The ␤-lactam resistance gene mecA of Staphylococcus 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 of mecAcarrying 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.

We have previously shown that the methicillin-resistance gene mecA 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 [ccrA and 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. aureus chromosome and site-specific as well as orientation-specific integration of the SCCmec into the S. aureus chromosome 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.Methicillin-resistant Staphylococcus aureus (MRSA) was first isolated in England in 1961 shortly after the development of methicillin, the first penicillinase-resistant semisynthetic penicillin (15). Since then, MRSA has become the most prevalent pathogen causing hospital infection throughout the world, and MRSA incidence is still increasing in many countries (1). MRSA is resistant to practically all ␤-lactam antibiotics, a class of antibiotics represented by penicillins and cephalosporins (3).The ␤-lactam resistance of MRSA is caused by the production of a novel penicillin-binding protein (PBP) designated PBP 2Ј (or PBP 2a), which, unlike the intrinsic set of PBPs (PBP 1 to 4) of S. aureus, has remarkably reduced binding affinities to ␤-lactam antibiotics (9,24,30). Despite the presence of otherwise inhibitory concentrations of ␤-lactam antibiotics, MRSA can continue cell wall synthesis solely depending upon the uninhibited activity of PBP 2Ј (21). PBP 2Ј is encoded by a mecA gene located on the chromosome of MRSA. In 1987, the mecA gene was cloned from a Japanese MRSA strain, and its sequence was determined (20,26). The mecA gene is widely distributed among S. aureus as well as coagulase-negative staphylococci (13, 28). Therefore, it has been speculated that the methicillin resistance determinant (mec determinant) is freely transmissible among staphylococcal species. However, with a detailed molecular epidemiological study, Kreiswirth et al. have proposed that MRSA originated from a single or two ancestral clones (16). This led to the view that the frequency of inter-or intraspecies transmission of mecA is a rather limited process and that mec transmission may not be due to specialized transmission machinery, such as a transposon.We have recently cloned and sequenced the entire chromosomal region surrounding the mecA gene, which is additionally present in the MRSA chromosome and is absent from the chromosome of methicillin-susceptible S. aureus (MSSA) (referred to herein as mecDNA), from a Japanese pre-MRSA strain, N315 (10). We identified the mecDNA-S. aureus chromosome junction points and the overall structure of me...

Summary Staphylococcus aureus commonly colonizes the epidermis, but the mechanisms by which the host senses virulent but not commensal S. aureus to trigger inflammation remain unclear. Using a murine epicutaneous infection model, we found that S. aureus expressed phenol-soluble modulin (PSM)α, a group of secreted virulence peptides, is required to trigger cutaneous inflammation. PSMα induces the release of keratinocyte IL-1α and IL-36α, and signaling via IL-1R and IL-36R was required for induction of the pro-inflammatory cytokine IL-17. The levels of released IL-1α and IL-36α, as well as IL-17 production by γδ T cells and ILC3 and neutrophil infiltration to the site of infection were greatly reduced in mice with total or keratinocyte-specific deletion of the IL-1R and IL-36R signaling adaptor Myd88. Further, Il17a−/−f−/− mice showed blunted S. aureus-induced inflammation. Thus, keratinocyte Myd88 signaling in response to S. aureus PSMα drives an IL-17-mediated skin inflammatory response to epicutaneous S. aureus infection.

In methicillin-resistant Staphylococcus aureus, the methicillin resistance gene mecA is localized within a large chromosomal region which is absent in the methicillin-susceptibleS. aureus chromosome. The region, designatedmec DNA, is speculated to have originated from the genome of another bacterial species and become integrated into the chromosome of the S. aureus cell in the past. We report here cloning and determination of the structure of the entire mec DNA sequence from a Japanese S. aureus strain, N315. Themec DNA was found to be 51,669 bp long, including terminal inverted repeats of 27 bp and a characteristic pair of direct repeat sequences of 15 bp each: one is situated in the right extremity ofmec DNA, and the other is situated outside themec DNA and abuts the left boundary of mec DNA. The integration site of mec DNA was found to be located in an open reading frame (ORF) of unknown function, designatedorfX. Clusters of antibiotic resistance genes were noted inmec DNA carried by transposon Tn554 and an integrated copy of plasmid pUB110. Both the transposon and plasmid were integrated in the proximity of the mecA gene, the latter being flanked by a pair of insertion sequence IS431elements. Many ORFs other than those encoding antibiotic resistance were considered nonfunctional because of the acquired mutations or partial deletions found in the ORFs. Two ORFs potentially encoding novel site-specific recombinases were found in mec DNA. However, there was no ORF that might encode mecDNA-specific transposase or integrase proteins, indicating that themec DNA is not a transposon or a bacteriophage in nature.

Staphylococcus (S.) aureus silently stays as our natural flora, and yet sometimes threatens our life as a tenacious pathogen. In addition to its ability to outwit our immune system, its multi-drug resistance phenotype makes it one of the most intractable pathogenic bacteria in the history of antibiotic chemotherapy. It conquered practically all the antibiotics that have been developed since 1940s. In 1961, the first MRSA was found among S. aureus clinical isolates. Then MRSA prevailed throughout the world as a multi-resistant hospital pathogen. In 1997, MRSA strain Mu50 with reduced susceptibility to vancomycin was isolated. Vancomycin-intermediate S. aureus (VISA), so named according to the CLSI criteria, was the product of adaptive mutation of S. aureus against vancomycin that had long been the last resort to MRSA infection. Here, we describe the genetic basis for the remarkable ability of S. aureus to acquire multi-antibiotic resistance, and propose a novel paradigm for future chemotherapy against the multi-resistant pathogens.