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

Area of Science:

• Microbiology
• Pathogenesis
• Cell Biology

Background:

• Epithelial cell invasion is crucial for enteric pathogens like Salmonella, Shigella, and Vibrio.
• Adherent-invasive Escherichia coli (AIEC) is an emerging pathogen linked to inflammatory bowel disease (IBD) pathogenesis.
• AIEC strains lack common virulence factors, complicating genetic identification and relying on phenotypic assays.

Purpose of the Study:

• To address the unreliability of gentamicin protection assays due to gentamicin-resistant AIEC isolates from IBD patients.
• To develop and validate an alternative amikacin-based protection assay for quantifying and visualizing AIEC cell invasion.
• To establish a reliable diagnostic tool for screening IBD patients for AIEC and assessing multidrug-resistant pathogen virulence.

Main Methods:

• Developed an amikacin-based protection assay to quantify AIEC invasion.
• Utilized amikacin, an extracellular antibiotic effective against aminoglycoside-resistant strains.
• Employed colony-forming-unit (CFU) counts for quantitative invasion scoring and confocal microscopy for qualitative visualization.

Main Results:

• The amikacin assay effectively quantifies AIEC strain invasiveness.
• Confocal microscopy with immunostaining provides a qualitative measure of AIEC invasion.
• The assay is a viable alternative for classifying AIEC, especially gentamicin-resistant strains.

Conclusions:

• The amikacin-based protection assay is a reliable method for assessing AIEC cell invasion.
• This assay overcomes limitations posed by antibiotic resistance in clinical isolates.
• The method can be broadly applied to evaluate the virulence of other multidrug-resistant pathogens.