Tylosin

Human bladder cancer cell lines UMUC3, J82, RT4 and immortalized urothelial cell line SV-HUC-1 were purchased from the American Type Culture Collection (Manassas, VA, USA), and bladder cancer cell line 5637 was kindly provided from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University. UMUC3 was cultured in Dulbecco's modified Eagle's medium (DMEM; Wako, Osaka, Japan), and 5637 and RT4 were cultured in RPMI 1640 medium (Wako). J82 was cultured in Eagle's minimum essential medium (EMEM; Wako), and SV-HUC-1 was cultured in Ham's F-12 (Wako), with 10% exosome-depleted fetal bovine serum (Thermo Fisher Scientific) and 5 μg/ml gentamaicin (MSD, NJ, USA) and 60 μg/ml tylosin (Sigma-Aldrich, St. Louis, USA), and incubated at 37°C in a humidified atmosphere containing 5% CO₂. Cells were grown to confluence for 72 h. Conditioned media were pooled and centrifuged to obtain EVs according to the same protocol as the urine samples.

CYA, bi-deoxy Cyadox (Cy1), N4-deoxy cyadox (Cy2), N1-deoxy cyadox (Cy10), QCT, carbonyl reduction bi-deoxy quinocetone (Q2), MEQ, bi-deoxy Mequindox (M1), carbonyl reduction N1-deoxy mequindox (M4), carbonyl reduction bi-deoxy mequindox (M5) and carbonyl reduction mequindox (M6) (Table 1) were provided by the National Reference Laboratory of Veterinary Drug Residues, Huazhong Agricultural University (Wuhan, Hubei, China). Amphotericin B, tetracycline, doxycycline, ketoconazole, enrofloxacin, danofloxacin, rifampicin, tilmicosin and kitasamycin were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Kanamycin, pyrazinamide, lincomycin, ethambutol, ribavirin and isoniazid were purchased from TRC (Toront, Canada). Amikacin, clindamycin, and tylosin were purchased from Sigma (St Louis, MO, USA). Stock solutions of the above compounds were prepared at a final concentration of 1280 μg/mL.Table 1

Chemical information of QdNOs and their metabolites

All chemicals were used as received without further purification. Methoxy-PEG-thiol with a molar mass of 5000 g mol⁻¹ (mPEG-SH5k) was purchased from Creative PEGWorks (Chapel Hill, NC, catalog # PLS-604). mPEG-SH5k was in powder form and dissolved in deionized (DI) water prepared using Milli-Q Academic water purification system (Billerica, MA) having an electric conductivity less than 0.7 μS cm⁻¹. All solutions were freshly made as needed and used within twelve hours. The bulk gold target (16 mm long, 8 mm wide, 0.5 mm thick, and 99.99% purity) used in the laser ablation experiment was purchased from Alfa Aesar (Ward Hill, MA). EndoGRO were ordered from Vec Technologies (Rensselaer, NY, USA). Complete MCDB-131, trypsin–ethylenediaminetetraacetic acid (trypsin-EDTA), antibiotics/antimycotics, fetal bovine serum (FBS), phosphate-buffered saline (PBS) were purchased from Gibco BRL, Life Technologies (Grand Island, NY, USA). Heparin, epidermal growth factor (EGF), fibronection, tylosin, sodium bicarbonate, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Hoechst 33342, and propidium iodide (PI) were obtained from Sigma-Aldrich (Sigma, St. Louis, Mo, USA).

The FF, TAP, oxytetracycline (OTC), doxycycline (DOX), erythromycin (ERY), and tylosin (TYL) were purchased from Sigma-Aldrich (St. Louis, MO, United States). All the drugs used were analytical reference standards with a purity ≥95%. Clinical isolates of A. pleuropneumoniae (58) and P. multocida (79) were obtained from the Department of Veterinary Medicine, National Chiayi University, Taiwan. All bacterial isolates were obtained originally from the lung tissue of naturally infected pigs and their species and serovars had been established beforehand using biochemical tests, PCR analysis and Multilocus Sequence Typing (MLST) (Yeh et al., 2017 (link); Liao et al., 2019 ). For routine culture, P. multocida was grown on chocolate agar (Creative Lifescience, New Taipei city, Taiwan) and incubated at 37°C for 16–18 h, while A. pleuropneumoniae was also grown on chocolate agar at 37°C with 5% CO₂ for 20–24 h.

The subset of isolates was tested for MICs as previously described in the CLSI M11-A8 document (CLSI, 2012 ). Briefly, colonies of C. perfringens, from a 24 h culture grown at 37°C on blood agar plates (Oxoid) under anaerobic conditions, were resuspended into 5 mL of saline to achieve a 0.5 McFarland turbidity. This adjusted inoculum was then diluted 1:75 in supplemented Brucella broth. 50 μL of this suspension was then transferred to individual wells on microplates containing antibiotics. The microplates were sealed and incubated in an anaerobic chamber for 48 h at 37°C. Antimicrobials tested were penicillin, lincomycin, virginiamycin, tylosin, salinomycin, narasin, and monensin (all from Sigma). For bacitracin susceptibility testing, the Etest technique was used as described earlier (Charlebois et al., 2012 (link)). Isolates with MIC higher than 256 μg/mL by Etest were tested with the microdilutions broth technique to determine the exact MIC. C. perfringens ATCC 13124 was used as a control. Breakpoint for penicillin (2 μg/mL) is available from CLSI M11-A8. There is a previously published breakpoint for bacitracin (16 μg/mL) that is widely used in the literature (Chalmers et al., 2008 (link); Charlebois et al., 2012 (link)). No other breakpoints are available for the antimicrobials tested in this study.

Antibiotics (amoxicillin, cephalexin, ceftiofur, oxytetracycline, doxycycline, neomycin, apramycin, tylosin and lincomycin) and sulfonamides (sulfamethazine and sulfadiazine) used as standards were supplied by Sigma-Aldrich (Poole, UK). For each of them, a stock solution of 1 mg mL⁻¹ was made in water or methanol (depending on solubility specifications given by the supplier), and aliquots were kept at −20 °C for no more than 2 months.
Spiked blood serum samples were used for the establishment of the limits of detection (LoD). Working dilutions were freshly made on a daily basis. Standards were prepared by spiking blank blood sera with the appropriate dilution to obtain the indicated levels for each molecule (Table 1), using as a reference the EU-MRLs and the LoDs described for muscle when tested with the Explorer^® test [15 (link)]. No more than 10% of antimicrobial dilution was added to the blood serum sample in order to avoid significant changes in the matrix composition.