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

Mezouarhi, C.; Vauchelles, R.; Abdallah, B.; Janel, R.; Ouadghiri, M.; Ait Benhassou, H.; Fechter, P.; CHOULIER, L.

doi:10.1101/2025.02.07.636672

Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.02.07.636672

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

Mezouarhi, C.·Vauchelles, R.·Abdallah, B.·Janel, R.·Ouadghiri, M.·Ait Benhassou, H.·Fechter, P.·CHOULIER, L.

View original source ↗Export metadata ↓

Abstract

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.

Source connections

Published article ↗

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Mezouarhi, C., Vauchelles, R., Abdallah, B., Janel, R., Ouadghiri, M., Ait Benhassou, H., Fechter, P., CHOULIER, L.. 2025-02-07. Bioimaging with fluorescent nucleic-acid aptamers for the specific detection and quantification of Pseudomonas aeruginosa alone and in heterogeneous bacterial populations. https://doi.org/10.1101/2025.02.07.636672

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Matrix-controlled emergence of biofilm architecture shapes antimicrobial survival

Biofilms are structured microbial communities whose extracellular matrix is widely regarded as a basis of their protection against antimicrobial compounds. Yet how matrix production by individual bacteria gives rise to collective architecture and antimicrobial protection remains poorly understood. Here, we systematically varied expression of the master biofilm regulator csgD in Salmonella enterica and found that increasing matrix production reorganizes biofilms from dense, isotropic packings into sparse, nematically aligned communities by altering cell-cell interactions. By combining experimentally measured biofilm architectures with reaction-diffusion modeling, we show that these structural changes produce distinct patterns of antimicrobial killing, ranging from preferential killing near the liquid-biofilm interface to more uniform killing throughout the community. Consequently, increasing matrix production unexpectedly reduces antimicrobial survival by shifting the biofilm into different transport regimes, while strain-specific physiological differences further modulate antimicrobial depletion. Rather than acting as a passive barrier, EPS therefore shapes antimicrobial susceptibility by reorganizing biofilm architecture and its transport properties. EPS thus provides a physical link between molecular regulation, collective architecture and antimicrobial survival, providing a quantitative framework for understanding how cellular matrix production generates emergent biofilm function.

microbiology↗

bioRxiv2026

Mapping virulence-associated protein interaction networks reveals regulators of thermotolerance in Cryptococcus neoformans

Protein-protein interactions (PPIs) influence critical biological processes in pathogenic microorganisms, such as the human fungal pathogen, Cryptococcus neoformans. Fungal thermotolerance and stress response pathways are key virulence determinants that directly impact pathogen adaptation and survival and the infection process. To establish a comprehensive baseline of PPIs in C. neoformans and explore these interactions to infer functional roles for uncharacterized proteins, we applied size exclusion chromatography coupled with mass spectrometry to the secreted and cellular proteomes of the fungi. As a result, 216 and 1699 unique proteins were identified across 24 secretome and proteome fractions, respectively. The predicted secretome networks included expected proteins associated with vesicles and virulence, indicating a role in extracellular defense. Whereas the cryptococcal proteome highlighted interactions among proteins with defined roles in fungal virulence for protein stability and thermotolerance, including two previously uncharacterized proteins, CNAG_00287 and CNAG_05199, putatively involved in complex formation with heat-shock proteins (HSP). Based on sequence and structure homology, we propose that CNAG_00287 is a tetratricopeptide repeat-containing co-chaperone that modulates Hsp 70 activity and CNAG_05199 functions as a Hsp70. We validated the thermotolerance role of CNAG_00287 in heat-related stress, as its absence significantly impaired fungal growth in nutrient-limited media at 37 {degrees}C. Together, this work resolves virulence-associated PPIs within C. neoformans and reveals new molecular regulators of thermotolerance that underpin fungal pathogenicity.

microbiology↗

bioRxiv2026

Environmental filtering and host identity collectively shape root-associated microbiomes of Ericaceae and ectomycorrhizal plants in fumarole fields

Background Symbiosis with microbes is a key strategy that has enabled plants to colonize extreme environments. Since the benefits conferred by root-associated microbes depend on both environmental conditions and host-microbe combinations, plant adaptation to harsh environments is closely linked to the assembly of root microbial communities. Understanding how environmental and host filtering jointly shape these communities is therefore fundamental to elucidating the mechanisms underlying plant adaptation to extreme environments. Results In this study, we investigated the differentiation of root-associated prokaryotic and fungal communities and individual operational taxonomic units (OTUs) across two contrasting habitats surrounding fumaroles, solfatara-field and forest-edge habitats, and six dominant Ericaceae and ectomycorrhizal plant taxa. Prokaryotic and fungal OTUs rarely exhibited strong preferences for both habitat and host identity. Instead, many of prokaryotic and fungal OTUs specialized to one of these niches, collectively generating root microbial communities differentiated by both factors. Nonetheless, striking specializations in habitat and host niches were observed in the fungal family Hyaloscyphaceae (Helotiales). To gain insight into the evolutionary basis of microbial specialization, we examined phylogenetic signals in preference phenotypes. The resulting weak phylogenetic signals in these preference phenotypes further suggest that this fungal clade has undergone substantial ecological divergence. Conclusion Overall, our findings indicate that root-associated microbial communities in extreme environments are assembled through the accumulation of microbial taxa specialized to either habitat or host, and that strong ecological specialization in fungi can arise with little phylogenetic constraint.

microbiology↗