A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria

Background: Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells. Results: Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R2), ranging from 0,92 to 0,99. Conclusions: The ICW assay offers several advantages including sensitivity, reproducibility, high speed, operatorindependent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches.