*Article Cupressus sempervirens* **Essential Oil: Exploring the Antibacterial Multitarget Mechanisms, Chemcomputational Toxicity Prediction, and Safety Assessment in Zebrafish Embryos**

**Sarra Akermi <sup>1</sup> , Slim Smaoui 1,\* , Khaoula Elhadef <sup>1</sup> , Mariam Fourati <sup>1</sup> , Nacim Louhichi <sup>2</sup> , Moufida Chaari <sup>1</sup> , Ahlem Chakchouk Mtibaa <sup>1</sup> , Aissette Baanannou <sup>2</sup> , Saber Masmoudi <sup>2</sup> and Lotfi Mellouli <sup>1</sup>**


**Abstract:** Nowadays, increasing interest has recently been given to the exploration of new food preservatives to avoid foodborne outbreaks or food spoilage. Likewise, new compounds that substitute the commonly used synthetic food preservatives are required to restrain the rising problem of microbial resistance. Accordingly, the present study was conducted to examine the chemical composition and the mechanism(s) of action of the *Cupressus sempervirens* essential oil (CSEO) against *Salmonella enterica* Typhimuriumand *Staphyloccocus aureus*. The gas chromatography analysis revealed α-pinene (38.47%) and δ-3-carene (25.14%) are the major components of the CSEO. By using computational methods, such as quantitative structure–activity relationship (QSAR), we revealed that many CSEO components had no toxic effects. Moreover, findings indicated that α-pinene, δ-3-carene and borneol, a minor compound of CSEO, could inhibit the AcrB-TolC and MepR efflux pump activity of *S. enterica* Typhimurium and *S. aureus*, respectively. In addition, our molecular docking predictions indicated the high affinity of these three compounds with active sites of bacterial DNA and RNA polymerases, pointing to plausible impairments of the pathogenic bacteria cell replication processes. As well, the safety profile was developed through the zebrafish model. The in vivo toxicological evaluation of (CSEO) exhibited a concentration-dependent manner, with a lethal concentration (LC50) equal to 6.6 µg/mL.

**Keywords:** *Cupressus sempervirens* essential oil; antibacterial activity; membrane permeability; replication and transcription inhibition; computational toxicology; molecular docking; zebrafish
