Molecular Signature of Pseudomonas aeruginosa with Simultaneous Nanomolar Detection of Quorum Sensing Signaling Molecules at a Boron-Doped Diamond Electrode

Abstract

Electroanalysis was performed using a boron-doped diamond (BDD) electrode for the simultaneous detection of 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline (HHQ) and pyocyanin (PYO). PQS and its precursor HHQ are two important signal molecules produced by Pseudomonas aeruginosa, while PYO is a redox active toxin involved in virulence and pathogenesis. This Gram-negative and opportunistic human pathogen is associated with a hospital-acquired infection particularly in patients with compromised immunity and is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. Early detection is crucial in the clinical management of this pathogen, with established infections entering a biofilm lifestyle that is refractory to conventional antibiotic therapies. Herein, a detection procedure was optimized and proven for the simultaneous detection of PYO, HHQ and PQS in standard mixtures, biological samples, and P. aeruginosa spiked CF sputum samples with remarkable sensitivity, down to nanomolar levels. Differential pulse voltammetry (DPV) scans were also applicable for monitoring the production of PYO, HHQ and PQS in P. aeruginosa PA14 over 8 h of cultivation. The simultaneous detection of these three compounds represents a molecular signature specific to this pathogen.

Electrochemical Characterizations of four Main Redox–metabolites of Pseudomonas Aeruginosa

“…On the other hand, Buzid et al. have reported the presence of three oxidation waves for PQS at + 0.759 V, + 1.103 V and + 1.590 V at pH 5 on boron doped diamond electrodes. As already reported for HHQ, the first wave may correspond to the one observed at GC electrodes with a potential decay due to the different pH and the different electrode material.…”

“…In the aforementioned pH range, the oxidation peaks potential shifts following a linear behavior (r = ‐0.998) with a slope of ‐54.5 ± 1.2mV/pH unit (Figure d). This value is close to the theoretical one given by the Nernst law of ‐59mV at 25 °C for an exchange of two protons and two electrons and close to previously reported values (‐55.1mV/pH unit ).…”

Direct and Rapid Electrochemical Detection of Pseudomonas aeruginosa Quorum Signaling Molecules in Bacterial Cultures and Cystic Fibrosis Sputum Samples through Cationic Surfactant‐Assisted Membrane Disruption

“…Without CTAB, the BDD electrode vs. Ag/AgCl in 50 mM acetate buffer, pH 5.0 containing 20 % ACN exhibits very low LOD values for PYO, HHQ, and PQS: 50 nM, 250 nM, and 250 nM, respectively as reported by Buzid et al . However, such corresponding values are only 0.15 μM, 0.62 μM, and 1.25 μM, when the analysis is performed in the CF sputum sample . The LOD values obtained for the above biomarkers are 2.06, 3.61, and 4.85 μM when the DPV measurement is performed in the LB medium with 1.0 mM CTAB (Table ), all well within the physiologically relevant range reported for CF sputum .…”

“…The resulting LOD values deserve a brief comment here since such values are highly dependent upon the electrolyte medium. Without CTAB, the BDD electrode vs. Ag/AgCl in 50 mM acetate buffer, pH 5.0 containing 20 % ACN exhibits very low LOD values for PYO, HHQ, and PQS: 50 nM, 250 nM, and 250 nM, respectively as reported by Buzid et al . However, such corresponding values are only 0.15 μM, 0.62 μM, and 1.25 μM, when the analysis is performed in the CF sputum sample .…”

“…Sputum samples were then spiked with aliquots of 8 h cultures of PAO1, equilibrated for 20 min, and analyzed for the presence of all three analytes (n=3). In our previous work, the production of the signaling molecules PYO, HHQ, and PQS in growing the cultures and the sputum samples was performed following 11 days incubation. In order to measure the effect of CTAB on standardized samples, spiking the sputum samples with equilibrated amounts of PA provided a fast, standardized and straightforward approach.…”