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[Display omitted] • PAW pre-exposure and subsequent US treatment has energy-efficient in MRSA inactivation. • The enhanced inactivation was partly due to the in-situ generation of RONS. • Elevated arginine levels induced the formation of VBNC bacteria. • Regrowth potential of MRSA cells was negligible. Methicillin-resistant Staphylococcus aureus (MRSA) and antibiotic resistance gene (mecA) are considered emerging contaminants that have been extensively identified in aqueous environments due to their high resistance to disinfectants. Herein, we report for the first time the inactivation of MRSA through a sequential application of plasma-activated water and ultrasound (PAW-SU). While the impact of PAW alone on bacterial culturability and mecA integrity was negligible, the combined PAW-SU treatment outperformed the inactivation method involving PAW with direct US (PAW-DU). It exhibited a shorter lag phase and a higher maximum inactivation rate constant. We numerically demonstrated that sonolysis processes induced by external ultrasound sources considerably improved the intracellular and extracellular reactive oxygen and nitrogen species (RONS) of MRSA in PAW system, thereby increasing the PAW reactivity and applicability. The highest inactivation efficiency of PAW-SU can be attributed to the synergistic effect of enhanced intracellular RONS (i.e., peroxynitrite and singlet oxygen) and membrane disruption induced by US, resulting in irreparable oxidative damage to the Save our Soul response and energy metabolic system. In particular, the elevated arginine metabolites in MRSA cells were primarily responsible for the formation of the viable but nonculturable state (VBNC) bacteria associated with high resistance to PAW system. Surprisingly, a decrease in arginine synthesis capacity was observed to expedite the flow of the tricarboxylic acid cycle, accelerating the detrimental effects of PAW-SU on intracellular components in VBNC bacteria. Finally, the limited regrowth potential of injured MRSA caused by cyclic inactivation of PAW-SU process was further confirmed in wastewater from meat and dairy models and therefore, advocates the possible real-world applications. [ABSTRACT FROM AUTHOR]