Cefalexin

Standards, certified reference material, for calibration and validation of biomimetic columns were: Acetylsalicylic acid, caffeine, cefalexin, chloramphenicol, cimetidine, codeine hydrochloride dihydrate, furosemide, ibuprofen, mesalazine, metronidazole, nifedipine, paracetamol, propranolol hydrochloride, salicylic acid, sulfadiazine, sulfamethoxazole, thiamine hydrochloride and warfarin purchased from Sigma-Aldrich (St. Louis, MO, USA). Azathioprine; folic acid; 6-mercaptopurine and 6-thioguanine, United States Pharmacopeia reference standard and 6-methylthioguanine (≥95%) have also been obtained from Sigma-Aldrich. Acetonitrile, ethanol, iso-propanol, methanol, formic acid, all HPLC grade; sodium hydroxide; ACS reagent (97.0%) pellets; sodium nitrate (≥99.0); phosphate-buffered saline tablets (0.01 M, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4 at 25 °C); potassium phosphate monobasic and potassium phosphate dibasic (HPLC grade) were provided by Sigma-Aldrich. Ultra-pure water was obtained using a MilliQ UF-Plus system (Millipore, Darmstadt, Germany); resistivity MΩcm⁻¹ >18 at 25 °C and TOC <5 ppb. The excipients included hydroxypropyl methylcellulose Methocel K100M Premium CR (Colorcon, Harleysville, PA, USA), magnesium stearate (Acros Organics, Princeton, NJ, USA), lactose monohydrate, stearic acid, wheat, rice and corn starch (Kemig, Zagreb, Croatia).

Tomatidine (TO) (Sigma Aldrich, Oakville, Ontario, Canada) (Fig. 1a) was solubilised in dimethylsulfoxide (DMSO) at a concentration of 2 mg/mL. The TO analog, FC04–100 (Fig. 1b) was synthesized [21 (link)] and solubilised in DMSO at 20 mg/mL. Cefalexin, kanamycin, gentamicin (GEN) (all from Sigma Aldrich), were solubilised in water at a concentration of 10 mg/mL. Menadione was solubilized in DMSO, hemin in 1.4 M NH₄OH and thymidine in water (all from Sigma Aldrich) and were prepared at a concentration of 10 mg/mL.

The minimum inhibitory concentration (MIC) was evaluated for ST541, ATCC29213, RC85, and RC85‐T strains. To confirm the role of MVs in interrupting antibiotic activity, several antimicrobials namely, ampicillin, cefotaxime, cefoperazone, colistin, gentamicin, and tetracycline were utilized. Eight β‐lactam antibiotics, i.e., ampicillin, amoxicillin, cefalexin, cefazolin, cefoperazone, cefotaxime, cloxacillin, and methicillin (Sigma‐Aldrich), and five other classes of antibiotics, i.e. colistin, gentamicin, nalidixic acid, streptomycin, and tetracycline (Sigma‐Aldrich) were selected to compare relative resistance levels of RC85‐T with that of RC85. The MIC was determined by broth‐dilution method in 96‐well plates (Andrews, 2001 (link); Kim et al., 2018 (link)). according to Clinical and Laboratory Standards Institute guidelines, except that cation‐adjusted Muller Hinton broth was substituted with LB. The MIC values were measured from three independent experiments.

Cefalexin (CFX, ≥99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium permanganate (PM, ≥99.5%) and sodium bisulfite (BS, ≥99.9%) were obtained from Aladdin (Shanghai, China). The HPLC grade of phosphoric acid and methanol were obtained from Sigma-Aldrich. Humic acid (HA) was purchased from ANPEL Laboratory Technologies Inc. (Shanghai, China). Other chemicals and reagents used were in analytical grade or higher, and obtained from Sigma-Aldrich (St. Louis, MO, USA) or Aladdin (Shanghai, China). All chemicals were used as received without any further purification. Reagent grade deionized (DI) water was produced by a Milli-Q Ultrapure Gradient A10 purification system (Millipore, Burlington, MA, USA).

Amoxicillin, cefalexin, clindamycin, and doxycycline were purchased from Sigma-Aldrich. Marbofloxacin was kindly provided by Vetoquinol. Antibiotics were dissolved in pure water, with NaOH added to Amoxicillin and cefalexin.
The tested drug concentrations were selected to be equal or slightly above the total maximum concentration attained in canine serum after administration of the approved or recommended standard doses (Silley et al., 1988 (link); Kung and Wanner, 1994 (link); Schneider et al., 1996 (link); Batzias et al., 2005 (link); Hillier et al., 2014 (link); KuKanich and KuKanich, 2015 (link)). The tested concentrations were 5 μg/mL for marbofloxacin, 10 μg/mL for clindamycin and doxycycline, 20 μg/mL for Amoxicillin, and 50 μg/mL for cefalexin.
Chlorhexidine was purchased as chlorhexidine digluconate (Hibitan Irrigation 20%ND, MSD, France).

Nine β-lactam antibiotics known to confer bactericidal effects by inhibiting cell wall biosynthesis, namely ampicillin, amoxicillin, cefalexin, cefazolin, cefoperazone, cefotaxime, cloxacillin, imipenem, and methicillin (Sigma-Aldrich, USA) and five other class antibiotics, such as chloramphenicol, gentamicin, kanamycin, streptomycin, and tetracycline (Sigma-Aldrich) were selected. The minimum inhibitory concentration (MIC) of each antimicrobial agent was determined in ST692 and ATCC29213 cells using the broth-dilution method in 96-well plates^{9 (link),54 (link)} according to Clinical and Laboratory Standards Institute (CLSI) guidelines, except that cation-adjusted Muller Hinton broth was substituted with LB. The MIC values were measured from three independent experiments.