Gaspak system

The colony morphology of the different S. aureus isolates was analyzed after plating of serially diluted stationary cells on Columbia blood agar and overnight incubation at 37°C. The colony size was quantified using the ImageJ Software (92 (link)). Growth of S. aureus at different temperatures was carried out as described earlier (93 (link)). Briefly, TSB was inoculated with 1% of a S. aureus overnight preculture and incubated at 37°C until reaching an optical density at 600 nm wavelength (OD₆₀₀) of 0.5. Serial dilutions (0.9% NaCl, 10-fold dilutions, 10 μL each) of the culture were spotted on TSA plates. The agar plates were incubated at different temperatures until visible growth occurred, usually overnight. Anaerobic growth conditions were generated using the GasPak system (Becton, Dickinson, Heidelberg, Germany).

In MI, the occlusion of a coronary artery blocks the oxygen and nutrient supply to the myocardium it supplied, and causes severe stress as well as cell death. Transplanted cells face the same severe surroundings. SMCs or DFBs (n = 5 per each groups) were seeded on 35 mm dishes and were incubated for four days. Cells were then incubated in normal (37 °C, 5 % CO₂) or hypoxic conditions for forty-eight hours with 10 % FBS DMEM. Hypoxic conditions (<2 % O₂) were prepared using a Gaspak system (Becton Dickinson, Bedford, MA). The hypoxic condition was verified by the color of anaerobic indicator which was inside the jar in all experiments. Enzyme-linked immunosorbent assay kits for rat VEGF (Quantikine, R&D, Minneapolis, MN), human bFGF (R&D) and rat HGF (Institute of Immunology, Osaka, Japan) were used according to the manufacturer’s protocols.

L. reuteri strain MM4-1A (obtained from the American Type Culture Collection [ATCC] strain PTA-6475) was grown anaerobically in a Gas-Pak system (Becton Dickinson, Franklin Lakes, NJ, United States) in MRS medium (Hardy Diagnostics, Santa Maria, CA, United States) at 37°C for 24 h. Under sterile conditions, cells were concentrated by centrifugation, washed and resuspended in phosphate-buffered saline (PBS)/10% glycerol at an optical density at 600 nm (OD₆₀₀) of 80–100, and immediately frozen in 2 mL aliquots at −80°C. At least 1 frozen aliquot from each live preparation was thawed and plated to MRS agar (BD-Difco, Sparks, MD, United States) to confirm live L. reuteri at a concentration of 4.0E09-1.3E10 colony-forming units (cfu) per aliquot. Aliquots from 2 separate batches of L. reuteri were used to construct 16S rRNA gene V3-V4 amplicon libraries. These were sequenced and analyzed as described below and found to be composed of only L. reuteri sequences (Supplementary Figure S1). Heat-killed (HK) aliquots of L. reuteri were prepared as above, but were killed by incubation at 80°C for 2–3 h and re-frozen. At least one aliquot of killed cells for each batch was tested for complete killing by plating to MRS agar. Aliquots were stored at −80°C until the time of use.

Bacteroides thetaiotaomicron VPI‐5482 (ATCC # 29148) was grown anaerobically in a rich medium based on brain heart infusion with other supplements added (see Supplementary Materials and Methods). The genomic library was maintained in an Escherichia coli K‐12 strain, NEB Turbo (New England Biolabs, Ipswich, MA, USA). Escherichia coli strains were grown in Luria broth (LB) and supplemented with carbenicillin (final concentration 100 μg/ml) as needed. For anaerobic growth, an anaerobic jar (GasPak System, Becton Dickinson, Franklin Lakes, NJ) was used. Mouse chow (MC) filtrate was prepared by adding 150 ml deionized water to 8 g of crushed mouse chow (Mouse Breeding Diet 5021, LabDiet, St. Louis, MO, USA). The mixture was heated at 95°C for 30 min with mixing, passed through a 0.22‐μm filter, and autoclaved. The sterility of the MC filtrate was confirmed by incubating at 37°C in aerobic and anaerobic conditions and observing no growth after several days.

Vaginal fluids collected from healthy women (after informed consent) were plated onto lactobacilli selective medium, namely MRS-agar (Oxoid) and incubated in anaerobic conditions (Gas-Pak System; BBL, Becton Dickinson Biosciences) for 48 h at 37°C.
Microorganisms were maintained in MRS-broth as suspended culture (stabs) at −80°C using glycerol (20% w/v) as cryoprotectant. These stabs were used to inoculate pyrex bottles (250 ml) completely filled with culture media to study cell growth and lactic acid production under microaerofilic conditions over a period of 24–30 h at 37°C, in a rotary shaker (HT Aquatron, Infors, Switzerland) at 160 rpm. Experiments were performed by adding different carbon sources (20 g∙l⁻¹) to the semi-defined medium, SDM [38 (link)]: in particular fructose, sucrose, lactose, trehalose and dextrins were used alternatively to analyze how microbial growth and organic acids production were affected. Shake flask experiments were also performed adding sodium lactate (0–60 g∙l⁻¹) at increasing concentrations in the SDM, to evaluate strain growth inhibition.