Anaerobic bag

Strain C1_020120 (representative strain of clone 1, see below) was subjected to a biofilm formation assay with strain 020130 (the closest strain of clone 1) and strain C2_020115 (representative strain of clone 2) used as controls under both aerobic and anaerobic conditions as described previously [58 (link), 59 (link)]. Briefly, bacterial cells were harvested from overnight cultures in LB broth by centrifugation at 2,500 r/min for 10 min and resuspended with saline and were adjusted to 0.5 McFarland standard. Aliquots (100 μl) were then pipetted into 96-well polystyrene culture plates and incubated for 3 h at 37 °C in an incubator or in an anaerobic bag (bioMérieux) to allow the formation of biofilms. The plates were washed twice with distilled water. Biofilms in the wells were fixed with 100 μl formalin per well for 15 min and were stained with 100 μl staining buffer containing 1% crystal violet for 5 min. The stained biofilms were washed again to remove the unbound stain and allowed to dry at room temperature. Biofilms were detected with 100 μl 30% glacial acetic acid by ELX800 Universal Microplate Reader (Bio-Tek, Winooski, VT, USA) at OD_{590 nm}. The biofilm formation assay was repeated a total of nine times for each strain.

Motility was examined using a CX21FS1 light microscope (Olympus, Tokyo, Japan). The Gram-staining reaction was performed as described previously (38 ). Growth at different temperatures (4, 15, 20, 25, 30, 35, 37, 45, and 50°C), at different pH values (3.0 to 12.0, at intervals of 1.0 pH unit), and at various salt concentrations (0 to 10% [wt/vol] NaCl) was determined in 15-ml test tubes containing 3 ml tryptic soy broth (TSB; Hopebio, Qingdao, China) after incubation for 2 days in a thermostatically controlled water bath as described previously (39 (link)). Anaerobic growth was performed by incubating cultures on nutrient agar for 7 days in an anaerobic bag (bioMérieux). Biochemical characteristics of the three strains were determined using the API 20E kit and API 50CH kit according to the manufacturer’s instructions (bioMérieux). Catalase activity was examined by bubble formation after dropping 3% (vol/vol) H₂O₂ on fresh biomass grown for 24 h on nutrient agar. Oxidase activity was determined using oxidase reagent (bioMérieux). All tests were carried out by incubating at 35°C unless indicated otherwise.