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

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 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 for establishing these as sensitivity strains SAL 109, SAL SAL biofilm combination than individual The surviving and dead biofilm populations the presence of the combination antibacterials were determined by imaging the BacLight live/dead-stained biofilm by CLSM. These results that in addition to being bacteriostatic, the GA-ceftiofur, hamamelitannin-erythromycin and GA-ampicillin combinations appeared to act against the biofilm matrix. The large effect of the GA-ceftiofur and GA-ampicillin combinations against the biofilm cells of S. Typhimurium and E. coli , respectively, might be due to the small molecular size of GA (170.12 g/mol), easily penetrates into the biofilm. combination antibacterials seem destroy detachment more

Typhimurium in presence of GA-ceftiofur, and E. coli in presence of hamamelitanninerythromycin 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.
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.
Background Livestock (farm animals) are one of the most important and rapidly expanding commercial agricultural sectors worldwide. Infectious diseases cause direct losses to this sector through increased mortality and reduced livestock productivity, as well as indirect losses associated with cost of control, loss of trade, decreased market values, and food insecurity [1]. Multidrug-resistant (MDR) bacteria are one of several vital aetiologic agents contributing to the emergence of infections [2].
The rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics [3][4][5][6][7][8]. The antibiotic resistance crisis has been attributed to the overuse and misuse of these medications, as well as a lack of new drug development by the pharmaceutical industry due to reduced economic incentives and challenging regulatory requirements [4][5][6][7][9][10][11][12][13][14][15][16]. A number of bacteria are classified for presenting urgent, serious, and concerning threats, many of which are already evident for placing substantial clinical and financial burden to the health care system [3,17].
The frequency of resistance is observed equally among Gram-negative and Grampositive organisms, although Gram-negative bacteria are more prone to develop the MDR phenotype [2]. Together with other bacterial species, Escherichia coli and Salmonella enterica serovar Typhimurium are severely antibiotic-resistant and were recently enlisted and designated as priority class bacterial pathogens in urgent need of effective antibiotics [18]. The gravity of the situation is highlighted by the fact that clinical isolates of these species have up to 1000-fold higher 50% growth inhibition concentrations (GIC 50 ) for a range of antibiotics with different mechanisms of action relative to the sensitive/resistant breakpoints recommended by the Clinical and Laboratory Standard Institute (CLSI). These trends show the urgent need for the development of new antimicrobials that can treat or potentiate current antibiotics against MDR bacteria [19]. The scientific community is continuously searching for new classes of disinfection systems that could act efficiently against these pathogens [20]. Certain naturally occurring phenolic compounds have antioxidant, anticarcinogenic, and antimicrobial activities [21,22].
The phenolic compound (methyl gallate and pyrogallol)-containing Nymphaea tetragona 50% methanol extract (NTME) was found to have quorum sensing and virulence factor inhibitory effects [23]. The synergistic antibacterial and quorum sensing (QS) inhibition effects of the phenolic compound-containing NTME were also evident in our earlier study [24]. The phenolic compound gallic acid demonstrated the potential to inhibit S. mutans biofilms [25]. Recently, we also reported that methyl gallate, a gallic acid (GA) derivative, can efficiently interfere with the QS 5 regulatory pathways of P. aeruginosa and inhibit the adhesion, invasion and intracellular survival of S. Typhimurium [26,27]. These properties of bacteria are known to have a significant role in increasing pathogenicity and antimicrobial resistance [28].
GA derivatives contain a large number of hydroxyls, which can form protonic and ionic bonds and combine with many biological proteins, such as enzymes, carriers, ion channels and receptors, deactivating them and consequently exhibiting bacterial inhibition. Additionally, many phenols can non-specifically affect molecular targets of microorganism [29]. These observations initiate the speculation that GA derivatives may also potentiate the efficacy of existing antibiotics. Thus, we intended in this study to evaluate the antibacterial potentials of GA and its 4 derivatives individually; and in combination with 8 commercially available antibiotics against S. Typhimurium and E. coli . Additionally, the effects of those combination antibacterials against selected virulence factors, including biofilm formation and motility were determined. Finally, the viability of in IEC-6 cells in presence of GA and hamamelitannin alone and in combination with phenolic compounds were investigated.
All these microbiological media were prepared before use by following 6 manufacturer's instructions. Quality control strains of E. coli (ATCC 25922) and S. . Antibiotics used in this study include amoxicillin, ampicillin, cefotaxime, ceftiofur, erythromycin, florfenicol, marbofloxacin, norfloxacin, penicillin G and thiamphenicol. GA, epicatechin, epicatechin gallate, epigallocatechin and hamamelitannin were utilized as antibacterial agent in this study. All the chemicals, reagents and media were from Sigma-Aldrich (St. Louis, MO, United States) unless otherwise mentioned. Stock solutions of epicatechin, epicatechin gallate and epigallocatechin were prepared by dissolving in water. Slight heat and sonication was applied to dissolve epicatechin gallate in water. Alcohol was used as co-solvent to dissolve GA and hamamelitannin in preparing stock solutions. All these stock solutions were further diluted to respective media (e.g., MHB, TSB, etc.) before using in experiment. Solvent controls were used where it was required. Respective growth media (e.g., MHB, TSB, etc.) were used as control medium in the combination experiment, unless mentioned otherwise.

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MICs of above mentioned commercial antibiotics and opportunistic antibacterial agents were determined by the standard broth microdilution method according to the CLSI guidelines [30] in CA-MHB using an inoculum concentration of ∼5 × 10 5 CFU/mL. Different antibacterial solutions were serially diluted in 96-well plates in 100 μL volumes. The cultures of different bacterial strains were diluted to adjust 0.5 McFarland units and, again diluted 100-times. Hundred microliters of these diluted bacterial suspensions were dispensed to all the wells of 96-well plates which contain 100 μL of antibacterial solution. After incubation at 35 °C overnight, the turbidity in each well was checked. The lowest concentrations of the antibacterial that completely inhibited any increase in turbidity were considered as the MICs.

Fractional inhibition concentration index of antibacterial agents
A slightly modified version of the previously described checkerboard microdilution method was utilized to determine the combination interactions of the commercial antibiotics and phenolic compounds [31]. One antibacterial agent was vertically diluted and the other antibacterial was horizontally diluted in 96-well plates to achieve a matrix of different combinations of the 2 antibacterials. Similar dilutions of individual drugs and the drug-free medium control were included in each test plate. Bacterial cultures in early log phase were diluted and 100 μL of the diluted bacterial suspension was added to each well of the 96-well plates, where the final inoculum concentration after transferring to each well would be ∼5 × 10 5 CFU/mL. drug divided by the MIC of the individual drug, and the FICI is the sum of the FICs of the individual drugs. An FICI of ≤ 0.5 is regarded as synergistic, 0.5 < FICI ≤ 1 is considered additive, 1 < FICI ≤ 2 is considered indifferent, and an FICI > 2 is considered antagonistic effects [32].

Effect of antibacterial combinations on bacterial inhibition rates
The time-dependent inhibition effects of GA-ceftiofur against S. Typhimurium and hamamelitannin-erythromycin and GA-ampicillin against E. coli were evaluated according to a previously reported method [24]. Drug compounds alone and in combination were supplemented in 10 mL MHB broth in 15 mL falcon tubes.
Bacterial cultures in early log phase were diluted and then resuspended in the drugsupplemented broth to a final inoculum concentration of 5 × 10 6 CFU/mL. A tube containing 5 × 10 6 CFU/mL of bacteria in 10 mL MHB without any drug was used as a control. The samples were incubated at 37 °C at 200 rpm in a shaking incubator.

Effect of antibacterial combinations on bacterial cell morphology
The effects of the GA-ceftiofur combination on the morphology of S. Typhimurium and the hamamelitannin-erythromycin and GA-ampicillin combinations on the 9 morphology of E. coli cells were evaluated. Drug compounds alone or in combination were supplemented into 10 mL of MHB broth in 15 mL falcon tubes. Bacterial cultures in early log phase were diluted and then resuspended in the drugsupplemented broth to a final inoculum concentration of 5 × 10 6 CFU/mL. A tube containing 5 × 10 6 CFU/mL of bacteria in 10 mL MHB without any drug was used as a control. The bacteria in tubes were incubated overnight at 37 °C and 200 rpm in a shaking incubator. Then, the cells were harvested, washed, and dehydrated according to a previously reported protocol [33]. The ultrastructural morphology of treated S. Typhimurium and E. coli cells was studied using a scanning electronic microscope (SEM; models S-4300 and EDX-350; Hitachi, Japan).

Effect of antibacterial combinations on biofilm growth and viability
The inhibitory effect of combination antibacterials on biofilm formation was determined using slightly modified version of a previously reported spectrophotometric method [34,35]. Briefly, test compounds were supplemented into TSB in three separate wells of a 96-well microplate for each concentration. The Previously reported biofilm viability assay methods were utilized to evaluate the effects of combination drugs on the viability of the biofilms produced by S.

Effect of antibacterial combinations on the motility of bacterial cells
The swarming and swimming motilities of E. coli (ATCC 25922) and S. Typhimurium (ATCC 14028) in the presence of the combination drugs were evaluated according to previously published methods with slight modifications [37,38]. The media used for the E. coli (ATCC 25922) swarming assay was composed of 0.8% Luria-Bertani (LB) supplemented with 0.5% glucose and 0.6% agar. Nutrient broth (NB) supplemented with 0.5% glucose and 0.5% agar was used for the evaluation of S. Typhimurium (ATCC 14028) swarming motility. The media used to evaluate E. coli swimming activity was composed of 1% tryptone broth supplemented with 0.5% NaCl and 0.3% agar. Nutrient broth supplemented with 0.5% glucose and 0.25% agar was used as the media for the S. Typhimurium swimming motility assay. Molten agar plates were supplemented with ¼MIC, ½MIC and 1MIC concentrations of test compounds alone or in combination (GA-ceftiofur, GA-ampicillin and hamamelitannin-erythromycin). A non-supplemented drug free plate was employed as the negative control. The plates were allowed to dry for 1 h and then 2 μL of E. coli and S. Typhimurium cultures were inoculated onto the respective swarming and swimming agar plates. For both strains, swarm plates were kept at 37 °C overnight, whereas swim plates were incubated at 37 °C for 10 h. After incubation, the swarm and swim zone diameters were measured using calibrated digital slide callipers (Mitotoyo, Japan), and photographs of the plates were captured.

Cell viability in the presence of antibacterial agents
The where OD is the optical density [39].

Statistical analysis
Results are presented as the means ± standard deviation (SD) of triplicate analysis.
Statistical analysis was carried out by using SAS software (SAS Institute Inc., Cary, NC, USA). One-way analysis of variance (ANOVA) followed by F-test was used to compare the results. Statistical significance was considered when the P-value was <0.05.

Antibacterial activities of commercial antibiotics and phenolic compounds
Antibacterial activities of different antibiotics and phenolic compounds were 14 Typhimurium ranged from 0.125~256 µg/mL and 0.062~128 µg/mL, respectively. In contrast, the MICs of the commercial antibiotics against the clinical isolates of S.
Typhimurium ranged from 0.25 to ≥1024 µg/mL. The results in Table 1 clearly demonstrate that the MICs of almost all of these commercial antibiotics against the clinical isolates were increased by several folds, which indicates that resistance has developed in these clinical strains [40][41][42][43][44]. The MICs of phenolic compounds (epicatechin, epicatechin gallate, epigallocatechin, GA and hamamelitannin) against the QC strains and clinical isolates of S. Typhimurium ranged from 256 to ≥1024 µg/mL, with GA being the most potent among all the compounds. The MICs of the phenolic compounds against the QC strains of E. coli ranged from 512~2048.00 µg/mL.

In vitro synergy with commercial antibacterials
Checkerboard microdilution assays were performed to evaluate the combination antibacterial interactions of the commercial antibiotics with the phenolic compounds. The results of the combined activities are presented in Table 2.

Effects of combination drugs on the morphology of bacterial cells
The ultrastructural morphologies of S. Typhimurium (ATCC 14028) treated with the combination of GA and ceftiofur and Escherichia coli (ATCC 25922) treated with 2 drug combinations (hamamelitannin-erythromycin and GA-ampicillin) were studied to assess whether the combination drugs had any impact on the cellular architecture. The representative SEM images of GA and ceftiofur-treated S.
Typhimurium cells are shown in Figure 2. The SEM images revealed that untreated and GA (1MIC)-treated S. Typhimurium cells had rod-like shape and were separated with perfect symmetry. In addition, binary fission of the bacteria was evident in the SEM images (Figure 2a and c). The cells treated with ceftiofur alone or in combination with GA were found in a long rope-like shape, and no binary fission was evident, which is completely different from control cells. None of the cells were pitted, deformed or broken and the antibacterials had no effect on the cell wall or cytoplasmic membrane of the bacteria. The E. coli cells treated with hamamelitannin-erythromycin and GA-ampicillin combinations also showed similar changes in cell length and binary fission without any effects on the cell wall or cytoplasmic membrane (data not shown).

Effects of combination drugs on the motility of bacterial cells
The effects of the antibacterial combinations on the swimming and swarming motilities of S. Typhimurium (ATCC 14028) and E. coli (ATCC 25922) were evaluated.
Representative photographs of drug-treated swim and swarm plates are displayed in Figure 5. Table 3

Effects of combination drugs on the viability of IEC-6 cells
The effects of GA and hamamelitannin alone, and GA-ampicillin, GA-ceftiofur and hamamelitannin-erythromycin combinations on the viability of IEC-6 cells are shown in Table 4 and Additional file 1. The IC 50

Discussion
The development of alternative antimicrobial drugs is urgently needed to combat infectious diseases associated with resistant pathogens [45,46]. The in vitro activities of the tested phenolic compounds against resistant strains of Salmonella to be resistant to one to eight out of ten currently available antibiotics ( Table 1).
The potentials of these phenolic compounds were further explored through their combined interactions with commercial antibiotics, where hamamelitannin possessed synergistic effects with thiamphenicol and erythromycin against E. coli.
Further, epicatechin gallate with erythromycin, and GA with thiamphenicol demonstrated synergistic effects against S. Typhimurium and E. coli, respectively.
The time-and concentration-dependent inhibition assays also exposed that hamamelitannin-erythromycin and GA-ampicillin combinations, and GA-ceftiofur combination more effectively inhibited the growth of E. coli and S. Typhimurium, respectively than the antibiotics alone. Furthermore, the combinations of these phenolic compounds and commercial antibiotics demonstrated improved inhibition of biofilm formation and motility in S. Typhimurium and E. coli.

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The MICs of the 5 phenolic compounds (epicatechin, epicatechin gallate, epigallocatechin, GA and hamamelitannin) were investigated against QC strains of S. Typhimurium and E. coli, and 5 clinical strains of S. Typhimurium. It has been reported that GA can restrain the growth of many bacteria, including methicillinsensitive S. aureus, MRSA, E. coli, P. aeruginosa, and Salmonella typhi [47]. The results in Table 1 indicate that GA possessed the strongest antibacterial activity among these phenolic compounds, followed by epigallocatechin, hamamelitannin, epicatechin gallate and epicatechin. The MIC values of GA against S. Typhimurium (256 µg/mL) and E. coli (1024 µg/mL) in this study are lower than those reported previously (2500 µg/mL) [47]. The mean MICs of plant-derived epigallocatechin against S. Typhimurium and E. coli were reported to be 572±186 and 733±121 µg/mL, respectively [48]. The MIC values of pure epigallocatechin against S.
Typhimurium (512 µg/mL) and E. coli (512 µg/mL) in our study were lower than the previously reported MIC values [48]. This lower MIC value of pure epigallocatechin against S. Typhimurium and E. coli in our study compared to the MIC value of plantderived epigallocatechin in the previous study is might be because of the purity of the compound used. However, the MIC values of plant-derived epigallocatechin against S. Typhimurium and E. coli were comparable with the results of our study.
Likewise, the MICs of epicatechin against S. Typhimurium and E. coli were 2500 µg/mL, which demonstrates the similarity between our results and previously published results [47]. g/mol), which easily penetrates into the biofilm. Subsequently, these combination antibacterials seem to destroy the biofilm matrix, resulting in the detachment of cells and thus the biofilm cells become more exposed and susceptible.
Motility is one of the pathogenic phenotypes of bacteria that contribute to the migration and dispersion of bacteria and their escape from the host immune response [37]. Flagella are known to be involved in swimming motility and play a role in biofilm formation, as well as swarming motility [48]. Recent reports mentioned that, similar to biofilms, swarming cells also show a higher degree of resistance to a variety of antibiotics [56,57]. In this study, we investigated the ability of the GA-ceftiofur, hamamelitannin-erythromycin and GA-ampicillin combinations to inhibit the swarming and swimming activities of S. Typhimurium and E. coli . The results (Table 3) showed significant inhibition of swimming and swarming motilities with the addition of GA-ceftiofur, hamamelitannin-erythromycin and GA-ampicillin combinations. The lack of swimming and swarming motilities in 24 the presence of the combination antibacterials suggest that these agents might have some effects on flagella-related processes, namely, flagella biosynthesis, rotation, and chemotaxis, which may lead to decreased swimming and swarming activities.
The evaluation of the safety/toxicity profiles of any drug is desirable and an essential part of the investigation of the pharmacological effects. Virtually all organs and tissues are exposed, once the ingested drugs cross the intestinal wall [58]. Many studies have demonstrated that insignificant amounts of orally administered phenolic compounds such as GA and hamamelitannin are absorbed through the gastrointestinal tract due to its low membrane permeability and poor water solubility [59,60]. Therefore, the pharmacological or toxicological effects of these phenolic compounds can be largely explained in terms of their local effects.
Moreover, this study found synergistic effects of these phenolic compounds against

Competing Interests
None of the authors have any conflicts of interest to declare.

Availability of Data and Materials
Data will be shared upon request to the corresponding author.

Ethics Approval and Consent to Participate
All procedures have been approved by the Bioethical Committee of Animal and Plant Quarantine Agency, Republic of Korea. FIC, fractional inhibitory concentration; Synergy, X ≤ 0.5; Additive, 0.5 < X ≤ 1; Indifferent, 1 < X ≤ 2; Antagonist, X > 2; (A), (I) and (S) stand for Additive, Indifferent and Synergy, respectively.