Quantification of Bacterial Adherence and Invasion in Human Bronchial Epithelial Cells

Human bronchial epithelial 16HBE14 cells (Gruenert et al., 1988 (link)), kindly provided by Dr. Kirsten Spann (Queensland University of Technology), were seeded in Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (sMEM) at an approximate density of 2 ^∗ 10⁵ cells/ml into 24-well culture dishes (Greiner Bio-One, Kremsmünster, Austria) (1 ml/well) for adherence and invasion assays or into 175-cm² flasks (Corning, Tewksbury, MA, United States) (40 ml/flask) for maintenance. Confluent 16HBE14 monolayers were washed once with prewarmed sMEM and then infected with Hi2019^WT, Hi2019^ΔdmsA, or Hi2019^ΔdmsA_c diluted in sMEM to 2 ^∗ 10⁷ bacteria/ml, giving a multiplicity of infection (MOI) of 1:100 (epithelial cells: bacteria). Bacterial adherence and invasion were determined as described previously (Dhouib et al., 2015 (link), 2016 (link)). Determination of intracellular bacteria used a standard gentamicin-protection assay (St Geme and Falkow, 1990 (link)). In brief, infected cells were washed to removed planktonic and loosely adherent bacteria and incubated for 1 h in sMEM containing 50 μg/ml gentamicin followed by saponin lysis and serial dilution for determination of bacterial CFU present. Assays used at least three biological replicates; replicate experiments were carried out on different days.