In vitro detection of MRSA strains were applied by cefoxitin (30 µg) disk diffusion test and oxacillin salt agar that compromised of Mueller-Hinton agar (MHA, Merck, Darmstadt, Germany) containing 6 µg/mL of oxacillin (Sigma, USA) supplemented with 4% NaCl following the Clinical and Laboratory Standards Institute (CLSI) instructions [12] . The plates were inoculated with S. aureus isolates at a concentration of 1.5 × 108 CFU/mL equal to the 0.5 McFarland tube and incubated at 35 °C for 16–18 h for cefoxitin (30 µg) disk diffusion and 24 h for oxacillin salt agar, respectively. In the cefoxitin (30 µg) disk diffusion, the isolates were considered MRSA if the inhibition zone around the disks was recorded ≤ 21 mm [12] . In the oxacillin salt agar, the existence of > 1 colony or light film of growth was considered as MRSA [12] . S. aureus ATCC® 29213™ and S. aureus ATCC® 43300™ were used as negative and positive quality control strains, respectively.
Oxacillin
Manufactured by Merck Group
Sourced in United States, Germany, Italy, Czechia, United Kingdom, Japan
Oxacillin is a semi-synthetic penicillin antibiotic used in laboratory settings. It is a crystalline powder that is soluble in water and alcohol. Oxacillin is commonly used in microbiological research and testing procedures.
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Lab products found in correlation
Vancomycin, by Merck Group (41 mentions)
Gentamicin, by Merck Group (34 mentions)
Ciprofloxacin, by Merck Group (30 mentions)
Ampicillin, by Merck Group (25 mentions)
Erythromycin, by Merck Group (18 mentions)
Chloramphenicol, by Merck Group (14 mentions)
Tetracycline, by Merck Group (13 mentions)
Kanamycin, by Merck Group (11 mentions)
Rifampicin, by Merck Group (11 mentions)
Amoxicillin, by Merck Group (10 mentions)
Linezolid, by Merck Group (10 mentions)
Penicillin g, by Merck Group (9 mentions)
Clindamycin, by Merck Group (9 mentions)
Cefoxitin, by Merck Group (8 mentions)
Streptomycin, by Merck Group (8 mentions)
Amikacin, by Merck Group (8 mentions)
Piperacillin, by Merck Group (6 mentions)
Cefotaxime, by Merck Group (6 mentions)
Methicillin, by Merck Group (5 mentions)
Norfloxacin, by Merck Group (5 mentions)
150 protocols using oxacillin
1
In vitro MRSA Detection Techniques
2
Preparation of MRSA Bacterial Samples
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MRSA USA300‐FPR3757 expressive of GFP was provided by Alice Prince at Columbia University (Diep et al, 2006 (link)). A single MRSA colony was cultured on Mueller‐Hinton agar plates (Sigma‐Aldrich Co.; catalog no.70191) containing oxacillin (6 μg/ml; Sigma‐Aldrich Co.; catalog no. 1481000), then planktonically cultured in lysogeny broth (LB; Invitrogen; catalog no. 10855021) containing oxacillin (6 μg/ml; Sigma‐Aldrich Co.) in a 35°C incubator for 24 h, after which absorbance (600 nm) was measured with a NanoDrop™ 2000c Spectrophotometer (Thermo Fisher Scientific, Inc.). MRSA (2 × 109 CFU/ml) was treated with vancomycin (20 mg/ml) for 24 h to make vancomycin‐treated MRSA (VT‐MRSA). MRSA (2 × 109 CFU/ml) incubated at 65°C for 2 h to make heat‐killed MRSA (HK‐MRSA). These samples were washed by Dulbecco's phosphate‐buffered saline solution (DPBS; Thermo Fisher Scientific, Inc.; catalog no. 14190144) at least thrice before experimentation.
3
Synergistic Evaluation of Compounds against MRSA
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The synergy of the two compounds and oxacillin (Sigma–Aldrich, St. Louis, Missouri, USA) against MRSA S. aureus CH 10850 was evaluated by the checkerboard assays [35 (link)], performed using 2-fold increasing concentrations of both compound (from 8 to 0.125µg/mL for compound 1 and from 512 µg/mL to 8 µg/mL for compound 2 ) and oxacillin (from 512 to 0.5 µg/mL). Since the two compounds were resuspended in DMSO, the upper limit of the concentrations range tested was determined considering a final concentration of 1% DMSO. The combinations of each compound and oxacillin were evaluated by fractional inhibitory concentration (FIC) index, interpreted as follows: ≤0.5, synergy; > 0.5 and ≤ 1.0, additive; >1.0 and <4, indifferent and ≥4, antagonistic.
4
Comparative Metabolomics of MRSA Strains
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The Staphylococcus aureus WKZ-1 and WKZ-2 strains,10 (link) which are isogenic except for SCCmec type IV, were used in this study. These bacterial strains were obtained through BEI Resources. Both bacterial strains were grown aerobically at 37 °C on 100 mL of tryptic soy broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with shaking at 250 rpm. First, we conducted an minimum inhibitory concentration (MIC) test for oxacillin (Sigma-Aldrich, St. Louis, MO, USA) using the broth microdilution method.11 (link) The MIC values for S. aureus WKZ-1 and WKZ-2 were determined to be 1 mg L−1 and 64 mg L−1, respectively, and we treated with oxacillin equal to 0.5 of the MIC as subinhibitory concentration. oxacillin was treated at the mid-exponential growth phase, and proteome and metabolome sample preparation were performed after 1 h 30 min.
5
Cultivation of Fluorescent MRSA Strain
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The USA300-FPR3757 strain of MRSA expressive of green fluorescent protein (GFP) was provided by Dr. Alice Prince at Columbia University, New York, USA. MRSA was transferred onto Mueller–Hinton agar plates (Sigma-Aldrich Co., St. Louis, MO, USA; cat. number: 70191) containing oxacillin (6 µg/mL; Sigma-Aldrich Co.; cat. number: 1481000) and incubated in a 35°C incubator for 24 h. Single MRSA colonies were also planktonically cultured in lysogeny broth (LB; Invitrogen, Carlsbad, CA, USA; cat. number: 10855021) containing oxacillin (6 µg/mL; Sigma-Aldrich Co.) for 24 h (Kwon et al., 2023 (link)).
6
Methicillin Resistance Determination
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Methicillin resistance was determined by Kirby-Bauer DD method using oxacillin 1 μg (Oxoid, England), cefoxitin 30 μg (Oxoid, England), and confirmed by oxacillin screen agar test supplemented with 4% NaCl and 6 μg/μl oxacillin (Sigma-Aldrich, USA) and cefoxitin E-test (AB Biodisc, Solna, Sweden) according to CLSI guidelines [ 15 ] and the manufacturer's instructions.
7
Antimicrobial Efficacy of DMF and Vancomycin
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Different concentrations of DMF (140, 420, and 700 μg/ml) and vancomycin (2.5 mg/ml) were added to LB (200 μl; Invitrogen) medium containing oxacillin (6 μg/ml; Sigma‐Aldrich Co.) in 96‐well plates (BD Biosciences). MRSA (4 × 106 CFU) was seeded into the medium and incubated for 24 h; DMSO was used as the negative control. MRSA (4 × 106 CFU) seeded into LB (200 μl; Invitrogen) medium containing oxacillin (6 μg/ml; Sigma‐Aldrich Co.) in 96‐well plates (BD Biosciences) and incubated at 35°C for 4 h. Different concentrations of DMF (140, 420, and 700 μg/ml) were applied to medium for 20 h; DMSO was used as the negative control.
The media was removed and slides were fixed with methanol (J.T. Baker, Center Valley, PA, USA; catalog no. 909302) for 10 min and then washed with DPBS (Thermo Fisher Scientific, Inc.) thrice. 200 μl of crystal violet solution (0.1%; Ward's Natural Science, NY, USA; catalog no. 548‐62‐9) was then applied and allowed to incubate for 15 min, after which the slides were washed with DPBS (Thermo Fisher Scientific, Inc.) thrice. 200 μl of acetic acid (33%; Sigma‐Aldrich Co.; catalog no. 695092) was added, and absorbance (570 nm) was measured using a BioTek Cytation™ 5 Cell Imaging Multi‐Mode Reader and analyzed using BioTek Gen5 software (BioTek Instruments Inc.).
The media was removed and slides were fixed with methanol (J.T. Baker, Center Valley, PA, USA; catalog no. 909302) for 10 min and then washed with DPBS (Thermo Fisher Scientific, Inc.) thrice. 200 μl of crystal violet solution (0.1%; Ward's Natural Science, NY, USA; catalog no. 548‐62‐9) was then applied and allowed to incubate for 15 min, after which the slides were washed with DPBS (Thermo Fisher Scientific, Inc.) thrice. 200 μl of acetic acid (33%; Sigma‐Aldrich Co.; catalog no. 695092) was added, and absorbance (570 nm) was measured using a BioTek Cytation™ 5 Cell Imaging Multi‐Mode Reader and analyzed using BioTek Gen5 software (BioTek Instruments Inc.).
8
Evaluating MRSA Susceptibility to Antibiotics
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A single MRSA colony was planktonically cultured in LB (Invitrogen) media containing oxacillin (6 μg/ml; Sigma-Aldrich Co.) for 24 hours, spread on Mueller-Hinton agar plates (Sigma-Aldrich Co.) containing oxacillin (6 μg/ml) using a sterile cotton-tipped applicator (McKesson Medical, San Francisco, CA, USA), and grown for 1 hour. Different concentrations of vancomycin, rifampin, and vancomycin/rifampin were prepared, and 20 μl of each solution was loaded onto sterile blank paper discs (6 mm; BD Biosciences) and dried for 1 hour. The dried discs were transferred to a Mueller-Hinton agar plate containing oxacillin (6 μg/ml) and MRSA and then incubated for 24 hours. After a day, the diameter of each inhibition zone was measured using a digital electronic caliper (Fine Science Tools, Heidelberg, Germany), and images were captured with the ChemiDoc Touch Imaging System (Bio-Rad Laboratories). For the antibacterial absorbance assay, MRSA (4 × 106 CFU) were seeded into LB (Invitrogen) media containing oxacillin (6 μg/ml; Sigma-Aldrich Co.) and incubated at 35°C. After 2 hours, we treated the LB media with three different concentrations of vancomycin (50 to 200 μg) or rifampin (75 to 300 ng). MRSA growth was measured by absorbance at 600 nm using the NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific Inc.) at 1-hour intervals for a total of 7 hours.
9
Hydrolysable Tannins and Antimicrobial Agents
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Two hydrolysable tannins (gallotannin and ellagitannin), 1,2,3,4,5-penta-O-galloyl-β-D-glucose (PGG) and 1,2,-di-O-galloyl-4,6-valoneoyl-β-D-glucose (dGVG), were obtained and characterized as described previously [12 (link)]. Briefly, 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and antibiotics (methicillin, oxacillin, and ampicillin) were obtained from Merck (KGaA, Darmstadt, Germany). Fluorescent probes—Sytox Green Nucleic Acid Stain and FM4-64 were from Thermo Fisher Scientific (Walthman, Massachusetts, USA).
All microbiological media used in the study were supplied by Oxoid (Basingstoke, England). All other reagents were purchased from POCH (Gliwice, Poland).
All microbiological media used in the study were supplied by Oxoid (Basingstoke, England). All other reagents were purchased from POCH (Gliwice, Poland).
10
Comprehensive Antibiotic Analytical Protocol
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All solvents of analytical LC-MS grade were provided by Merck (Darmstadt, Germa-ny). Sixty-six antibiotics belonging to different classes: Enrofloxacin, Difloxacin, Danofloxacin, Levofloxacin, Lomefloxacin, Marbofloxacin, Norfloxacin, Enoxacin, Flumequine, Nadifloxacin, Oxolinic acid, Nalidixicacid, Amoxicillin, Ampicillin, Phe-noxymethylpenicillin, Benzylpenicillin, Cefadroxil, Cefalexin, Cefalonium, Cefalothin, Cefazolin, Cefoperazone, Cefquinome, Cefapirin, Ceftiofur, DesfuroylCeftiofur, ClOxacillin, DiclOxacillin, Benethamine penicillin, Nafcillin, Oxacillin, Piperacillin, Tylosin, Tilmicosin, Oleandomycin, Spiramycin, NeoSpiramycin, Kitasamycin, Josamycin, Tu-lathromycin, Erythromicyn A, Rifaximin, Sulfadiazine, Sulfadimethoxine, Sulfadimidine, Sulfamerazine, Sulfamethoxazole, Sulfamonomethoxine, Sulfapirydine, Sulfatiazole, Tri-methoprim, ChloroTetracycline, OxyTetracycline, Tetracycline, Doxyicline, Lincomycin, Chloramphenicol, Tiamphenicol, Florfenicol, Florfenicol amine, Tiamulin, Valnemulin, Dimetridazole, Ronidazole, Tinidazole), and Enrofloxacin-d5 (internal standard) were bought from Merck (Darmstadt, Germany).
The Solid Phase extraction cartridges SPE Oasis HLB (3 mL, 60 mg) were provided by Waters (Milford, MA, USA).
The Solid Phase extraction cartridges SPE Oasis HLB (3 mL, 60 mg) were provided by Waters (Milford, MA, USA).
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