This study used well-characterized reference strains for preclinical investigations, ensuring consistency and accuracy in evaluating inhibition zones. Also, using these strains enhances the validity and reproducibility of research findings in AST. The experiment comprised a total of 44 strains,, including 11 strains each of Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, and StaphylococcuS. aureus. Multiple strains of Escherichia coli were obtained from ATCC (American Type Culture Collection) and CCARM (Culture Collection of Antimicrobial Resistant Microbes, Republic of Korea). The specific strains of E. coli obtained were ATCC 25922, CCARM 1A050, CCARM 1A076, CCARM 1A525, CCARM 1B669, CCARM 1B695, CCARM 1G109, CCARM 1G490, CCARM 1G517, NCCP (National Culture Collection for Pathogens, Republic of Korea) 14,629, and NCCP 16283. Similarly, multiple strains of Pseudomonas aeruginosa were obtained from ATCC and CCAR. The specific strains of P. aeruginosa obtained were ATCC 27853, CCARM 2092, CCARM 2095, CCARM 2244, CCARM 2271, CCARM 2191, CCARM 2198, CCARM 2206, CCARM 2235, CCARM 2282, and CCARM 2325. The specific strains of Enterococcus faecalis obtained were ATCC 29212, CCARM 5115, CCARM 5172, CCARM 5185, CCARM 5204, CCARM 5466, CCARM 5515, CCARM 5531, CCARM 5543, CCARM 5555, and CCARM 5560. Likewise, multiple strains of StaphylococcuS. aureus, such as ATCC 25923, ATCC 29213, CCARM 3795, CCARM 3799, CCARM 3800, CCARM 3807, CCARM 3808, CCARM 3816 2325, NCCP 14558, NCCP 14565, and NCCP 15869 were obtained from TCC, CCARM, and NCCP.

The DD method was carried out in accordance with the CLSI M02 guidelines [19]. Briefly, three to five well-isolated colonies of the test organism were cultured on a tryptic soy agar (TSA, Bacto™, BD, USA) plate. Next, a suspension equivalent to a 0.5 McFarland standard was prepared in 0.85% saline prepared from overnight cultures. Using a swab, the organism was inoculated onto a Mueller–Hinton agar (MHA, Asan Pharmaceutical, Hwaseong, Korea) plate. For each MHA plate, corresponding antimicrobial disks were applied. Disks are positioned equidistantly on the agar surface to achieve uniform exposure of microbes to antibiotics. The arrangement adheres to standardized guidelines, such as those provided by the Clinical and Laboratory Standards Institute (CLSI), to ensure that the arrangement remains consistent and enables reproducibility between tests. Disk spacing is meticulously planned in order to avoid overlap and assure the presence of distinct inhibition zones for each antibiotic. To perform RDD, the plates were put in the LS-AST device, and the initial measurement was taken. Then plates were incubated for 4 h at 35 °C, and the secondary measurement was taken on an LS-AST device. The inhibition zone was estimated by comparing the results of the initial and secondary measurements. The plates were then put back into the incubator and incubated for up to 16 to 18 h at 35 °C to perform the final manual measurement. Specifically, the initial and second measurements were merged with the final measurement to forecast the projected bacterial inhibition values produced by the equipment. These observations were used to compare the visual assessment. Schematic diagram of the work flow is shown in Fig. 1D.

The disks meant for E. coli contain different concentrations of different antimicrobials. The E. coli disks contain the following concentrations of antimicrobials. 300 μg of nitrofurantoin, 100 μg of nitrofurantoin; 30 μg of amikacin, aztreonam, chloramphenicol, ceftazidime, cefotaxime, cefepime, cefoxitin, and cefazolin; 20/10 μg of amoxicillin-clavulanic acid; 10 μg of ampicillin, gentamicin, imipenem; 5 μg of ciprofloxacin; 10/10 μg of ampicillin-sulbactam; 1.25/23.75 μg of trimethoprim-sulfamethoxazole. Similarly, The P. aeruginosa disks contain varying amounts of distinct antimicrobials. The P. aeruginosa disks contain the following concentrations of antimicrobials. 100/10 μg of piperacillin-tazobactam; 30 of μg amikacin, aztreonam, ceftazidime, cefepime; 10 μg of gentamicin, imipenem, and tobramycin; 5 μg of ciprofloxacin, levofloxacin. Likewise, E. faecalis disks contain the following concentrations of antimicrobials. 300 μg of nitrofurantoin and streptomycin; 100 μg of nitrofurantoin; 120 μg of gentamicin; 30 μg of linezolid and vancomycin; 10 μg of ampicillin and norfloxacin; 5 μg of ciprofloxacin, levofloxacin, and vancomycin. Similarly, S. aurues disks contain the following concentrations of antimicrobials. 30 μg of cefoxitin, linezolid, and tetracycline; 15 μg of erythromycin; 10 μg of gentamicin; 5 μg of rifampin; 2 μg of clindamycin; 0.01 μg of penicillin; 1.25/23.75 μg of trimethoprim-sulfamethoxazole. DD results were evaluated using 2022 CLSI disk breakpoints [20].