Synergistic Interaction Between Paired Combinations of Natural Antimicrobials Against Poultry-Borne Pathogens

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 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.