Eight Mtb clinical strains and the reference strain H37Rv were used in this study (Table S1). Antimycobacterial drug susceptibility testing under standardized guidelines, in silico lineage determination, and spoligotyping were previously performed for the clinical strains at the Portuguese National Institute of Health (10 (link)). Screening for bedaquiline and linezolid susceptibility was not part of standard routine during the timeframe the strains were tested. Regarding antimycobacterial drug resistance profile, clinical strains were classified according to the most recent definitions by WHO as susceptible, MDR (resistance to both rifampicin and INH), or pre-extensively drug-resistant (pre-XDR; resistance to rifampicin and any fluoroquinolone) (22 ). Bacteria were grown in Middlebrook 7H9 medium (BD Biosciences) or in Middlebrook 7H10 medium (BD Biosciences), supplemented with 0.2% or 0.5% of glycerol, respectively. Both media were supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) (BD Difco), and tyloxapol (Sigma-Aldrich) was added to liquid medium to a final concentration of 0.05%. Stocks of amoxicillin (AMX), EMB, meropenem (MEM), and vancomycin (VAN) (Sigma-Aldrich) were prepared in purified water. Potassium clavulanate (CLA) (Sigma-Aldrich) was prepared in phosphate buffer pH 6.0, 0.1 M, and INH was prepared in dimethyl sulfoxide (DMSO; PanReac AppliChem).
Vancomycin van
Manufactured by Merck Group
Sourced in United States
Vancomycin (VAN) is a laboratory product used for the detection and quantification of vancomycin, a type of antibiotic. It is commonly used in diagnostic and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Gentamicin gen, by Merck Group (3 mentions)
Ciprofloxacin cip, by Merck Group (3 mentions)
Tyloxapol, by Merck Group (2 mentions)
Potassium clavulanate cla, by Merck Group (2 mentions)
Middlebrook 7h9 medium, by BD (2 mentions)
Middlebrook 7h10 medium, by BD (2 mentions)
Ampicillin amp, by Merck Group (2 mentions)
Amikacin ami, by Chem-Impex (2 mentions)
Tobramycin tob, by Thermo Fisher Scientific (2 mentions)
Cefepime cef, by Chem-Impex (2 mentions)
Ciprofloxacin cip, by Enzo Life Sciences (2 mentions)
Imipenem imi, by Combi-Blocks (2 mentions)
Synergy neo2 multi mode plate reader, by Agilent Technologies (2 mentions)
Gentamicin, by Merck Group (2 mentions)
Cefotaxime, by Merck Group (1 mentions)
Ertapenem, by Merck Group (1 mentions)
Doripenem, by Merck Group (1 mentions)
Biapenem, by Merck Group (1 mentions)
Meropenem, by Merck Group (1 mentions)
Ethambutol, by Merck Group (1 mentions)
13 protocols using vancomycin van
1
Antimycobacterial Drug Susceptibility of Mtb Strains
2
Antimicrobial Susceptibility Profiling
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Mtb H37Rv, Msm mc2-155 and Msm PM965 (ept-1 rpsL4 ΔblaS1), a mutant strain deficient for the major beta-lactamase BlaS (Raymond et al., 2005 (link)), were used in this study. Bacteria were grown in Middlebrook 7H9 medium (BD Biosciences) or in Middlebrook 7H10 medium (BD Biosciences), supplemented with 0.2% or 0.5% of glycerol, respectively. Both media were supplemented with 10% oleic acid-albumin-dextrose-catalase (BD Difco) for Mtb and 0.5% glucose for Msm. Tyloxapol (Sigma-Aldrich) was added to liquid medium to a final concentration of 0.05% to prevent clump formation. Stocks of amoxicillin (AMX), biapenem (BIA), cefotaxime (CTX), doripenem (DOR), ertapenem (ETP), ethambutol (EMB), meropenem (MEM) and vancomycin (VAN) (Sigma-Aldrich) were prepared in purified water. Potassium clavulanate (CLA) (Sigma-Aldrich) was prepared in phosphate buffer pH 6.0, 0.1 M. Isoniazid (INH) and rifampicin (RIF) (Sigma-Aldrich) were prepared in dimethyl sulfoxide or methanol, respectively.
3
Antibacterial Agents Characterization
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Antimicrobial agent Vancomycin (VAN) was purchased from Sigma Aldrich (St. Louis, MO, USA), and Linezolid (LNZ) was supplied by Pfizer (Rome, Italy). BTZ2e was freshly synthesized and characterized at the Department of Pharmacy of University “A. Moro” of Bari. BTZ2e purity was analytically estimated to be > 99% by performing elemental analysis as reported in Franchini et al. [21 (link)].
4
Isolation and Culture of Streptococcus suis CZ130302
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S. suis strain CZ130302 from the novel serotype Chz was isolated from a diseased piglet in Changzhou, China [18 (link), 45 (link)]. Plasmid pSET-4S with Spc resistance gene was generously provided by Professor Daisuke Takamatsu. Plasmid pUC19 was maintained in the OIE Reference Laboratory for Swine Streptococcosis. S. suis were cultured in Todd Hewitt Broth (THB, BD) or agar comprising 6% (v/v) sheep blood at 37 °C and 5% CO2. E. coli strains were cultured in Luria-Bertani (LB, BD) medium at 37 °C supplemented with 100 μg/mL Spc (Sigma) per requirement for S. suis and 50 μg/mL Spc or 100 μg/mL ampicillin (Amp, Sigma) for E. coli. Different types of antibiotics, especially bacitracin (Bac, Sigma), were used to determine the minimum inhibitory concentration. In total, 100 μg/mL lysozyme (Lzm, Sigma) and 1 μg/mL vancomycin (Van, Sigma) were used for phagocytosis assays.
5
Antibacterial Compound Preparation Protocol
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Berberine chloride (BBR) (chemical formula C20H18CINO4) purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA) was dissolved in DMSO (Biomus, Poland) and filtered through a 0.22-μm Millipore filter (Corning, USA). Vancomycin (VAN) was obtained from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). BHI medium was obtained from (BIOMAXIMA, Lublin, Poland).
6
Antibiotic Susceptibility Profiling of P. aeruginosa
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All MICs were determined using serial two-fold microdilution method in 96-well microtiter plates as done previously68 (link). Antibiotics used in this study were: kanamycin (Kan; OmniPur), amikacin (Ami; ChemImpex), gentamicin (Gen; Sigma), tobramycin (Tob; Alfa Aesar), cefepime (Cef; ChemImpex), ciprofloxacin (Cip; Enzo), tigecycline (Tig; Astatech), imipenem (Imi; Combi-Blocks), and vancomycin (Van; Sigma). MIC values were determined at 18 hours post-inoculation by reading optical density at 600 nm (OD600) on a Synergy Neo2 Multi-Mode Plate Reader (Agilent BioTek), and inhibition of growth defined as OD600<0.5 above background. All MIC assays were performed at least twice starting from fresh streak plates on separate days and each assay was performed as two technical replicates. Assays were performed for all P. aeruginosa strains to determine starting MICs in the absence of berberine or berberine analogs. All MIC assays with berberine ligands (Ber, Ber-C3 [5 ], Ber-C12 [9 ], or Ber-pAr [10 ]) were adjusted for a final concentration of 1% DMSO in each well.
7
Antimicrobial Susceptibility Testing Using Standard Media
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Mueller-Hinton broth (MHB) (Sigma, Mendota Heights, MN, USA) was used for the determination of MIC and cultures of bacterial isolates. Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB) (Becton, Dickinson and Company, San Diego, CA, USA), Mueller-Hinton agar (MHA) (Sigma, Mendota Heights, MN, USA), and Brain Heart Infusion (BHI) broth (Sigma-Aldrich, St. Louis, MO, USA) were also used for bacterial cultures. Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), crystal violet (CV) and antibiotics (ATBs): ciprofloxacin (CIP), amikacin (AMK), ampicillin/sulbactam (SAM), gentamicin (GEN), meropenem (MEM), vancomycin (VAN), and trimethoprim/sulfamethoxazole (SXT) used in this study were obtained from Sigma-Aldrich, St. Louis, MO, USA; glacial acetic acid was obtained from Carlo Erba Reagents, Milano, Italy.
8
Preparation of Iron-Chelating Compounds
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DIBI and FEC1 were supplied by Chelation Partners Inc. Gentamicin (GEN), ciprofloxacin (CIP), and vancomycin (VAN) (Sigma-Aldrich) were prepared as 10 mg/mL stocks in water while deferiprone (DFP) from Sigma-Aldrich was dissolved in RPMI. DIBI stocks were prepared in either water (10 mg/mL) or RPMI (200 mg/mL). Exogenous iron was added to growth media as ferric citrate (Sigma-Aldrich) in RPMI. All stock solutions were filter-sterilized (0.2 μm filter) before use.
9
Antimicrobial and Antibiofilm Activities of Cu(II) Schiff Base Complexes
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Reference strains of S. aureus ATCC 29213 and ATCC 43300 representing methicillin-susceptible (SA) and methicillin-resistant S. aureus (MRSA), respectively, and a non-cancerous human cell line MRC5 (normal lung tissue) were used in the study. Two Cu(II) Schiff base complexes (SBD2 and SBD4) which have been prepared previously were also used (Table 1 ). Copper was chosen to complex with the SBD ligands as it was known to increase the potency [7 (link)]. Vancomycin (VAN) and oxacillin (OXA) which were used as controls to compare the antimicrobial and antibiofilm activities were obtained from Sigma-Aldrich.
Cu(II) Schiff base complexes used in the study
| Name | Compound description | Molecular structure | Molecular weight (g/mol) |
|---|---|---|---|
| SBD2 | Cu(SB4CB)2: Copper complex of SB4CB | 722.37 | |
| SBD4 | Cu(SBFH)2: Copper complex of SBFH | 754.37 |
10
Antibiotic Susceptibility Profiling of S. aureus
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S. aureus NCTC 8325-4 was grown in tryptic soy broth (TSB; BD, NJ, USA) at 37°C with shaking (120 rpm). Amoxicillin (AMX; Sigma-Aldrich, MO, USA), vancomycin (VAN; Sigma-Aldrich, MO, USA), gentamycin (GEN; Sigma-Aldrich, MO, USA), chloramphenicol (CHL; TCI, Tokyo, Japan), tetracycline (TET; Sigma-Aldrich, MO, USA), ciprofloxacin (CIP; Sigma-Aldrich, MO, USA), norfloxacin (NOR; Sigma-Aldrich, MO, USA), and rifampicin (RIF; TCI, Tokyo, Japan) were used as the antibiotics in this study. S. aureus was treated with these antibiotics at MICs. The modes of action of these antibiotics include inhibition of cell wall synthesis, protein synthesis, and DNA gyrase or RNA synthesis inhibition. The detailed information of the antibiotics and their MICs are summarized inTable 1
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S. aureus NCTC 8325-4 was grown in tryptic soy broth (TSB; BD, NJ, USA) at 37°C with shaking (120 rpm). Amoxicillin (AMX; Sigma-Aldrich, MO, USA), vancomycin (VAN; Sigma-Aldrich, MO, USA), gentamycin (GEN; Sigma-Aldrich, MO, USA), chloramphenicol (CHL; TCI, Tokyo, Japan), tetracycline (TET; Sigma-Aldrich, MO, USA), ciprofloxacin (CIP; Sigma-Aldrich, MO, USA), norfloxacin (NOR; Sigma-Aldrich, MO, USA), and rifampicin (RIF; TCI, Tokyo, Japan) were used as the antibiotics in this study. S. aureus was treated with these antibiotics at MICs. The modes of action of these antibiotics include inhibition of cell wall synthesis, protein synthesis, and DNA gyrase or RNA synthesis inhibition. The detailed information of the antibiotics and their MICs are summarized in
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