Kashi Milad, Farahani Abbas, Ahmadi Azam, Shariati Aref, Akbari Majid
Student Research Committee, Arak University of Medical Sciences, Arak, Iran.
Molecular Medicine Research Center, Khomein University of Medical Sciences, Khomein, Iran.
Mol Biol Rep. 2025 May 21;52(1):480. doi: 10.1007/s11033-025-10564-6.
Colistin-resistant Klebsiella pneumoniae (CR K. pneumoniae) is considered one of the bacteria with the highest levels of antibiotic resistance, necessitating the discovery of alternative therapeutic strategies. This study aimed to evaluate the ability of eugenol, carvacrol, and cinnamaldehyde to inhibit CR K. pneumoniae and the biofilm community of this bacterium.
The minimum inhibitory concentration (MIC) and antibiofilm effects of compounds were assessed using broth macrodilution and microtiter plate assays, respectively. Time-kill assays determined the bactericidal effects of natural compounds on CR K. pneumoniae isolates. Protein and nucleic acid leakage were examined to assess metabolic disruption and cell membrane integrity. Real-time PCR was used to evaluate the effect of natural compounds on the expression of biofilm-related genes (mrkA, treC, and luxS).
In the disk diffusion test, the inhibition zones of eugenol, carvacrol, and cinnamaldehyde were 15 ± 1, 29 ± 2, and 30 ± 1 mm, respectively. The MIC of eugenol, carvacrol, and cinnamaldehyde were 260, 119, and 128 µg/mL, respectively. The time-kill assay demonstrated the rapid bactericidal effects of eugenol, carvacrol, and cinnamaldehyde at 2× MIC, with kill times of 6, 2, and 3 h, respectively. At the MIC, the kill times were 12, 10, and 12 h, respectively. These compounds significantly released proteins and nucleic acids from the treated bacteria. They also inhibited biofilm formation and disrupted mature biofilms. Furthermore, mrkA and treC expression levels were significantly reduced in the presence of eugenol and cinnamaldehyde.
These natural compounds demonstrated significant antibacterial and antibiofilm activity against CR K. pneumoniae, emerging as a promising natural alternative for treatment.
耐黏菌素肺炎克雷伯菌（CR肺炎克雷伯菌）被认为是抗生素耐药性最强的细菌之一，因此需要发现替代治疗策略。本研究旨在评估丁香酚、香芹酚和肉桂醛抑制CR肺炎克雷伯菌及其生物膜群落的能力。
分别采用肉汤稀释法和微量滴定板法评估化合物的最低抑菌浓度（MIC）和抗生物膜作用。时间杀菌试验确定天然化合物对CR肺炎克雷伯菌分离株的杀菌作用。检测蛋白质和核酸泄漏以评估代谢紊乱和细胞膜完整性。采用实时荧光定量PCR评估天然化合物对生物膜相关基因（mrkA、treC和luxS）表达的影响。
在纸片扩散试验中，丁香酚、香芹酚和肉桂醛的抑菌圈直径分别为15±1、29±2和30±1毫米。丁香酚、香芹酚和肉桂醛的MIC分别为260、119和128微克/毫升。时间杀菌试验表明，丁香酚、香芹酚和肉桂醛在2倍MIC时具有快速杀菌作用，杀菌时间分别为6、2和3小时。在MIC浓度下，杀菌时间分别为12、10和12小时。这些化合物能使处理过的细菌显著释放蛋白质和核酸。它们还能抑制生物膜形成并破坏成熟生物膜。此外，在丁香酚和肉桂醛存在的情况下，mrkA和treC的表达水平显著降低。
这些天然化合物对CR肺炎克雷伯菌具有显著的抗菌和抗生物膜活性，有望成为一种有前景的天然替代治疗药物。
