Data_Sheet_1_Synergistic Interaction Between Paired Combinations of Natural Antimicrobials Against Poultry-Borne Pathogens.docx

Natural antimicrobials (NAM) are promising candidates for the successful control of poultry-borne bacteria, carrying potent antimicrobial activity (AMA) against a wide range of multidrug-resistant pathogens. Individual activities of carvacrol, eugenol, trans-cinnamaldehyde, oregano, and thymol, along with the combined activity of paired compounds, were examined using broth microdilution and checkerboard techniques. The characteristic interactions between the compounds were calculated using an improved method, based on combination index (CI) values. The bacteria examined herein were selected due to their known genetic resistance to at least one antibiotic. Our results indicated that thymol was most effective, exhibiting the lowest minimum inhibitory concentration (MIC) value against Salmonella pullorum, Escherichia coli, and Klebsiella pneumoniae, establishing the order of antimicrobial efficacy as: thymol > oregano > carvacrol > trans-cinnamaldehyde > eugenol. In the interaction study, the paired combination of carvacrol and thymol showed synergistic effects and was highly effective in reducing the antibiotic resistance of all the evaluated pathogens. Notably, all CI values were <1.0 in evaluations of S. pullorum, indicating the absence of antagonism between eugenol and thymol (or oregano). In K. pneumoniae, majority of CI values, which had a few concentration points, were smaller than 1.0, indicating a synergistic effect between eugenol and carvacrol (oregano or thymol), and trans-cinnamaldehyde and carvacrol. In E. coli, apart from some concentration points, some CI values were smaller than 1.0, demonstrating a synergistic effect between eugenol and carvacrol, and thymol and carvacrol (eugenol or oregano). It is therefore of great significance to investigate and illuminate the minimal effect concentration of these five components when they are used in combination as feed additives. Moreover, the improved evaluation method of this study provides a precise and extensive means to assess the synergistic effects of NAM.

天然抗菌剂（Natural Antimicrobials, NAM）是防控禽源细菌的理想候选制剂，对多种多重耐药病原体均具有强效抗菌活性（Antimicrobial Activity, AMA）。本研究采用肉汤微量稀释法与棋盘格法，检测了香芹酚、丁香酚、反式肉桂醛、牛至以及百里酚的单独抗菌活性，同时考察了化合物两两联合后的活性表现。基于联合指数（Combination Index, CI）值，通过改良方法计算各化合物间的特征相互作用。本研究所选用的细菌均已知对至少一种抗生素存在遗传抗性。实验结果表明，百里酚的抗菌效果最佳，针对鸡白痢沙门氏菌、大肠埃希氏菌与肺炎克雷伯菌的最低抑菌浓度（Minimum Inhibitory Concentration, MIC）最低，由此得出五种物质的抗菌效力排序为：百里酚 > 牛至 > 香芹酚 > 反式肉桂醛 > 丁香酚。在相互作用研究中，香芹酚与百里酚的两两组合表现出协同效应，可显著降低所有受试病原体的抗生素耐药性。值得注意的是，针对鸡白痢沙门氏菌的所有联合指数值均小于1.0，表明丁香酚与百里酚（或牛至）之间不存在拮抗作用。针对肺炎克雷伯菌的实验中，多数联合指数值（仅少数浓度点除外）小于1.0，提示丁香酚与香芹酚（牛至或百里酚）、反式肉桂醛与香芹酚之间存在协同效应。针对大肠埃希氏菌的实验中，除部分浓度点外，部分联合指数值小于1.0，表明丁香酚与香芹酚、百里酚与香芹酚（丁香酚或牛至）之间存在协同效应。因此，探究并明确这五种成分作为饲料添加剂联合使用时的最低有效浓度具有重要的研究价值。此外，本研究采用的改良评估方法，可为天然抗菌剂的协同效应评估提供精准且全面的技术手段。