In vitro synergistic potentials of novel antibacterial combination therapies against Salmonella Typhimurium and Escherichia coli

Abstract

Background: The antibiotics generally used in farm animals are rapidly losing their effectiveness all over the world as bacteria develop antibiotic resistance. New strategies are needed to block the development of resistance and to prolong the life of traditional antibiotics. This study aimed to increase the efficacy of existing antibiotics by combining them with the opportunistic phenolic compounds gallic acid (GA), epicatechin, epicatechin gallate, epigallocatechin and hamamelitannin. Fractional inhibitory concentration index (FICI) of phenolic compound-antibiotic combinations against Salmonella enterica serovar Typhimurium (S. Typhimurium) and Escherichia coli (E. coli) were determined. Based on the FICI and clinical importance, 3 combinations were selected to evaluate their effects on the virulence factors of these bacteria. The in vitro cytotoxicity of GA and hamamelitannin in Rattus norvegicus (IEC-6) cell lines were evaluated. Results: Minimum inhibitory concentrations (MICs) of epigallocatechin, GA and hamamelitannin found against different strains were (512–1024), (256–1024) and (512–2048) μg/mL, respectively. Synergistic effects were obtained from combinations of thiamphenicol-GA (FICI: 0.28), erythromycin-hamamelitannin (FICI: 0.38) and thiamphenicol-hamamelitannin (FICI: 0.50) against E. coli, and erythromycin-epicatechin gallate (FICI: 0.50) against S. Typhimurium. Moreover, additive effects were obtained from 33 combinations against S. Typhimurium (FICI: 0.502~0.750) and E. coli (FICI: 0.502~0.625). The time-kill assays and ultrastructural morphology showed that GA-ceftiofur, and hamamelitannin-erythromycin and GA-ampicillin combinations more efficiently inhibited the growth of S. Typhimurium and E. coli, respectively, compared to individual antibiotics. Biofilm viability and swimming and swarming motilities of S. Typhimurium in presence of GA-ceftiofur, and E. coli in presence of hamamelitannin-erythromycin and GA-ampicillin combinations were more competently inhibited than individual antimicrobials. The inhibitory concentrations 50% (IC50) of GA and hamamelitannin in IEC-6 cells were 564.55 μM and 988.54 μM, respectively. Conclusions: This study suggest that GA-ceftiofur combination can be potential medication to treat S. Typhimurium-associated diarrhea and prevent S. Typhimurium-associated blood-stream infections (e.g.: fever) in farm animals. Hamamelitannin-erythromycin and GA-ampicillin combinations can be effective in restricting E. coli contamination in farm animals, and ultimately its transmission from animal to human. Further study to confirm these effects and safety profiles in in vivo system should be undertaken for establishing these combinations as medications.