Synergistic antimicrobial potential of EGCG and fosfomycin against biofilms associated with endodontic infections

Abstract Objective This study aimed to evaluate the cytotoxicity and synergistic effect of epigallocatechin gallate (EGCG) and fosfomycin (FOSFO) on biofilms of oral bacteria associated with endodontic infections. Methodology This study determined minimum inhibitory and bactericidal concentration (MIC/MBC) and fractionated inhibitory concentration (FIC) of EGCG and FOSFO against Enterococcus faecalis, Actinomyces israelii, Streptococcus mutans, and Fusobacterium nucleatum. Monospecies and multispecies biofilms with those bacteria formed in polystyrene microplates and in radicular dentin blocks of bovine teeth were treated with the compounds and control chlorhexidine (CHX) and evaluated by bacterial counts and microscopy analysis. Toxicity effect of the compounds was determined on fibroblasts culture by methyl tetrazolium assays. Results The combination of EGCG + FOSFO demonstrated synergism against all bacterial species, with an FIC index ranging from 0.35 to 0.5. At the MIC/FIC concentrations, EGCG, FOSFO, and EGCG+FOSFO were not toxic to fibroblasts. EGCG+FOSFO significantly reduced monospecies biofilms of E. faecalis and A. israelli, whereas S. mutans and F. nucleatum biofilms were eliminated by all compounds. Scanning electron microscopy of multispecies biofilms treated with EGCG, EGCG+FOSFO, and CHX at 100x MIC showed evident biofilm disorganization and substantial reduction of extracellular matrix. Confocal microscopy observed a significant reduction of multispecies biofilms formed in dentin tubules with 84.85%, 78.49%, and 50.6% of dead cells for EGCG+FOSFO, EGCG, and CHX at 100x MIC, respectively. Conclusion EGCG and fosfomycin showed a synergistic effect against biofilms of oral pathogens related to root canal infections without causing cytotoxicity.


Introduction
The endodontic treatment aims to disinfect the root canal system and obturate the space to eliminate any residual microorganisms and prevent reinfection in teeth with apical periodontitis. 1 New technologies, such as nickel-titanium files and rotary instrumentation, have simplified instrumentation procedures, reduced treatment time, and increased the predictability of endodontic treatments. These procedures favor the retention of essential irrigants, which are necessary for their proper activation and dispersion throughout the endodontic system, resulting in better cleaning, especially in the apical portion, better treatment outcome. 2, 3 Although a significant reduction in the number of bacteria can be achieved by cleaning, shaping, and irrigating the canal during endodontic procedures, [1][2][3][4]5 it is not possible to attain complete disinfection of the root canal system by any of the available irrigants due to its anatomical complexity and bacterial resistance. 1,4,5 Some microorganisms are resistant to antimicrobial treatment and can survive in the root canal after chemo-mechanical preparation.
The most common bacteria are the Gram-negative anaerobic bacteria, such as Fusobacterium nucleatum, Prevotella spp., and Campylobacter rectus, and some Gram-positive bacteria, such as Streptococcus, Lactobacillus, Actinomyces, and Enteroccocus faecalis, among others. 6 Supplementary approaches, such as intracanal medications, have been used to eliminate surviving microorganisms inside dentin tubules and inhibit bacterial growth. 7 Calcium hydroxide is widely used as intracanal medication due to its bactericidal action and ability to neutralize the pH of remaining pulp tissues. However, calcium hydroxide may not be effective against all bacteria found in the root canal and requires multiple sessions to increase its antimicrobial action. 7,8 Triple antibiotic paste (TAP), a combination of metronidazole, ciprofloxacin, and minocycline, has been used as an intracanal dressing to eliminate pathogens in teeth with pulp necrosis and incomplete root formation in regenerative endodontic protocols.
However, the use of TAP has generated some concerns regarding bacterial resistance, crown discoloration, and possible allergic reactions. 9 New therapies have emerged to improve the antimicrobial efficiency and to enhance the penetration of irrigants into the complex root canal anatomy. These include antimicrobial photodynamic therapy (aPDT), laser-activated irrigation, and the use of sonic and ultrasonics activation. 10 Additionally, natural products, such as polyphenols, have been explored as alternative endodontic medicaments due to their wide range of biological properties, such as antimicrobial, antioxidant, anti-inflammatory, and anti-tumoral activities, among others. 11 Polyphenols are classified into four categories based on the presence of phenolic groups and structural elements: flavonoids, stilbenes, lignans, and phenolic acids. 11 Epigallocatechin-3-gallate (EGCG) is a major flavonoid compound extracted from green tea leaves, which represents approximately 59% of the total catechins. 11 In addition to its anti-oxidative, anti-tumor, and antiinflammatory properties, EGCG has demonstrated effectiveness against both Gram-positive and Gramnegative bacteria, 12,13 including multidrug-resistant bacteria such as Pseudomonas aeruginosa and Escherichia coli. 14 EGCG has also been shown to be efficacious against Enterococcus faecalis, both in planktonic cells and biofilm, 15 and it is also efficacious at inhibiting the adhesion of Streptococcus mutans in a dose-dependent manner. 16 Furthermore, EGCG also demonstrated potent inhibition of metalloproteinases (MMP), mainly MMP-2 and MMP-9, 17 which are hostderived enzymes associated with the self-degradation of dentin collagen and the progression of dental caries, periodontitis, and apical periodontitis. 17,18 Studies have demonstrated that low concentrations of EGCG can enhance antimicrobial activity of β-lactam antibiotics against multidrug-resistant bacteria by attacking the same site on cell walls as the antibiotics, specifically the peptidoglycan. 19 In addition, the combination of antibiotics with EGCG has been shown to increase the antibacterial effect on oral biofilms. 20 The antibiotic fosfomycin is a phosphonic acid derivative with wide-spectrum activity against several bacteria, including multidrug-resistant bacteria, since it inhibits an enzyme-catalyzed reaction in the first step of bacteria cell wall synthesis. [21][22] Synergistic effect of fosfomycin can be observed when it is combined with other antimicrobial agents that present different mechanism of action, thereby allowing for reduced dosages and lower toxicity. 21 This study aimed to evaluate the cytotoxicity and antibacterial effect of EGCG and fosfomycin, alone and in combination, on endodontic bacteria in both planktonic and biofilm conditions. Due to the large Synergistic antimicrobial potential of EGCG and fosfomycin against biofilms associated with endodontic infections 2023;31:e20220282 3/13 number of species involved in endodontic infections and the need for a broad-spectrum medicament to be used in root canals between appointments, the combinations of these drugs could be promising in controlling infection and inflammation before root canal obturation. We hypothesized that the combined use of EGCG and fosfomycin will present superior antibacterial effect without affecting cell viability.
This novel combination could be used as an effective bioactive agent for both irrigants and intracanal medication in endodontic treatments.

Preparation of the compounds
The compounds tested were fosfomycin sodium  Plates were incubated at 37°C in 5% CO 2 for 48 h, except for F. nucleatum, which was incubated for 72 h in an anaerobic condition. After these periods, the culture medium was removed and the wells were The microplates were incubated for 24 h under the same conditions as previously described. Next, the treatments were removed, the biofilms were scraped off with sterile plastic cell scrapers and saline solution, serially diluted and plated in BHIA. After 48h of incubation, CFU/mL was determined.

Multispecies biofilm assays in polystyrene microplates and SEM analysis
The multispecies biofilm assays were conducted following the same steps described previously for monospecies biofilms; however, all bacteria (E. faecalis, A. israelii, S. mutans, and F. nucleatum) were mixed in equal aliquots at the same concentration (1-5× 10 3 CFU/mL) in supplemented BHI broth containing 1% glucose. The inoculum was inserted in 24 wells microplates and incubated for one week in anaerobic conditions. After that, biofilms were washed twice with sterile saline solution and the following treatments were performed: 1) EGCG at 10× the highest MIC (2.5 mg/mL) and 2) EGCG + FOSFO at 10× the highest FIC (0.625mg/mL EGCG and 0.078 mg/mL FOSFO). Ltda, Lake Bluff, IL). After enlarging the root canals with a Gates Glidden (#6, 1.5 mm in diameter) drill, dentin blocks were fractured into two semi cylindrical halves (3×3×2 mm) using a using a handpiece diamond disc. After that, they were autoclaved and

Monospecies biofilms
The biofilm of E. faecalis was significantly reduced by EGCG + FOSFO at 10x FIC (-6.2 log), followed by CHX at 10× MIC (-5.    which can be seen in Figure 3C (CHX 10×) and Figure   3E (control without the antimicrobial agents). Figure   3F presents the effect of all compounds on bacterial counts (in log CFU/mL) in the multispecies biofilms, confirming that CHX 100×, EGCG+FOSFO, and EGCG were the most effective treatments, reducing 9.35 log, 7.79 log, and 6.43 log UFC/mL, respectively.

Multispecies biofilms in dentin tubules and CLSM analysis
The effect of EGCG+FOSFO and CHX was also observed on multispecies biofilm formed in dentinal tubules of bovine teeth, as shown in Figure 4. There is no statistical difference between EGCG and EGCG+FOSFO, however, the concentrations of EGCG (0.62 mg/mL) in combination with FOSFO were much lower than EGCG alone (2.5 mg/mL). A significant reduction in the bacterial counts can be observed in Figure 4F for all groups, with 84.85%, 78.49%, and 50.6% of dead cells in dentinal tubules for EGCG+FOSFO at 10× FIC, EGCG 10× MIC, and CHX at 100× MIC, respectively.   Additionally, EGCG has also been shown to suppress the expression and activities of virulence factors of S. mutans associated with acidogenicity and aciduricity, making it potentially attractive anti-caries agent. 16 However, different from our results, a previous study reported that EGCG was able to inhibit the growth or In addition to ECGC, fosfomycin was also used in the present study as an antimicrobial agent.
Fosfomycin interferes with the first stage of bacterial cell wall biosynthesis, specifically the formation of the peptidoglycan precursor UDP N-acetylmuramic acid (UDPMurNAc), which is linked to reduced bacterial adhesion to epithelial cells. 22 Clinically, fosfomycin in combination with daptomycin, vancomycin, rifampin, and tigecycline has been shown to significantly reduce and modify the structure of Staphylococcus aureus (MRSA) and Enterococcus faecalis biofilms. 33,34 In this study, the combination of EGCG and