Sulfamethoxazole

Finafloxacin was supplied by MerLion Pharmaceuticals GmbH. Sulfamethoxazole and trimethoprim were purchased from Sigma Aldrich (UK) for the in vitro assays and an oral suspension of co-trimoxazole (Septrin) was purchased from GlaxoSmithKline (UK) for use in the in vivo studies.
For the in vitro assays, working concentrations of finafloxacin at 10 mg/mL were prepared by adding 100 mg of antibiotic to 9 mL of sterile water and 1 mL of 1 M sodium hydroxide (NaOH). Co-trimoxazole (10 mg/mL) was prepared by adding 16.5 mg of trimethoprim to 4.985 mL distilled water and 15 μL acetic acid, and 83.5 mg of Sulfamethoxazole was added to 4.5 mL of distilled water and 0.5 ml of 1M NaOH, and the 2 components mixed. Bacteria grown in the equivalent concentration of sodium hydroxide used to prepare the antibiotics was included as a control.
For the in vivo studies a 15 mg/mL solution of finafloxacin was prepared by adding 2.1 mL of 0.01 M Tris buffer to 44 mg of finafloxacin powder (containing 37.5 mg of active ingredient). 200 μL of 1 M NaOH was added to dissolve the antibiotic followed by 200 μL of 0.01 M hydrochloric acid. The pH of the resulting solution was pH 8; a new solution was prepared for each time point. Co-trimoxazole was diluted in PBS to obtain a concentration of 1.56 mg per 50μL dose.

Graphite was purchased from AVONCHEM; glucosamine (GLA) was purchased from Merck; 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride (EDC) was purchased from Ambeed; triethyl amine (Et₃N), sulfuric acid (H₂SO₄- 98%), and hydrochloric acid (HCl) were obtained from BDH laboratories; KMnO₄ was used of analytical grade; hydrogen peroxide H₂O₂ (35%) was purchased from Scharlau chemicals; phosphoric acid, ethanol, and diethyl ether were purchased from Sigma Aldrich (St. Louis, MO, USA); and ethacridine lactate (EL) and sulfamethoxazole (Sul) were kind gifts from the local pharma. All these compounds were prepared and their working concentrations were used in different assays.

Bacterial strains resistant to different antimicrobials were isolated from water samples in the second sampling campaign (S2, low rainfall sampling). The triplicates were mixed (750 mL of water from each replicate), and 1000 mL of water from each pond was filtered through 0.45 μm pore size Millipore^® (Barueri, SP, Brazil) membranes using a Kitassato connected to a vacuum pump. The four membranes were washed individually in 20 mL of saline (NaCl 0.85%).
Aliquots of 100 µL of each sample and their respective dilutions (10⁻¹ and 10⁻²) were plated in Petri dishes containing CHROMagar Orientation culture medium (BD Diagnostics) supplemented with 50 μg/mL ciprofloxacin (Sigma^®, Saint Louis, MI, USA), 60 μg/mL sulfamethoxazole (Sigma^®, Buchs, Switzerland) or 8 μg/mL ceftriaxone (Sigma^® Saint Louis, MI, USA) [43 (link),44 (link)]. The plates were incubated at 37 °C for 24 h. After determination of colony forming units per milliliter (CFU/mL), three colonies of each morphology were reinoculated on CHROMagar and further identified at the genus level by MALDI-TOF (Bruker Daltonics Bremen, Germany). All isolated strains were maintained at −80 °C in tryptic soy broth medium (TSB) supplemented with the antibiotic used for their isolation, and 20% glycerol.

Halloysite nanoclay (diameter × length- 30–70 nm × 1–3 μm, nanotube), sulfamethoxazole (SMX, analytical standard), laccase from Trametes versicolor (form- powder), sodium sulfite (Na₂SO₃, ACS reagent grade), sodium hydroxide (NaOH, ACS reagent grade), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, assay- ≥98% high-performance liquid chromatography (HPLC)), syringaldehyde (SA, assay- ≥98%), guaiacol (GUA, assay- ≥98%), methanol (HPLC grade), glutaraldehyde (GTA, grade II, 25% in H₂O), and chitosan (75–85% deacetylated, low molecular weight, molecular weight- 50,000–190,000 Da) were obtained from Sigma-Aldrich (St. Louis, MO, USA). FeCl₃-6H₂O was obtained from JUNSEI (Kyoto) Japan. laccase from Trametes versicolor have a molecular mass of 70 kDa and an isoelectric point (pI) of 3.5 [23 (link)].

Peptide dendrimer G3KL was synthesized by solid-phase peptide synthesis and purified as described earlier [32 (link)]. Vancomycin, ampicillin, novobiocin, azithromycin, sulfamethoxazole, and trimethoprim were purchased from Sigma Aldrich (Buchs, Switzerland), erythromycin and ciprofloxacin were purchased from Acros Organics (Geel, Belgium), and chloramphenicol and gentamicin were purchased from AppliChem (Darmstadt, Germany). All compounds were conditioned as 8 or 10 mg/mL stock solutions in water (G3KL, ampicillin, novobiocin, chloramphenicol, and gentamicin), 1% acetic acid (ciprofloxacin), and DMSO (erythromycin, azithromycin, sulfamethoxazole, and trimethoprim).