Api 20c aux kit

The isolates were identified using the API 20C AUX kit (Biomérieux, Marcy l’Etoile, Rhône, France) and maintained in Sabouraud dextrose agar at 4°C.
For the experiments, the microorganisms were plated in brain heart infusion (BHI) agar (Himedia, Mumbai, Maharashtra, India) and cultured for 24 h. A suspension was prepared in 0.9% saline solution (NaCl; Vetec, Duque de Caxias, RJ, Brazil), according to the McFarland 0.5 scale (approximately 5x10⁶ colony forming units (CFU)/ml).
The next step was to inoculate 100 mL of the suspensions in 10 mL of BHI broth (Himedia, Mumbai, Maharashtra, India) supplemented with PIPES buffer (1,4-piperazinediethanesulfonic acid; Sigma Aldrich, Saint Louis, Missouri, USA) and 0.2% glucose (Vetec, Duque de Caxias, RJ, Brazil). For the analysis of the production of acids, extracellular polysaccharides and proteins, biofilms were grown for five days in the bottom of 24-wells plates (TPP, Trasadingen, Schaffhausen, Switzerland) containing 3 mL of the inoculum. For the metabolic activity test, biofilms were grown in the bottom of 96-wells plates (TPP, Trasadingen, Schaffhausen, Switzerland) containing 200 µL of the inoculum. The culture medium was renewed once a day by carefully removing the old culture medium and adding a fresh one. All incubations were performed at 37°C in aerobiosis, and all experiments were performed in duplicate.

At the central laboratory, 50 ml of PDE was centrifuged at 3500g for 15 minutes, discarding the supernatants. The remaining solution (about 5 ml) was mixed up with pellet and injected into Bactec Plus Aerobic/F vial (Dun Laoghaire, Ireland) and was also spread onto several agar plates, including blood agar, MacConkey agar (Oxoid, Basingstoke, United Kingdom), and Chocolate agar (Oxoid, Basingstoke, United Kingdom) for 7 days at 37 °C for bacterial culture. For fungal culture, the pellet from another 50 ml of centrifuged PDE was streaked on Sabouraud dextrose agar, blood agar (Oxoid), and specific agar plates (as needed) then incubated at 25 °C and 37 °C for 15 to 30 days. Bacterial pathogens were identified by Gram stain and Vitek MS system (bioMérieux, USA),⁷ (link) whereas fungi were identified by API20c AUX kit (bioMérieux, Marcy l’Etoile, France) based on biochemical reactions and stained by Lactophenol Cotton Blue technique to classify mold-form fungi based on the morphology of their sexual spores and conidia. To exclude coincidental infection with Mycobacterium organisms, the pellet from an additional 50 ml PDE was inoculated in Ogawa medium slants and BACTEC MGIT 960 media for 2 months.⁸ (link)