0.45 μm pore size

To obtain the organic acid profile of selected bacteria, each strain was cultured anaerobically at 37°C in 1L fermentors (Biobundle; Applikon), in the MRS. For each strain, two conditions were assayed: with and without pH control. In both cases, pH levels were monitored during the assay. Samples were collected throughout the assay.
For organic acid quantification, 0.8 mL of cell-free supernatant was mixed by vortexing with 0.2 mL of a mixture containing 5% meta-phosphoric acid, copper sulfate (1.56 mg/mL) and 50 mM 4-methyl valeric acid as an internal standard. Samples were then filtered by 0.45 μm pore size (Millipore) and diluted 1/2 and 1/10 in MilliQ water.
Organic acids were evaluated by HPLC chromatography. An aliquot of 10 μL of processed samples was injected onto a HPLC Alliance 2695 (Waters) equipped with a Rezex column [ROA Organic Acid (H+) 8% 300 × 7.8 mm, 8 um [Premium]] under conditions defined by manufacturer. Detection was achieved by an index of refraction detector (2414 Waters). The eluent was degassed H₂SO₄ 2.5 mM at an isocratic flow rate of 0.6 mL/min. In all cases, quantification curves were constructed adding the mixed previously defined.

Plasmid conjugal transfer experiments were carried out with the rifampin-resistant E. coli EC600 or the sodium azide-resistant E. coli J53 being used as a recipient and each of the 14E509, 11011 and 61806 isolates as a donor. Three milliliters of overnight cultures of each of donor and recipient bacteria were mixed together, harvested and resuspended in 80 μl of Brain Heart Infusion (BHI) broth (BD Biosciences, NJ, United States). The mixture was spotted on a 1 cm² hydrophilic nylon membrane filter with a 0.45 μm pore size (Millipore, MA, United States) that was placed on BHI agar (BD Biosciences, NJ, United States) plate and then incubated for mating at 37°C for 12 to 18 h. Bacteria were washed from filter membrane and spotted on Muller–Hinton (MH) agar (BD Biosciences, NJ, United States) plates containing 1000 μg/ml rifampin or 200 μg/ml sodium azide together with 200 μg/ml ampicillin for selecting an E. coli transconjugant that carrying bla_CTX-M.