Next Article in Journal
Mechanical Performance and Stress Redistribution Mechanisms in Photovoltaic Support Connections: A Finite-Element-Driven Design Optimization Study
Previous Article in Journal
A Comparative Study of UCS Results Obtained from Triaxial Tests Under Multiple Failure State Conditions (Test Type II)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Applied Sciences
Volume 15
Issue 6
10.3390/app15063178
applsci-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Anti-Planktonic, Antibiofilm, and Synergistic Effects of Nasturtium officinale and Psidium guajava Hydroethanolic Extracts Against Standard and Clinical Strains of Enterococcus faecalis

by
Lara Steffany de Carvalho
1,
Livia Ramos Dorta da Silva
2,
Cláudio Antonio Talge Carvalho
2,
Maria Cristina Marcucci
1,
Luciane Dias de Oliveira
1 and
Amjad Abu Hasna
2,3,*
1
Department of Bioscience and Oral Diagnosis, Institute of Science and Technology, São José dos Campos, São Paulo State University (UNESP), São Paulo 12245-000, Brazil
2
Department of Restorative Dentistry, Endodontics Division, Institute of Science and Technology, São José dos Campos, São Paulo State University (UNESP), São Paulo 12245-000, Brazil
3
School of Dentistry, Universidad Espíritu Santo, Samborondón 092301, Ecuador
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(6), 3178; https://doi.org/10.3390/app15063178
Submission received: 12 February 2025 / Revised: 7 March 2025 / Accepted: 12 March 2025 / Published: 14 March 2025
(This article belongs to the Special Issue Recent Developments in Endodontics and Dental Materials)
Browse Figures
Versions Notes

Abstract

:
Enterococcus faecalis is strongly associated with secondary/persistent root canal infections, being the most prevalent bacterium in cases of apical periodontitis in previously treated teeth. This study was elaborated to evaluate the anti-planktonic, antibiofilm, and synergistic effects of Nasturtium officinale and Psidium guajava hydroethanolic extracts against standard and clinical strains of E. faecalis. Firstly, the N. officinale extract was prepared from watercress leaves, and P. guajava extract was prepared from guava tree leaf shoots. Then, the content of soluble solids was quantified in both. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the isolated N. officinale and P. guajava extracts for each bacterial strain were determined using the broth microdilution method, following the Clinical and Laboratory Standards Institute (CLSI) guideline M7-A9. The MTT assay was used to evaluate the antibiofilm activity, and the fractional bactericidal concentration index (FBCI) was utilized to evaluate the synergistic effect of the N. officinale and P. guajava extracts using the checkerboard technique. Again, the MTT assay was used to evaluate the antibiofilm activity of the combined extracts this time. The data were subjected to statistical analysis using ANOVA and Tukey’s test, with a significance level of p ≤ 0.05. It was found that the soluble solid content of N. officinale was 50 mg/mL, and of P. guajava was 33.5 mg/mL. The MBC value of N. officinale was 12.5 mg/mL, and of P. guajava was 0.52 mg/mL against all the tested strains of E. faecalis. The combined 0.1 mg/mL N. officinale + 0.1 mg/mL P. guajava, and 0.1 mg/mL N. officinale + 0.5 mg/mL P. guajava hydroethanolic extracts effectively reduced the biofilm formation of the standard and clinical strain 4 of E. faecalis. Therefore, these combined extracts may be considered as endodontic irrigants in future studies.

1. Introduction

The oral cavity naturally contains various species of microorganisms that can invade the root canal system and cause endodontic infections [1]. These infections are associated with pulp necrosis resulting from caries, trauma, iatrogenic damage, and periodontal disease, known as primary infections, or they may occur in teeth that have undergone previous root canal treatment, known as secondary/resistant infections [2]. At least 500 microbial species, mostly bacteria, are linked to endodontic infections, including primary, secondary, persistent, and extraradicular infections. Some fungal species are also present [3].
Microorganisms can organize into a biofilm that adheres to the dentinal walls of the root canal system [4]. In biofilm form, bacteria become more resistant than their planktonic counterparts [5]. Around 20 to 30 species of oral microorganisms are frequently found in primary infections, the majority being strict anaerobic and Gram-negative bacteria, along with some Gram-positive bacteria. In secondary and persistent infections, microbial diversity is more restricted if the previous endodontic treatment was adequate. However, if the treatment was inadequate, the microbiota resembles that of a primary infection in both load and diversity [1].
Enterococcus faecalis is a Gram-positive, facultative anaerobic, fermentative coccus. Its shape is ovoid, ranging from 0.5 to 1 µm in diameter [6]. The presence of E. faecalis is strongly associated with secondary/persistent root canal infections, being the most prevalent bacterium in cases of apical periodontitis in previously treated teeth [7]. Its ability to survive in the root canal system despite conventional antimicrobial protocols is attributed to several key mechanisms. One of the primary factors is its ability to invade dentinal tubules and adhere to collagen, allowing it to persist in areas inaccessible to irrigants and mechanical instrumentation. Additionally, it has the capacity to withstand inhospitable conditions, such as nutrient scarcity and the highly alkaline environment of the root canal following endodontic treatment [8]. This resistance is partly due to its proton pump activity, which helps maintain intracellular pH homeostasis. E. faecalis was detected in different types of endodontic infections, including chronic asymptomatic periapical lesions, acute apical periodontitis, acute dentoalveolar abscesses, and teeth requiring retreatment due to chronic asymptomatic periapical lesions. Furthermore, E. faecalis was significantly more associated with asymptomatic cases, and was present in 33% of asymptomatic periradicular lesions and 67% of retreatment cases with persistent infections, highlighting its strong association with failed endodontic treatments [9].
Commonly, sodium hypochlorite (NaOCl) is used as an endodontic irrigant, it exhibits antimicrobial efficacy against E. faecalis, antiendotoxin activity against its lipoteichoic acid (LTA), and dissolving capacity [10]; however, it has serious contraindications in endodontics represented by its cytotoxicity, genotoxicity, and the potential for allergic reactions in some patients [10]. Thus, the search for alternative irrigants in endodontics is constant. The use of herbal medicines is increasing as these are valuable sources of bioactive molecules with antimicrobial properties and biocompatibility [11]. Studies have reported synergistic effects when combining herbal medicines, revealing promising potential for new therapeutic products [12,13,14].
Nasturtium officinale W.T. Aiton, commonly known as watercress, is a perennial plant from the Brassicaceae family, native to West Asia, Europe, India, and Africa, but cultivated in various regions worldwide [15]. N. officinale contains flavonoids, phenolic acids, and glucosinolates, which, through natural hydrolysis by the enzyme myrosinase, produce isothiocyanate (ITC) compounds [16]. It was added to calcium hydroxide to improve its efficiency against Candida albicans as intracanal medicament, and it was effective [17].
Psidium guajava L., commonly known as guava, is a tropical tree found predominantly in South American countries [18,19]. Studies have shown that guava leaf extract is rich in polyphenols, including quercetin, a flavonoid with antimicrobial and antifungal activity. It has demonstrated strong efficacy against Gram-positive and Gram-negative bacteria [20,21]. In a pilot study, P. guajava was indicated as an alternative endodontic irrigant [22].
To the best of our knowledge, the synergistic effect of N. officinale and P. guajava hydroethanolic extracts was not tested against planktonic and biofilms of the standard (ATCC 29212) and clinical strains of E. faecalis. Therefore, the aim of this study was to evaluate the anti-planktonic, antibiofilm, and synergistic effects of these extracts against E. faecalis. The null hypothesis states that these extracts have no anti-planktonic, antibiofilm, or synergistic effects against the tested bacteria.

2. Materials and Methods

2.1. Production of N. officinale and P. guajava Hydroethanolic Extracts

N. officinale hydroethanolic extract was prepared, following a standardized protocol to ensure consistency across experiments, from watercress leaves, and P. guajava hydroethanolic extract was prepared from guava tree leaf shoots. Fresh plant materials were collected, washed, and dried in a controlled environment in the dark at room temperature between 20 and 27 °C for 5 days. The extraction solvent consisted of absolute ethanol (ethyl alcohol 99.5%—Merck Darmstadt, Darmstadt, Germany) and ultrapure water obtained from a Milli-Q® system (EtOH:H2O) in a 50:50 ratio.
The dried materials (30 g) after grounded into a fine powder were subjected to hydroethanolic extraction by 100 mL of the extraction solvent for 48 h at room temperature with constant agitation. Finally, the N. officinale and P. guajava hydroethanolic extracts were filtered in two stages: paper filter to remove solid residues followed by sterilization using a 0.22 µm membrane filter [13].
A formal power analysis was not performed for this study. Instead, the sample size was determined based on a pilot study [22] conducted prior to the main experiments. The pilot study allowed for an assessment of variability and feasibility, ensuring that the chosen sample size was adequate for reliable statistical comparisons.

2.2. Quantification of Soluble Solid Contents

Six empty 25 mL beakers were weighed, and their weights were recorded. Then, 5 mL of each extract was pipetted into them (triplicate for each extract), and they were dried in an oven at 80 °C for 24 to 48 h. After drying, the beakers were placed in a desiccator until completely cooled and then weighed [13,22]. The number of soluble solids in the N. officinale and P. guajava hydroethanolic extracts was quantified using the following formulas:
% Soluble Solids (m/v) = (m − b) × 100/Va
% Soluble Solids (m/m) = % soluble solids (m/V)/density
where b = the mass of the beaker, m = final mass of the extract after drying, V = the volume pipetted into the beaker, and extract density = m/V (mass of the 5 mL aliquot weighed, and V is the volume of 5 mL).

2.3. Anti-Planktonic Activity

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the isolated N. officinale and P. guajava hydroethanolic extracts for each bacterial strain were determined using the broth microdilution method, according to the Clinical and Laboratory Standards Institute (CLSI) guideline M7-A9. In this study, a standard strain (ATCC 29212) and two clinical strains (nominated 2 and 4) were used.
The reference strains sourced from the American Type Culture Collection (ATCC) (Rio de Janeiro, RJ, Brazil), and the clinical strains were obtained from the National Institute of Quality Control in Health (INCQS) of the Oswaldo Cruz Foundation (FIOCRUZ). The antibiotic sensitivity profile for clinical isolates of E. faecalis and E. faecium was published in a previous study by our research group [14], where E. faecalis 3 from that study corresponds to E. faecalis 4 in the present research.
The bacterial strains were cultured on Brain Heart Infusion (BHI) agar (HiMedia® Mumbai, India) at 37 °C for 24 h. Subsequently, bacterial inoculums were prepared in which colonies of the respective strains were diluted in sterile saline solution (0.9% NaCl) and homogenized in a vortex for 1 min to standardize the microbial solution according to the requirements of each protocol. The inoculums were standardized using a spectrophotometer (Micronal B-582, São Paulo, Brazil) at a wavelength of 625 nm and an optical density of 0.080, resulting in a standard suspension of 1 × 106 cells/mL.
Then, in separate 96-well plates (Kasvi K12-096, São José dos Pinhais, Brazil), 100 µL/well of Mueller Hinton broth (HiMedia® Mumbai, India) was added. A 100 µL aliquot of one of the prepared N. officinale and P. guajava hydroethanolic extracts was added only in the first well, followed by 10 serial dilutions of the extract (1:2) in Mueller Hinton broth. Then, 100 µL of each microbial inoculum was added to all wells in different 96-well plates for each bacterial strain. The plates were incubated at 37 °C for 24 h, in which the MIC values were determined as the last well in the microplate without turbidity, indicating no microbial growth (the test was performed in triplicate).
To determine the MBC values of the N. officinale and P. guajava hydroethanolic extracts, aliquots from all wells were plated on BHI agar and incubated at 37 °C for 48 h. The MBC of each extract for each bacterial strain was determined in plates without colony growth. A control group treated with the vehicle (EtOH:H2O/50:50) was included to assess any possible interference with the anti-planktonic activity of the extract [14].

2.4. Antibiofilm Activity

Different inoculums of the standard and clinical strains of E. faecalis were prepared and standardized in saline solution (0.9% NaCl) at a concentration of 1 × 108 CFU/mL using a spectrophotometer. Then, 100 µL/well of the corresponding inoculum and 100 µL/well of BHI broth were added in 96-well plates. The plates were kept at 37 °C for 48 h to form biofilms, in which the BHI broth was changed after 24 h [14].
After biofilm formation, the N. officinale and P. guajava hydroethanolic extracts were placed in contact with the biofilms, and the plates were incubated again at 37 °C for 24 h. Saline solution (Eurofarma, São Paulo, SP, Brazil) and 2.5% sodium hypochlorite (NaOCl) (Biodinâmica, Ibiporã, PR, Brazil) were used as the control groups. Each experimental group consisted of n = 10.
The experimental groups were evaluated by introducing 100 µL of MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA). The samples were incubated at 37 °C for 1 h, shielded from light. After incubation, the MTT solution was discarded, and 100 µL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) was added. The plates underwent a second incubation at 37 °C for 10 min, followed by agitation on a shaker for an additional 10 min. Absorbance was then recorded at 570 nm using a microplate reader (BIO-TEK Instruments, Highland Park, Winooski, VT, USA). The resulting optical density (OD) values were used to determine microbial cell viability percentages [13].
% Viability = (OD Treated Group × 100)/(Mean OD Control Group)

2.5. Synergistic Anti-Planktonic Activity

For the evaluation of the synergistic effect of the N. officinale and P. guajava hydroethanolic extracts, the “checkerboard” technique was used. This technique is based on the broth microdilution sensitivity test and determines the type of interaction between the extracts as synergistic, additive, antagonistic, or indifferent [13,23,24].
Based on the results found in the anti-planktonic analysis, the MBC values were used as the determination of the MIC values was not succeeded. For the double combinations of the extracts, 50 μL of each dilution of the N. officinale hydroethanolic extract was added to the x-axis (horizontal) of the microplate, while 50 μL of the dilutions of the P. guajava hydroethanolic extracts was added to the y-axis (vertical) of the microplate so that different concentrations of both extracts were associated. Then, 100 μL/well of Mueller Hinton broth and 100 μL/well of the bacterial inoculum, standardized at 1 × 106 cells/mL, were added in each well. Subsequently, the plate was incubated at 37 °C for 24 h before reading. Then, an aliquot from each well was plated on BHI agar. The plates were incubated for 24 h, and the Fractional Bactericidal Concentration Index (FBCI) was used, classifying the combinations as synergistic when FBCI ≤ 0.5, additive when FBCI > 0.5 and ≤1.0, indifferent when FBCI > 1 and ≤4, and antagonistic when FBCI > 4.0. The FBCI is calculated by the following formula:
FBC index = FBC 1st + FBC 2nd = (concentration of the 1st extract in combination/MBC of the 1st extract alone) + (concentration of the 2nd extract in combination/MBC of the 2nd extract alone).

2.6. Synergistic Anti-Biofilm Activity

After evaluating the possible combinations of plant extracts against E. faecalis strains, the selection criterion for combinations to be studied on biofilms was the lowest concentrations of the combined extracts that demonstrated effectiveness against all analyzed strains. This criterion was established since, for the future development of a dental product, it is necessary to focus on the combinations that achieved the greatest success.
The analysis of the combination of N. officinale and P. guajava hydroethanolic extracts on biofilms was performed in 96-well microplates, in which 100 μL/well of BHI broth was added, and 100 μL/well of the bacterial inoculum of each bacterial strain was standardized in saline solution (0.9% NaCl) at a concentration of 1 × 108 CFU/mL using a spectrophotometer. The plates were incubated at 37 °C for 48 h to allow for biofilm formation, with the broth being replaced after 24 h of incubation. Later, the mature biofilms were treated with 100 μL/well of the synergistic or additive concentration of the extracts for 5 min. The saline solution and 2.5% NaOCl were used as the control groups. Each experimental group consisted of n = 10. Finally, the experimental groups were analyzed by MTT assay as described above.

2.7. Statistical Analysis

The anti-planktonic and antibiofilm activity tests of the isolated extracts and their combinations were subjected to statistical analysis using ANOVA and Tukey’s test, with a significance level of p ≤ 0.05.

3. Results

3.1. Soluble Solid Contents

It was found that the soluble solid content of N. officinale hydroethanolic extract was 50 mg/mL (5%) and was 33.5 mg/mL (3.35%) for the P. guajava hydroethanolic extract.

3.2. Anti-Planktonic Activity

The MIC value could not be determined because of the intense coloration of the extracts, which hindered visual evaluation. Instead, the MBC value of the N. officinale hydroethanolic extract, identified by the last well found without turbidity, corresponds to a concentration of 12.5 mg/mL, and the P. guajava hydroethanolic extract corresponds to a concentration of 0.52 mg/mL for all the tested strains of E. faecalis.

3.3. Anti-Biofilm Activity

The N. officinale hydroethanolic extract at 25 mg/mL was effective in reducing the biofilm formation of E. faecalis ATCC, clinical strain 2, and clinical strain 4 by 63.41, 86.24, and 91.25%, respectively, with a statistically significant difference in the control group (p < 0.0001) and was as effective as NaOCl without a statistically significant difference (p > 0.05), as shown in Figure 1.
The P. guajava hydroethanolic extract was not effective against E. faecalis ATCC; however, it was effective in reducing the biofilm formation of the clinical strains of E. faecalis at different concentrations in a percentage ranging from 41.68 to 55.05% against the clinical strain 2, with the concentration of 1.04 and 0.52 mg/mL as effective as NaOCl, and in a percentage ranging from 40.46 to 45.08% against the clinical strain 4, with the concentrations of 2.09 and 1.04 mg/mL as effective as NaOCl, all with a statistically significant difference in the control group (p < 0.0001), as shown in Figure 1.

3.4. Synergistic Anti-Planktonic Activity

According to the calculation of the Fractional Bactericidal Concentration Index (FBCI), the combination of the N. officinale and P. guajava extracts exhibited synergistic and additive effects (Table 1).

3.5. Synergistic Anti-Biofilm Activity

The hydroethanolic extracts of 0.1 mg/mL N. officinale + 0.1 mg/mL P. guajava, and 0.1 mg/mL N. officinale + 0.5 mg/mL P. guajava were effective in reducing biofilm formation in both the standard and clinical strain 4 of E. faecalis, with a statistically significant difference compared to the control group (p < 0.0001). Additionally, the 0.1 mg/mL N. officinale + 0.1 mg/mL P. guajava, and 0.1 mg/mL N. officinale + 0.2 mg/mL P. guajava hydroethanolic extracts also significantly reduced biofilm formation in the standard and clinical strain 2 of E. faecalis (p < 0.0001), as shown in Figure 2.

4. Discussion

The search for new endodontic irrigants is constant. Recent studies have increasingly focused on alternative irrigants and bio-inspired solutions for the disinfection of E. faecalis in root canals [14,15,16]. For instance, Puleio et al. (2024) investigated the use of 2′-Fucosyllactose and Lacto-N-Neotetraose as alternative irrigants. Although their study found no bactericidal effect against E. faecalis, it highlights the growing scientific interest in exploring novel, bio-based agents for endodontic disinfection [25]. The aim of this study was to evaluate the anti-planktonic, antibiofilm, and synergistic effects of N. officinale and P. guajava hydroethanolic extracts against planktonic and biofilms of the standard (ATCC 29212) and clinical strains of E. faecalis, in which it was found that the extracts had effective anti-planktonic and antibiofilm activities at determined concentrations. Moreover, the combination of both extracts exhibited synergistic and additive effects; thus, the null hypothesis was rejected.
In this study, it was not possible to visually read the MIC values of the tested extracts due to the intense coloration of the extracts. Instead, the MBC value of the N. officinale hydroethanolic extract, represented by the last well found without turbidity, corresponds to a concentration of 12.5 mg/mL. These results are very similar to the findings of another study that reported the MBC of the N. officinale essential oil at a concentration of 1.25 mg/mL [26]. In addition, in the present study, the MBC value of the P. guajava hydroethanolic extract corresponds to a concentration of 0.52 mg/mL for all the tested strains of E. faecalis. In the literature, the aqueous and ethanolic extracts of P. guajava exhibited MIC values ranging from 4 to 24% at different concentrations [27].
Furthermore, the N. officinale hydroethanolic extract at 25 mg/mL effectively reduced the biofilm formation of E. faecalis ATCC, clinical strain 2, and clinical strain 4 by 63.41, 86.24, and 91.25%, respectively, exhibiting a statistically significant difference in the control group (p < 0.0001). Its effectiveness was comparable to that of NaOCl without a statistically significant difference (p > 0.05). In the present study, the MTT assay was used like in different previous studies [12,13,28]. Still, to the best of our knowledge, no studies in the literature have evaluated the antibiofilm activity of N. officinale against E. faecalis. In contrast, the P. guajava hydroethanolic extract was not effective against E. faecalis ATCC; however, it was effective in reducing the biofilm formation of the clinical strains of E. faecalis at different concentrations in percentages ranging from 41.68 to 55.05% against the clinical strain 2, with the concentration of 1.04 mg/mL as effective as NaOCl, and in percentages ranging from 40.46 to 45.08% against the clinical strain 4, with the concentrations of 2.09 and 1.04 mg/mL as effective as NaOCl, all with a statistically significant difference in the control group (p < 0.0001). In the literature, P. guajava was effective against different fungi like Candida albicans, Candida tropicalis, and Candida krusei based on the tested microdilution method [21].
This study was pioneered by the idea of testing the combined effect of N. officinale and P. guajava hydroethanolic extracts, making a comparison with other studies in the literature inviable, in which it was found that the extracts at different concentrations were effective in reducing the biofilm formation of the standard and clinical strains 2 and 4 of E. faecalis, with a statistically significant difference with the control group (p < 0.0001). However, it is important to acknowledge that NaOCl and CHX remain widely used in endodontics due to their well-established properties. NaOCl exhibits broad-spectrum antimicrobial efficacy, an anti-endotoxin effect, and pulp tissue-dissolving capabilities, while CHX is valued for its antimicrobial activity and biocompatibility [10]. Despite these advantages, certain drawbacks associated with these irrigants have led to the search for alternative, more biocompatible agents. While the potential of plant-based extracts as endodontic irrigants is promising, direct comparisons with NaOCl and CHX remain premature. Further studies, particularly in vivo and clinical settings, are necessary to validate their efficacy and practical applicability.
This antibiofilm action is attributed to the fact that vegetables from the Brassicaceae family, such as watercress, have been studied for their richness in vitamins, minerals, and phytochemicals, particularly phenolics, which include flavonoids and sulfur-containing compounds. Among the sulfur-containing compounds, glucosinolates are the most prominent and are found alongside the enzyme myrosinase. This enzyme hydrolyzes glucosinolates, converting them into isothiocyanates, nitriles, epithionitriles, and thiocyanates [29]. In addition, Isothiocyanates are recognized as key inhibitors of microbial activity, a property attributed to their ability to bind to key enzymes involved in microbial growth and survival. In the species N. officinale, as well as in other plants from the same family, the glucosinolate known as gluconasturtiin is present. This compound is the precursor of 2-phenylethyl isothiocyanate (PEITC), which exhibits biological effects such as antimicrobial activity, particularly against bacteria, and serves as a barrier to microbial growth [29].
It was shown that the antimicrobial mechanism of action of isothiocyanates involves affecting the integrity of the cell membrane, enzymes related to the redox balance, and bacterial metabolism [30]. According to a review by Nguyen and Bhattacharya (2022), quercetin, a compound found in both N. officinale and Psidium guajava, acts against bacteria through four main mechanisms: disruption of the cell wall and membrane, inhibition of nucleic acid synthesis, prevention of biofilm formation, and reduction in virulence factor expression [31]. Additionally, a study by Qayyum et al. demonstrated quercetin’s effectiveness in inhibiting Enterococcus faecalis biofilm formation by altering the expression of 19 proteins—10 of which were overexpressed and 9 suppressed—primarily involved in the glycolytic pathway, protein translation and elongation, and protein folding. Furthermore, scanning electron microscopy in this study revealed that E. faecalis treated with quercetin did not exhibit shape distortion or external damage [32].
Finally, given the increasing interest in biocompatible and natural antimicrobial agents, the use of herbal medicines is gaining a greater space constantly [12,13,14]; thus, the use of a herbal medicine extract would be considered in endodontics because of the positive results obtained in these basic studies. However, further in vitro and animal studies are highly recommended, and then clinical studies, to consider the use of these extracts as endodontic irrigants, and to explore the optimal concentration, formulation, and possible synergistic effects of these plant extracts when combined with existing irrigants. Their potential role in reducing the cytotoxic effects of NaOCl or enhancing the efficacy of CHX presents a promising avenue for future research.

5. Conclusions

  • The N. officinale hydroethanolic extract at 25 mg/mL exhibited the best results against the standard and clinical biofilms of E. faecalis.
  • The combined 0.1 mg/mL N. officinale + 0.1 mg/mL P. guajava, and 0.1 mg/mL N. officinale + 0.5 mg/mL P. guajava hydroethanolic extracts effectively reduced the biofilm formation of the standard and clinical strains of E. faecalis.

Author Contributions

Conceptualization, L.S.d.C., L.R.D.d.S., C.A.T.C., M.C.M., L.D.d.O. and A.A.H.; methodology, L.S.d.C., L.R.D.d.S., C.A.T.C., M.C.M., L.D.d.O. and A.A.H.; software, L.S.d.C., L.R.D.d.S., C.A.T.C., M.C.M., L.D.d.O. and A.A.H.; validation, L.S.d.C., L.R.D.d.S., C.A.T.C., M.C.M., L.D.d.O. and A.A.H.; formal analysis, L.S.d.C., L.R.D.d.S., C.A.T.C., M.C.M., L.D.d.O. and A.A.H.; investigation, L.S.d.C., L.R.D.d.S., C.A.T.C., M.C.M., L.D.d.O. and A.A.H.; resources, L.S.d.C., L.R.D.d.S., C.A.T.C., M.C.M., L.D.d.O. and A.A.H.; data curation, L.S.d.C., L.R.D.d.S., C.A.T.C., M.C.M., L.D.d.O. and A.A.H.; writing—original draft preparation, L.S.d.C., L.R.D.d.S., C.A.T.C. and M.C.M.; writing—review and editing, L.S.d.C., L.D.d.O. and A.A.H.; visualization, L.S.d.C., L.R.D.d.S., C.A.T.C., M.C.M., L.D.d.O. and A.A.H.; supervision, L.D.d.O. and A.A.H.; project administration, L.D.d.O. and A.A.H.; funding acquisition, L.R.D.d.S. and A.A.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Institutional Scientific Initiation Scholarship Program (PIBIC) of National Council for Scientific and Technological Development (CNPq) (No. 10195).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request (d.d.s.amjad@gmail.com).

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Wong, J.; Manoil, D.; Näsman, P.; Belibasakis, G.N.; Neelakantan, P. Microbiological aspects of root canal infections and disinfection strategies: An update review on the current knowledge and challenges. Front. Oral Health 2021, 2, 672887. [Google Scholar] [CrossRef] [PubMed]
  2. Gomes, B.P.F.dA.; Herrera, D.R. Etiologic role of root canal infection in apical periodontitis and its relationship with clinical symptomatology. Braz. Oral Res. 2018, 32, e69. [Google Scholar] [CrossRef] [PubMed]
  3. Siqueira, J.F.; Rôças, I.N. Present status and future directions: Microbiology of endodontic infections. Int. Endod. J. 2022, 55 (Suppl. 3), 512–530. [Google Scholar] [CrossRef]
  4. Jhajharia, K.; Parolia, A.; Shetty, K.V.; Mehta, L.K. Biofilm in endodontics: A review. J. Int. Soc. Prev. Community Dent. 2015, 5, 1–12. [Google Scholar] [CrossRef] [PubMed]
  5. Jayakumar, S.; Sridhar, D.; John, B.M.; Arumugam, K.; Ponnusamy, P.; Pulidindi, H. Biofilm in Endodontic Infection and its Advanced Therapeutic Options—An Updated Review. J. Pharm. Bioallied Sci. 2024, 16, S1104–S1109. [Google Scholar] [CrossRef]
  6. Said, M.S.; Tirthani, E.; Lesho, E. Enterococcus Infections. In StatPearls; StatPearls Publishing: Treasure Island, FL, USA, 2025. [Google Scholar]
  7. Alghamdi, F.; Shakir, M. The Influence of Enterococcus faecalis as a Dental Root Canal Pathogen on Endodontic Treatment: A Systematic Review. Cureus 2020, 12, e7257. [Google Scholar] [CrossRef]
  8. Yang, S.; Meng, X.; Zhen, Y.; Baima, Q.; Wang, Y.; Jiang, X.; Xu, Z. Strategies and mechanisms targeting Enterococcus faecalis biofilms associated with endodontic infections: A comprehensive review. Front. Cell. Infect. Microbiol. 2024, 14, 1433313. [Google Scholar] [CrossRef]
  9. Rôças, I.N.; Siqueira, J.F.; Santos, K.R.N. Association of Enterococcus faecalis with different forms of periradicular diseases. J. Endod. 2004, 30, 315–320. [Google Scholar] [CrossRef]
  10. Khoury, R.D.; de Carvalho, L.S.; do Nascimento, M.F.R.; Alhussain, F.; Abu Hasna, A. Endodontic irrigants from a comprehensive perspective. World J. Clin. Cases 2024, 12, 4460–4468. [Google Scholar] [CrossRef]
  11. Domingues, N.; Ramos, L.d.P.; Pereira, L.M.; do Rosário Estevam Dos Santos, P.B.; Scorzoni, L.; Pereira, T.C.; Abu Hasna, A.; Carvalho, C.A.T.; de Oliveira, L.D. Antimicrobial action of four herbal plants over mixed-species biofilms of Candida albicans with four different microorganisms. Aust. Endod. J. 2023, 49, 262–271. [Google Scholar] [CrossRef]
  12. de Lima, P.M.N.; Pereira, T.C.; de Carvalho, L.S.; Dos Santos, L.F.; Oliveira, C.E.R.; Ramos, L.d.P.; Marcucci, M.C.; Abu Hasna, A.; de Oliveira, L.D. Antimicrobial and synergistic effects of lemongrass and geranium essential oils against Streptococcus mutans, Staphylococcus aureus, and Candida spp. World J. Crit. Care Med. 2024, 13, 92531. [Google Scholar] [CrossRef]
  13. Meccatti, V.M.; Martins, K.M.C.; Ramos, L.d.P.; Pereira, T.C.; de Menezes, R.T.; Marcucci, M.C.; Abu Hasna, A.; de Oliveira, L.D. Synergistic Antibiofilm Action of Cinnamomum verum and Brazilian Green Propolis Hydroethanolic Extracts against Multidrug-Resistant Strains of Acinetobacter baumannii and Pseudomonas aeruginosa and Their Biocompatibility on Human Keratinocytes. Molecules 2023, 28, 6904. [Google Scholar] [CrossRef] [PubMed]
  14. Santos, T.d.S.A.; Meccatti, V.M.; Pereira, T.C.; Marcucci, M.C.; Hasna, A.A.; Valera, M.C.; de Oliveira, L.D.; Carvalho, C.A.T. Antibacterial Effect of Combinations of Salvia officinalis and Glycyrrhiza glabra Hydroalcoholic Extracts against Enterococcus spp. Coatings 2023, 13, 1579. [Google Scholar] [CrossRef]
  15. Zeb, A. Phenolic profile and antioxidant potential of wild watercress (Nasturtium officinale L.). Springerplus 2015, 4, 714. [Google Scholar] [CrossRef] [PubMed]
  16. Klimek-Szczykutowicz, M.; Szopa, A.; Ekiert, H. Chemical composition, traditional and professional use in medicine, application in environmental protection, position in food and cosmetics industries, and biotechnological studies of Nasturtium officinale (watercress)—A review. Fitoterapia 2018, 129, 283–292. [Google Scholar] [CrossRef]
  17. Abdul Kareem, M.W.; Al Dhaher, Z.A. Evaluation of The Antifungal Activity of Nasturtium officinale (watercress) Oil with Calcium Hydroxide against Candida albicans Isolated from Root Canal. JBCD 2021, 33, 1–5. [Google Scholar] [CrossRef]
  18. Morais-Braga, M.F.B.; Carneiro, J.N.P.; Machado, A.J.T.; Dos Santos, A.T.L.; Sales, D.L.; Lima, L.F.; Figueredo, F.G.; Coutinho, H.D.M. Psidium guajava L., from ethnobiology to scientific evaluation: Elucidating bioactivity against pathogenic microorganisms. J. Ethnopharmacol. 2016, 194, 1140–1152. [Google Scholar] [CrossRef]
  19. Gutierrez-Montiel, D.; Guerrero-Barrera, A.L.; Chávez-Vela, N.A.; Avelar-Gonzalez, F.J.; Ornelas-García, I.G. Psidium guajava L.: From byproduct and use in traditional Mexican medicine to antimicrobial agent. Front. Nutr. 2023, 10, 1108306. [Google Scholar] [CrossRef]
  20. Metwally, A.M.; Omar, A.A.; Harraz, F.M.; El Sohafy, S.M. Phytochemical investigation and antimicrobial activity of Psidium guajava L. leaves. Pharmacogn. Mag. 2010, 6, 212–218. [Google Scholar] [CrossRef]
  21. Bezerra, C.F.; Rocha, J.E.; Nascimento Silva, M.K.d.; de Freitas, T.S.; de Sousa, A.K.; Dos Santos, A.T.L.; da Cruz, R.P.; Ferreira, M.H.; da Silva, J.C.P.; Machado, A.J.T.; et al. Analysis by UPLC-MS-QTOF and antifungal activity of guava (Psidium guajava L.). Food Chem. Toxicol. 2018, 119, 122–132. [Google Scholar] [CrossRef]
  22. Baldoni, G.d.A.; Meccatti, V.M.; De Carvalho, L.S.; Carvalho, C.A.T.; Oliveira, L.D.d.; Hasna, A.A. Antimicrobial action of Psidium guajava L. extract against Enterococcus faecalis and Candida albicans strains: A pilot study. Arq. Ciênc. Saúde Unipar 2023, 27, 3484–3493. [Google Scholar] [CrossRef]
  23. Fratini, F.; Mancini, S.; Turchi, B.; Friscia, E.; Pistelli, L.; Giusti, G.; Cerri, D. A novel interpretation of the Fractional Inhibitory Concentration Index: The case Origanum vulgare L. and Leptospermum scoparium J. R. et G. Forst essential oils against Staphylococcus aureus strains. Microbiol. Res. 2017, 195, 11–17. [Google Scholar] [CrossRef] [PubMed]
  24. Moreno, M.A.; Zampini, I.C.; Isla, M.I. Antifungal, anti-inflammatory and antioxidant activity of bi-herbal mixtures with medicinal plants from Argentinean highlands. J. Ethnopharmacol. 2020, 253, 112642. [Google Scholar] [CrossRef] [PubMed]
  25. Puleio, F.; Pirri, R.; Tosco, V.; Lizio, A.S.; Tripodi, P.; La Spina, I.; La Fauci, V.; Squeri, R. Assessment of 2′-Fucosyllactose and Lacto-N-Neotetraose Solution as an Irrigant in E. faecalis-Infected Root Canals: An In Vitro Study. Clin. Pract. 2024, 14, 1348–1356. [Google Scholar] [CrossRef]
  26. Al-Shekhli, M.; Al-Dhaher, Z. Evaluation of The Antibacterial Activity of Nasturtium officinale (watercress) Essential Oil with Calcium Hydroxide against Enterococcus faecalis Isolated from Root Canal (In vitro study). J. Res. Med. Dent. Sci. 2021, 9, 78–84. [Google Scholar]
  27. Chandran, K.D.; Karthikeyan, K.; Sujatha, V.; Mahalaxmi, S. Antibacterial evaluation of guava leaves extract and its effect on reactive oxygen species formed by calcium hydroxide and chlorhexidine mixture. J. Conserv. Dent. 2021, 24, 389–392. [Google Scholar] [CrossRef]
  28. Shi, D.; Xu, W.; Wong, M.; Popovich, D.G. An Improved Purification Method for Removing Colour Interference from 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide (MTT) Antibacterial Assays. Appl. Sci. 2023, 13, 5067. [Google Scholar] [CrossRef]
  29. Freitas, E.; Aires, A.; de Santos Rosa, E.A.; Saavedra, M.J. Antibacterial activity and synergistic effect between watercress extracts, 2-phenylethyl isothiocyanate and antibiotics against 11 isolates of Escherichia coli from clinical and animal source. Lett. Appl. Microbiol. 2013, 57, 266–273. [Google Scholar] [CrossRef]
  30. Romeo, L.; Iori, R.; Rollin, P.; Bramanti, P.; Mazzon, E. Isothiocyanates: An Overview of Their Antimicrobial Activity against Human Infections. Molecules 2018, 23, 624. [Google Scholar] [CrossRef]
  31. Nguyen, T.L.A.; Bhattacharya, D. Antimicrobial activity of quercetin: An approach to its mechanistic principle. Molecules 2022, 27, 2494. [Google Scholar] [CrossRef]
  32. Qayyum, S.; Sharma, D.; Bisht, D.; Khan, A.U. Identification of factors involved in Enterococcus faecalis biofilm under quercetin stress. Microb. Pathog. 2019, 126, 205–211. [Google Scholar] [CrossRef] [PubMed]
Applsci 15 03178 g001
Figure 1. The microbial viability of the standard and clinical strains of E. faecalis after treatment with the experimental groups. Different letters (a, b, and c) indicate a statistically significant difference among the tested groups.
Figure 1. The microbial viability of the standard and clinical strains of E. faecalis after treatment with the experimental groups. Different letters (a, b, and c) indicate a statistically significant difference among the tested groups.
Applsci 15 03178 g001
Applsci 15 03178 g002
Figure 2. The microbial viability of the standard and clinical strains of E. faecalis after treatment with the combined extracts. Different letters (a, b, and c) indicate a statistically significant difference among the tested groups.
Figure 2. The microbial viability of the standard and clinical strains of E. faecalis after treatment with the combined extracts. Different letters (a, b, and c) indicate a statistically significant difference among the tested groups.
Applsci 15 03178 g002
Table 1. The results obtained from the Fractional Bactericidal Concentration Index calculation.
Table 1. The results obtained from the Fractional Bactericidal Concentration Index calculation.
E. faecalisIsolated MBC mg/mLCombined MBC mg/mLResult
N. officinaleP. guajavaN. officinaleP. guajavaFBCIEffect
ATCC12.50.520.10.11.5Synergistic
12.50.520.10.51.25Synergistic
12.50.520.10.55.5Additive
12.50.520.050.55.2Additive
Clinical strain 212.50.521.50.29.5Synergistic
12.50.521.50.18.5Synergistic
12.50.520.1110.5Additive
12.50.520.05110.25Additive
Clinical strain 412.50.520.10.11.5Synergistic
12.50.520.10.051.25Synergistic
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

de Carvalho, L.S.; da Silva, L.R.D.; Carvalho, C.A.T.; Marcucci, M.C.; de Oliveira, L.D.; Abu Hasna, A. Anti-Planktonic, Antibiofilm, and Synergistic Effects of Nasturtium officinale and Psidium guajava Hydroethanolic Extracts Against Standard and Clinical Strains of Enterococcus faecalis. Appl. Sci. 2025, 15, 3178. https://doi.org/10.3390/app15063178

AMA Style

de Carvalho LS, da Silva LRD, Carvalho CAT, Marcucci MC, de Oliveira LD, Abu Hasna A. Anti-Planktonic, Antibiofilm, and Synergistic Effects of Nasturtium officinale and Psidium guajava Hydroethanolic Extracts Against Standard and Clinical Strains of Enterococcus faecalis. Applied Sciences. 2025; 15(6):3178. https://doi.org/10.3390/app15063178

Chicago/Turabian Style

de Carvalho, Lara Steffany, Livia Ramos Dorta da Silva, Cláudio Antonio Talge Carvalho, Maria Cristina Marcucci, Luciane Dias de Oliveira, and Amjad Abu Hasna. 2025. "Anti-Planktonic, Antibiofilm, and Synergistic Effects of Nasturtium officinale and Psidium guajava Hydroethanolic Extracts Against Standard and Clinical Strains of Enterococcus faecalis" Applied Sciences 15, no. 6: 3178. https://doi.org/10.3390/app15063178

APA Style

de Carvalho, L. S., da Silva, L. R. D., Carvalho, C. A. T., Marcucci, M. C., de Oliveira, L. D., & Abu Hasna, A. (2025). Anti-Planktonic, Antibiofilm, and Synergistic Effects of Nasturtium officinale and Psidium guajava Hydroethanolic Extracts Against Standard and Clinical Strains of Enterococcus faecalis. Applied Sciences, 15(6), 3178. https://doi.org/10.3390/app15063178

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop