Jilani Saoussen, Ferjeni Mohamed, Al-Shammery Kholoud, Rashid Mohammed AlTamimi Haya, Besbes Malek, Ahmed Lotfi Salwa, Farouk Amr, Ben Selma Walid
Department of Biology, College of Science, University of Ha'il, Ha'il, Saudi Arabia.
Laboratory of Analysis, Treatment and Valorization of Pollutants of the Environmental and Products, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia.
Front Pharmacol. 2025 May 22;16:1582102. doi: 10.3389/fphar.2025.1582102. eCollection 2025.
Carbapenem-resistant (CRAB) is one of the most predominant causative agents of nosocomial infections, especially in the intensive care unit patients.
The current study investigates the antibacterial activities of Tunisian essential oil (Thyme-EO) alone and in combination with imipenem against CRAB.
Thyme-EO antimicrobial activities were evaluated by disc diffusion and microdilution assays. Synergism between imipenem and Thyme-EO was determined by combined disc diffusion and checkerboard technique. The synergistic effect of the combined use of carvacrol and imipenem was evaluated by checkerboard assay. Interaction between the major compound identified by Gas Chromatography-Mass Spectrometry (GC/MS) of Thyme-EO and eight bacterial vital enzymes was analyzed by molecular docking and checked by molecular simulation for their stability.
According to GC/MS analysis, carvacrol (78.83%) was the major component. The inhibition zones' diameter by Thyme-EO varied from 18 to 36 mm. Importantly, the values of minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were of low level and ranged between 0.312 and 1.25 mg/mL. Interestingly, the MBC/MIC was equal to 1 for most tested bacterial strains, confirming a bactericidal effect of Thyme-EO. Combining imipenem and Thyme-EO diminished importantly the MIC of imipenem by 8- to 16-fold in the CRAB [fractional inhibitory concentration indexes (FICI) ˂ 0.5, synergy)]. Carvacrol showed antibacterial activities at low MIC levels of 64 and 128 μg/mL and advanced bactericidal effect justified by the MBC/MIC ratio, which was equal to 1 for most tested CRAB. Moreover, carvacrol interacts synergistically with imipenem against all bacterial isolates (FICI ˂ 0.5). The docking study demonstrated that carvacrol seemed to have high binding free energies (-8.1 kcal/mol) against D-alanine: D-alanine ligase (2ZDQ), which is implicated in the pathway of peptidoglycan' biosynthesis. A 100-ns dynamic simulation investigation confirmed binding interactions and stability between carvacrol and the active residues of 2ZDQ.
The current results demonstrated that carvacrol alone or combined with imipenem may constitute a promising opportunity as a novel strategy to treat infections caused by CRAB.
耐碳青霉烯类鲍曼不动杆菌（CRAB）是医院感染最主要的病原体之一，尤其是在重症监护病房患者中。
本研究调查突尼斯百里香精油（百里香-EO）单独及与亚胺培南联合对CRAB的抗菌活性。
采用纸片扩散法和微量稀释法评估百里香-EO的抗菌活性。通过联合纸片扩散法和棋盘法确定亚胺培南与百里香-EO之间的协同作用。采用棋盘法评估香芹酚与亚胺培南联合使用的协同效应。通过分子对接分析百里香-EO经气相色谱-质谱联用（GC/MS）鉴定的主要化合物与8种细菌重要酶之间的相互作用，并通过分子模拟检查其稳定性。
根据GC/MS分析，香芹酚（78.83%）是主要成分。百里香-EO的抑菌圈直径在18至36毫米之间。重要的是，最低抑菌浓度（MIC）和最低杀菌浓度（MBC）值较低，范围在0.312至1.25毫克/毫升之间。有趣的是，大多数受试菌株的MBC/MIC等于1，证实了百里香-EO的杀菌作用。在CRAB中，亚胺培南与百里香-EO联合使用可使亚胺培南的MIC显著降低8至16倍[分数抑菌浓度指数（FICI）˂0.5，协同作用]。香芹酚在64和128微克/毫升的低MIC水平下显示出抗菌活性，且MBC/MIC比值证明其具有显著的杀菌作用，大多数受试CRAB的该比值等于1。此外，香芹酚与亚胺培南对所有细菌分离株均有协同作用（FICI˂0.5）。对接研究表明，香芹酚对参与肽聚糖生物合成途径的D-丙氨酸：D-丙氨酸连接酶（2ZDQ）似乎具有较高的结合自由能（-8.1千卡/摩尔）。一项100纳秒的动态模拟研究证实了香芹酚与2ZDQ活性残基之间的结合相互作用和稳定性。
目前的结果表明，香芹酚单独或与亚胺培南联合使用可能是治疗CRAB感染的一种有前景的新策略。
