Antibacterial and Antibiofilm Activity of Juglone Derivatives against Enterococcus faecalis: An In Silico and In Vitro Approach

Enterococcus faecalis is a Gram-positive bacterium that is normally found in the gastrointestinal tract of humans and animals. E. faecalis is an opportunistic pathogen that causes a number of invasive and noninvasive infections. The emergence of multidrug resistance and biofilm formation by the bacterium have rendered the treatment of E. faecalis infections very difficult. Due its high rate of resistance and biofilm formation, there are very few options of treatment. Therefore, the current study was designed to evaluate the antibacterial and biofilm activities of juglone derivatives such as 2-methoxy-6-acetyl-7-methyljuglone and 2-ethoxy-6-acetyl-7-methyljuglone against multidrug-resistant (MDR) and biofilm-producing strains of E. faecalis. Agar well diffusion and broth microdilution methods were used to determine the antibacterial activities. Biofilm attachment and preformed biofilm inhibition were determined using crystal violet staining assay. Both juglone derivatives displayed promising antibacterial and antibiofilm activities against E. faecalis. Among these compounds, 2-ethoxy-6-acetyl-7-methyljuglone possessed better inhibitory activity with minimum inhibitory concentration (MIC) of 9.7 ± 3 μM as compared to 2-methoxy-6-acetyl-7-methyljuglone (MIC, 19.5 ± 2 μM). Additionally, 2-ethoxy-6-acetyl-7-methyljuglone also showed stronger antibiofilm activity than 2-methoxy-6-acetyl-7-methyljuglone. Furthermore, both the ligand molecules were docked into the binding site of the enterococcal surface protein, and the results revealed that both the molecules are actively binding in the target site. Based on these findings, juglone derivatives may be considered useful for the treatment of E. faecalis infections; however, further studies are required to elucidate the mechanism of action.


Introduction
Enterococcus faecalis is a noncapsulated, non-spore-forming Gram-positive bacterium, a member of group D Streptococci [1,2]. Morphologically, these bacteria are round to oval in shape and arranged mostly in chains but are also found single or in pairs and groups [3,4]. E. faecalis bacteria are found ubiquitously in the environment including dairy products, fermented food, plants, water, and soil and the intestinal tracts of animals [2]. Their ubiquitous presence is largely due to their ability to survive in various environmental conditions. They can grow in a wide temperature range of 10-45°C in the presence of high NaCl content and 40% bile and can survive for 30 min at 60°C [5]. Being a member of normal gastrointestinal, oral, and vaginal microbiota, E. faecalis can also become an opportunistic pathogen especially in hospitalized and immunocompromised individuals [6].
E. faecalis is responsible for 80−90% of hospital-acquired enterococcal infections [7]. A variety of virulence factors are responsible for the pathogenesis of E. faecalis, including cytolysin, aggregation substances, enterococcal surface protein (Esp), adhesins to collagen of E. faecalis (Ace), E. faecalis antigen A (EfaA), endocarditis and biofilm-associated pili (Ebp), and gelatinase and serine protease [5]. E. faecalis is an important Enterococcus that causes a range of infections in both humans and animals. Invasive infections include bacteremia endocarditis and peritonitis. Noninvasive infections include urinary tract infections and postsurgery wound infections [4].
E. faecalis possesses a high rate of antibiotic resistance with intrinsic resistance to cephalosporins and quinupristin-dalfopristin, and acquired resistance to quinolones, glycopeptides, and aminoglycosides by horizontal gene transfer (HGT) and mutation has led to the emergence of multi-drug-resistant (MDR) strains of this bacterium [8]. E. faecalis has the potential to inhabit the site of infection and secrete extracellular matrices that lead to biofilm development [9]. Biofilms have been linked to the development of chronic diseases that are difficult to treat with available antimicrobial drugs [10]. Biofilms exhibit strong resilience to antibiotics and other stressors, as well as a high rate of HGT and distinct gene expression patterns compared to planktonic bacteria [11]. Adaptive resistance to antibiotics, which is on the rise globally, poses a barrier to treating biofilm-associated chronic and acute infections [12]. Biofilm production increases resistance to antibiotics increased 100 −1000-fold, and approximately 65−80% infections are mediated by biofilms [13,14]. All these factors make E. faecalis treatment rather challenging; therefore, it is important to focus on newer therapeutic measures.
Reynoutria japonica, commonly called the Japanese knotweed, is a well-known medicinal plant that belongs to the family Polygonaceae [15]. The roots of Reynoutria japonica have been traditionally used for the treatment of infection, inflammation, jaundice, hyperlipidemia, and burns [16]. The roots of this plant are rich source of many biologically active compounds such as polydatin, emodin, resveratrol, physcion, and naphthoquinones [17]. Naphthoquinone and its derivatives especially juglone derivatives exert a variety of pharmacological activities [16,17]. Recently, it has been reported that juglones such as 2-methoxy-6-acetyl-7-methyljuglone and 2-ethoxy-6-acetyl-7methyljuglone show promising inhibitory activity against Helicobacter pylori 13 . Therefore, the current study was designed to evaluate the antibacterial and antibiofilm activities of these juglone derivatives against the MDR and biofilm-producing strains of E. faecalis. The bactericidal nature of these juglone derivatives was also determined.

Isolates of Enterococci.
A total of forty enterococcal isolates were collected from the microbiology laboratories of three tertiary care hospitals in Peshawar, Pakistan (Rehman Medical Institute, Northwest Hospital, and Khyber Teaching Hospital), from September 2020 to May 2021, after ethical approval was granted by the concerned ethical committees. The isolates were transferred in transport media in leak proof sterile container to the microbiology laboratory of the Institute of Basic Medical Sciences (IBMS), Khyber Medical University Peshawar, for further processing.

Identification of Enterococci.
For validation and confirmation, the isolates were cultured on selective media (bile esculin agar) and identified by colony characteristics, Gram staining, growth on 6.5% NaCl media, catalase test, and esculin hydrolysis. The identification to species level was done using carbohydrate utilization tests (E. faecalis is positive for sorbitol and mannitol and E. faecium is positive for arabinose and raffinose utilization). Additionally, only E. faecium can grow at 4°C [18].

Biofilm Assay.
A fresh overnight E. faecalis culture was suspended in 5 mL of Luria-Bertani (LB) broth and incubated aerobically at 37°C for 24 h 19 . After incubation tubes were centrifuged for 3-5 min, supernatant was discarded. The precipitate was suspended in normal saline and adjusted to 0.5 McFarland standard. To each well with 150 μL of LB broth of 96-well polystyrene microtiter plate, 50 μL of 0.5 McFarland cell suspension was added. E. faecalis TX-82 was used as a positive control while LB broth without inoculum was used as negative control. After 48 h of aerobic incubation, the plate was washed three times with phosphate-buffered saline and stained with 1% crystal violet for 15 min at room temperature (25°C). The quantification of biofilm was measured at OD 570 nm after solubilization of biofilm with 95% ethanol [19]. The result of the biofilm was interpreted as follows:

Minimum Inhibitory Concentration (MIC).
The antibacterial activity of tested compounds was determined with modified microbroth dilution method. The MIC assay was determined using the method described earlier with some modifications [21]. A standard bacterial inoculum of 0.5 McFarland was prepared in tryptic soya broth. Two hundred microliter of broth was added to each well of a 96-well plate, and 100 μL of 0.5 McFarland bacterial suspension was added to each well. Juglone derivatives were serial-double fold diluted and added to the well in different concentrations followed by incubation at 37°C for 24 h. After incubation, the MIC was defined as the lowest concentration of juglone derivatives which inhibit the growth of E. faecalis. All the assays were performed in triplicate.

Minimum Bactericidal Concentration (MBC).
To determine the MBC of juglone derivatives, 2 μL of each suspension from the 96-well plate streaked on the blood agar plate along with the positive and negative controls. The plate was incubated aerobically at 37°C for 24 h, and the results for growth were observed [9]. MBC is the lowest concentration of molecules at which no growth is observed after incubation. No visible growth signified the bactericidal nature of juglone derivatives.
2.9. Biofilm Attachment Inhibition Assay. The standard 0.5 McFarland bacterial suspension was prepared by adding fresh E. faecalis colonies into 5 mL of LB broth. To the 96well plate, 100 μL of bacterial suspension was added. Sterile distilled water and bacterial inoculum were added for positive and negative controls, respectively. To the test the wells, 100 μL of juglone derivatives were added in different concentrations, i.e., from higher to lower. The plate was incubated for 24 h at 37°C without shaking. The plate was washed three times with phosphate buffer and quantified using crystal violet staining assay as described earlier [22].

Preformed Biofilm Inhibition
Assay. Standard bacterial suspension of 100 μL was added to the 96-well plate along with the positive and negative controls and incubated aerobically at 37°C for 24 h. After incubation, juglone derivatives were added in different concentrations in amount of 100 μL to test wells and incubated. In the next day, the plate was washed three times with phosphate buffer and stained with 1% crystal violet as describer earlier. All the assays were performed in triplicate for the purpose of validity and authenticity. For the result interpretation, the following formula was used to determine the inhibitory ability of molecules and expressed in percentage. The absorbance was measured spectrophotometrically at 570 nm [23].
2.11. Statistical Analysis. All data of the present study were obtained from at least triplicate assays performed on different days. The data are given with the mean value followed by the standard deviation. 2.14. Protein Preparation. Our research relied on the protein molecules enterococcal surface protein (Esp; PDB ID: 6ori), which could be found in the database known as Protein Data Bank. Following the elimination of water molecules, the three-dimensional protonation of the protein molecule was carried out. The energy minimization algorithm of the MOE tool was utilized to bring the energies of the protein molecules down to their lowest possible levels. For the purpose of minimizing the amount of energy required, the following parameters were utilized: gradient: 0.05, force field: MMFF94X+solvation, and chiral constraint: current geometry. When the root mean square gradient dropped below the 0.05 threshold, the energy minimization process came to an end. Docking was modelled after the minimized structures, which served as the template [26].
2.15. Docking. The binding of the ligand molecules with the protein molecules was analyzed using MOE docking program to find the correct conformation of the ligands, so as to obtain minimum energy structure. After the completion of docking, we analyze the best poses for hydrogen bonding/π − π interactions and root mean square deviation (RMSD) calculation by using MOE applications [24].

Antimicrobial Susceptibility Testing. The modified
Kirby-Bauer disc diffusion method was performed to identify the MDR strains of E. faecalis. Strains of E. faecalis that exhibited resistance to three or more antibiotics were categorized as MDR. Among the tested antibiotics, erythromycin showed the lowest potency, followed by ciprofloxacin and tetracycline with 13%, 22%, and 26% sensitivity, respectively. The most potent antibiotics against E. faecalis were linezolid, vancomycin, and fosfomycin, with 97%, 87%, and 79% sensitivity, respectively. Gentamycin and amoxicillin showed   2-Ethoxy-6-acetyl-7methyljuglone 9:7 ± 3 9:7 ± 2 Data are expressed as the mean and SD of three independent experiments. 4 BioMed Research International moderate activity, with 61% and 39% sensitivity, respectively ( Figure 1).

Antibacterial Activity Using Agar of Juglone Derivative
Well Diffusion Method. The antibacterial activities of juglone derivatives against the MDR biofilm-producing strains of E. faecalis and ATCC 29212 were evaluated using the agar well diffusion method. It was observed that 10 mM of 2-methoxy-6-acetyl-7-methyljuglone produced inhibitory zones of 15 ± 1 mm and 16:2 ± 0:8 mm against the MDR biofilmproducing strains of E. faecalis and ATCC 29212 strain, respectively; a decrease in the zone of inhibition was observed with a decrease in concentration. Furthermore, 2ethoxy-6-acetyl-7-methyljuglone showed better inhibitory activity than the other juglone with inhibitory zones of 17:3 ± 0:5 mm and 18:1 ± 0:9 mm MDR strains and ATCC strain, respectively, at 10 mM concentration (Table 1).
3.6. Preformed Biofilm Inhibition Assay. Both juglones were applied against the preformed biofilm of E. faecalis; 2- ethoxy-6-acetyl-7-methyljuglone showed higher inhibitory efficacy than 2-methoxy-6-acetyl-7-methyljuglone with the same pattern. The highest activity was observed at highest concentration against low biofilm producers of the strain as compared to moderate and high biofilm producers (Figures 2-5).
In the case of 2-methoxy-6-acetyl-7-methyljuglone, a total of four hydrogen bond interactions were observed (Figure 6(a)). The binding site residues Arg211, Gln241, Asp243, and Phe244 were found to interact with ligand molecules, whereas ligand molecule 2-ethoxy-6-acetyl-7-methyljuglone was observed to interact with Arg211, Asp243, and Phe244, respectively (Figure 6(b)). The docking scores and binding energies of both ligand molecules are listed in Table 3. A scoring function known as the generalized-Born volume integral/weighted surface area estimates the free energy required to bind ligand in a given pose. For each of the scoring functions, a lower score indicates a more advantageous pose. When conducting molecular docking research, the binding energies and the docking scores are the primary criteria used to determine which compounds are active and which are inactive.
For the validation of the MOE docking program, RMSD value was calculated. For this purpose, the cocrystallized ligand was removed from the protein and redocked in the same site. The found RMSD value was 1.3561 Å. Figure 7 shows the validity of our docking method. It proves that MOE is reliable for docking studies.

Discussion
Enterococci are normal inhabitants of intestinal tract of humans and animals and constitute a predominant part of the environmental microbiota, found in food, water, sewage, soil, and plants. They are important opportunistic pathogens and pose a major threat for hospitalized and immunocompromised patients. Among enterococci, Enterococcus faecalis is the most prevalent and responsible for a number of infections, such as urinary tract infections, soft tissue and wound infections, bacteremia, peritonitis, and endocarditis [3].

BioMed Research International
On account of the high occurrence of nosocomial infections, E. faecalis is constantly exposed to multiple antibiotics, resulting in the emergence of its antibiotic resistance. E. faecalis possesses intrinsic resistance to some antibiotics including quinupristin-dalfopristin, lincosamides, cephalosporin, and low levels of aminoglycosides [18]. Over time, it has acquired resistance genes via HGT and mutation resulting in resistant phenotypes that are very difficult to treat. It has acquired resistance against quinolones, glycopeptides, and aminoglycosides, thereby leading to the emergence of MDR superbugs, which have resulted in few options for treatment [27].
MDR strains of E. faecalis arise from the excessive use of antibiotics in livestock, plants, and humans. Continuous use of antibiotics eliminates the sensitive bacterial population, but the resistant ones remain alive, grow, and multiply [29]. MDR strains of E. faecalis increased substantially over the past decade and present a major healthcare burden today [30].
In our study, agar well diffusion assay was performed to evaluate antibacterial activity. A 15 ± 1 mm zone of inhibition was observed against the MDR and biofilm-producing strains and 16:2 ± 0:8 mm zone against ATCC 29212 by 2methoxy-6-acetyl-7-methyljuglone. We found that 2-ethoxy-6-acetyl-7-methyljuglone produced a 17:3 ± 0:5 mm zone of inhibition against MDR strains and 18.1 ± 0.9 mm against ATCC 29212. Some studies have shown that juglones have the potential to target bacterial DNA, RNA cell wall, 7 BioMed Research International and cell membrane. A similar assay with very similar results was performed in India to check the antibacterial activity of a medicinal plant against Escherichia coli, P. aeruginosa, Bacillus cereus, and staphylococcus aureuss [22]. The clear zones of inhibition in our study supported such earlier work, wherein bioactive compounds extracted from medicinal plants were found to have strong antibacterial potential even against MDR strains.
The antibacterial activity of juglone derivatives was further evaluated through broth microdilution method. In current study, both molecules exhibited excellent antienterococcal activity against MDR E. faecalis and ATCC 29212 strain. Between the two compounds, 2-ethoxy-6-acetyl-7-methyljuglone exhibited stronger antienterococcal activity than 2-methoxy-6-acetyl-7-methyljuglone, with MIC values of 9:7 ± 3 μM and 19:5 ± 2 μM, respectively. In the current study, the MBC and MIC values were same for both the compounds which showed that these compounds were bactericidal at lower concentrations. A similar study was performed in Malaysia to determine the antibacterial activity of juglone derivatives against E. coli, B. cereus, K. pneumoniae, and SS. aureuss [30]. This indicated that juglone derivatives displayed a wide spectrum of antibacterial activity against different bacterial species.
In biofilms, bacterial populations grows in a community attached to biotic surfaces. It is an important phenomenon in the life cycle of microorganisms, which provides protection against harsh condition, desiccation, antiseptics, antibodies, antibiotics, and abrupt changes in pH. Biofilms are mostly composed of polysaccharides, hydrated exopolymeric substances, proteins, and nucleic acids [9,12]. E. faecalis has strong biofilm formation potential that renders it 100−1000 times more resistant to antiseptics, antibodies, and antibiotics. In a majority of chronic infections caused by E. faecalis, biofilm-producing strains were involved. Biofilm formation not only provides protection but also enhances HGT giving rise to MDR strains and making the treatment more challenging [27,31]. To control chronic infections and antibiotic resistance, it is thus crucial to focus on biofilm inhibition. While earlier studies confirmed the antibacterial  BioMed Research International activity of juglone derivatives, in this study, we tested their ability to inhibit the biofilm formation of E. faecalis. The MDR strains of E. faecalis were sensitive to juglone derivatives used in the study in planktonic state. These compounds were also checked for their inhibition of biofilm attachment and preformed biofilms. The biofilm attachment inhibition rate of 2-ethoxy-6-acetyl-7-methyljuglone at 70% was found more promising as compared to that of 2-methoxy-6-acetyl-7-methyljuglone at 63.1% while their disruption rates of preformed biofilm were 69.3% and 59%, respectively. Wang et al. stated that juglones produced oxidative environment in the cell which resulted in cell wall dis-ruption and increased cellular permeability leading to cell death. They also interfered with cell signaling leading to biofilm inhibition [32]. Our study found that the inhibitory effect of juglone derivatives reduced with a reduction in concentration. A similar study reported that 31.4% biofilm attachment inhibition and 7.5% of preformed biofilm inhibition were recorded against E. faecalis biofilm-producing strains [33]. Moreover, 2-ethoxy-6-acetyl-7-methyljuglone, which has an ethoxy group at the C-2 position, was found to have a slightly more potent activity than 2-methoxy-6acetyl-7-methyljuglone, which has a methoxy group at the same position [13]. Qayyum et al. also evaluated the antibiofilm activity of quercetin against E. faecalis and recorded biofilm inhibition rates of 95%, 85%, and 70% at concentrations of 256, 128, and 64 mg/L, respectively [9]. Our study proves that juglone derivatives have a remarkable antibiofilm potential and can be used to eradicate biofilms, which represent a key cause of antibiotic resistance and chronic infections.

Conclusion
The findings of our study demonstrated that two juglone derivatives (2-methoxy-6-acetyl-7-methyljuglone and 2-ethoxy-6-acetyl-7-methyljuglone) isolated from Reynoutria japonica have very promising antibacterial activity against the MDR strains of E. faecalis. Furthermore, these compounds also have the potential to prevent biofilm formation    BioMed Research International by E. faecalis. According to our findings, 2-ethoxy-6-acetyl-7-methyljuglone has better antibacterial and antibiofilm activity as compared to 2-methoxy-6-acetyl-7-methyljuglone. Therefore, it is concluded that these natural compounds can be a good alternative to commercial antibiotics to treat infections caused by the MDR and biofilmproducing strains of E. faecalis.

Data Availability
All data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors report no conflicts of interest.