4.8. Chemotaxis Assay

Chemotaxis assays were carried as described by Mazumder [41] and revised by Cerda et al. [28]. Bacteria were grown as described above, collected from the plate and inoculated in BHI broth supplemented with 10% FBS, starting from 0.8 OD₆₀₀. After 24 h, bacteria were diluted in chemotaxis buffer (10 mM potassium phosphate, pH 7.0; 3.0% polyvinylpyrrolidone) up to a concentration of 4 × 10⁷ bacteria/mL (0.4 OD₆₀₀) and 100 μL of bacterial suspension was drawn into a disposable 200 μL pipette tip. A 100 μL volume of solution to be tested (10 mM HCl, hrTFF1 6 μg/mL, hrTFF1 0.6 μg/mL diluted in chemotaxis buffer) was drawn up into a 1-mL tuberculin syringe and a 23 G × 1¼ stainless-steel needle (0.6 × 30 mm) was used as the chemotaxis capillary. Chemotaxis buffer alone was included as control. The needle-syringe system was positioned into the pipette tip and incubated horizontally at 37 °C under microaerophilic conditions for 45 min. At the end of incubation, bacteria recovered from the syringe were appropriately diluted and plated onto Columbia Blood Agar (Oxoid, Basingstoke, Hampshire, UK) plates supplemented with 10% FBS (Euroclone) and DENT (Oxoid, Basingstoke, Hampshire, UK). Colonies were counted after 4–5 days of incubation in capnophilic atmosphere with 10% CO₂. The experiment was repeated three independent times in technical duplicates.

The experiment was similar to the above described except for starting bacteria that were diluted up to a concentration of 1 × 10⁷ bacteria/mL (OD₆₀₀ = 0.1) and preincubated for 1.5 h w/w hrTFF1 (6 μg/mL), in order to favor the formation of aggregates.

Proper dilutions of the syringe content were plated for CFU (colony forming unit) counting. The experiment was repeated three independent times in technical duplicates.