Microbank system

Salmonella enterica subsp. Enterica (S.) serovar Typhimurium 9098 and serovar Infantis 1326 [6 (link),18 (link)] were used to inoculate avian macrophages. Both Salmonella strains were stored in the Microbank system (PRO-LAB Diagnostics, Ontario, Canada) at -20°C. The bacterial suspensions for infection were cultivated by shaking in nutrient broth (SIFIN, Berlin, Germany) for 18 h at 37°C. Infection doses were estimated by measuring extinction at 600 nm against a calibration graph determined for each strain and subsequent plate counting on nutrient agar (SIFIN).

The fungal strains used in this study were isolated from a foam sampled from the Baltic Sea environment, Windebyer Noor in Schleswig-Holstein, Germany [12 (link)]. The isolates were identified based on the morphology and molecular analysis of the ITS1-5.8S-ITS2 regions and 18S rRNA gene whenever the amplification of the ITS region failed. The six strains were maintained on PDA medium (potato starch 4 g, glucose monohydrate 4 g, agar 15 g, 1000 mL H₂O), and also cryo-conserved using the Microbank system (PRO-LAB Diagnostics, Richmond Hill, Ontario, Canada).

Five strains of LAB were used for the fermentation of BWP—Levilactobacillus brevis LUHS173, Liquorilactobacillus uvarum LUHS245, Lactiplantibacillus plantarum LUHS135, Lacticaseibacillus paracasei LUHS244 and Lacticaseibacillus casei LUHS210—which were isolated from spontaneous sourdough and provided from the collection of the Lithuanian University of Health Sciences [16 (link)]. Prior the use, LAB strains were maintained at −80 °C in a 25% glycerol solution in a Microbank system (Pro-Lab Diagnostics, Merseyside, UK) and before the fermentation strains were activated in MRS broth (de Man, Rogosa, and Sharpe, CM 0359, Oxoid Ltd., Hampshire, UK) for 48 h at 30 °C.

All samples were isolated from clinical cases that occurred between February 2022 and May 2023, with the assistance of the National Food Chain Safety Office, Veterinary Diagnostic Directorate. The isolates were stored at −80 °C in a Microbank™ system (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) until use. Information regarding the samples was recorded, including the species (duck, goose), the organ from which they were obtained (liver, lung, bone marrow), and the geographic origin. The species identification of the strains was determined using MALDI-TOF mass spectrometry (Flextra-LAB Kft., Budapest, Hungary) and the Biotyper software vs. 12.0 (Bruker Daltonics GmbH, Bremen, Germany, 2024) [21 (link)].

Antifungal activity of new N-phenacyldibromobenzimidazole derivatives was carried out against two C. albicans strains: reference C. albicans SC5314 from American Type Culture Collection (ATCC) and clinical SPZ176 strain (resistant to antifungal drugs: fluconazole Flu and itraconazole Itr) and clinical C. neoformans SPZ173 strain (naturally resistant to echinocandins). Fungal strains were stored at −80 °C in Microbank system (ProLab Diagnostics, Richmond Hill, ON, Canada) and cultured for 24 h at 30 °C with shaking at 100 rpm prior to each examination in liquid medium: YEPD (Yeast Extract Peptone Dextrose) or YNB (Yeast Nitrogen Base 0.67% w/v, glucose 2% w/v, CSM-URA 0.077% w/v, sterile water). After centrifugation at 3000 rpm at 4 °C for 5 min, cells were washed twice with sterile water and resuspended to prepare suspensions for experiments (ranging from 1.9 × 10⁷ to 2.0 × 10¹¹ cfu/mL; where cfu/mL = (number of colonies) × (inverse dilution of coefficient plated) × 10.

The study included a total of 197 black grouse fecal samples, collected in the Karkonosze National Park of approximately 25 km² area and 1000–1250 m above sea level, from places where the birds spent the night, during the winter months of 2017 and 2018. The collected feces samples were placed in sterile containers and immediately transported in an isothermal box with freezing inserts to the “Epi-Vet” Veterinary Diagnostic Laboratory. For microbiological examination, 1 g of fresh feces was incubated for 16–20 h at 37^oC in BHI (Brain Heart Infusion broth) (Oxoid, Hampshire, United Kingdom). The suspension was then inoculated onto McConkey’s agar (Oxoid, Hampshire, United Kingdom) and incubated for 18 h at 37^oC. The single, lactose-positive colonies characteristic of E. coli were transferred to nutrient agar medium (Merck, Darmstadt, Germany). The isolates were preserved for further testing at -70^oC, using the Microbank system (Pro-lab Diagnostics, Richmond Hill, Canada). Furthermore, isolated bacteria were identified as E. coli by observing their cultural characteristics, morphology by Gram’s stain, oxidase test, EMB (eosin methylene blue) agar and motility test. Presumptive E. coli strains were selected for further PCR analysis.

Antifungal activity of benzoxazole derivatives was tested against the reference Candida albicans SC5314 strain from American Type Culture Collection (ATCC). Additionally, two clinical isolates were used in biological activity testing: C. albicans (fluconazole- and itraconazole-resistant) and C. glabrata. The strains were stored at −80 °C in Microbank system (Pro-Lab Diagnostics, Round Rock, TX, USA). Prior to biological examinations, yeasts were cultured overnight in YEPD (Yeast Extract Peptone Dextrose) or in RPMI 1640 (Roswell Park Memorial Institute, NY, Buffalo, USA) at 30 °C with shaking at 100 rpm. Then, cells were harvested at 3,000 rpm for 5 min at 4 °C and washed twice with sterile water. The final inoculum ranged from 1.9 × 10⁷ to 4.0 × 10⁸ CFU/mL.