Ofloxacin

Rifampicin (RMP), isoniazid (INH), ethambutol (EMB), streptomycin (SM), ofloxacin (OFX) and kanamycin (KAN) were purchased from Sigma. Racemic R,S-1′-acetoxychavicol acetate (rac-ACA) was purchased from LKT laboratories (St. Paul, MN, USA; Product Number A0817). The MICs were determined as described previously [16 (link)]. Briefly, all mycobacterial strains were prepared from scoops of solid culture and suspended in PBS plus Tween-80, to reach a concentration equivalent to McFarland Standard No. 1 (approximately 3 × 10⁷ Colony Forming Units (CFU)/mL) and then diluted by Middlebrook 7H9 medium supplemented with 10% OADC to about 10⁵ CFU/mL. One hundred microliters of the bacterial suspensions were added to microtiter plates containing, in each well, two-fold diluted drugs in 100 µL of Middlebrook 7H9 medium with 10% OADC. The volume of drug-free and bacteria-free wells was adjusted to 200 µL and used as the control. The plates were incubated at 37 °C for 5 days. Twenty microliters of Alamar Blue dye (Accumed International, Westlake, OH, USA) and 12.5 µL of 20% sterilized Tween 80 reagent were added. The plates were re-incubated at 37 °C for 24 h, and the colors of all wells were recorded. The MIC was defined as the lowest drug concentration that prevented a color change from blue to pink.

All of the chemicals used in this investigation were of analytical grade. Danofloxacin (DAN), ofloxacin (OFL), levofloxacin (LEV), garenoxacin (GAR), pefloxacin (PEF), gatifloxacin (GAT), clinafloxacin (CLI), sarafloxacin (SAR), lomefloxacin (LOM), tosufloxacin (TOZ), sparfloxacin (SPA), difloxacin (DIF), pazufloxacin (PAZ), marbofloxacin (MAR), moxifloxacin (MOX), rufloxacin (RUF), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENR), pipemidic acid (PIP), nalidixic acid (NAL), oxolinic acid (OXO), orbifloxacin (ORB), enoxacin (ENO), nadifloxacin (NAD), flumequine (FLU), bovine serum albumin (BSA), ovalbumine (OVA), casein, 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), N,N-dimethylformamide (DMF), ethylenediamine hydrochloride, triethylamine, sodium borohydride, glutardialdehyde, 4-aminomethylfluorescein (4-AMF), 3,3′,5,5′-tetramethylbenzidine (TMB), and Tween-20 were Sigma-Aldrich (St. Louis, MO, USA) products. Complete and incomplete Freund’s adjuvants were produced by Becton Dickinson (Franklin Lakes, NJ, USA). Peroxidase-labeled anti-rabbit immunoglobulins were from the Gamaleya Institute of Microbiology and Epidemiology (Moscow, Russia). All other chemicals (salts and solvents of analytical grade) were from Khimmed (Moscow, Russia).

A total of 56 P. aeruginosa strains were selected from the Pseudomonas collection of the Molecular Microbiology Area (Centre for Biomedical Research in La Rioja, CIBIR, Logroño, Spain) (Table S1). These strains were obtained from clinical (n = 28) and non-clinical (n = 28) samples from Spain. P. aeruginosa PAO1, PA7 and PA14 reference strains were also included in the study.
Minimum inhibitory concentrations (MIC) of ciprofloxacin, levofloxacin, norfloxacin and ofloxacin (Sigma-Aldrich, St. Louis, MO, USA) were determined by agar dilution method using P. aeruginosa ATCC27853 strain as control strain and interpreted according to CLSI (2020) [13 ]. Furthermore, to assess the role of efflux pumps, MIC of ciprofloxacin was determined in presence and absence of the inhibitor Phe-Arg-β-naphthylamide (PAβN, 40 mg/L, Sigma-Aldrich, St. Louis, MO, USA). Isolates were defined as efflux pump overproducers when MIC_{without PAβN}/MIC_PAβN was > 2, as previously described [18 (link)].

All chemicals and solvents were purchased from Sigma-Aldrich (Milan, Italy) and used without purification. The hosts tetra-dodecoxy-p-tetra-(N,N-dimethyl-N-hydroxyethylammonium)-methylene-calix[4]arene (CholineC4dod) (Rodik et al., 2015 (link), Granata et al., 2017 (link)) and tetra-propoxy-p-tetra-(N,N-dihydroxyethyl-N-methylammonium)-methylene-calix[4]arene (MedeaC4prop) (Consoli et al., 2018b (link)) were synthesized as previously reported. The guests (chloramphenicol, ofloxacin, and tetracycline hydrochloride) were purchased from Sigma-Aldrich and used as received. MOPS salt was purchased from Sigma-Aldrich and was of the highest purity commercially available. High-purity water (Millipore, Milli-Q Element A 10 ultrapure water) and A grade glassware were employed throughout.

All chemicals used were of analytical-reagent grade. Melamine, cyanuric acid, ofloxacin, and ampicillin were purchased from Sigma-Aldrich (Darmstadt, Germany). Deionized water with the conductivity of 0.1 μS cm⁻¹ was used for the preparation of all solutions and experiments.

CHO-expressed recombinant human lactoferrin (rHLF; >98% purity; <10% iron saturated; <0.5 EU·mg⁻¹; Cat# LFH-101) was kindly provided as lyophilized powder by PharmaReview Corporation (Houston, TX) (Choi et al. 2008 (link); Kruzel et al. 2021 (link)). The rHLF was reconstituted in dH₂O to a concentration of 10 mg·mL⁻¹. From day 14 to day 28 after Mtb infection, mice were given 1 mg·(100 μL)⁻¹·mouse⁻¹ of rHLF by oral gavage every other day as prophylatic treatment, similar to reported use in models of mycobacterial granulomatous responses (Hwang et al. 2017 (link); Welsh et al. 2010 (link), 2011 (link)). One mg/ml was found to be more productive than a 100 μg/ml dose (Fig. S.1). From day 21 to day 28 after Mtb infection, another group of mice were given 1 mg·(100 μL)⁻¹·mouse⁻¹ of recombinant human lactoferrin by oral gavage every other day as therapeutic treatment. Ofloxacin (Sigma Life Science; O8757-1G) was given at 100 μL of 30 mg·mL^·1·mouse⁻¹, solubilized in DMSO and diluted 1:10 with phosphate buffered saline (PBS), was intraperitoneal administered 30 min prior to sacrifice (Batard et al. 2011 ; Hwang et al. 2008 (link)).

Stock solutions of the target compounds (Atenolol CAS 29122-68-7, Azithromycin CAS 83905-01-5, Caffeine CAS 58-05-2, Erythromycin CAS 114-07-8, Diclofenac sodium salt CAS 15307-79-6, Cyclophosphamide CAS 50-18-0, CiprOfloxacin CAS 85721-33-1, Sulfamethoxazole CAS 723-46-6, Carbamazepine CAS 298-46-4, Ketoprofen CAS 22071-15-4, Trimethoprim CAS 738-70-5, Clarithromycin CAS 81103-11-9, Ofloxacin CAS 82419-36-1, Estrone CAS 53-16-7, 17-beta Estradiol CAS 50-28-2, Ibuprofen CAS 15687-27-1; Sigma-Aldrich, St. Louis, MO, USA) were prepared in UHPLC-grade MeOH, purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA). MilliQ water was obtained from MilliPore (MA, USA), LiChropur Formic Acid 98–100% and LiChropur Ammonia solution 25% for LC-MS were purchased from Merck KGaA (Darmstadt, Germany), Hydrochloric Acid (HCl), and Ethylenediaminetetraacetic acid trisodium salt dihydrate (Na₄EDTA) were obtained from Fluka Analytical (Sigma-Aldrich, MO, USA), while Ammonium acetate for LC-MS was purchased from Fisher Chemical Scientific (Geel, Belgium).