Worldwide occurrence of methicillin-resistant Staphylococcus aureus (MRSA) poses enormous challenges for both communities and health care settings. Cassette chromosome recombinases (Ccr) specifically perform excision and acquisition of a staphylococcal cassette chromosome mec (SCCmec) in staphylococci and are responsible for the spread of methicillin resistance. This study explored the roles of CcrC2, a recently discovered Ccr, in the horizontal transfer of SCCmec and developed a potential means to control the spread of methicillin resistance. Knockout of CcrC2 completely aborted the excision of SCCmec, while overexpression of CcrC2 partially removed the SCCmec from the genome and transformed methicillin-resistant Staphylococcus aureus (MRSA) into methicillin-susceptible Staphylococcus aureus (MSSA). Moreover, two nucleotide residues (G₅C₆) in the direct repeat sequence within an att site were found to be critical for excision and acquisition efficiencies. To block the horizontal transfer of methicillin resistance, a SCCmec killer system was developed by combining the CcrC2-mediated SCCmec excision and the mecA-targeting CRISPR–Cas9 machinery. The SCCmec killer transformed MRSA to MSSA and disrupted the mecA-carrying SCCmec intermediate, thereby eliminating methicillin resistance determinant mecA gene inside a MRSA cell and blocking the horizontal transfer of SCCmec. The SCCmec killer was versatile for efficiently removing multiple types of SCCmec elements. It is envisioned that this approach could offer a new means to control the spread of methicillin resistance.

中文翻译：

---

盒式染色体重组酶C2（CcrC2）的功能分析及其在结合CRISPR–Cas9消除甲氧西林抗性中的应用

耐甲氧西林的金黄色葡萄球菌（MRSA）在世界范围内的出现对社区和医疗机构都构成了巨大的挑战。盒式染色体重组酶（Ccr）专门进行葡萄球菌中葡萄球菌盒式染色体mec（SCC mec）的切除和获取，并负责甲氧西林耐药性的传播。这项研究探索了最近发现的Ccr CcrC2在SCC mec的水平转移中的作用，并开发了控制甲氧西林耐药性扩散的潜在手段。CcrC2的敲除完全中止了SCC mec的切除，而CcrC2的过表达则部分去除了SCC mec。从基因组中分离出来，并将耐甲氧西林的金黄色葡萄球菌（MRSA）转化为对甲氧西林敏感的金黄色葡萄球菌（MSSA）。此外，发现一个att位点内直接重复序列中的两个核苷酸残基（G ₅ C ₆）对于切除和采集效率至关重要。为了阻止耐甲氧西林的水平转移，通过结合CcrC2介导的SCC mec切除和靶向mecA的CRISPR–Cas9机制，开发了SCC mec杀手系统。SCC mec杀手将MRSA转变为MSSA，并破坏了携带mecA的SCC mec中间体，从而消除了MRSA细胞内甲氧西林抗性决定簇mecA基因，并阻止了SCC mec的水平转移。SCC mec杀手具有多种功能，可有效删除多种类型的SCC mec元素。可以预见，该方法可以提供控制甲氧西林耐药性扩散的新手段。