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

Background: Bacteria have remarkable abilities to acquire resistance against antibiotics by several mechanisms. 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 compound gallic acid (GA) and its derivatives. Fractional inhibitory concentration (FIC) indexes of phenolic compound-antibiotic combinations against Salmonella enterica serovar Typhimurium, Escherichia coli and Staphylococcus aureus were determined. Based on the FIC indexes 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 the Rattus norvegicus (IEC-6) cell line were evaluated. Results: Phenolic compounds demonstrated considerable antibacterial effects as the minimum inhibitory concentrations (MICs) of epigallocatechin, GA and hamamelitannin found against different strains were (32–1024), (128–1024) and (512–≥2048) μg/mL, respectively. The FIC indexes of the combined antibacterials against these strains were 0.281–1.016. The ultrastructural morphology and time-kill assays showed that the GA-ceftiofur combination, and hamamelitannin-erythromycin and GA-ampicillin combinations more efficiently inhibited the growth of S. Typhimurium and E. coli, respectively, compared to the individual antibiotics. Biofilm viability and the swimming and swarming motilities of S. Typhimurium in the presence of GA-ceftiofur and E. coli in the presence of the hamamelitannin-erythromycin and GA-ampicillin combinations were more competently inhibited than individual antimicrobials. The 50% inhibitory concentrations (IC50) of GA and hamamelitannin in IEC-6 cells were 564.55 μM and 988.54 μM, respectively. Conclusions: The compounds virulence conclude that The in vitro viability of a small intestine cell line of Rattus norvegicus (IEC-6; American Type Culture Collection CRL-1592, VA, United States) in the presence of combination drugs were evaluated according to standard EZ-cytox (EZ-1000; Daeillab Service Co. Ltd., Jeonju, South Korea) assay method. In brief, the 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 foetal bovine serum (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 the abovementioned DMEM 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. One hundred microliters of the test compounds at various concentrations in the abovementioned DMEM medium were dispensed into each well and the cells in the drug-supplemented medium were allowed to incubate at 37 °C under 5% CO 2 for 24 h. A total of 10 µL of EZ-cytox were added to each well. After incubation for 2 h, the absorbances in each well were measured at 450 nm using a plate reader. Cells not treated with any drugs were assigned as the control. The cell viability (%) was calculated by the following formula:

safe and can be potential medications to treat S. Typhimurium, E. coli and S. aureus infections in animals and humans. Further study to confirm this effect in in vivo system and to determine the precise mechanism of action should be undertaken to establish these combinations as medications.

Background
Infectious diseases are the third most significant cause of mortality around the world according to the World Health Organization (WHO) [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]. The Centers for Disease Control and Prevention (CDC) has classified a number of bacteria as presenting urgent, serious, and concerning threats, many of which are already responsible for placing substantial clinical and financial burden on the health care system, patients, and their families [3,7,12,17].
The frequency of resistance is observed equally among Gram-negative and Gram-positive organisms, although Gram-negative bacteria are more prone to develop the MDR phenotype [2]. Together with other bacterial species, Escherichia coli, Salmonella Typhimurium and Staphylococcus aureus are severely antibiotic-resistant and were recently enlisted and designated as priority class bacterial pathogens in urgent need of effective antibiotics by the WHO [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 4 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 derivative, can efficiently interfere with the QS regulatory pathways of P. aeruginosa and inhibit the adhesion, invasion and intracellular survival of S. Typhimurium [26,27]. These properties of bacterial are known to have a significant role in increasing pathogenicity and antimicrobial resistance [28].
Gallic acid 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 microorganism molecular targets [29]. These observations initiate the speculation that gallic acid derivatives may also potentiate the efficacy of existing antibiotics. Thus, we intended to evaluate the synergistic antibacterial potentials of gallic acid and its 4 derivatives in this study with 8 currently available antibiotics against E. coli, S. Typhimurium and S. aureus . Additionally, the effects of those combination antibacterials against selected virulence factors, including biofilm formation and motility, were determined. Finally, in vitro cytotoxicity tests of potent phenolic compounds were conducted to determine the safety profile of these compounds for further commercial use.

Chemicals, Reagents and Bacterial Strains
Unless otherwise mentioned, all the chemicals, reagents and media were from Sigma-  Fractional Inhibition Concentration (FIC) 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 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. Plated bacteria were incubated at 35 °C for 16 to 20 h. The fractional inhibitory concentration (FIC) and the FIC index (FICI) were calculated from the MICs of the drugs alone and in combination. The FIC is the MIC of a drug in presence of another 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 gallic acid-ceftiofur against S. Typhimurium and hamamelitannin-erythromycin and gallic acid-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 drug-supplemented 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. At different time points (0, 1, 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 Phenolic Compounds on Quorum Sensing (QS) Inhibition
The potentials of QS inhibition of phenolic compounds were verified in accordance with the method described by Alvarez et al. [34]. The standard disk diffusion assay using the biomonitor C. violaceum strain (ATCC 12472) was performed for this evaluation. C.
violaceum ATCC 12472 was dispensed onto molten Luria-Bertani (LB, Becton Dickinson and Company, Becton Drive, NJ, United States) agar medium in a (90 × 15 mm) petri dish as to make the bacterial density 5 × 10 6 CFU/mL. Sixty microliter solutions of antibiotics that contain 60 μg of each compound were loaded onto sterile paper disks, and then the drugcontaining paper disks were allowed to air dry and placed on the solidified agar. The bacteria on the agar plates were incubated overnight at 30 °C and examined for violacein production. QS inhibition was determined by the presence of a colourless, opaque, but viable halo around the disks.

Effect of Antibacterial Combinations on Biofilm Growth and Viability
The inhibitory effect of combination antibacterials on biofilm formation was determined using a slightly modified version of a previously reported spectrophotometric method [ 35 , 36 ]. Briefly, test compounds were supplemented into trypticase soy broth (TSB; Becton Previously, reported biofilm viability assay methods were utilized to evaluate the effects of combination drugs on the viability of the biofilms produced by S. Typhimurium and E.

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. 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 [41][42][43][44][45] Figure 2. The SEM images revealed that untreated and gallic acid (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).

Results
The cells treated with ceftiofur alone or in combination with gallic acid 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 gallic acid-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 2 shows

Discussion
There are recent reports indicating the resistance of several bacterial strains to different antibiotics that have been used in the treatment of infectious diseases of human and animals [46]. Thus, to combat infectious diseases associated with resistant pathogens, the development of alternative antimicrobial drugs is urgently needed [47,48]. The in vitro activities of all the tested phenolic compounds against resistant strains of Salmonella coli and S. aureus, which have been shown to be resistant to one to eight out of ten antibiotics ( Table 1). The potentials of these phenolic compounds were further explored through their combined interactions with commercial antibiotics, where they possessed synergistic effects with thiamphenicol and erythromycin against S. Typhimurium and E.
coli and additive and/or indifferent effects with the other tested antibiotics. The time-and concentration-dependent inhibition assays also exposed that the combinations of phenolic compounds and commercial antibiotics more effectively inhibited the growth of S. aureus, MRSA, E. coli, P. aeruginosa, and Salmonella typhi [49]. The results in Table 1 indicate that gallic acid possessed the strongest antibacterial activity among these phenolic compounds, followed by epigallocatechin, hamamelitannin, epicatechin gallate and epicatechin. Moreover, very convincing MIC values were obtained for gallic acid and epigallocatechin against the Gram-positive bacterium S. aureus. The MIC value of gallic acid against the QC strain of S. aureus (128 µg/mL) in this study is lower than those reported previously for five strains (630 µg/mL) [49], one single strain (3200 µg/mL) [50], or 18 strains isolated from clinical cases of human impetigo and furuncle lesions (8000 µg/mL) [51], while the MIC value was higher than that reported in another study using only two strains (62.5 µg/mL) [52]. The MIC values of gallic acid against S. Typhimurium (256 µg/mL) and E. coli (1024 µg/mL) in this study are lower than those reported previously (2500 µg/mL) [49]. The mean MICs of plant-derived epigallocatechin against S. aureus, S.
Typhimurium and E. coli were reported to be 162±44, 572±186 and 733±121 µg/mL, respectively [53]. The MIC value of pure epigallocatechin against S. aureus (32 µg/mL) in our study was lower than the previously reported MIC value (162±44 µg/mL) [53]. This lower MIC value for plant-derived epigallocatechin against S. aureus in the previous study compared to the MIC value for pure epigallocatechin in our study is might be because of the purity of the compound used. However, the MIC values for plant-derived epigallocatechin against S. Typhimurium and E. coli were comparable with the results of our study. Likewise, the MICs of epicatechin against S. aureus, S. Typhimurium and E. coli were 2500 µg/mL, which demonstrates the similarity between our results and previously published results [49].
It is always recommended to treat bacterial infections with a combination of antimicrobial agents to prevent the development of drug resistance and to improve efficacy. Drug combinations with synergistic interactions are generally considered to be more effective and, therefore, preferable [54]. Incidentally, synergistic effects were obtained from the CLSM. These results revealed that in addition to being bacteriostatic, the gallic acidceftiofur, hamamelitannin-erythromycin and gallic acid-ampicillin combinations also appeared to act against the biofilm matrix. The large effect of the gallic acid-ceftiofur and gallic acid-ampicillin combinations against the biofilm cells of S. Typhimurium and E. coli, respectively, might be due to the small molecular size of gallic acid (170.12 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 [38]. Flagella are known to be involved in swimming motility and play a role in biofilm formation, as well as swarming motility [53]. Recent reports mentioned that, similar to biofilms, swarming cells also show a higher degree of resistance to a variety of antibiotics [39,59]. In this study, we investigated the ability of the gallic acid-ceftiofur, hamamelitanninerythromycin and gallic acid-ampicillin combinations to inhibit the swarming and swimming activities of S. Typhimurium and E. coli. The results ( Figure 3) showed significant inhibition of swimming and swarming motilities with the addition of gallic acidceftiofur, hamamelitannin-erythromycin and gallic acid-ampicillin combinations. The lack of swimming and swarming motilities in 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. Especially considering that the combinations of gallic acid-ceftiofur, hamamelitannin-erythromycin and gallic acid-22 ampicillin were found to be active against intestinal bacteria. Moreover, humans are very often exposed to ingested contaminants or toxins. Although virtually all organs and tissues are exposed, once the ingested toxins cross the intestinal wall, the gut is the first organ exposed and experiences the highest concentrations/doses of toxins. Many of these toxins are able to affect intestinal functions in both animals and humans [60].

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.

Authors' Contributions
MAH conceived the study, and involved in conception, design, acquisition of data, analysis, interpretation, drafting and critically revising the manuscript; JWK and HCP were

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