Advances in antimicrobial activity analysis of fluoroquinolone, macrolide, sulfonamide, and tetracycline antibiotics for environmental applications through improved bacteria selection

Abstract The widespread use of antibiotics has led to their ubiquitous presence in water and wastewater and raised concerns about antimicrobial resistance. Clinical antibiotic susceptibility assays have been repurposed to measure removal of antimicrobial activity during water and wastewater treatment processes. The corresponding protocols have mainly employed growth inhibition of Escherichia coli. The present work focused on optimizing bacteria selection to improve the sensitivity of residual antimicrobial activity measurements by broth microdilution assays. Thirteen antibiotics from four classes (i.e., fluoroquinolones, macrolides, sulfonamides, tetracyclines) were investigated against three gram-negative organisms, namely E. coli, Mycoplasma microti, and Pseudomonas fluorescens. The minimum inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC50) were calculated for each antibiotic-bacteria pair. P. fluorescens produces a fluorescent siderophore, pyoverdine, that was used to assess sublethal effects and further enhance the sensitivity of antimicrobial activity measurements. The optimal antibiotic-bacteria pairs were as follows: fluoroquinolone-E. coli (growth inhibition); macrolide- and sulfonamide-M. microti (growth inhibition); and, tetracycline-P. fluorescens (pyoverdine inhibition). Compared to E. coli growth inhibition, the sensitivity of antimicrobial activity analysis was improved by up to 728, 19, and 2.7 times for macrolides (tylosin), sulfonamides (sulfamethoxazole), and tetracyclines (chlortetracycline), facilitating application of these bioassays at environmentally-relevant conditions.