Schaedler agar

Clinical specimens were collected in sterile containers and then transferred to a microbiology laboratory for immediate processing. The samples were processed by sonication according to the methodology previously described by our group (44 (link)). In summary, 50 mL of phosphate saline buffer was added, and the tubes were vortexed and sonicated in an Ultrasons-H 3000840 low-power bath sonicator (J.P. Selecta, Barcelona, Spain) at 22°C for 5 min, followed by an additional centrifugation. After centrifugation, the resulting pellet was cultured in different growth agar plates: tryptic soy agar with 5% sheep blood, chocolate agar, MacConkey agar, Schaedler agar with 5% sheep blood, and Sabouraud-chloramphenicol agar, all from bioMérieux (Marcy-l’Étoile, France) and incubated at 37°C and 5% CO₂ for at least 24 h and up to 7 days, with the exception of Sabouraud-chloramphenicol agar, which was maintained at 30°C for 4 weeks. All of the microorganisms isolated were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) (VitekMS, bioMérieux, Marcy-l’Étoile, France) and subsequently frozen and stored at –80°C.

Samples were tested for the presence of urease with the use of RUT—Rapid Urease Test (CLO test Kimberly-Clark) and placed in tubes with 0.9% NaCl solution and transported to the Department of Pharmaceutical Microbiology with Laboratory for Microbiological Diagnostics, Medical University of Lublin, Poland. Biopsies were smashed with the use of two microscope slides and spread onto Schaedler agar (BioMerieux, Marcy-l’Étoile, France) with 5% sheep blood plates and Schaedler agar with 5% sheep blood plates additionally supplemented with DENT (Oxoid), incubated for 3–7 days at 35 °C in microaerophilic (5–10% CO₂, 80–90% N₂, 5–10% O₂, Generbag microaer, BioMerieux) conditions. H. pylori isolates were identified by colony morphology, Gram-staining and positive catalase (3% H₂O₂), urease and oxidase (BBL DrySlide Oxidase—Becton Dickinson, Franklin Lakes, NJ, USA) tests.
DNA of biopsy sample and bacterial isolates were extracted using QIAGEN QIAamp DNA Mini Kit (Qiagen, USA) according to the manufacturer’s instructions. The identification in biopsy samples and isolates were further confirmed as H. pylori using ureC gene amplification with specific primers [14 (link)]. Extracted DNA was frozen to −70 °C until its use.

Sewage and sludge samples were taken directly into 50 mL sterile, screwed-off Falcon tubes and transported to a laboratory for further analysis.
Air samples were stationary collected using 6-stage Andersen impactor (model 10-710, Graseby-Andersen, Inc., Smyrna, USA), which can separate particles of the following aerodynamic diameters: > 7/4.7/3.3/2.1/1.1/0.65 µm. The impactor was set at a height of approx. 0.5 m above the floor or the ground. The sampling time was 5 min, a flow rate of the air was 28.3 L/min and the volume of each collected air sample was 0.1415 m³. Calibration of the flow rate was carried out before and after each measurement using a digital flow meter (model Gilibrator-2, Sensidyne, Inc., Clearwater, USA). Between the sampling sessions, an impactor was subjected to disinfection and cleaning with isopropyl alcohol. For sampling of bacterial aerosols, impactor was loaded with Petri plates containing Schaedler agar with 5% additive of sheep blood (bioMérieux, Marcy l’Etoile, France). The graph including size distribution results was created with Microsoft Excel 2010 software (Microsoft Corp., Redmond, USA).
Simultaneously with bioaerosol measurements, at each sampling point, the temperature and relative humidity were measured with the use of portable thermo-hygrometer (model TFA 30.5024, Conrad Electronic GmbH, Hirschau, Germany).