Amikacin

Polymyxin-B sulfate, amikacin, and sulbactam (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) solutions were prepared according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2020 ). The stock solutions of Polymyxin-B sulfate, amikacin, and sulbactam were dissolved in pure water at a concentration of 5210 μg/mL for all three antibiotics and stored at −80°C. Working solutions were diluted in Millipore water and filtered before use. Clinical A. baumannii isolate 12 was obtained from the affiliated hospital of Qingdao University and grown in cation-adjusted Mueller-Hinton broth (CAMHB; LAND BRIDGE, Beijing, China). The quality control strain for antimicrobial susceptibility test was E. coli ATCC 25922 and A. baumannii ATCC 19606. The resistance genes carried by the clinical isolate 12 are shown in Table 1.

Amikacin, moxifloxacin, clarithromycin, tigecycline, azithromycin, rifabutin, meropenem, cefoxitin, ciprofloxacin and linezolid were purchased from Macklin (Shanghai, China). Water-soluble antibiotics such as Amikacin, meropenem, and cefoxitin were dissolved in ddH2O, while other antibiotics, which are less soluble in water, were dissolved in DMSO. Cinnamaldehyde and carvacrol were obtained from Acmec (Shanghai, China). A collection of 80 commercially available EOs (Table S1) were purchased from Ningbo Ge Blueprint Electronic Industry and Trade Co., Ltd (Ningbo, China), Poli Aromatic Pharmaceutical Technology Co., Ltd (Shanghai, China), Guangzhou La Gu Na Biotechnology Co., Ltd (Guangzhou, China), Prime Time Commerce (Scottsdale Arizona, USA), Healing solutions (USA), Fabulous Frannie (USA) and Plant Therapy (USA). EOs and the EO active components Cinnamaldehyde and Carvacrol were dissolved in DMSO at 20% (v/v) and then diluted using stationary phase or log phase cultures to evaluate their activity against M. abscessus in MIC and drug exposure tests (see below). When the concentration of the EO is 0.25%, the concentration of DMSO is 1%.

The broad-spectrum aptamer for AAs was synthesized and purified from Sangon Biotech Co., Ltd. (Shanghai, China) and the sequence was as follows: 5′-CGGATCCCCAGCTCGGGGTGCTATGGAGGCTGTATCGGAGACCTGCAGG-3′. AAs including neomycin (NEO), kanamycin (KAN), gentamicin (GEN), streptomycin (STR), dihydrostreptomycin (DH-STR), tobramycin (TOB), spectinomycin (SPE), apramycin (APR), paromomycin (PAR), amikacin (AMI) and other antibiotics including tetracycline (TET), sulfadiazine (SUL), erythromycin (ERY), chloramphenicol (CHL) and ampicillin (AMP) were bought from Macklin Biochemical Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) and chitosan (CS) were purchased from Aladdin industrial Co. (Shanghai, China). Bovine serum albumin (BSA) was obtained from BioDev-Tech. Co. (Beijing, China). Ti₃AlC₂ was got from XFNANO Materials Tech Co., Ltd. (Nanjing, China). OMC was purchased from Yoshikura Nanotechnology Co. (Nanjing, China). The [Fe(CN)₆]^3−/4− solution containing 5.0 mM K₄[Fe(CN)₆], 5.0 mM K₃[Fe( CN)₆] and 0.1 M KCl was prepared with ultrapure water as solvent. All chemicals were of analytical grade. All aqueous solutions in the experiment were prepared with ultrapure water obtained by water purification system (PALL, New York, NY, USA, 18.2 MΩ·cm at 25 °C).

Broth-microdilution assays were performed to determine the antibiotic susceptibility and minimum inhibitory concentrations (MICs) for 10 antibiotics, including ceftiofur, cefquinome, ceftazidime, ceftriaxone, meropenem, norfloxacin, ciprofloxacin, gentamycin, kanamycin and amikacin (Shanghai Macklin Biochemical Co., Ltd, China) as recommended by the Clinical and Laboratory Standards Institute and the European Committee on Antimicrobial Susceptibility Testing. The above antibiotics are commonly used in veterinary clinical treatment of E. coli infection. Susceptibility to ceftiofur and cefquinome was determined in reference to previous research (23 (link)). All drugs were diluted 2-fold in MHB and mixed with an equal volume of bacterial suspension in a 96-well microtiter plate. Each test was repeated three times. Escherichia coli ATCC^® 25922™ was used as the quality-control strain (without magnolol). According to a previous report, bacteria resistant to ≥3 different classes of antibiotics were considered MDR (24 (link)).