Triclosan is the Predominant Antibacterial Compound in Ontario Sewage Sludge

Sewage treatment plants (STPs) accumulate both antibiotic and nonantibiotic antimicrobial compounds that can select for antibiotic resistant bacteria. Herein, we aimed to identify the predominant antibacterial compounds impacting E. coli from Ontario sewage sludge consisting of thousands of unknown compounds. Among the 10 extracted sludge samples, 6 extracts exerted significant growth inhibition effects in E. coli. A total of 103 compounds were tentatively detected across the 10 sludge samples by suspect screening, among which the bacterial enoyl-ACP reductase (FabI) inhibitor triclocarban was detected at the highest abundance. A hypomorphic FabI knockdown E. coli strain was highly susceptible to the sludge extracts, confirming FabI inhibitors as the primary antibacterial compounds in the sludge. Protein affinity pulldown identified tridosan as the major ligand binding to a His-tagged FabI protein from the sludge, despite the higher abundance of triclocarban in the same samples. Effect-directed analysis was used to determine the contributions of tridosan to the observed antibacterial potencies. Antibacterial effects were only detected in F-17 and F-18 across 20 fractions, which was consistent with the elution of tridosan and tridocarban in the same two fractions. Further, potency mass balance analysis confirmed that tridosan explained the majority (58-113%) of inhibition effects from sludge extracts. This study highlighted tridosan as the predominant antibacterial compound in sewage sludge impacting E. coil despite the co-occurrence of numerous other antibiotics and nonantibiotics.