Amikacin Protection Assay for Quantification and Visualization of Escherichia coli Cell Invasion

Epithelial cell invasion is a key phenotype for many enteric pathogens, including Salmonella, Shigella, and Vibrio. An emerging invasive pathogen is adherent-invasive Escherichia coli (AIEC), which is implicated in the pathogenesis of inflammatory bowel disease (IBD). AIEC strains lack virulence factors commonly associated with other invasive pathogens, making genetic identification challenging. As a result, AIEC strains are classified phenotypically based on their ability to invade epithelial cells using a field-standard gentamicin protection assay. However, since AIEC strains are often isolated from IBD patients who receive various antibiotics as part of their treatment regimen, the rise of gentamicin-resistant isolates has made this assay less reliable for AIEC classification. To circumvent this issue, an alternative amikacin-based protection assay was developed to quantify and visualize AIEC cell invasion. This assay utilizes amikacin, a broad-spectrum semisynthetic aminoglycoside that effectively targets strains resistant to other aminoglycosides while remaining extracellular to eukaryotic host cells. Thus, extracellular bacteria are killed during infection while intracellular bacteria are "protected". This forms the basis of a quantitative assay to compare strain invasiveness, measured as the percent invasion score using colony-forming-units (CFU) recovered post-infection. This assay can also be used as a qualitative measurement of invasion by immunostaining infected epithelial cells and visualizing these cells through confocal microscopy. This method serves as a diagnostic tool to screen IBD patients for AIEC that can also be applied to other multidrug-resistant pathogens to assess cell invasion, a proxy for virulence and pathogenicity.