Effects of Essential Oil Treatment on Clostridium perfringens Cells

Clostridium perfringens type F is a Gram-positive, spore-forming bacterium that causes food poisoning (FP) in humans. Our previous findings demonstrated that essential oils (EOs), including clove, rosemary, and peppermint oils, significantly inhibited the germination, outgrowth, and vegetative growth of C. perfringens spores. We hypothesized that cytoplasmic membrane damage could be one mechanism by which EOs exert their inhibitory effects against C. perfringens vegetative cells. To test this hypothesis, vegetative cells of two clinical FP strains of C. perfringens, SM101 and E13, were treated with EOs, and the leakage of intracellular molecules was compared between EO-treated and untreated cells. Our results demonstrated increased leakage of intracellular molecules, including ATP, 260-nm-absorbing materials, and proteins, from EO-treated cells relative to untreated cells. In addition, EO-treated cells exhibited greater crystal violet uptake than untreated cells. These results suggest that EOs induced cytoplasmic membrane damage, thereby promoting the leakage of intracellular molecules and the uptake of extracellular crystal violet. Scanning electron microscopy further confirmed cytoplasmic membrane damage by revealing substantially greater membrane disruption in EO-treated cells than in untreated cells. In conclusion, EOs may exert their anti-C. perfringens effects by damaging the cytoplasmic membrane, thereby disrupting membrane permeability and suppressing cell multiplication.