Gaspak jar

After serial dilutions of the samples stomached 1 min in BPW (Buffered Peptone Water, bioMerieux, Marcy l’Etoile, France), decimal dilutions of the samples were plated and incubated in appropriate conditions: for aerobic bacteria, soy agar plates were incubated at 37 °C for 24 h; for LAB, MRS (deMan Rogosa Sharpe; Difco) agar plates were incubated in a GasPak jar (BBL, Becton Dickinson Microbiology Systems) at 30 °C for 48 h, complemented with a gas generating sachet (BD GasPak EZ Gas Generating Sachet). For enumeration of O157_EDL933 and O26₂₁₇₆₅ strains, dilutions were spread respectively onto ChromID O157:H7 agar (ChromID O157:H7 + 0.05 mg/l cefixime +2.5 mg/l tellurite, bioMérieux) and chromogen agar E. coli Brillance. The plates were incubated at 37 °C for 24 h. All counts were performed in duplicate.

The same minimal medium that was used for growth of the planktonic nitrate-reducing cultures was supplemented with 1% agar, pH adjusted to 10, flushed with N₂ for 30 min, autoclaved and poured into petri dishes inside a laminar flow cabinet. After solidification of the agar, the plates were transferred into an anaerobic chamber and left upside down for 4 days. A subsample from the last subculture of the nitrate-reducing culture (the same one that was used for pyrosequencing studies) was spread onto the plates with a sterile disposable plastic spreader. The inoculated plate and a control plate were put in a sealed GasPak jar (Becton Dickinson, Franklin Lakes, NJ, USA), closed and incubated at 20 °C under anaerobic conditions. After 3 days, small transparent colonies were observed and five representative colonies were isolated using a sterile 5 μl microbiology loop and transferred to 20 ml of sterile minimal medium supplemented with 2 mM Ca(ISA)₂ and 24 mM NaNO₃. These bottles were incubated at 20 °C for 10 days, during which they were monitored for bacterial growth, pH change, ISA degradation and nitrate reduction. After the stationary growth phase was reached, 10 ml of the liquid culture was centrifuged at 4000 g for 10 min and the pellet was used for DNA extraction and microbial identification using Sanger sequencing of PCR amplified 16S rRNA gene.

To test sensitivity to Zn in disc diffusion assays, 25 μl of exponentially grown cultures (OD₆₀₀ ~ 0.5) were spread onto agar plates using a sterile swab and topped with sterile Whatman filter paper discs (6-mm diameter) saturated with 20 μl of a 1 mM ZnSO₄ solution. The diameter of the zone of growth inhibition was measured after 24 h of incubation at 37°C in 5% CO₂. The minimum inhibitory concentration (MIC) of ZnSO₄ was determined by a broth microdilution method using two-fold serial dilutions of ZnSO₄. Plates were incubated at 37°C in 5% CO₂ for 16 h and the concentration of ZnSO₄ at which the absorbance values were 10% of the control condition was determined to be the MIC. For plate titration assays, exponentially grown BHI cultures (OD₆₀₀ ~ 0.5) were serially diluted and 8 μl of each 10-fold dilution spotted on BHI plates supplemented with selected trace metals. Plates were photographed after 24 h incubation at 37°C in 5% CO₂ or under anaerobiosis (GasPak jar, BD Biosciences).