Quorum Sensing as Potential Biomarker Targets to Diagnose Bacterial Infections

Abstract

[eng] Pseudomonas aeruginosa is a Gram-negative bacterium that causes infections in individuals with weakened immune systems, such as those with cystic fibrosis. This opportunistic pathogen is common in infections acquired in hospital and community settings, with multidrug-resistant strains exacerbating the situation. P. aeruginosa's ability to adapt and evade host defenses makes it a global health issue, further complicated by the lack of effective therapeutic options. Diagnosing bacterial infections remains difficult due to the slow pace of traditional culture methods, leading to the unnecessary use of broad-spectrum antibiotics. This contributes to the rise in antimicrobial resistance, worsening patient outcomes. This issue is especially relevant in P. aeruginosa infections, where delayed or incorrect treatments can worsen the situation and promote the spread of resistant strains. One of the main factors behind P. aeruginosa's pathogenicity is its bacterial communication system, known as Quorum Sensing (QS), which regulates gene expression and bacterial behavior based on cell density. QS controls processes like biofilm formation, virulence factor production, and antibiotic resistance. This reliance on QS makes it a potential target for new diagnostic and treatment strategies. This doctoral thesis proposes the development of novel diagnostic tools to improve the detection of P. aeruginosa infections, based on the hypothesis that QS signaling molecules (QSsm) can serve as useful biomarkers for early diagnosis and monitoring of these infections. The project developed high-affinity antibodies to detect key QSsm and virulence factors (VF) of the bacterium. ELISA assays were created to detect these molecules in complex clinical samples, such as sputum from patients with lower respiratory tract infections (LRTI) and urinary tract infections (UTI). The results include the successful development of monoclonal antibodies against PQS, a key molecule in P. aeruginosa's Pqs QS system. A specific and sensitive ELISA was designed with a detection limit below 1 nM, demonstrating robustness in various conditions. Additionally, polyclonal antibodies against autoinducers of the Las and Rhl QS systems were developed, achieving good detection levels. Immunochemical assays to analyze sputum samples were complex due to the variability in sputum composition, but they ultimately enabled the detection of PQS and HHQ, two important molecules of the Pqs QS system, in samples from LRTI patients. Furthermore, the analysis of bacterial isolates from LRTI and UTI patients showed clear differences in the QS molecular signature between acute and chronic infections. Thresholds were identified that could distinguish these types of infections, with important implications for diagnosis and treatment. It was also observed how the QS molecular signature evolves in chronic infections, although no correlation with the patient's clinical status could be established due to lack of access to medical records. Another significant finding was the correlation between QSsm and VF levels with antibiotic resistance patterns, suggesting that QS profiles could predict resistance and guide more precise treatments. HQNO was also identified as a promising biomarker for detecting bacteremia in urinary infections, though further studies are needed to validate its clinical use. In summary, this thesis demonstrates the potential of QS biomarkers in P. aeruginosa to improve early diagnosis and infection management, allowing for more personalized and effective treatments. It is suggested that future research delves deeper into the dynamics of these biomarkers and explores their broader clinical applications.

[spa] Pseudomonas aeruginosa es una bacteria gramnegativa conocida por causar infecciones, especialmente en individuos inmunocomprometidos o con condiciones subyacentes, como fibrosis quística. Este patógeno oportunista es común en infecciones adquiridas en el entorno hospitalario y en la comunidad, con cepas multirresistentes que complican los casos graves y persistentes. La carga global de P. aeruginosa se agrava por la limitada efectividad de las terapias convencionales. El diagnóstico bacteriano es complejo debido a la lentitud de los métodos tradicionales de cultivo, lo que retrasa el tratamiento y conlleva el uso de antibióticos de amplio espectro, impulsando la resistencia antimicrobiana. Esto es especialmente problemático en las infecciones por P. aeruginosa, donde un tratamiento tardío o inadecuado empeora los resultados clínicos y fomenta la propagación de cepas resistentes. Un factor clave en la patogenicidad de P. aeruginosa es su sistema de Quorum Sensing (QS), que regula la expresión génica y el comportamiento bacteriano según la densidad poblacional. El QS controla procesos como la formación de biopelículas, la producción de factores de virulencia y la resistencia a los antibióticos, lo que lo convierte en un objetivo prometedor para nuevas estrategias diagnósticas y terapéuticas. Esta tesis doctoral se centra en el desarrollo de herramientas diagnósticas para mejorar la detección y manejo de infecciones por P. aeruginosa. La hipótesis principal es que las moléculas de señalización del QS (QSsm) pueden servir como biomarcadores para un diagnóstico temprano. El proyecto desarrolló anticuerpos de alta afinidad para detectar QSsm clave y factores de virulencia, creando ELISAs para su detección en muestras clínicas como esputo y orina. Entre los principales hallazgos se incluye el desarrollo de anticuerpos monoclonales contra el PQS, un QS crucial, y la creación de ELISAs con alta especificidad y sensibilidad, además de correlaciones entre perfiles de QS y patrones de resistencia antibiótica, lo que sugiere potenciales terapias dirigidas.