Quantifying Membrane Alterations with Tailored Fluorescent Dyes: A Rapid Antibiotic Resistance Profiling Methodology

Area of Science:

• Microbiology
• Biophysics
• Biochemistry

Background:

• Accurate detection of bacterial antibiotic sensitivity is vital for managing antibiotic resistance and guiding theranostic strategies.
• Quantifying antibiotic-induced changes in bacterial membranes and their link to resistance mechanisms remains a significant challenge.
• Current methods struggle to precisely measure subtle, subnanometer alterations in membrane structure.

Purpose of the Study:

• To develop and validate novel fluorescent probes for quantifying antibiotic-induced bacterial membrane damage.
• To correlate membrane alterations with bacterial antibiotic sensitivity and resistance.
• To assess the applicability of these probes for clinical pathogen detection.

Main Methods:

• Utilized novel 4-aminophthalimide (4AP)-based fluorescent dyes (4AP-C9, 4AP-C13) with environment-sensitive properties.
• Employed single-cell imaging to monitor dye positioning and fluorescence changes within bacterial membranes.
• Quantified antibiotic-induced membrane damage via the peak maxima difference ratio (PMDR) and correlated it with lipid peroxidation.

Main Results:

• 4AP-Cn dyes accurately detected stress-mediated alterations in *E. coli* membranes at a subnanometer scale.
• The PMDR of the dyes correlated with cytoplasmic membrane damage and lipid peroxidation, quantifying antibiotic sensitivity.
• The method demonstrated effectiveness on clinical isolates of *E. coli*, *K. pneumoniae*, and *Enterobacter* species.

Conclusions:

• 4AP-Cn probes serve as precise nanoscale scales for detecting antibiotic-induced membrane thinning.
• These probes show significant promise for the rapid and specific detection of bacterial antibiotic resistance in clinical diagnostics.
• The developed method can distinguish between sensitive and resistant bacterial infections with high specificity.