Bacterial Adhesion and Invasion Assay

The adhesion and invasion test was performed as described previously (Wang et al., 2018 (link)). The human bladder carcinoma 5,637 cells were cultured in Roswell Park Memorial Institute (RPMI) medium, supplemented with 10% fetal bovine serum (FBS) and 0.1% (w/v) urea, at 37°C and 5% CO₂ and inoculated (2 × 10⁵ cells/well) in a 24-well plate. The UTI-ST1 and UTI-ST5 isolates were cultivated in TSB, with or without urea supplementation, to the mid-logarithmic growth phase and washed twice with PBS. Thereafter, the epithelial cells were infected with the UTI isolates at a 1:10 ratio. The adhesion assay was performed by co-incubating bacterial cells and epithelial cells for 2 h at 37°C and 5% CO₂. The cells were washed three times with PBS to remove the planktonic bacteria and lysed with 0.1% sodium deoxycholate (Sangon) to release the adhered bacteria. Bacterial CFU was determined by serial dilutions of epithelial cell lysates on TSA plates. For the invasion test, the cells were incubated with bacteria for 4 h, and gentamicin (100 μg/mL) was added to the culture for 30 min to digest the bacteria outside the cells. Thereafter, the bacterial CFU was enumerated to assess the invasion ability.