Abstract
The development of a highly sensitive, specific, and reliable immunochemical assay to detect pyocyanin (PYO), one of the most important virulence factors (VFs) of Pseudomonas aeruginosa, is here reported. The assay uses a high-affinity monoclonal antibody (mAb; C.9.1.9.1.1.2.2.) raised against 1-hydroxyphenazine (1-OHphz) hapten derivatives (PC1; a 1:1 mixture of 9-hydroxy- and 6-hydroxy-phenazine-2-carobxylic acids). Selective screening using PYO and 1-OHphz on several cloning cycles allowed the selection of a clone able to detect PYO at low concentration levels. The microplate-based ELISA developed is able to achieve a limit of detection (LoD) of 0.07 nM, which is much lower than the concentrations reported to be found in clinical samples (130 μM in sputa and 2.8 μM in ear secretions). The ELISA has allowed the investigation of the release kinetics of PYO and 1-OHphz (the main metabolite of PYO) of clinical isolates obtained from P. aeruginosa-infected patients and cultured in Mueller–Hinton medium. Significant differences have been found between clinical isolates obtained from patients with an acute or a chronic infection (~6,000 nM vs. ~8 nM of PYO content, respectively) corroborated by the analysis of PYO/1-OHphz levels released by 37 clinical isolates obtained from infected patients at different stages. In all cases, the levels of 1-OHphz were much lower than those of PYO (at the highest levels 6,000 nM vs. 300 nM for PYO vs. 1-OHphz, respectively). The results found point to a real potential of PYO as a biomarker of P. aeruginosa infection and the possibility to use such VF also as a biomarker for patient stratification[2] and for an effective management of these kinds of infections.
Highlights
Pseudomonas aeruginosa is a common Gram-negative opportunistic multidrug-resistant pathogen that causes acute and chronic infections especially in immunocompromised patients (Barbier et al, 2013; Pieters et al, 2019)
We reported for the first time the development of polyclonal antibodies and microplatebased ELISA for 1-hydroxyphenazine (1-OHphz), the main metabolite of PYO, with an excellent detectability (IC50 = 0.53 nM) (Pastells et al, 2016)
Aiming at simplifying the procedure, we attempted to accomplish a high-affinity monoclonal antibody (mAb) against PYO using the hybridoma technology that allows selecting cell clones with tailored features based on a rational selective screening approach
Summary
Pseudomonas aeruginosa is a common Gram-negative opportunistic multidrug-resistant pathogen that causes acute and chronic infections especially in immunocompromised patients (Barbier et al, 2013; Pieters et al, 2019) It is one of the main pathogens causing nosocomial infections such as hospital-acquired pneumonia (HAP), health care-associated pneumonia (HCAP), ventilator-associated pneumonia (VAP), and ventilator-associated tracheobronchitis (Williams et al, 2010), contributing to a high mortality and morbidity (Gaynes et al, 2005; Sadikot et al, 2005; Pang et al, 2019). Defective expression of CFTR induces mucus hypersecretion that obstructs airways and triggers morbidity and mortality in CF patients (Puchelle et al, 2002)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
