ABSTRACTStaphylococcus aureusoften leads to severe skin infections. However,S. aureusis facing a crisis of antibiotic resistance. The combination of phage and antibiotics is effective for drug-resistantS. aureusinfections. Therefore, it is worth exploiting novel antibacterial agents to cooperate with antibiotics againstS. aureusinfections. Herein, a novel chimeric lysin ClyQ was constructed, which was composed of a cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) catalytic domain fromS. aureusphage lysin LysGH15 and cell wall-binding domain (CBD) fromEnterococcus faecalisphage lysin PlyV12. ClyQ had an exceptionally broad host range targeting streptococci, staphylococci,E. faecalis, andE. rhusiopathiae. ClyQ combined with mupirocin (2.64 log reduction) was more effective at treatingS. aureusskin infections than ClyQ (0.46 log reduction) and mupirocin (2.23 log reduction) alone. Of equal importance, none ofS. aureusATCC 29213 or S3 exposed to ClyQ developed resistance, and the combination of ClyQ and mupirocin delayed the development of mupirocin resistance. Collectively, chimeric lysin ClyQ enriches the reservoirs for treatingS. aureusinfections. Our findings may provide a way to alleviate the current antibiotic resistance crisis.IMPORTANCEStaphylococcus aureus, as anEnterococcus faecium,Staphylococcus aureus,Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa, andEnterobacterspecies (ESKAPE) pathogen, can escape the elimination of existing antibiotics. At present, phages and phage lysins againstS. aureusinfections are considered alternative antibacterial agents. However, the development of broad-spectrum chimeric phage lysins to cooperate with antibiotics againstS. aureusinfections remains at its initial stage. In this study, we found that the broad-host-range chimeric lysin ClyQ can synergize with mupirocin to treatS. aureusskin infections. Furthermore, the development ofS. aureusresistance to mupirocin is delayed by the combination of ClyQ and mupirocinin vitro. Our results bring research attention toward the development of chimeric lysin that cooperates with antibiotics to overcome bacterial infections.