In vitro synergistic potentials of novel antibacterial combination therapies against Salmonella Typhimurium and Escherichia coli CURRENT STATUS: REVIEW

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 (GA-ampicillin, GA-ceftiofur and hamamelitannin-erythromycin) were selected to evaluate their effects on the virulence factors of these bacteria. Viabilities of Rattus norvegicus (IEC-6) cell in presence of these combination antibacterials 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. Viabilities of IEC-6 cells were significantly enhanced by GA-ceftiofur, GA-ampicillin combinations than these antibacterials alone. Conclusions: This study suggest that GA-ceftiofur combination can be potential medication to treat S. ampicillin (½MIC) agar evaluate their on swimming and swarming motilities of E. coli A non-supplemented drug free the negative The for 1 h and then 2 μL of E. coli and S. Typhimurium were inoculated onto the respective swarming and swimming agar plates. For both strains, swarm plates kept at 37 °C whereas swim plates incubated at 37 for 10 h. After incubation, the swarm and zone diameters using calibrated digital slide callipers and the CRL-1592, United States) cell lines in the presence of GA, hamamelitannin, ampicillin, ceftiofur and erythromycin alone, and combinations of GA-ceftiofur, GA-ampicillin and hamamelitannin-erythromycin were evaluated according to standard EZ-cytox (EZ-1000; Daeillab Service Co. Ltd., Jeonju, South Korea) assay method. In brief, IEC-6 cells were cultured at 37 °C under a humidified atmosphere of 5% carbon dioxide (CO 2 ) in Dulbecco's Modified Eagle's medium (DMEM; ThermoFisher Scientific, Waltham, MA, United States) with 4 mM L-glutamine (ThermoFisher Scientific, Waltham, MA, United States), adjusted to contain 1.5 g/L sodium bicarbonate (Carolina Biological Supply Company, Burlington, NC, United States) and 4.5 g/L glucose and supplemented with 0.1 Unit/mL bovine insulin (90%) and FBS (10%). The cells were subpassaged at a ratio of 1:5 twice a week. One hundred microliters of suspended cells (2 × 10 4 cells/mL in their respective culture medium) were acclimated in 96-well plates at 37 °C under 5% CO 2 for 24 h. The medium from each well was aspirated and the cells were washed twice. Test compounds were serially diluted in cell culture medium to make a concentration range as ampicillin (500-31) µg/mL, ceftiofur (500-31) µg/mL, erythromycin (500-31) µg/mL, GA (500-31) µg/mL and hamamelitannin (500-31) µg/mL. One hundred microliters of the test compounds alone, and combinations of GA-ceftiofur, GA-ampicillin and hamamelitannin-erythromycin at concentrations mentioned above were dispensed the swim and swarm zones. The results demonstrated that the hamamelitannin-erythromycin combination and the GA-ampicillin combination significantly inhibited both the swimming and the swarming motilities of E. coli . Similarly, the swimming and swarming motilities of S. Typhimurium were presence of the hamamelitannin-erythromycin and GA-ampicillin combinations were evaluated. The inhibition of both planktonic and biofilm cells of these bacteria was induced by the combination drugs more than the individual drugs in most cases. The surviving and dead biofilm populations in the presence of the combination antibacterials were determined by imaging the BacLight live/dead-stained biofilm by CLSM. These results revealed that the GA-ceftiofur, hamamelitannin-erythromycin and GA-ampicillin combinations more efficiently S. Typhimurium and E. coli , respectively than the antibiotics alone, which is crucial to develop new antimicrobials and/or improve the efficacy of existing antimicrobials for reducing the pathogenicity associated with these bacteria. This study also suggest that gallic acid-ceftiofur combination can be potential medication to treat S. Typhimurium-associated diarrhea and prevent S.