Campygen envelopes

Each H. pylori strain was co-cultured with each Candida spp. strain. A suspension of each strain of each kind of microorganism was independently prepared in 0.89% saline solution (SS) and adjusted to an OD of 0.1 at 600 nm. Then, 500 µL of each suspension of the bacterial strains were placed in wells of 12-well plates (Thomas Scientific, Swedesboro, NJ, USA) previously loaded with 4 mL de BB-5%FBS. Then, 500 µL of yeast cells from each strain were independently added to wells previously inoculated with H. pylori. Four wells were required for each bacterial strain in order to be independently co-cultured with each of the yeast strains. Plates containing the co-cultures were incubated under microaerobic conditions for 48 h at the following temperatures: 4 °C, 20 °C, 37 °C or 40 °C. Microaerobiosis atmosphere was achieved using CampyGen envelopes (Thermo Scientific, Waltham, MA, USA). All assays were repeated thrice. As controls for this assay, pure inocula of all H. pylori and Candida strains were used.

In order to make sure to start the co-cultures of H. pylori and Candida strains when both microorganisms were in their respective exponential phase of growth, this assay was necessary to determine the incubation time required by each microorganism to reach the desired phase of growth. Each strain of either H. pylori or Candida spp. used in the present work was suspended at an optical density (OD) of 0.1 at 600 nm in Brucella broth (BB) (Difco, Wokingham, UK) supplemented with 5% FBS (BB-5%FBS), and 200 µL of each suspension were placed in a well of 96-well plates (Thomas Scientific, Swedesboro, NJ, USA) and plates incubated at 4 °C, 25 °C, 37 °C or 40 °C in an Infinite M200 PRO equipment (TECAN, Männedorf, Switzerland), in which the microaerobic conditions were obtained using CampyGen envelopes (Thermo Scientific, Waltham, MA, USA). The growth of each strain was monitored by OD at 600 nm every 8 h during 72 h for the bacterial strains and every 2 h during 50 h in the case of yeast strains. Additionally, in the case of H. pylori, 5 µL aliquots were obtained at each time OD was measured, and a Gram-staining was prepared to monitor possible morphological changes in bacterial cells caused by the incubation conditions.