Sabouraud dextrose agar

The reference strain of the American Type Culture Collection (ATCC) Candida albicans ATCC^® 10231 (National Collection of Pathogenic Fungi (NCPF), Salisbury, UK) was used in this study. A 24-h culture of the fungus for further studies was prepared by uniformly spreading 100 μL of phosphate buffer solution (PBS) containing 5 × 10⁷ CFU/mL (5 units of the McFarland scale, determined with a DEN-1B type densitometer, Biosan, Riga, Latvia) onto a Petri dish with Sabouraud dextrose agar (bioMérieux, Marcy-l’Etoile, France), after which it was exposed to the plasma treatment for 1 min and then incubated at 37 °C for 24 h. C. albicans cells that were not killed in the plasma-induced growth inhibition zone were the protoplasts of growing spot colonies, which were the starting material for further studies. They also served as the basis for the preparation of the next culture and its plasma treatment, exactly as it was done the first time. This procedure was repeated 12 times in each measurement series. Then, subsequent screenings without plasma treatment were performed to determine the durability of occurring changes that had taken place under the influence of the plasma. As control samples, a 24-h culture of the fungus was used.

The study analyzed all pathogens identified in UTIs from patients of all ages, who were investigated in outpatient or inpatient settings, one-day hospitalization, as well as the Intensive Care Unit. Our hospital mainly treats patients from the southern area of Romania; therefore, the results of our study reflect the antibiotic resistance trends of bacteria in this region. Informed consent was obtained from all individual participants (or legal guardians) included in the study on admission to the hospital, and data were anonymized. Urine samples were collected in sterile containers and arrived in the Microbiology Laboratory within a maximum of 2 h after collection. In the laboratory, samples were inoculated on blood agar, cystine–lactose–electrolyte-deficient (CLED) agar, and Sabouraud Dextrose Agar (BioMerieux, Salt Lake City, Utah) using a calibrated loop (1 µL) under sterile conditions, followed by an 18 ± 2 h incubation period in atmospheric conditions at 35 ± 2 °C. Plates were examined according to laboratory procedures, and samples in which the number of colonies was higher or equal to 100,000 CFU/mL were considered positive for urinary tract infection. Bacteria were identified using the Vitek2 Compact (BioMerieux, Salt Lake City, Utah) and the automated matrix-assisted laser desorption ionization (MALDI) TOF MS (Bruker, Billerica, MA, USA).

Species identification was performed using the Vitek 2 yeast identification card (Vitek 2 Yeast ID; bioMérieux, Marcy l'Etoile, France) and Phoenix 100 Yeast ID panel (Becton-Dickinson Microbiology Systems, Sparks, MD, USA), according to the manufacturer's instructions [15] (link). Prior to testing, the isolates were subcultured onto Sabouraud dextrose agar (SDA, bioMérieux) and trypticase soy agar on 5% sheep blood agar plates (BAP, BD Microbiology System) and were incubated at 35°C in an aerobic atmosphere for 24–48 h. Extended incubation to 72 h was needed to ascertain adequate growth. C. albicans ATCC 14053 (Vitek 2 YST), C. albicans ATCC 24433, and C. parapsilosis ATCC 22019 (Phoenix Yeast ID panel) were used as control strains.

The following standard strains to determine the direct antimicrobial activity were used in the study: (1) Gram-positive cocci: Staphylococcus aureus ATCC 6538P MSSA, S. aureus subsp. aureus ATCC 43300 MRSA, S. epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, E. faecium ATCC 6057, Bacillus subtilis ATCC 6633; (2) Gram-negative bacteria from Enterobacteriales order: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453, Enterobacter cloacae DSM 6234, Serratia marcescens ATCC 13880; (3) Gram-negative non-fermentative rods: Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Stenotrophomonas maltophilia ATCC 12714, S. maltophilia ATCC 13637, Burkholderia cepacia ATCC 25416, Bordetella bronchiseptica ATCC 4617; (4) yeasts: Candida albicans ATCC 90028, C. parapsilosis ATCC 22019, C. tropicalis IBA 171, C. tropicalis (Castellani) Berkhout ATCC 750, C. guilliermondii IBA 155, C. krusei ATCC 6258 and Saccharomyces cerevisiae ATCC 9763.
All strains were stored at −80 °C. Prior to testing, each bacterial strain was subcultured twice on tryptic soy agar TSA (bioMerieux) medium and yeast strains on Sabouraud dextrose agar (bioMerieux) for 24–48 h at 30 °C to ensure viability.

The culture media used in the experiments were the following: cation-adjusted Mueller-Hinton broth (MHB II; Sigma-Aldrich, St- Louis, MO, USA and Biokar Diagnostics, Allone, Beauvais, France), Tryptic Soy broth (TSB; Scharlau Chemie S. A., Barcelona, Spain), and Trypto-Casein Soy agar (TSA; Biokar Diagnostics) were purchased. Sabouraud Dextrose Agar (SDA) was purchased from Bio-Mérieux (Marcy L’Etoile, France), RPMI-1640 broth medium from Biochrom AG (Berlin, Germany), which was buffered with 3-(N-morpholino) propanesulfonic acid (MOPS), purchased from Sigma-Aldrich, to pH 7.0.
Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), phosphate-buffered saline (PBS; pH 7.4), ethidium bromide (EB), reserpine, and crystal violet (CV) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Doxorubicin 2 mg/mL was purchased from Teva Pharmaceuticals, Budapest, Hungary. The antibiotics cefotaxime (CTX) was purchased from Duchefa Biochemie, Haarlem, The Netherlands, and vancomycin (VAN) from Oxoid, Basingstoke, England.

AZM in the form of dihydrate was a donation from PLIVA Croatia Ltd. (Zagreb, Croatia). Egg phosphatidylcholine (EPC), egg phosphatidylglycerol sodium salt (EPG), hydrogenated soybean phosphatidylcholine (SPC-3), and monoacyl phosphatidylcholine from soybean (SLPC-80) were kindly gifted by Lipoid GmbH (Ludwigshafen, Germany). Medium molecular weight (MMW) chitosan with 82.0% deacetylation degree and viscosity of 420 CPS (c = 1%, 1% acetic acid) as well as Sephadex G-50, urea, glucose, lactic acid, propylene glycol, Dulbecco’s modified Eagle medium, fetal bovine serum, L-glutamine, and bovine serum albumin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, ethanol, and methanol were of HPLC grade and procured from BDH Prolabo (Lutterworth, UK). Tryptic Soy Agar, Sabouraud Dextrose Agar and Tryptic Soy Broth were purchased from BioMérieux (Marcy-l’Étoile, France). Dey/Engley (D/E) Neutralizing Broth and Difco Antibiotic Medium 11 were obtained from Becton Dickinson (East Rutherford, NJ, USA).
Phosphate buffer, pH 8.0 (used in microbiological studies), was prepared by dissolving 16.73 g of potassium hydrogen phosphate and 0.523 g of potassium dihydrogen phosphate in demineralized water up to 1000 mL.
Vaginal fluid simulant (VFS, pH 4.5) and phosphate buffer (pH 7.5) were prepared as previously reported [19 (link)].

The following strains were used during the experiments: Gram-negative bacteria E. coli ATCC 8739, P. aeruginosa ATCC 9027; Gram-positive bacteria S. aureus ATCC 6538, B. subtilis ATCC 6633; yeast C. albicans ATCC 10231 and moulds A. brasiliensis ATCC 1640. All the microorganisms were the reference strains originated from the American Type Culture Collection ATCC. The bacteria were cultured on Trypticase Soy Agar (TSA, Oxoid, Basingstoke, UK) and yeast and moulds were cultured on Sabouraud Dextrose Agar (SDA, bioMerieux, Warsaw, Poland). All the microorganisms were twice subcultured in the respective media before each experiment. The broths of inoculated E. coli, S. aureus, and P. aeruginosa at 37 °C were incubated for 24 h, B. subtilis was incubated at 30 °C for 24 h, and yeast and moulds were incubated at 25 °C for 24–72 h. The physiological salt solution (0.85% NaCl) was used for inoculum preparation of each strain.