Gaspak

P. freudenreichii KCTC 1063 was obtained from the Korean Collection for Type Cultures (KCTC), and Escherichia coli OP50 was provided by the Caenorhabditis Genetics Centre (CGC) at the University of Minnesota and used as a standard food for C. elegans. E. coli OP50 was grown in Luria-Bertani (LB) broth (Difco, Detroit, MI, USA) at 37 °C with shaking overnight. P. freudenreichii was cultured using reinforced clostridial medium (RCM) broth (Oxoid, Hampshire, United Kingdom) for 48–72 h in anaerobic conditions using a BD GasPak (Becton Dickinson, Franklin Lakes, NJ, USA) at 30 °C. E. coli OP50 and P. freudenreichii were collected by centrifugation and washed twice with M9 buffer. Then, the bacteria were diluted to a final concentration of 0.1 mg (wet weight) per mL in M9 buffer37 .

Participants collected stool at home 12–24 h before the baseline and end of study visits using an anaerobic collection kit (BD Gaspak, Becton Dickinson and Company, Sparks, MD)^{66 (link)}. Blood was collected at baseline and end of the study and was processed for serum and plasma within one hour of collection and was stored at −80 °C until analysis.

For induction of hypoxia, shPLC-γ1 and shSCR transduced kasumi-1 cells were kept in an air tight with a pouch (Gaspak, Becton Dickinson) without oxygen for 48h at 37°C as described previously [45 (link)]. The rate cell proliferation was measured by absolute cell counting after 48h using a Coulter Counter (Beckman Coulter, Fullerton, CA, USA) to assess cell growth under hypoxia and normoxia.

Valve culture proceeded according to the Duke Criteria and microbiological routine standards [15 (link)]. Columbia, chocolate, and MacConkey agars were incubated at 37 °C in 5% CO₂ for 24 to 48 h. Schaedler and neomycin-vancomycin agars were incubated at 37 °C in an anaerobic chamber (GasPak; Becton, Dickinson, Franklin Lakes, NJ) for 48 h. Chromogenic Candida agar was incubated at 37 °C in 5% CO₂ for 24 to 48 h. Plates were reviewed after 24 and 48 h. Samples with no growth were further incubated under the same conditions for 120 h. Colonies were identified by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) (Bruker Daltonics, Billerica, USA). Antibiotic susceptibility was determined routinely by Vitek 2 (bio-Mérieux, Marcy l’Étoile, France) using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines for interpretation. If different microorganisms were detected on the same valve, these were all represented in the results.

Eleven fecal samples were donated by healthy Estonian adult subjects (mean age 32 ± 12 years).
The fecal samples were collected into sterile 25 ml feces sampling containers (APTACA, Italy) and kept under anaerobic environment in a gas generating pouch system (GasPak™, Becton Dickinson & Co, USA) at -20°C. Frozen samples were delivered to the laboratory within 48 h and stored at -80°C until processing (no longer than three weeks). The storage cultures were prepared aseptically in a glove box flushed with nitrogen gas. The stool sample was diluted five times with sterile anaerobic PBS (mM): NaCl (160), KCl (3), Na₂HPO₄ (8), NaH₂PO₄ (1), containing 5% dimethylsulfoxide (DMSO) (vol/vol), pH 7.2, supplemented with freshly made and filter sterilized Cys-HCl (0.05% in final solution). The diluted samples were stored at -80°C as aliquots (5 ml) sufficient to start a cultivation experiment.
Levan was produced from sucrose using Lsc3 from P. syringae DC3000 and precipitated with ethanol as shown previously in Adamberg et al. [17 (link)]. To remove low molecular weight compounds including mono- and disaccharides, levan was dissolved in MQ water and dialyzed for 36 h at room temperature in membrane tubing of 20 kDa molecular weight cut-off against sterile milliQ water. The dialyzed levan preparation was freeze-dried (VirTis freeze dryer, SP Industries Inc., Warminster, USA).