Mueller hinton broth (mhb)

The following media were used throughout this study: viability-maintaining microbiostatic medium, anaerobically prepared (VMGA III) transport medium (Doan et al. 1999 (link)), tryptone soya serum bacitracin vancomycin agar (TSBV, HiMedia Laboratories, India) (Slots 1982 (link)), tryptone soya broth (TSB, Biolife, Italy), tryptone soya agar (TSA, Biolife, Italy), Müller–Hinton broth (MHB, Biolife, Italy) and Müller–Hinton agar (MHA, Biolife, Italy). All chemicals used in this work were of analytical grade (HiMedia Laboratories, India). CEO was purchased from Frey & Lau GmbH, Henstedt-Ulzburg, Germany.

N-acetylcysteine (NAC), bromelain, ascorbic acid, Ribes nigrum, resveratrol, and pelargonium were kindly donated by Anvest Health s.r.l. (Castel San Giorgio, Salerno, Italy). Deoxyribonucleic acid (DNA), mucin from the porcine stomach, diethylenetriaminepentaacetic acid (DTPA), RPMI 1640 amino acids solution, egg yolk emulsion, sodium chloride, and potassium chloride, used for artificial mucus preparation, and barbital buffer were purchased from Sigma-Aldrich (Milan, Italy), while the hydroxyethylcellulose was obtained from A.C.E.F. SpA (Piacenza, Italy). Mueller–Hinton broth (MHB) and Mueller–Hinton agar (MHA) were acquired from Biolife (Milan, Italy); trypticase soy broth (TSB) was obtained from Difco Laboratories, and phosphate-buffered saline (PBS) from GIBCO, Thermo Fisher Scientific. All other solvents and chemicals were of analytical grade.

Standardized inoculum of Staphylococcus aureus ATCC 29213, S. aureus 393, Pseudomonas aeruginosa L2, and P. aeruginosa L4L were used for the experiments. The strains were plated on Mueller Hinton Agar (Biolife Italiana srl, Milano, Italy) and incubated at 37 °C for 19 h. Pure colonies were suspended in 10 mL Mueller Hinton Broth (MHB, Biolife Italiana srl, Milano, Italy) and diluted until the concentration was 5.0 Log10 colony forming unit/mL (CFU/mL). The microbroth dilution technique of antimicrobial susceptibility was performed to test the inhibitory effect of the formulations on the bacterial growth in comparison to controls. Dilutions were carried out using sterile deionized water in 96-well flat-bottom microtiter plates and were incubated with standardized inoculum (100 mL). Control experiments on bacteria were carried out by simple addition of sterile deionized water. The microtiter assays were incubated at 37 °C and the optical density (OD₆₀₀), namely the UV-vis absorbance (ABS) at 600 nm, was continuously monitored by spectrophotometry (Multiskan GO Microplate Readers, Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 24 h. Three biological replicates were performed for each strain and two different experiments were duplicated.

The bacteria were recent clinical isolates, belonging to the Institute of Microbiology (University of Genova) collection. They comprised of: (i) ten S. aureus strains, including five methicillin-resistant (MRSA) and five methicillin-susceptible (MSSA) strains. Of the MRSA strains, three were multi-resistant (resistant to at least three classes of antibiotics); (ii) ten multi-resistant Escherichia coli strains; (iii) ten Pseudomonas aeruginosa strains; (iv) ten vancomycin-resistant and susceptible Enterococcus faecalis and Enterococcus faecium strains; and (v) ten group A streptococci (Streptococcus pyogenes) strains, which remain universally susceptible to penicillin. All isolates were identified at the species level using clinical methods and an API STAPH, API20E, API NE and API STREP system (bioMèrieux, Marcy l’Etoile, France) for S. aureus, E. coli, P. aeruginosa, Enterococcus spp. and S. pyogenes respectively. The antibiotype was determined using the disk diffusion test, as according to the latest Clinical and Laboratory Standards Institute (CLSI) guidelines [57 ]. Strains were cultured in Mueller-Hinton Broth, Mueller-Hinton agar, MacConkey agar and Columbia blood agar (Biolife, Milan, Italy) at 37 °C.

Petroleum ether (boiling point ranges: 40 °C-60 °C), methylene chloride, ethyl acetate, deuterochloroform (CDCl₃), dimethyl sulfoxide (DMSO) and HPLC grade methanol were obtained from Sigma-Aldrich (Steinheim, Germany). Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Brea, CA, USA). Mueller Hinton agar was obtained from Merck (Germany). Nutrient liquid medium, Mueller Hinton broth was purchased from Biolife (Italy). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from SERVA (Heidelberg, Germany). Annexin V-FITC/7-AAD Kit was purchased from Beckman Coulter (USA). Standard antibiotics, Cisplatin, Phosphate-buffered saline (PBS), Ribonuclease A (RNAse A), fetal bovine serum (FBS) and 7-Aminoactinomycin D (7-AAD) were obtained from Sigma-Aldrich (Steinheim, Germany).

Minimal inhibitory concentrations (MICs) were determined by the microbroth dilution method according to the Clinical and Laboratory Standards Institute document (CLSI, 2009[6 ]) in Mueller Hinton broth (Biolife, Italy). Briefly, the DMSO solutions of isolated naphthoquinones (or standard antibiotics) were prepared as serial two-fold dilutions with the final concentrations ranging between 0.25-512 μg/mL. After that, each well was inoculated with microbial suspension at the final density of 0.5 McFarland. After 24 h of incubation at 37 °C, the inhibition of microbial growth was evaluated by measuring the well absorbance at 450 nm in an absorbance microplate reader Biotek EL808 with shaker (Biotek Instruments, USA). The 96-microtiter plates were read before and after experiment. Measurement error was established for 0.05 values of absorbance. Wells without tested naphthoquinones (or standard antibiotics) were used as negative controls of growth. Pure DMSO was used as a negative control. This experiment was done in eight-replicates for a higher accuracy of the MICs of tested compounds.