**1. Introduction**

Cold, or non-thermal, atmospheric-pressure plasma jets (CAPJ) have gained attention in biomedical applications [1–6] due to unique characteristics, comprising of a complex plasma chemistry without the need for elevated gas temperatures as required for traditional thermal plasma [7,8]. In addition, the high energy electrons of CAPJ can produce reactive oxygen species (ROS) with slightly higher temperature than ambient environment in an open air. The ROS produced by CAPJ plays a significant role in promising inactivation of bacteria [9,10], which can be used in developing antimicrobial treatments for infectious diseases or in sterilization of reusable thermal sensitive medical devices to prevent the outbreak of antibiotic-resistant bacteria [11]. Although these properties have led to extensive use of CAPJ in material processing and biomedical applications, consistent use of CAPJ without risks remains di fficult. As the performance of CAPJ is dependent on the operational parameters, understanding how each parameter impacts the plasma's properties is necessary and can allow for adjustment of the plasma for use in di fferent applications.

*E. coli* is often used as an indicator of hygiene and safety in food products [12], and it is commonly found in community and hospital-acquired infections. *E. coli* is one of the most common and deadly pathogens causing pediatric urinary tract infection, intra-abdominal infection, or acute lung injury [13–16] and can lead to life-threatening intestinal infections in immunocompromised patients [17]. In addition, empiric antibiotic therapy may be ine ffective with multidrug-resistant *E. coli* which will delay care and may cause further harm to the patient [18,19]. Therefore, we selected *E. coli* to investigate the antimicrobial e fficiency of CAPJ treatment. The aim of this work is to develop the interventions by CAPJ treatment to prevent *E. coli*-induced diseases.

The influence of operational parameters of CAPJ, such as operation power, CAPJ-sample distance, gas mixtures composition, and treatment periods need to be determined to generate consistent results when using CAPJ. In general, a large number of experiments are required to optimize the processing parameters to improve the antimicrobial e fficiency by changing one parameter value at a time. Such approach is time consuming, costly, and labor intensive. In this study, a systematic and e fficient approach using the Taguchi experimental method design [20–23] is applied to the CAPJ system in determining the optimal process parameters for the highest antimicrobial e fficiency. The optimal condition of CAPJ was determined experimentally and the antimicrobial mechanism of CAPJ was explored using DNA damage assay and microbial microstructure analysis.
