Previous studies have employed antimicrobial susceptibility assays to identify the residual antimicrobial activity of water/wastewater solutions containing antibiotics. The standard protocol uses optical density as the primary metric for assessing growth inhibition of a specific microorganism. Most studies have employed Escherichia coli in this regard. In this work, Pseudomonas fluorescens, which is a gram-negative bacteria that secretes the fluorescent siderophore, pyoverdine, was employed to investigate growth inhibition (optical density) and sub-lethal impacts (fluorescence). Pyoverdine is used by Pseudomonas spp. as a virulence factor that supports biofilm formation. This attribute suggests that pyoverdine secretion may be linked to a stress response resulting from quorum-sensing cascades. Due to its strong fluorescence characteristics, pyoverdine can be used to sensitively detect sub-lethal effects of antibiotics on P. fluorescens cell vitality. Solutions containing antibiotics from four major classes (i.e., two fluoroquinolones, two macrolides, six sulfonamides, and two tetracyclines) were serially diluted by microdilution. P. fluorescens or E. coli inocula were then added before incubation for 16-20 hours at 35 ± 2°C. For example, the bacteria were exposed to 0.1-200 mg/L sulfamethoxazole to determine the following: P. fluorescens IC50 (optical density at 600 nm), 11.18 ± 0.49 mg/L; P. fluorescens EC50 (fluorescence; excitation at 405 nm, emission at 455 nm), 1.55 ± 0.45 mg/L; and, E. coli IC50 (optical density at 600 nm), 10.10 ± 0.84 mg/L. For P. fluorescens, the fluorescence-based EC50 value was lower than the optical density-based IC50 for all tested antibiotics. For most antibiotics, inhibition followed the Hill dose-response model. However, low concentrations of some antibiotics (e.g., erythromycin) exhibited higher fluorescence than the positive control, suggesting hormetic effects, whereby the presence of low antibiotic concentrations stimulate a bacterial response. The sensitive fluorescent signature from pyoverdine, therefore, facilitated new knowledge on sub-lethal impacts of environmentally-relevant antibiotic concentrations.

Antimicrobial susceptibility, stress responses, and hormesis in Pseudomonas fluorescens as metrics for assessing impacts of antibiotics in the environment

255th National Meeting (2018)

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