Bioimaging with fluorescent nucleic-acid aptamers for the specific detection and quantification of Pseudomonas aeruginosa alone and in heterogeneous bacterial populations

The rising prevalence of bacterial infections, antibiotic resistance, and emerging pathogens underscores the urgent need for innovative diagnostic approaches. 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. In this study, we present a fluorescent aptamer-based bioimaging approach for detecting Pseudomonas aeruginosa, a multidrug-resistant pathogen of significant clinical concern. Conjugated with fluorescent dye, the detection efficacy of the F23 aptamer was evaluated on 15 different Gram-negative and Gram-positive bacteria, including fixed and lived cells, as well as homogeneous and heterogeneous population. To quantify microscopy images, we developed an automated, open-access identification software using ImageJ. Its high sensitivity provides a robust platform for accurately quantifying bacteria labeled with aptamers and potentially other fluorescent ligands. For instance, 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 almost 200,000 analyzed bacteria and an exceptionally clear signal-to-noise ratio, we demonstrated that the F23 aptamer effectively detects various reference and clinical strains of Pseudomonas aeruginosa, while failing to detect Gram-positive Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis and Corynebacterium striatum, as well as Gram-negative Klebsiella pneumonia, Acinetobacter baumannii, and Escherichia coli. The F23 aptamer is therefore a promising tool to distinguish Pseudomonas 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.