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

Meysam Sarshar,Marisa Di Pietro,Simone Filardo,Cecilia Ambrosi,Daniela Scribano,Giulia Tranquilli,Anna Teresa Palamara,Rosa Sessa,Carlo Zagaglia

doi:10.1186/s12866-019-1625-1

Abstract

BackgroundQuantification 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.ResultsHerein, 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.ConclusionsThe ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent 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.

Highlights

Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections
In the early phase of S. flexneri (1 h post-infection (HPI)) and late phase of E. coli (72 HPI) infections, the In-Cell Western (ICW) technique detected a number of bacteria ranging from 5 × 104 and 1 × 103 bacteria/ml, respectively, indicating a Quantification of the obligate intracellular C. trachomatis L2 strain 434/Bu we evaluated the applicability of the ICW technique for determining the number of Inclusion forming units (IFU) of the obligate intracellular bacterium C. trachomatis strain 434/ Bu
In summary, the automatic, fast, reliable and highthroughput laser scan-based technique ICW assay was applied for the first time for quantifying intracellular bacteria in in vitro infection models

Summary

Introduction

Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Some bacterial pathogens have evolved a complex interplay with the host mediated by bacteria-derived factors These factors enable bacteria to colonize and invade host cells as well as evade host immune systems, thereby representing the hallmark of pathogenesis [1,2,3,4,5,6]. These techniques are mainly used for the quantification of obligate intracellular pathogens, such as Chlamydia trachomatis This is usually achieved by counting the number of membrane-bound vacuoles (inclusions), expressed as chlamydial inclusion forming units (IFUs), using direct immunofluorescence [18, 22, 23]. It provides high sensitivity and specificity, this method is time-consuming, tedious and requires extensive operator’s technical skills.

Methods

Results

Conclusion