The avermectin, emamectin benzoate, kills gram-positive bacteria and targets the cell envelope of <i>Bacillus subtilis</i>

The avermectin family of anthelmintics is largely considered to lack antibacterial activity against gram-positive and gram-negative bacteria. Here, we screened six avermectins (ivermectin, eprinomectin, doramectin, abamectin, selamectin, and emamectin benzoate) and a structurally related milbemycin (moxidectin) for antibacterial activity against a panel of representative gram-positive and gram-negative bacteria. We report that emamectin benzoate exhibited activity against several species of gram-positive bacteria, whereas selamectin was active against <i>Staphylococcus aureus</i> and <i>Staphylococcus epidermidis</i>. Emamectin benzoate was the only avermectin with activity against hyper-permeabilized <i>Escherichia coli</i> cells. Using the model bacterium, <i>Bacillus subtilis</i>, we demonstrated that emamectin benzoate is bactericidal, causing rapid lysis and extensive cell envelope damage. To gather insight into its molecular target, several mutant selection methods were employed but failed to yield emamectin benzoate-resistant mutants. Macromolecular synthesis assays indicated that emamectin benzoate inhibited peptidoglycan synthesis. Transcriptome sequencing showed that emamectin benzoate activates the Lia and SigM cell envelope stress-responsive systems and induces the expression of peptidoglycan biosynthetic genes. Although the inactivation or activation of the Lia system did not alter the susceptibility of <i>B. subtilis</i> to emamectin benzoate, CRISPRi-mediated repression of individual essential peptidoglycan biosynthetic genes sensitized <i>B. subtilis</i> to emamectin benzoate. Emamectin benzoate synergized with cell wall-targeting antibiotics (nisin, fosfomycin, daptomycin, and tunicamycin) against <i>B. subtilis</i>. Phosphatidylglycerol antagonized the antibacterial activity of emamectin benzoate, suggesting that EMB likely interacts with membrane phospholipids to exert its effect. Altogether, avermectins exhibit anti-gram-positive activity, albeit with differing levels of potency and selectivity, and, mechanistically, emamectin benzoate targets the cell envelope.IMPORTANCEAvermectins and the structurally related milbemycins are thought to lack antibacterial activity against gram-positive and gram-negative bacteria. Using antimicrobial susceptibility testing, we showed that avermectins and the milbemycin, moxidectin, possess anti-gram-positive activity, with the avermectin, emamectin benzoate, exhibiting the greatest spectrum of activity. Using <i>B. subtilis</i> as a model organism, we showed that emamectin benzoate is bactericidal, causes extensive cell envelope damage, and inhibits peptidoglycan synthesis. Transcriptome analysis of <i>B. subtilis</i> cells treated with emamectin benzoate showed that this avermectin activates two envelope stress-responsive systems and induces the expression of peptidoglycan biosynthetic genes, likely to counteract emamectin benzoate-mediated cell envelope damage. As some avermectins and milbemycins are approved for human and animal use, these drugs may be repurposed for the treatment of gram-positive bacterial infections. Furthermore, given the extensive use of these agents in agriculture, aquaculture, and medicine, examination of their off-target effects on various bacterial communities is warranted.