Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15–0.6 μM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 μM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 μg/cm²). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections.

金黄色葡萄球菌引起人类和动物的各种感染，皮肤是这种病原体的主要储存库。耐甲氧西林普遍存在金黄色葡萄球菌(MRSA) 限制了这种病原体的消除和治疗。噬菌体裂解蛋白已被证明是有效的抗微生物剂金黄色葡萄球菌. 在这里，一组 12 种基于内溶素的工程蛋白被概念化，以通过逐步漏斗方法评估参数，包括浊度降低、最小抑制浓度 (MIC)、时间-杀伤曲线和抗生物膜测定，以及测试它们的在广泛的储存条件（pH、温度和离子强度）中保持稳定性。工程噬菌体溶素 LysRODIΔAmi 和 ClyRODI-H5 显示出最高的比裂解活性（比其他产品高 5 至 50 倍），保质期长达 6 个月，并在高达 50°C 的温度和 pH 范围内保持稳定从 3 到 9。LysRODIΔAmi 对所有测试的葡萄球菌菌株显示出较低的 MIC 值。两种蛋白质都能够杀死 6 个对数单位的菌株金黄色葡萄球菌Sa9 在 5 分钟内可以去除预先形成的生物膜（分别为 76% 和 65%）。此外，LysRODIΔAmi 可以在低蛋白质浓度（0.15-0.6 μM）下防止生物膜形成。由于其增强的抗生物膜特性，LysRODIΔAmi 被选择用于有效去除金黄色葡萄球菌完整和破碎的角质形成细胞单层中的污染。值得注意的是，即使在高浓度 (22.1 μM) 下，这种蛋白质也没有表现出对人类角质形成细胞的任何毒性。最后是猪皮离体模型用于评估使用 LysRODIΔAmi (16.5 μg/cm ² )对人为污染的猪皮肤的处理。在早期减少之后金黄色葡萄球菌，第二剂蛋白质完全根除 金黄色葡萄球菌. 总体而言，我们的结果表明 LysRODIΔAmi 是预防和治疗葡萄球菌皮肤感染的合适抗微生物剂。