Diagnosis and Stratification of Pseudomonas aeruginosa Infected Patients by Immunochemical Quantitative Determination of Pyocyanin From Clinical Bacterial Isolates

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.

Combining hyperspectral imaging and electrochemical sensing for detection of Pseudomonas aeruginosa through pyocyanin production

“…43 This has been linked to P. aeruginosa exoproduct synthesis being heightened to establish infection during acute phases, 31 further evolving towards a decrease at chronic stages. 56 Overall, pyocyanin production trends were in agreement with previous reports. 48,50,57…”

Label-free multimodal imaging of infected Galleria mellonella larvae

“…This is because the implants are thicker than the tissue section. The strong bacterial TPEF signal was from the pyocyanin pigment (excitation/emission at 360/460 nm 63 , 64 ), known to be produced by P. aeruginosa , and this was confirmed by imaging bacterial suspension samples 65 , 66 . The TPEF signal observed in Fig.…”

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

“…To explain these data more, QS biomarkers were analyzed. In this sense, the same group had measured PYO levels in the same bacterial isolates reporting high levels of this virulence factor in most of them, presenting undetectable HQNO levels ( 49 ). This would indicate a possible correlation between the levels of both QS molecules, which is a very interesting aspect that should be studied in more detail.…”