Electrochemical Monitoring of the Impact of Antibiotic Agents on Pseudomonas aeruginosa Phenazine Metabolite Production Rates

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a high percentage of nosocomial infections, particularly posing a threat to immunocompromised patients. The increased use of antibacterial agents has led to the emergence of antibiotic resistance mechanisms in this pathogen, exacerbating challenges in treating P. aeruginosa infections. Previous studies have correlated the endogenous production of phenazines in P. aeruginosa to the development of antibiotic resistance in the bacterium. Phenazines are nitrogen-containing heterocyclic compounds endogenously produced by P. aeruginosa as redox-active biogenic signaling molecules during early infection stages. While several studies have provided qualitative insights into the effects of different antibiotics on P. aeruginosa growth, quantitative reports on the changes in P. aeruginosa cellular communication and metabolite production dynamics following antibiotic exposure are limited. Addressing this knowledge gap, this study reports real-time electrochemical quantification of phenazine virulence metabolites in both wild-type PA14 and clinical PA2114 strains of P. aeruginosa with varying levels of antibiotic resistance. Specifically, the impact of antibacterial agent ciprofloxacin (CIP) at different concentrations (0, 0.05, and 1 mg L⁻¹ CIP) on phenazine production in these P. aeruginosa strains was electrochemically monitored directly from bacterial cultures over a 48-h period. Square-wave voltammetry was utilized for quantitative monitoring of two phenazines, namely pyocyanin (PYO) and 5-methylphenazine-1-carboxylic acid (5-MCA), correlating electrical current signal at a unique redox potential to the phenazine concentration. The results indicate distinct phenazine redox responses of the clinical isolate PA2114 strain and wild-type PA14 strain due to antibiotic stress. Under optimal cell growth conditions without CIP exposure, the PA2114 strain with higher antibiotic resistance produced increasing PYO concentrations over 48 h, peaking at 84 ± 9 μM PYO. In contrast, the PYO levels produced by the wild-type strain plateaued and decreased after 24 h of growth, reaching a maximum PYO concentration of 65 ± 3 μM. Exposure to 0.05 mg L⁻¹ CIP led to marginally higher PYO concentrations in the clinical strain (88 ± 9 μM) while the wild-type strain displayed a notable decrease in PYO concentration (54 ± 4 μM). Additionally, cellular growth with 1 mg L⁻¹ CIP resulted in a decreased maximum PYO concentration for the clinical strain (70 ± 3 μM) and a substantially decreased PYO concentration for the wild-type strain (11 ± 5 μM). These differences in PYO production are attributed to distinctive changes in phenazine biosynthesis dynamics in different P. aeruginosa strains and the addition of CIP. Furthermore, this study shows the monitoring of phenazine biosynthesis dynamics through the detection of the reactive, redox-active intermediate 5-MCA in the bacterial cultures under antibiotic stress conditions. Our results indicate noteworthy differences in 5-MCA production in all samples, suggesting that antibiotics contribute to variation in the dynamics of the phenazine biosynthetic pathway.