Resazurin sodium

Compounds 1, 5, and 9c were synthesized as described below. The biotin-diol-azide linker was obtained from Click Chemistry Tools (Scottsdale, United States). Activated charcoal, sodium resazurin, sodium periodate, Triton X-114, isoniazid, rifampicin (Rif), and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich, United States. The tris-(2-carboxyethyl)phosphine HCl (TCEP) and streptavidin-agarose beads used for enrichment and purification were from Thermo Fisher Scientific, United States.

Acetic acid, methanol, sodium chloride, and sucrose were purchased from Chembur (Piekary Śląskie, Poland), trifluoroacetic acid purchased from Honeywell Riedel-de Haen (Seelz, Germany). HPLC-grade methanol and acetonitrile were purchased in Merck (Darmstadt, Germany). Plant culture media components, plant growth regulators NAA (naphthalene-1-acetic acid), BAP (6-benzylaminopurine) and IBA (indole-3-butyric acid) and agar were purchased in Duchefa Biochemie (Haarlem, Netherlands). The following commercially available standards for HPLC analyses were used: verbascoside, isoverbascoside, verbenaline and hastatoside (ChromaDex^®, Los Angeles, California, USA) and caffeic acid, chlorogenic acid, ferulic acid, protocatechuic acid, rosmarinic acid, vanillic acid (Sigma-Aldrich Co., Munich, Germany). Scutellarin, scutellarein and apigenin 7-glucuronide obtained from ChemFaces (Wuhan, Hubei, China). Bacterial culture media: nutrient agar (NA), Müller-Hinton Broth (MHB) and Müller-Hinton agar (MHA) were purchased in BTL (Łódź, Poland). Resazurin sodium was purchased from Sigma-Aldrich Co. (St. Louis, USA). Ferrous chloride (FeCl₂) was obtained from Carlo Erba (Milan, Italy). Unless indicated otherwise all chemicals were purchased from Sigma-Aldrich (Milan, Italy).

To verify the occurrence of efflux pump-mediated resistance, MIC values of amikacin, ceftazidime or norfloxacin were determined in the presence or absence of a carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma–Aldrich) solution at the subinhibitory concentration. CCCP is a decoupler from oxidative phosphorylation that interrupts the proton gradient of membranes. Microtitration plates were incubated at 37°C for 24 hours, and following this time 20 μl of a 0.01% (w/v) aqueous Resazurin sodium (Sigma–Aldrich) solution was added to each well. These plates were incubated for 1 hour at room temperature, where following this period a reading was performed taking into account that a change in coloration from blue to pink indicated the occurrence of bacterial growth due to resazurin reduction [42 (link), 43 (link)]. As a criterion for classifying the occurrence of resistance mediated by the efflux pump phenotype, a minimum of a 2-fold antibiotic MIC reduction in the presence of CCCP was necessary [22 ].

Silver nanoparticles solution (4000 µg/ mL with the particle size of 35 nm) was purchased from Pars Nano Nasb Co (Tehran, Iran) and sterilized by filtration through 0.22 µm filters before use. Peptone water, phosphate buffered saline (PBS), Luria-Bertani (LB) broth and agar, Agar-agar, and resazurin sodium were obtained from Sigma Chemical Co (St. Louis, MO., USA). All other chemicals were purchased from Merck (Darmstadt, Germany). The plant, Zataria multiflora Boiss, was purchased from local groceries. Staph. aureus ATCC 25923 and Salm. Typhimurium ATCC 14028 were obtained from the Department of Food Hygiene and Quality Control, Urmia University, Urmia, Iran.