Dimethyl-sulfoxide (DMSO) 0.2% (Pure Pharma Grade, USP) and TWEEN-20 (Kremer Pigmente Gmbh and CoKG) were used to dissolve plant extracts. para-Iodonitrotetrazolium chloride (INT) (Sigma-Aldrich, Germany) was used as a microbial growth indicator. Five commonly prescribed antibiotics from different classes were employed for plant extract/antibiotic combination assays, including ciprofloxacin, cefixime, imipenem, tetracycline, and vancomycin (Sigma-Aldrich, Germany).
Ciprofloxacin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, France, Australia, Italy, Spain, China, India, Switzerland, Poland, Canada, Sao Tome and Principe, Ireland, Portugal, Czechia, Belgium, Japan, Mexico, Israel, Macao, Brazil, Egypt, Senegal
About the product
Ciprofloxacin is a broad-spectrum antibiotic that belongs to the fluoroquinolone class of antimicrobial agents. It is used in the treatment of various bacterial infections. Ciprofloxacin functions by inhibiting the activity of bacterial DNA gyrase and topoisomerase IV, which are essential enzymes for bacterial DNA replication and transcription.
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Metronidazole, by Merck Group (63 mentions)
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Colistin, by Merck Group (62 mentions)
Check compatibility
1 548 protocols using ciprofloxacin
1
Antimicrobial Screening of Plant Extracts
2
Synthesis and Characterization of Lipid-Based Formulations
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Reagents were obtained from commercial sources and used directly; exceptions are noted. BIC was purchased from BOC Sciences (Shirley, NY, USA). Myristoyl chloride, stearoyl chloride, behenic acid, octadecanedioic acid, pyridine, dimethylformamide (DMF), N,N-diisopropylethylamine (DIEA), polysorbate 20 (Tween 20), polyethylene glycol-3350 (PEG-3350), Pluronic F127 (poloxamer 407; P407), ciprofloxacin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 1-octanol, paraformaldehyde (PFA), and 3,3’-diaminobenzidine (DAB), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Diethyl ether, acetonitrile (ACN), methanol, optima-grade water, ethyl acetate, hexanes, dichloromethane (DCM), Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate-buffered saline (PBS), gentamicin, L-glutamine, potassium phosphate monobasic (KH2PO4), bovine serum albumin (BSA), and Triton X-100 were purchased from Thermo Fisher Scientific/Gibco (Waltham, MA, USA). Cell culture grade water (endotoxin-free) was purchased from Cytiva (Logan, UT, USA). Heat-inactivated pooled human serum was purchased from Innovative Biologics (Herndon, VA, USA).
3
Antibacterial Activity of Spt EOs
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The P. aeruginosa PAO1 strain used in this study was obtained from the Department of Microbiology, Faculty of Pharmacy, Zagazig University. Microbiological media, including Mueller Hinton (MH) broth, Tryptone soya broth (TSB) and agar (Oxoid, Hampshire, UK). Ciprofloxacin was purchased from Sigma Chemical Co. (St. Louis, MO, United States). All chemicals utilized were of pharmaceutical grade. For each experiment, S. trilobata EOs (Spt 1 and Spt 2) were solubilized in the respective media using 1% Tween-20. Control groups from bacterial culture (without adding the EO) were prepared for each experiment.
4
Modulating Gut Microbiome for Tumor Suppression
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Antibiotic treatment
An antibiotic mixture was given to mice ad libitum with sterilized fresh drinking water every two days. This mixture contained 100 μg/ml neomycin (Sigma, N6386), 50 μg/ml streptomycin (MCE, HY-B0472), 100 μg/ml ampicillin (Sigma, A9518), 50 μg/ml vancomycin (Sigma, V2002), 100 μg/ml metronidazole (Sigma, M3761), 1 mg/ml bacitracin (MCE, HY-B0278), 125 μg/ml ciprofloxacin (Sigma, PHR1167), and 100 μg/ml ceftazidime (Solarbio, C9731).
Sodium butyrate oral gavage
We randomly assigned mice to the following experimental groups: normal saline (0.9% w/v), sodium butyrate (NaBu) (sigma, B5887) treatments. In the NaBu treatment group, mice were orally gavaged with NaBu solution (50, 100, 200 mg/kg) per day for 21 days, which was dissolved in saline and filtered through a 0.22 μm membrane. Equal volume of sterilized saline was added to control mice.
High fiber diets
We randomly assigned mice to two diets: a diet added with inulin as a source of fiber or a control diet (mFD/mCD; TP 23300-X1/LAD 3001 G, Trophic Animal Feed High-tech, China). Both mCD and mFD diets contained 19.4% kcal from protein, 63.9% kcal from carbohydrates, and 16.7% kcal from fat. All mice received mCD for the first week of the experiment, after which mice were given either the mCD or mFD diets. During the procedure, murine body weights were measured every two days. Feed disappearance was measured to confirm that mice consumed a similar amount of feed between the mCD group and mFD group.
Akkermansia muciniphila supplementation
To evaluate the tumor suppressive role of Akkermansia muciniphila (A. muciniphila strain #1, AKK) (ATCC number: BAA-835) and (A. muciniphila strain #2, AKK-2) (DSMZ number: DSM26127) supplementation on tumor growth, A. muciniphila was first anaerobically incubated in media with brain heart infusion (BHI) at 37°C. The bacterial cultures were then incubated with 25% glycerol in saline and adjusted to a final concentration of 1 × 1010 colony-forming units (CFU)/ml. Cultures were immediately frozen and stored at -80°C. Before gavage, the glycerol stock solution was thawed under anaerobic conditions and resuspended in anaerobic normal saline to a concentration of 3 × 108 CFU in 200 μl and each mouse was treated with 200 μl. As the bacterial associated with stress, Alistipes shahii (ATCC number: BAA-1179) was utilized as negative control. Oral gavage with the 200 μl A. muciniphila (3 × 108 CFU) suspension in mice once a day for 21 days. For dead-A. muciniphila (heat-killed) experiments, 3 × 108 CFU bacterial A. muciniphila per mice were heated at 95°C for 2.5 h before gavage.
5
Antibiotic Susceptibility Assay on MAP
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The antibiotics were mixed into separate dilution series before being transferred to the pads of the MAP. They were sourced as follows: ampicillin (Sigma-Aldrich 10835242001), carbenicillin (Sigma-Aldrich C1389), cecropin A (Bachem AG, 4030488.1000), ciprofloxacin (Sigma-Aldrich 17850), chloramphenicol (Sigma-Aldrich C0378), gentamycin (Sigma-Aldrich G1397), kanamycin (Sigma-Aldrich K4000), mecillinam (Sigma-Aldrich 33447), neomycin (Sigma-Aldrich N1142), norfloxacin (Sigma-Aldrich N1142), tetracycline (Sigma-Aldrich T3258), trimethoprim (Sigma-Aldrich T7883), rifampicin (Sigma-Aldrich 557303) and vancomycin (Sigma-Aldrich V2002).
Stock solutions were prepared by dissolving the antibiotics in 10 mL of milliQ water (ampicillin, carbenicillin, cecropin A, ciprofloxacin, gentamycin, kanamycin, mecillinam, neomycin, tetracycline, vancomycin), 100% methanol (rifampicin, trimethoprim), or 95% ethanol (chloramphenicol, norfloxacin). These were stored at −20°C until use. Some antibiotics were subjected to ultrasonication to aid in their dissolution process.
Antibiotics were homogeneously mixed with the agarose and growth medium at 60°C prior to preparing the dilution series. Subsequently, the mixture was dispensed onto the MAP platform using an Opentrons OT2 pipetting robot [47 ], where it solidified to form the small assay pads.
Stock solutions were prepared by dissolving the antibiotics in 10 mL of milliQ water (ampicillin, carbenicillin, cecropin A, ciprofloxacin, gentamycin, kanamycin, mecillinam, neomycin, tetracycline, vancomycin), 100% methanol (rifampicin, trimethoprim), or 95% ethanol (chloramphenicol, norfloxacin). These were stored at −20°C until use. Some antibiotics were subjected to ultrasonication to aid in their dissolution process.
Antibiotics were homogeneously mixed with the agarose and growth medium at 60°C prior to preparing the dilution series. Subsequently, the mixture was dispensed onto the MAP platform using an Opentrons OT2 pipetting robot [47 ], where it solidified to form the small assay pads.
6
Antimicrobial Activity of Thymbra Spicata Essential Oils
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In the antimicrobial activity assays, the bacterial strains Staphylococcus aureus ATCC 29213 (methicillin-susceptible, MSSA), S. aureus ATCC 43300 (methicillin-resistant, MRSA), Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were used. Fungal strains included Candida parapsilosis RSKK 994, C. parapsilosis ATCC 22019, C. glabrata RSKK 4019, C. krusei RSKK 3016, and C. albicans ATCC 10231. The broth microdilution method was performed to determine the Minimum Inhibitory Concentration (MIC) values of the T. spicata EOs [37 ,38 ].
In accordance with the recommendations of Onder et al. (2024), essential oil samples were prepared for antibacterial and antifungal activity tests [32 (link)]. For the antibacterial activity test, TS-EO1 and TS-EO2 were serially diluted 2-fold to obtain concentrations ranging from 50 mg/mL to 0.78 mg/mL in Mueller-Hinton Broth (MHB; Difco Laboratories, Detroit, MI, USA), which was supplemented with 0.5% (v/v) Tween 80 (Merck, Darmstadt, Germany) to facilitate dissolution of the samples. The wells containing only inoculum and MHB with Tween 80 served as the control group. The inoculums were prepared from overnight cultures and the final concentrations of the test bacteria were adjusted to 5 × 105 CFU/mL in each well. The microtiter plates were then incubated at 35 °C for 18–24 h. The MIC values were determined as the lowest concentration (mg/mL) of EOs at which no visible bacterial growth was observed. Ciprofloxacin (Sigma, St. Louis, MO, USA) was used as the reference antibiotic for comparison.
For the antifungal activity tests, the EOs were dissolved in a solution of 10% dimethyl sulfoxide (DMSO) with 0.02% Tween 80. The concentration range was then prepared in RPMI 1640 broth (ICN-Flow, Aurora, OH, USA) supplemented with glutamine but without bicarbonate and pH indicator as previously described. The control wells contained the modified culture medium and inoculum. The fungal inoculum was adjusted to a final concentration of 0.5–2.5 × 103 CFU/mL in each well. The plates were incubated at 35 °C for 48 h. The MIC values were determined as the lowest concentration (mg/mL) of EOs at which no visible fungal growth was observed. Amphotericin B (Sigma, USA) was used as the reference standard for comparison.
In accordance with the recommendations of Onder et al. (2024), essential oil samples were prepared for antibacterial and antifungal activity tests [32 (link)]. For the antibacterial activity test, TS-EO1 and TS-EO2 were serially diluted 2-fold to obtain concentrations ranging from 50 mg/mL to 0.78 mg/mL in Mueller-Hinton Broth (MHB; Difco Laboratories, Detroit, MI, USA), which was supplemented with 0.5% (v/v) Tween 80 (Merck, Darmstadt, Germany) to facilitate dissolution of the samples. The wells containing only inoculum and MHB with Tween 80 served as the control group. The inoculums were prepared from overnight cultures and the final concentrations of the test bacteria were adjusted to 5 × 105 CFU/mL in each well. The microtiter plates were then incubated at 35 °C for 18–24 h. The MIC values were determined as the lowest concentration (mg/mL) of EOs at which no visible bacterial growth was observed. Ciprofloxacin (Sigma, St. Louis, MO, USA) was used as the reference antibiotic for comparison.
For the antifungal activity tests, the EOs were dissolved in a solution of 10% dimethyl sulfoxide (DMSO) with 0.02% Tween 80. The concentration range was then prepared in RPMI 1640 broth (ICN-Flow, Aurora, OH, USA) supplemented with glutamine but without bicarbonate and pH indicator as previously described. The control wells contained the modified culture medium and inoculum. The fungal inoculum was adjusted to a final concentration of 0.5–2.5 × 103 CFU/mL in each well. The plates were incubated at 35 °C for 48 h. The MIC values were determined as the lowest concentration (mg/mL) of EOs at which no visible fungal growth was observed. Amphotericin B (Sigma, USA) was used as the reference standard for comparison.
7
Quantification of Quinolone Antibiotics
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Quinoflox (100 mg/mL, Global vet Health, S.L. – Spain). Enrofloxacin (ENRO, purity: 99.0%), ciprofloxacin (CIPRO, purity: 99.9%) and norfloxacin (internal standard - IS, NOR, purity: 99.7%) were obtained from Sigma Aldrich (Steinheim, Germany). Enrofloxacin (Bayer Animal Health, Milano, Italy). Acetonitrile (ACN) and methanol (MeOH) were purchased from Carlo Erba Reagents (Carlo Erba, Milan, Italy). Formic acid (FA, 98%), ammonium acetate (98%) and potassium phosphate monobasic KH2PO4 were obtained from Sigma Aldrich (Steinheim, Germany). All reagents were of analytical grade. Ultrapure water was generated using a Milli-Q system (Millipore). Phenex-RC (regenerated cellulose) syringe filters 0.22 mm (Phenomenex, Torrance, CA, United States) were used to filter the extracts before injection into the LC–MS system.
8
Ciprofloxacin Aptamer Purification and Preparation
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The deoxyribonucleic acid (DNA) aptamer of Ciprofloxacin was purchased from Microsynth, Switzerland. It is modified by adding an amine at the 5′ end of the oligonucleotide sequence. An additional purification step was requested, due to the high chain length of the sequence (98 nt). The purification method performed in this case is PAGE purification (PolyAcrylamide Gel Electrophoresis). Hence the biotechnology company sent the oligonucleotide (aptamer) sequence in its purified form. This final sequence, similar to the Ciprofloxacin oligonucleotide (aptamer) previously described in published references [29 ], is as follows:
5′-(NH2 modified) ATA CCA GCT TAT TCA ATT GCA GGG TAT CTG AGG CTT GAT CTA CTA AAT GTC GTG GGG CAT TGC TAT TGG CGT TGA TAC GTA CAA TCG TAA GTT AG-3′.
Sterile water (commercial grade, purchased from Sigma-Aldrich France) is the solution used to prepare aliquots of the aptamer. Sterile water is an appropriate solvent for the aptamer solution because it is purified from RNA-ase and protease, making it suitable to be used as a solvent for a biological entity. It does not interfere with the oligonucleotide’s DNA sequence, nor does it decrease its activity. Offering a biological advantage for solvent preparation.
Ciprofloxacin hydrochloride pharmaceutical secondary standard was purchased from Sigma-Aldrich (France). The range of concentrations, proposed for this standard, is based on electrochemical works [30 (link)] published for Ciprofloxacin detection in pharmaceutical dosage form.
5′-(NH2 modified) ATA CCA GCT TAT TCA ATT GCA GGG TAT CTG AGG CTT GAT CTA CTA AAT GTC GTG GGG CAT TGC TAT TGG CGT TGA TAC GTA CAA TCG TAA GTT AG-3′.
Sterile water (commercial grade, purchased from Sigma-Aldrich France) is the solution used to prepare aliquots of the aptamer. Sterile water is an appropriate solvent for the aptamer solution because it is purified from RNA-ase and protease, making it suitable to be used as a solvent for a biological entity. It does not interfere with the oligonucleotide’s DNA sequence, nor does it decrease its activity. Offering a biological advantage for solvent preparation.
Ciprofloxacin hydrochloride pharmaceutical secondary standard was purchased from Sigma-Aldrich (France). The range of concentrations, proposed for this standard, is based on electrochemical works [30 (link)] published for Ciprofloxacin detection in pharmaceutical dosage form.
9
Antibiotic Susceptibility Testing Protocol
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The broad-spectrum antibiotics bacitracin, ciprofloxacin, colistin, rifampicin, and tetracycline were purchased from Sigma-Aldrich (Saint-Louis, MI, USA).
10
Microdilution MIC and MBC Determination
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Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by a microdilution method with some modifications [75 (link)]. In 96-well microplates, 100 μL of MH broth was added to 10 μL of bacterial inoculum (1.5 × 108 UFC/mL). Sterility control (MH broth), growth control (broth + inoculum), and positive control (Ciprofloxacin, Sigma-Aldrich®, St. Louis, MO, USA; 1 mg/mL) were set. Samples and positive control were serially diluted (100, 50, 25, 12.5, and 6.25 μg/mL) and 50 μL was added to each well. Microplates were incubated with constant shaking (37 °C/150 rpm for 18 h, Scientific CVP-250®, Bohemia, NY, USA). A total of 15 μL of aqueous MTT solution (1 mg/mL) was added to wells as a growth indicator and was incubated for 2 h. The lowest concentration of unstained wells was considered MIC. MBC was determined by culturing 50 μL of samples of each well without color change on MH agar and incubating them at 37 °C for 24 h. The lowest concentration that did not produce growth after this subculturing was considered as the MBC.
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