Bioimaging With Fluorescent Nucleic‐Acid Aptamers for the Specific Detection and Quantification of <i>Pseudomonas aeruginosa</i> Alone and in Heterogeneous Bacterial Populations

Abstract

Aptamers, short nucleic acid sequences with high specificity and affinity for their targets, are promising candidates for diagnostic applications due to their ability to detect a wide range of pathogens. We present a fluorescent bioimaging approach for detecting Pseudomonas aeruginosa, based on aptamer F23. Conjugated with fluorescent dye, its detection efficacy was evaluated on 15 Gram-negative and -positive bacteria, including fixed and live cells, as homogeneous and heterogeneous populations. We developed an automated, open-access software for quantifying microscopy images. Its high sensitivity enables accurate quantification of bacteria labeled with aptamers. For example, it successfully detected 1122 P. aeruginosa cells labeled with aptamer F23 out of a total of 1123 P. aeruginosa cells in a single image. With 200,000 analyzed bacteria, we demonstrated that the aptamer effectively detects various reference and clinical strains of P. aeruginosa, while failing to detect Gram-positive Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, and Corynebacterium striatum, as well as Gram-negative Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli. This aptamer is therefore a promising tool to distinguish P. aeruginosa from different strains of the skin microbiota. However, our quantitative method also revealed partial labeling to other bacterial cells, highlighting the issue of refining aptamer selection to improve selectivity.