Chloramphenicol

The susceptibility of the WT and ΔsdiA mutant strains to different antibiotics was evaluated by the disc diffusion method. The bacterial cells were diluted with PW to 10⁸CFU/mL, and 0.1 mL of bacterial culture was plated onto a TSA plate. The antibiotic resistance was checked against 6 different antibiotics (200 μg/mL and 500 μg/mL): kanamycin, chloramphenicol, rifampicin, erythromycin, tetracycline hydrochloride and penicillin (Macklin Inc., Shanghai, China). Sterilized deionized water was used as a blank control. The sterilized circular filter paper (Φ = 8 mm) was soaked in antibiotic solutions and deionized water for 10 min. Then, the filter papers were removed with sterile tweezers, affixed to a dried TSA plate and incubated for 24 h at 37°C, and the diameter of the antibacterial coil was measured. The tolerance and resistance assay was repeated in three separate experiments. Results are presented as the mean ± SD.

The E. coli strain used in this work is a standard DH5α strain which is commonly used in genetic engineering. The antibiotics tetracycline, streptomycin, roxithromycin, and nalidixic acid were formulated as 2-mg/mL stock solutions in water. Rifampin (Macklin, Beijing) was formulated as a 2-mg/mL stock in dimethyl sulfoxide (DMSO). Azithromycin and chloramphenicol (Macklin, Beijing) were formulated as 2-mg/mL stock solutions in ethanol. Ciprofloxacin (Sigma, Shanghai) was dissolved in 0.15 M sodium hydroxide at 1 mg/mL, and it was neutralized before addition to the medium. Cyanide-m-chlorophenylhydrazone (CCCP; Sigma, Shanghai) was dissolved in DMSO to reach a final concentration of 5 mg/mL, and 20 μg/mL of CCCP was used in the medium to inhibit the activity of efflux pumps (10 (link), 54 (link)). The MFS-specific inhibitor verapamil (Sigma, Shanghai) was formulated at 5 mg/mL in water. Berberine (Sigma, Shanghai) was dissolved in water (1 mg/mL) or DMSO (10 mg/mL) for different applications. LB medium (containing per liter, 10 g NaCl, 10 g peptone, 5 g yeast extract, 15 g agar if necessary; Bioroyee, Beijing) was prepared as broth according to the manufacturer’s instructions. All strains were grown overnight in a 37°C shaking incubator (200 rpm) prior to initiating the experiment.

2-aminoterephthalic acid (ATA, 98%), tetracycline (TET, ≥98%), doxycycline (DOX, ≥98%), oxytetracycline (OTC, ≥98%), chlortetracycline (CTC, ≥98%), chloramphenicol (CAP, 98%), tyrosine (Tyr), histidine (His), serine (Ser), phenylalanine (Phe), arginine (Arg) and cysteine (Cys) were provided by Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Zirconium chloride (ZrCl₄, 99%) was purchased from Beijing Yinuokai Technology Co., Ltd. (Beijing, China). Methacrylic acid (MAA, 99%), 2,2-azobisisobutyronitrile (AIBN, 99%) and ethylene glycol dimethacrylate (EGDMA, 98%) were obtained from TCI Development Co., Ltd. (Shanghai, China). N, N-dimethylformamide (DMF), ethanol, glucose, sucrose, MnCl₂·4H₂O, MgCl₂·6H₂O, Zn(NO₃)₂·6H₂O, CaCl₂, NaCl and KCl were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All reagents and chemicals used in this work were of at least analytical grade. The apparatuses utilized in this study are described in detail in the supporting information.

Arginine (Arg, 98%), chloramphenicol (CAP, 98%), potassium chloride (KCl), potassium ferricyanide (K₃[Fe(CN)₆]), potassium hexacyanoferrate (II) (K₄[Fe(CN)₆]∙3H₂O), metronidazole (MTR, 99%), fluconazole (FLU, 98%), clarithromycin (CLA, 98%), and tetracycline (TC, 98%) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Sodium phosphate dibasic dodecahydrate (Na₂HPO₄·12H₂O) and Sodium phosphate monobasic dehydrate (NaH₂PO₄·2H₂O) were purchased from Aladdin Chemistry Co., Ltd. (Shanghia, China). All the solutions were prepared with ultrapure water.

K. pneumoniae was previously isolated from the biogas slurry residue, and ampicillin (10 μg/mL) was used in the following steps to separate this strain. The genome sequencing of this strain was completed and the genomic information is available from NCBI (Accession: ASM1583203v1) [40 (link)]. Tetracycline (Macklin, Shanghai, China), streptomycin (Sigma, Shanghai, China), hygromycin (Macklin, Shanghai, China), kanamycin (Macklin, Shanghai, China), and roxithromycin (Macklin, Shanghai, China) were formulated as a 2 mg/mL stock solution in distilled water and stored at 4 °C. Rifampicin (Macklin, Shanghai, China) was formulated as a 2 mg/mL stock in DMSO, and azithromycin (Sigma, Shanghai, China) and chloramphenicol (Macklin, Shanghai, China) were formulated as a 2 mg/mL stock solution in ethanol. Ciprofloxacin (Macklin, Shanghai, China) was dissolved in 0.15 M of sodium hydroxide at 1 mg/mL and was neutralized before use. Concentrations of antibiotics and extra carbon sources used for the treatment of K. pneumoniae are listed in the Supplementary Materials, Table S1. MHB medium was purchased from Bioyee (Beijing, China) and prepared as broth according to the manufacturer’s instructions. All strains were grown overnight in a 37 °C shaking incubator (200 rpm) prior to initiating the experiment.

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).