Staphylococcus epidermidis is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical S. epidermidis strains is poorly understood due to the strong genetic barrier that precludes the efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical S. epidermidis isolates is primarily due to the type I and IV restriction-modification systems, which act as genetic barriers. Here, we show that efficient plasmid transformation of clinical S. epidermidis isolates from clonal complexes 2, 10, and 89 can be realized by employing a plasmid artificial modification (PAM) in Escherichia coli DC10B containing a Δdcm mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of S. epidermidis and hence improve our understanding of their pathogenesis in human infections.IMPORTANCE Staphylococcus epidermidis is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of S. epidermidis are poorly understood. Genetic manipulations of clinically relevant strains of S. epidermidis are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation of clinically relevant S. epidermidis isolates has now become possible, thus improving our understanding of S. epidermidis as a pathogen.KEYWORDS Staphylococcus epidermidis, transformation, restriction system, DC10B, clonal complexes, HsdS, efficiency, methylation, restriction barrier S taphylococcus epidermidis is a ubiquitous human commensal that normally colonizes the human skin and mucous membranes (1). S. epidermidis can also assume an invasive lifestyle by colonizing indwelling medical devices, which in some cases can lead to invasive bacteremia. Indeed, due to the common use of implanted medical devices in modern medicine, S. epidermidis is now the number one cause of nosocomial bacteremia in the United States (2). Although these device-related infections are less severe than those due to their Staphylococcus aureus counterparts, these diseases tend to be more persistent, with most being described as subacute or chronic in nature. In many cases, infection cannot be eliminated unless the offending medical devices, such as catheters, prostheses, or pacemakers, are removed, thus leading to high morbidity rates (3). Treatment of S. epidermidis infections is also complicated by the high level of innate antibiotic resistance as well as robust biofilm formation, which can render host defenses and antibiotics ineffective (1).