Norfloxacin

The antibiotic sensitivity of L. reuteri PSC102 was determined against 14 antibiotics, including cephalexin, colistin sulfate, enrofloxacin, cefalonium, amoxicillin trihydrate, penicillin G procaine, norfloxacin, spectinomycin, tylosin base, cefuroxime sodium, florfenicol, penicillin G benzathine, gentamicin sulfate, and streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA), as previously described [26 (link)]. Briefly, 100 µL of cultured L. reuteri PSC102 was mixed with 100 µL of diluted antibiotic solution in a 96-well plate. After finally adjusting the concentration to 10⁶ CFU/mL, the bacteria were incubated for 24 h at 37 °C. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by measuring the OD value at 600 nm using Gen5 microplate reader version 3.08 (BioTek, Winooski, VT, USA) and by streaking on Mueller–Hinton agar plate, respectively.

The minimum inhibitory concentration was determined using broth microdilution according to the CLSI standards (47 (link), 48 (link)). The experiment was conducted in 96-well round-bottom plates with a 200-µL total reaction volume. Twofold serial dilutions of the drugs [ciprofloxacin (256–2 µg/mL) and norfloxacin (256–2 µg/mL)] were prepared. The OD_600nm of the bacteria grown to the mid-exponential phase was adjusted to 0.25–0.3, and it was diluted such that each well finally contained 5 × 10⁵ CFU/mL, and the plates were incubated at 37°C for 18 h.
The synergistic efficacy of indole derivatives with norfloxacin (Sigma Aldrich) and ciprofloxacin (Sigma Aldrich) was identified by checkerboard assay (47 (link), 48 (link)). The MIC of antibiotics and the indole derivatives were determined by varying the concentration of the indole derivatives and the drugs’ varying concentrations. The fractional inhibitory concentration index was calculated to identify the synergistic efficacy of indole derivatives using the following equation:
FICI = MIC (antibiotic in the presence of EPI) / MIC (antibiotic alone) + MIC (EPI in the presence of antibiotic) / MIC (EPI alone).
The FICI values of ≤0.5, >0.5–4, and >4 were considered to be synergistic, indifferent, and antagonistic, respectively (10 (link)).

Three representative antibiotics with various modes of action in the cell and medically relevant for the species utilized in this work were selected for cross-resistance evaluation. The antibiotics selected were: ampicillin (Roche), chloramphenicol (Calbiochem), and norfloxacin (Sigma-Aldrich). ampicillin acts in the cell membrane, chloramphenicol affects protein synthesis (ribosomal action), and norfloxacin blocks DNA replication (DNA gyrase). Stock solutions (20 mg/mL) were prepared by dilution in sterile demineralized water, ethanol, and 0.1M HCl, respectively, and stored at −20°C. Working solutions were prepared as needed by dilution in sterile demineralized water.

Periplanetasin-2 was purchased from Anygen Co. (Republic of Korea). The detailed procedure of peptide synthesis was described in a previous study [8 (link)]. Norfloxacin, chloramphenicol, vancomycin, amikacin, kanamycin, rifampicin, ciprofloxacin, cefotaxime, and hygromycin B were purchased from Sigma-Aldrich (USA). Escherichia coli wild type (WT) BW25113 was obtained from the Coli Genetic Stock Center. For all assays, bacterial cells were grown in Luria-Bertani (LB) broth (BD Biosciences, USA) under aerobic conditions at 37°C while shaking at 120 ×g. The cells were centrifuged and suspended in phosphate-buffered saline (PBS). Afterward, the cells were incubated with periplanetasin-2 or the conventional antibiotics at 37°C.

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).

Antibiotics (Figure 1) enrofloxacin (ENRO), norfloxacin (NOR), lomefloxacin (LOME), enoxacin (ENOX), levoxacin (LEVO), ciprofloxacin (CIPRO), methacrylic acid (MAA), 2-trifluoromethacrylic acid (TFMAA) and 2-hydroxyethyl ethylene glycol dimethacrylate (EDMA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2,2′-azobis(2,4-dimethylvaleronitrile) (ABDV) was purchased from Wako (Neuss, Germany) and used as received. Sarafloxacin hydrochloride (SARA), was a gift from Fort Dodge veterinaria (Girona, Spain). Danofloxacin (DANO) was purchased from Riedel-de-Haën (Seelze, Germany).
Acetonitrile (ACN) and methanol (MeOH) (HPLC-grade) were provided by SDS (Peypin, France) and trifluoroacetic acid (TFA) (HPLC-grade, 99%) was from Fluka (Buchs, Switzerland).
Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). The monomers were purified, as required, by chromatography immediately before use, using an inhibitor–remover from Aldrich (Milwaukee, WI, USA). All solutions prepared for the HPLC were passed through a 0.45 µm nylon filter before use. 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) was supplied by Aldrich (Steinheim, Germany). Trifluoroacetic acid (TFA) (HPLC-grade) was from Fluka (Buchs, Switzerland).

Analytical solvents, such as n-hexane, chloroform, ethyl acetate, acetone, and methanol were purchased from Fisher Scientific, Loughborough, UK. Dimethyl sulfoxide, sodium chloride, norfloxacin and 3-[4,5-dimethylthiazol-2-yl]-2,5-iphenyltetrazolium bromide (MTT) used during antibacterial assay were purchased from Sigma Aldrich (Dorset, UK). Silica gel 60H used for vacuum liquid chromatography was purchased from Merck Millipore, UK. Sephadex LH 20 used for gel filtration chromatography was purchased from GE healthcare, Uppasala, Sweden. Prepacked silica column (normal phase) used for solid phase extraction (SPE) was purchased from Phenomenex, Cheshire, UK. Analytical and preparative TLC carried out on 0.2 mm silica gel 60 F₂₅₄ was purchased from Merck, Darmstadt, Germany. Spots on the TLC plates were visualized under short UV (254 nm) and long UV (366 nm), and also by spraying them with 1% vanillin in concentrated H₂SO₄ followed by heating at 100 °C for 3–6 min. The NMR spectroscopy was performed with Bruker AMX 600 NMR spectrometer, Coventry, UK (600 MHz for ¹H, and 150 MHz for ¹³C) in the Department of Chemistry at University College London (London, UK). High Resolution Mass Spectrometry (HRMS) was performed in Liverpool John Moores University (Liverpool, UK). IR spectroscopy was recorded on Agilent FT-IR (Cary 630, Stockport, UK).