elements were identified in the tested strains, and limited numbers were identified by their multilocus sequence typing genotypes. These data suggest that numerous MRSA clones are disseminated in Asian hospitals, and these consist of minor clones that are presumed to have arisen locally and major clones that are presumed to have been introduced from other countries.

Dissemination of Methicillin-Resistant Staphylococci among Healthy Japanese Children

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA), regarded as a tenacious pathogen in the hospital, has recently become increasingly prevalent as a community pathogen. We evaluated the prevalence and characteristics of methicillin-resistant staphylococci in the Japanese community by testing nasal samples of 818 children of five day care centers and two kindergartens in three districts. We found that methicillin-resistant staphylococci are already prevalent among healthy children. Among 818 children, 35 children (4.3%) carried MRSA and 231 children (28.2%) carried methicillin-resistant coagulase-negative staphylococci (MRC-NS). The types of staphylococcal cassette chromosome mec (SCCmec) found among 44 MRSA isolates were as follows: type IIa, 11 isolates; type IIb, 19 isolates; and type IV, 14 isolates. The type IIb SCCmec element was a new SCCmec element found in this study. Eleven (25%) strains which belonged to clonal complex 5 (CC5) carried type IIa SCCmec, and they produced type 2 coagulase and toxic shock syndrome toxin 1. They were indistinguishable from health care-associated MRSA (H-MRSA) strains in Japan, represented by strain N315. On the other hand, 33 (75%) strains, most of which belonged to CC78 or CC91, carried small SCCmec elements, such as type IIb or type IV, and they produced type 1 or type 3 coagulase and exfoliative toxin. The data indicated that MRSA clones distinct from H-MRSA have disseminated in healthy children. The fact that MRC-NS strains were prevalent in the community suggested that they might serve as a reservoir for the SCCmec element carried by MRSA strains disseminated in the community.

Identification in Methicillin-Susceptible Staphylococcus hominis of an Active Primordial Mobile Genetic Element for the Staphylococcal Cassette Chromosome mec of Methicillin-Resistant Staphylococcus aureus

We previously reported that the methicillin resistance gene mecA is carried by a novel type of mobile genetic element, SCCmec (staphylococcal cassette chromosome mec), in the chromosome of methicillin-resistant Staphylococcus aureus (MRSA). These elements are precisely excised from the chromosome and integrated into a specific site on the recipient chromosome by a pair of recombinase proteins encoded by the cassette chromosome recombinase genes ccrA and ccrB. In the present work, we detected homologues of the ccr genes in Staphylococcus hominis type strain GIFU12263 (equivalent to ATCC 27844), which is susceptible to methicillin. Sequence determination revealed that the ccr homologues in S. hominis were type 1 ccr genes (ccrA1 and ccrB1) that were localized on a genetic element structurally very similar to SCCmec except for the absence of the methicillin-resistance gene, mecA. This genetic element had mosaic-like patterns of homology with extant SCCmec elements, and we designated it SCC(12263) and considered it a type I staphylococcal cassette chromosome (SCC). The ccrB1 gene identified in the S. hominis strain is the first type 1 ccrB gene discovered to retain its function through the excision process as judged by two criteria: (i) SCC(12263) was spontaneously excised during cultivation of the strain and (ii) introduction of the S. hominis ccrB1 into an MRSA strain carrying a type I SCCmec whose ccrB1 gene is inactive generated SCCmec excisants at a high frequency. The existence of an SCC without a mec determinant is indicative of a staphylococcal site-specific mobile genetic element that serves as a vehicle of transfer for various genetic markers between staphylococcal species.

Novel Types of Staphylococcal Cassette Chromosome mec Elements Identified in Clonal Complex 398 Methicillin-Resistant Staphylococcus aureus Strains

ABSTRACT The structures of staphylococcal cassette chromosome mec (SCCmec) elements carried by 31 clonal complex 398 (CC398) methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from the participants at a conference were analyzed. The SCCmecs were classified into novel types, namely, IX, X, V(5C2&5) subtype c, and IVa. Type V(5C2&5) subtype c, IX, and X SCCmecs carried genes conferring resistance to metals. The structures of SCCmecs from CC398 strains were distinct from those normally found in humans, adding to the evidence that humans are not the original host for CC398.

Community- acquired Methicillin-resistant Staphylococcus aureus, Uruguay

A novel, methicillin-resistant Staphylococcus aureus clone (Uruguay clone) with a non–multidrug-resistant phenotype caused a large outbreak, including 7 deaths, in Montevideo, Uruguay. The clone was distinct from the highly virulent community clone represented by strain MW2, although both clones carried Panton-Valentine leukocidin gene and cna gene.