An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates.

Understanding quorum sensing (QS) and its role in the development of pathogenesis may provide new avenues for diagnosing, surveillance, and treatment of infectious diseases. For this purpose, the availability of reliable and efficient analytical diagnostic tools suitable to specifically detect and quantify these essential QS small molecules and QS regulated virulence factors is crucial. Here, we reported the development and evaluation of antibodies and an enzyme-linked immunosorbent assay (ELISA) for HQNO (2-heptyl-4-quinoline N-oxide), a QS product of the PqsR system, which has been found to act as a major virulence factor that interferes with the growth of other microorganisms. Despite the nonimmunogenic character of HQNO, the antibodies produced showed high avidity and the microplate-based ELISA developed could detect HQNO in the low nM range. Hence, a limit of detection (LOD) of 0.60 ± 0.13 nM had been reached in Müeller Hinton (MH) broth, which was below previously reported levels using sophisticated equipment based on liquid chromatography coupled to mass spectrometry. The HQNO profile of release of different Pseudomonas aeruginosa clinical isolates analyzed using this ELISA showed significant differences depending on whether the clinical isolates belonged to patients with acute or chronic infections. These data point to the possibility of using HQNO as a specific biomarker to diagnose P. aeruginosa infections and for patient surveillance. Considering the role of HQNO in inhibiting the growth of coinfecting bacteria, the present ELISA will allow the investigation of these complex bacterial interactions underlying infections. IMPORTANCE Bacteria use quorum sensing (QS) as a communication mechanism that releases small signaling molecules which allow synchronizing a series of activities involved in the pathogenesis, such as the biosynthesis of virulence factors or the regulation of growth of other bacterial species. HQNO is a metabolite of the Pseudomonas aeruginosa-specific QS signaling molecule PQS (Pseudomonas quinolone signal). In this work, the development of highly specific antibodies and an immunochemical diagnostic technology (ELISA) for the detection and quantification of HQNO was reported. The ELISA allowed profiling of the release of HQNO by clinical bacterial isolates, showing its potential value for diagnosing and surveillance of P. aeruginosa infections. Moreover, the antibodies and the ELISA reported here may contribute to the knowledge of other underlying conditions related to the pathology, such as the role of the interactions with other bacteria of a particular microbiota environment.

Direct Quantitative Immunochemical Analysis of Autoinducer Peptide IV for Diagnosing and Stratifying Staphylococcus aureus Infections.

An immunochemical strategy to detect and quantify AIP-IV, the quorum sensing (QS) signaling molecule produced by Staphylococcus aureusagr type IV, is reported here for the first time. Theoretical calculations and molecular modeling studies have assisted on the design and synthesis of a suitable peptide hapten (AIPIVS), allowing to obtain high avidity and specific antibodies toward this peptide despite its low molecular weight. The ELISA developed achieves an IC50 value of 2.80 ± 0.17 and an LOD of 0.19 ± 0.06 nM in complex media such as 1/2 Tryptic Soy Broth. Recognition of other S. aureus AIPs (I-III) is negligible (cross-reactivity below 0.001%), regardless of the structural similarities. A pilot study with a set of clinical isolates from patients with airways infection or colonization demonstrates the potential of this ELISA to perform biomedical investigations related to the role of QS in pathogenesis and the association between dysfunctional agr or the agr type with unfavorable clinical outcomes. The AIP-IV levels could be quantified in the low nanomolar range in less than 1 h after inoculating agr IV-genotyped isolates in the culture broth, while those genotyped as I-III did not show any immunoreactivity after a 48 h growth, pointing to the possibility to use this technology for phenotyping S. aureus. The research strategy here reported can be extended to the rest of the AIP types of S. aureus, allowing the development of powerful multiplexed chips or point-of-care (PoC) diagnostic devices to unequivocally identify its presence and its agr type on samples from infected patients.

Biological and clinical significance of quorum sensing alkylquinolones: current analytical and bioanalytical methods for their quantification.

Quorum sensing (QS) is a sophisticated bacterial communication system which plays a key role in the virulence and biofilm formation of many pathogens. The Pseudomonas aeruginosa QS network consists of four sets of connected systems (las, rlh, pqs and iqs) hierarchically organized. The pqs system involves characteristic autoinducers (AI), most of them sharing an alkylquinolone (AQ) structure, and is able to carry out several relevant biological functions besides its main signalling activity. Their role in bacterial physiology and pathogenicity has been widely studied. Indeed, the presence of these metabolites in several body fluids and infected tissues has pointed to their potential value as biomarkers of infection. In this review, we summarize the most recent findings about the biological implications and the clinical significance of the main P. aeruginosa AQs. These findings have encouraged the development of analytical and bioanalytical techniques addressed to assess the role of these metabolites in bacterial growth and survival, during pathogenesis or as biomarkers of infections. The availability of highly sensitive reliable analytical methods suitable for clinical analysis would allow getting knowledge about pathogenesis and disease prognosis or progression, supporting clinicians on the decision-making process for the management of these infections and guiding them on the application of more effective and appropriate treatments. The benefits from the implementation of the point-of-care (PoC)-type testing in infectious disease diagnostics, which are seen to improve patient outcomes by promoting earlier therapeutic interventions, are also discussed.

An Immunochemical Approach to Quantify and Assess the Potential Value of the Pseudomonas Quinolone Signal as a Biomarker of Infection.

Quorum sensing (QS) is a bacterial cell density-based communication system using low molecular weight signals called autoinducers (AIs). Identification and quantification of these molecules could provide valuable information related to the stage of colonization or infection as well as the stage of the disease. With this scenario, we report here for the first time the development of antibodies against the PQS (pseudomonas quinolone signal), the main signaling molecule from the pqs QS system of Pseudomonas aeruginosa, and the development of a microplate-based enzyme-linked immunosorbent assay (ELISA) able of quantifying this molecule in complex biological media in the low nanometer range (LOD, 0.36 ± 0.14 nM in culture broth media). Moreover, the PQS ELISA here reported has been found to be robust and reliable, providing accurate results in culture media. The technique allowed us to follow up the PQS profile of the release of bacterial clinical isolates obtained from patients of different disease status. A clear correlation was found between the PQS immunoreactivity equivalents and the chronic or acute infection conditions, which supports the reported differences on virulence and behavior of these bacterial strains due to their adaptation capability to the host environment. The results obtained point to the potential of the PQS as a biomarker of infection and to the value of the antibodies and the technology developed for improving diagnosis and management of P. aeruginosa infections based on the precise identification of the pathogen, appropriate stratification of the patients according to their disease status, and knowledge of the disease progression.

High-Throughput Immunochemical Method to Assess the 2-Heptyl-4-quinolone Quorum Sensing Molecule as a Potential Biomarker of Pseudomonas aeruginosa Infections.

Bacterial quorum sensing (QS) is being contemplated as a promising target for developing innovative diagnostic and therapeutic strategies. Here we report for the first time the development of antibodies against 2-heptyl-4-quinolone (HHQ), a signaling molecule from the pqs QS system of Pseudomonas aeruginosa, involved in the production of important virulent factors and biofilm formation. The antibodies produced were used to develop an immunochemical diagnostic approach to assess the potential of this molecule as a biomarker of P. aeruginosa infection. The ELISA developed is able to reach a detectability in the low nM range (IC50 = 4.59 ± 0.29 nM and LOD = 0.34 ± 0.13 nM), even in complex biological samples such as Müeller Hinton (MH) culture media. The ELISA developed is robust and reproducible and has been found to be specific to HHQ, with little interference from other related alkylquinolones from the pqs QS system. The ELISA has been used to analyze the HHQ production kinetics of P. aeruginosa clinical isolates grown in MH media, pointing to its potential as a biomarker of infection and at the possibility to use the technology developed to obtain additional information about the disease stage.