Invest Clin 61(2): 105 - 116, 2020 https://doi.org/10.22209/IC.v61n2a01

Antibacterial effect of a hyperosmotic solution containing sorbate and ethanol on Enterococcus faecalis in planktonic form and as biofilm: an in vitro study.

Mónica Elizabeth Rojas Briones1, Ricardo Oliva Rodríguez1, Omar González Ortega2, Ana María González Amaro1, Jairo Mariel Cárdenas1, Francisco Javier Avelar González3 and Alma Lilián Guerrero Barrera3

1Endodontics Postgraduate Program, Faculty of Dentistry, Autonomous University of San Luis Potosí, San Luis Potosí, SLP, México.

2Laboratory of Bioseparations, Faculty of Chemical Sciences, Autonomous University of San Luis Potosí, San Luis Potosí, SLP, México.

3Laboratory of Cell and Tisular Biology, Basic Sciences Center, Autonomous University of Aguascalientes, Aguascalientes, México.

Key words: biofilm; hyperosmolarity; E. faecalis; irrigant; endodontics.

Abstract. The antibacterial effect of a hyperosmotic solution containing sor- bate and ethanol on E. faecalis in planktonic state and in biofilm was evaluated. Three hyperosmotic solutions (HS-A, HS-B y HS-C) were obtained from different formulations of potassium sorbate and sodium chloride, which were tested as an- timicrobials against planktonic forms of E. faecalis, in McFarland standards from

0.5 to 7, using the sedimentation technique and colony forming units (CFU) count. Afterwards an E. faecalis biofilm was produced in the palatal roots of up- per first molars, by a static method in 21 days; subsequently they were prepared biomechanically by the Universal Protaper system, using the hyperosmotic solu- tion B as an irrigant to evaluate the bacterial load reduction. One pre-instru- mentation sample and one post-instrumentation sample were taken, and then were processed and cultivated to count CFU. Consecutively, roots were observed by scanning electron microscopy. The hyperosmotic solution had an important antibacterial effect when used against E. faecalis in planktonic state; solutions HS-A and HS-B were effective in eliminating E. faecalis up to 7 McFarland, while a statistical difference (p˂0.001) was observed in reducing the bacterial load in the

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biofilm, based on the log

CFU count. The final solution tested seemed not to

harm the dentinal structure and was capable of causing morphological changes to the bacterial cell consistent with a hyperosmotic shock. Thus, the solutions tested could be an option to be considered as irrigating agents; nonetheless fur- ther research is required regarding its biocompatibility.

Corresponding author: Alma Lilián Guerrero Barrera. Laboratory of Cell and Tisular Biology, Basic Sciences Cen- ter, Autonomous University of Aguascalientes, México. Blvd Universidad N° 940, Cd. Universitaria, Zip Code 20100; Aguascalientes, Ags. Mexico. Phone: +52 (449) 1960192. Email: alguerre@correo.uaa.mx

Efecto antibacteriano de una solución hiperosmótica a base de sorbato y etanol sobre Enterococcus faecalis en forma planctónica y como biofilm: estudio in vitro.

Invest Clin 2020; 61 (2): 105-116

Palabras clave: biopelícula; hiperosmolaridad; E. faecalis; irrigante; endodoncia.

Resumen. Se evaluó el efecto de una solución hiperosmótica sobre E. fae- calis en forma planctónica y en forma de biofilm. A partir de formulaciones de sorbato de potasio y cloruro de sodio se obtuvieron tres soluciones hiperosmóti- cas (HS-A, HS-B y HS-C) con ellas se realizaron pruebas antimicrobianas contra

E. faecalis en forma planctónica en concentraciones desde 0,5 hasta 7 McFar- land mediante técnica de sedimentación y cuenta de UFC. Posteriormente se formó biofilm de E. faecalis por método estático por 21 días en raíces palatinas de primeros molares superiores, las cuales se prepararon biomecánicamente mediante el sistema Protaper Universal usando la solución hiperosmótica HS-B como irrigante para luego evaluar la disminución de carga bacteriana, compa- rando el conteo de UFC de muestras pre-instrumentación con muestras post- instrumentación. Consecutivamente las raíces fueron observadas al microsco- pio electrónico de barrido. En las pruebas contra microorganismos planctónicos las soluciones HS-A y HS-B fueron efectivas en eliminar E. faecalis hasta un Mc Farland de 7. En la reducción de carga bacteriana mediante conteo de UFC de los microorganismos embebidos en biofilm, se identificó una diferencia estadís- tica (p˂0,001). La solución hiperosmótica tiene un importante efecto antibac- teriano contra E. faecalis en forma planctónica y en forma de biofilm, no parece dañar la estructura dentinaria y es capaz de provocar cambios morfológicos a la célula bacteriana consistentes con shock hiperosmótico. De manera que la solución hiperosmótica parecería ser una opción a ser considerada como agen- te irrigante o coadyuvante de la irrigación; sin embargo, se requiere investigar más profundamente con respecto a su biocompatibilidad.

Received: 31-05-2019 Accepted: 24-03-2020

INTRODUCTION

An effective chemo-mechanical instru- mentation of the root canal system is crucial for the long-term success of the endodontic treatment. Modern endodontic therapy in- volves the combination of mechanical de- bridement of dentine with chemical agents for irrigation and disinfection, with the goal being the removal of all microorganisms from the root canal system (1).

Since the anatomy of the root canal system is complex and its access is limited,

microorganisms can remain in the dentinal tubules and other irregular spaces. When these microorganisms find a favorable envi- ronment; they can proliferate and reinfect the entire root canal system, and thus infect- ed dentinal tubules and the presence of bio- films make disinfection much more difficult. Biofilms are microbial communities embed- ded within an extracellular matrix, forming a highly organized structure associated with a surface such as the root canal wall. There is convincing evidence that microorganisms organized in this way are much less suscepti-

ble to antimicrobial agents than their plank- tonic counterparts (2).

Decades of research have clearly dem- onstrated that pathogens have developed an arsenal of mechanisms to enhance the virulence potential of the biofilm, the struc- tural and biochemical properties of the ma- trix provide the emergent properties of bio- films that include surface adhesion, spatial and chemical heterogeneities, synergistic/ competitive interactions and increased tol- erance to antimicrobials. Despite this, how pathogens modify the spatial–temporal or- ganization and communal behavior to create localized pathological niches remains still widely unknown (3).

At present it is known that clinical endodontic and oral Enterococcus faecalis strains, in contrast to strains from other clinical sources (e.g. particular endocardi- tis strains), have lower inherent capacity to form biofilms (4). Thus the conditions under which biofilms might occur in infected root canals in vivo are not yet well understood.

E. faecalis has been related to oral dis- eases such as endodontic infections, peri- odontitis, and peri-implantitis. It has been frequently implicated in failure of the end- odontic treatment, due to its high resistance and ability to produce recalcitrant biofilms both in treated and untreated root canals (5).

Recently an innovative approach has been attempted to inactivate bacterial bio- films through hypertonic saline solutions. These salts are used to prevent bacterial growth in food or cosmetic products that when used at high concentrations; a syn- ergistic effect is induced by hyperosmotic stress (6).

The rationale of the antibacterial effect of hyperosmotic solutions is based on the fact that hyperosmotic stress causes loss of cellular water and cellular contraction, el- evates the intracellular ionic force, and gen- erates macromolecular overcrowding and protein denaturation; thus inducing cellular plasmolysis that results in inhibition of a va- riety of physiological processes, from nutri-

ent absorption to DNA replication. Certain agents such as inorganic ions (e.g. K+, Na+, and Cl–) have the ability to destabilize the secondary structure of proteins and disturb enzymatic activity (7).

Van der Waal et al. tested the survival of 48 h E. faecalis biofilm expressed as log col- ony forming units (log UFC) after challenge with a series of combinations of potassium sorbate (KS) and sodium chloride (NaCl) so- lutions; getting good results with a combi- nation named modified salt solution, formed with 3 M NaCl and 1 M KS (8).

Recently our group carried out an eval- uation of the effect of a hyperosmotic aque- ous solution composed of 1.5 M NaCl, 1 M KS, 0.051 M HCl, and 38.4% ethylic alcohol against a combination of Enterococcus fae- calis and Candida albicans in planktonic state; showing a potent anti-microbial effect for both endodontic microorganisms (9).

The aim of this study was to evaluate the antibacterial effect of a hyperosmotic so- lution against E. faecalis, in planktonic sus- pension and embedded in a biofilm, in terms of bacterial load reduction.

MATERIALS AND METHODS

Formulation of hyperosmotic solu- tions. Three different hyperosmotic solu- tions: hyperosmotic solution A (HS-A), hyperosmotic solution B (HS-B) and hyper- osmotic solution C (HS-C) were evaluated against E. faecalis in planktonic state. Based on the results the most promising solution was chosen to carry out tests against the E. faecalis biofilm.

Initially we were not able to prepare the hyperosmotic solution reported by Van der Waal et al. (3 M NaCl and 1 M KS) due to incomplete dissolution of the salts, thus the concentration of NaCl was decreased to

1.5 M for full solubilization; despite reduc- ing the osmotic value. Looking to compen- sate this situation, the speciation of sorbate was favored towards the protonated species (sorbic acid) to have a synergetic effect in

the solutions tested. This was accomplished by simply adding HCl to solutions HS-A and HS-B. Given the low solubility of sorbic acid in aqueous solutions, ethanol was added as co-solvent and vehicle to favor the entrance of sorbic acid to the bacterial cell. Solution HS-C was composed of NaCl, ethanol, and sorbic acid the composition of solutions HS- A, HS-B and, HS-C is shown in Table I.

Antimicrobial capacity tests of the hy- perosmotic solutions against planktonic microorganisms. This study was registered and approved by the Institutional Ethics Committee of the Faculty of Stomatology at San Luis Potosi Autonomous University (ap- proval code no. CEIFE-057-015) and the pa- tient signed an informed consent form.

A strain of Enterococcus faecalis from a patient with persistent apical periodonti- tis was identified by the conventional API 20 STREP® Test (Biomerieux, France) and main- tained in trypticase soy agar (BD Sparks, MD, USA) until its use. It was reactivated in brain heart infusion broth (BHI); the purity of the culture was verified regularly by gram staining.

Centrifugation sedimentation tech- nique. An inoculum of E. faecalis in a tube with BHI broth was made; afterwards it was incubated for 24 h until reaching a concen- tration of 7.5 McFarland, allowing this previ- ous incubation time to carry out the experi- ment in the stationary phase, from which the corresponding volume was withdrawn to achieve suspensions of microorganisms in the 0.5 to 7 McFarland concentrations range. Each suspension was centrifuged for 20 min at 2500 rpm (Solvat J-600, México). Subsequently, the supernatant of each tube

was discarded and the microbial pellet com- bined with 10 mL of the corresponding hy- perosmotic test solution (HS-A, HS-B, or HS- C); each tube was stirred and kept at room temperature for periods of 5, 15, and 25 min. Finally, 100 µL samples from of each solution were spread on BHI agar plates sow- ing by surface dissemination and incubating at 35 ± 2°C during 24-48 h. The grade of mi- crobial inhibition was based on the count of microbial colonies (CFU). All these experi- ments were performed in triplicate using a purifier class II Biosafety Cabinet (Labconco Corp. Kansas City, MO, USA).

Test of antimicrobial capacity against

E. faecalis biofilm. 25 palatal roots of upper first molars adjusted with a carbide disk at a length of 14 mm were used, 15 were used with the hyperosmotic solution irrigation protocol and 10 for the positive and negative controls (5 each). Each canal was widened with a Protaper SX instrument (Dentsply- Maillefer, Ballaigues, Switzerland) at 250 rpm in order to facilitate bacterial coloniza- tion. The organic and inorganic rests includ- ing the smear layer were removed by ultra- sonic bath treatment (BioSonic UC50) with 17% EDTA (J.T. Baker), followed by 5.25% NaOCl for 4 min according to the Saleh pro- tocol (10). Samples were sterilized in an au- toclave at 121°C for 20 min.

The biofilm of E. faecalis was produced in the roots using a static method. The inocu- lation system, based on the system proposed by Hockett et al. (11), was carried out as fol- lows: from a culture of E. faecalis in a plate of BHI agar, an inoculum was produced in a tube with BHI broth (J.T. Baker); followed by incubation at 37°C for 8 h in aerobic condi-

TABLE I

SOLUTIONS FORMULATED TO TEST AGAINST PLANKTONIC E. FAECALIS

Solution Components

HS: hyperosmotic solution, KS: Potassium sorbate.

tions. Five drops of 8 h cultures were with- drawn and inoculated in 10 mL of BHI broth, followed by incubation at 37°C for 4 h. After- wards, turbidity was adjusted to 0.5 McFar- land. Five drops of bacterial suspension were placed in the cultured tubes containing the samples. The time of biofilm formation was 21 days, within which refills of fresh broth were made every 48 h incubating it at 37°C. The purity of the strain was monitored for 21 days by gram staining every 48 h.

Instrumentation and irrigation of sam- ples. The chemo-mechanical phase was car- ried out on an adaptor, which contained 2 inserts to place roots; allowing the absolute isolation of each root. Once isolated, the oper-

canal for 1 min and transferred to a tube with 8 mL of BHI broth; the same procedure was performed with 2 subsequent paper points.

Evaluation of the antimicrobial effect on biofilm by quantification of colony form- ing units (CFU). Once the sampling of the root canal was performed, a 100 µL aliquot of the broth containing the paper points was withdrawn; with it two sowings were carried out in blood agar by surface dissemination, the first direct discharge of 100 mL at 15 min of incubation and the second by making a 0.5×10–2 dilution in McFarland scale with 24 h of incubation. The microbial inhibition was calculated based on the number of mi- crobial colonies, the CFU values were trans-

ative field was disinfected with 30% hydrogen

ferred to log

. The control experiments

peroxide for 1 min, 5.25% sodium hypochlo- rite for 1 min, and both inactivated with 10% sodium thiosulfate. Before pre-instrumenta- tion sampling, the canal was moistened with a drop of BHI broth. A #35 sterile paper point (Viarden, México) was placed into the root ca- nal for 1 min and subsequently transferred to a tube with 8 mL of BHI broth, the same pro- cedure was performed with two more paper points for each root.

The cleaning and shaping of the roots were made with Protaper Universal instru- ments (Dentsply Maillefer, Ballaigues, Swit- zerland) following the sequence recom- mended by the manufacturer, irrigation was performed with 2 mL of each irrigation solution between each instrument. The hy- perosmotic solution B (HS-B) was used in 15 roots, 5.25% sodium hypochlorite in 5 roots as bacterial growth inhibition control, and distilled water in 5 roots as bacterial growth control. Using a total of 12 mL of solution per root. Each instrument was used to pre- pare no more than 4 canals. The final irriga- tion was performed inactivating the hyper- osmotic and NaOCl solutions with 2 mL of distilled water, 2 mL of 17% EDTA solution for 1 min, and finally 2 mL of distilled water. Post-instrumentation sampling was carried out by moistening the canal with BHI broth, a sterile #35 paper point was placed in the

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behaved as expected: countless growth for

distilled water and total elimination with the 5.25% NaOCl solution. All these experi- ments were performed in a purifier class II Biosafety Cabinet (Labconco Corporation, Kansas City, MO, USA).

2

Scanning electron microscopy. Five of the treated samples were randomly selected and splitted to be observed in the scanning elec- tron microscope (JEOL, JSM-6610LV, Peabody, MA, USA). The samples were fixed with glutar- aldehyde/cyan blue, dehydrated with increas- ing concentrations of alcohol, and subjected to critical point drying with CO . Afterwards they were gold-coated and a general scanning of the canal wall was performed. The cervical, middle, and apical thirds of the root canal wall were observed at 30×, 200×, 1000×, and 5000× magnifications.

Statistical analysis. Data were ana- lyzed using the statistical software GraphPad Prism v5.0 (GraphPad, San Diego, CA. USA) by a specialist with data blinding. The nor- mality of the variables was analyzed with the Shapiro Wilk test. Comparison among the study groups was carried out applying a Stu- dent T test and the statistical significance was determined. To reduce the bias of the measurements, the coefficient of reliability (95%) was calculated with a statistical sig- nificance of p<0.05.

RESULTS

Antimicrobial capacity test against planktonic cells. Table II shows the mean

10

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in the pre-treatment group; as well as for the difference between the pre-treatment and post-treatment number of log UFC on plate and total log UFC that were statisti-

10

log

CFU present in the different McFarland

cally significant (p<0.024).

concentrations in contact with the test so- lutions at different exposure periods (5, 15, and 25 min), in which it is observed that solutions HS-A and HS-B were highly effec- tive at eliminating E. faecalis in plankton- ic suspension; unlike solution HS-C, which stopped working at 6 McFarland.

Antimicrobial capacity test against E. faecalis biofilm. The number of CFU devel- oped in the pre-instrumentation and post- instrumentation samples are shown in Table

1. Average values were 7.4267 for pre-treat- ment and 1.9130 for post-treatment, which represents an 80% of reduction in the McFar- land lecture.

   Once the normality of the variables was analyzed, the Student t test showed that the

   Scanning Electronic Microscopy of treated roots. In order to observe the effect of the solution on bacterial cells and the dentin- al structure in the root canal wall by means of electronic scanning microscopy; we observed the cervical, medium, and apical thirds to dif- ferent magnifications (Figs. 1 - 4).

   In the cervical third with a 1000× mag- nification we observed open dentinal tubules without damage in the dentinal structure, as well as low count of microorganisms at a 5000× magnification. In the middle third at 5000× we observed the presence of bacteria with morphology modification, at 1000× the dentinal structure remained without modi- fications and dentinal tubules were wide open, unlike the apical third where most of

   10

   mean log

   CFU in the post-treatment group

   dentinal tubules were blocked; the presence

   was significantly lower (p<0.001) than that

   of bacteria is also evident.

   
   

   TABLE II

   EFFECT OF THE HYPEROSMOTIC SOLUTIONS HS-A, HS-B AND HS-C ON E. FAECALIS

   IN PLANKTONIC STATE

   
   

   McFarland 0.5 1 2 3 4 5 6 7

   

   5 min	0	0	0	0	0	0	0	0
   15 min	0	0	0	0	0	0	0	0
   25 min	0	0	0	0	0	0	0	0

   Solution HS-A

   
   

   HS-B

   5 min	4	0	0	0	0	0	0	0
   15 min	0	0	0	0	0	0	0	0
   25 min	13	2	0	0	0	0	0	0

   HS-C

   5 min	0	0	11	972	4	1120	1804
   15 min	0	0	0	37	0	2	114
   25 min	0	0	0	0	0	0	15

   

   The mean of three readings of log10 CFU resulting from the contact between the different concentrations of E. faecalis in McFarland scale (0.5 – 7) in contact with the test solutions in the three exposure times (5, 15 and 25 minutes) is shown.

   
   

   TABLE III

   EFFECT OF THE HYPEROSMOTIC SOLUTION HS-B ON E. FAECALIS BIOFILM

   
   

   Variable	Time	n	Mean	Mean error	SD	Min Value	Max Value	Pre- treatment vs. Post- treatment
   McFarland	Pre-treatment	15	7.4267	0.0605	0.2344	6.6000	7.50000	p 0.001*
   	Post-treatment	15	1.9130	0.5940	2.3020	0.4000	7.30000
   UFC plate	Pre-treatment	15	151.90	59.900	232.00	1.0000	724.000	p 0.024*
   	Post-treatment	15	0.2000	0.1450	0.5610	0.0000	2.00000
   UFC total	Pre-treatment	15	15186	59899	231986	1000.0	724000	p 0.024*
   	Post-treatment	15	200.00	145.00	561.00	0.0000	2000.00

   Student t test was done to compare pre-treatment and post-treatment log10 CFU count.

   * Statistically significant difference (p<0.05).

   
   

   

   
   

   Fig. 1. Photomicrograph of sample #1 where the action of the hyperosmotic solution on biofilm is observed:

   1. Sample observed to 30x, in the circle the cervical area observed to greater magnifications. B. Area highlighted in cervical third, 200x. C. Area of cervical third showing predominantly open dentinal tubules, 1000x; and D. Cervical third showing scarce presence of microorganisms and no damage to the dentinal structure, 5000x.

Fig. 2. Photomicrograph of sample #1: A. Observed sample to 30x, in the circle the area of the middle third observed to greater magnifications. B. Highlighted area in middle third to 200x. C. Middle third area showing all open dentine tubules, 1000x and D. Area of middle third where the presence of E. faeca- lis is observed, some of them present alteration in their morphology; no damage is observed in the dentinal structure, 5000x.

Fig. 3. Photomicrograph of sample #1. A. Panoramic view of the root canal and in the circle the apical area to be observed at greater magnifications, 30x. B. Root canal wall in apical third, 200x. C. Apical third showing a large majority of blocked dentin tubules, 1000x, and D. It is observed the presence of microorganisms in the apical third and some disruptions in the dentinal structure, in form of micro-cracks, 5000x.

Fig. 4. Photomicrograph showing in A. Dentinal wall in the middle third of sample #1 where the irregular morphology of E. faecalis is observed (arrows), 12,000x. B. Middle third of sample #2 where a bacte- rial agglomeration is observed, showing the irregular morphology of bacterial cells (arrows).

DISCUSSION

Biofilms are defined as structured com- munities of microorganisms that are at- tached to a surface and enmeshed in an ex- tracellular polymeric matrix (12).

The fight against bacterial biofilms has led us to investigate several methods to eliminate or control them. In this last regard biological agents have been tested. In periodontology, Pa- tini et al. (13) used the genus Bdellovibrio with predatory selectivity against Gram-negative bacteria; they obtained a favorable response from this microorganism versus periodontopa- togenic bacteria, which promotes research in this arena to find something similar in other areas of dentistry.

In the endodontic area mechanical methods have been implemented to remove biofilms inside the root canal during the ir- rigation protocol using sonic and ultrasonic devices. Actually, the gold standard is the PUI (passive ultrasonic irrigation), which has been compared to conventional needle irrigation (CNI) and passive sonic irrigation (PSI). Eneide et al. (14) found no difference between PUI and PSI in terms of CFU reduc- tion, while CNI was less effective.

A final irrigation protocol was carried out, which includes the use of EDTA for the smear layer removal; allowing the permeability of den- tinal tubules. This procedure does not influ- ence our results of antimicrobial efficacy since EDTA with mild agitation or CNI do not show important antimicrobial properties (15).

It is vital to consider bacterial biofilm models as essential models for in vitro mi- crobiological investigations that assess dif- ferent disinfectants and disinfection strate- gies in endodontics.

The methodology to generate biofilm in the present study was a monoculture un- der static growth conditions as reported by Paranjpe et al. (16).

Although some authors privilege the relevance of a polymicrobial biofilm over a mono-species biofilm; it has been reported that biofilms produced in pure cultures of bacteria under laboratory conditions and the mixed-species biofilms produced in natural ecosystems show similar basic organization (17). Thus the E. faecalis biofilm produced in this work, with a degree of maturity of 21 days, can be considered as suitable model to test the efficacy of the hyperosmotic solu- tion in vitro.

Van der Waal et al. developed an aque- ous solution based on 3 M NaCl + 1 M KS that proved to be extremely effective at in- activating a 48 h E. faecalis biofilm within 1 h of contact (8), while the hyperosmotic solution tested here showed an accentuated bacterial reduction when used as irrigating agent for a shorter period of time and with a small volume (12 mL) per root.

Analyzing the significant antibacte- rial effect that took place in this evaluation for both solutions, HS-A and HS-B, against bacteria in planktonic state and the effect shown by solution B against the E. faecalis biofilm, it can be considered that a synergy is occurring between the hyperosmolarity of the solution and the presence or sorbic acid molecules at low pH. The antimicrobial prop- erties of potassium sorbate are observed at a pH near or less than 4 (18,19); where the protonated species dominates (sorbic acid). Sorbic acid is a monoprotic acid with a pKa equal to 4.76; thus, in solutions HS-A and HS-B only 2.8 and 1.4%, respectively, of the dissolved sorbate molecules exist as sorbic acid. Sorbic acid, being more hydropho- bic than the sorbate ion, can penetrate the lipid membrane of the bacterial cell and act as inhibitor of various enzymes involved in the carbohydrate metabolism and the citric acid cycle. Despite having low percentages or sorbic acid molecules in solutions HS-A and HS-B, once they are contacted with E. faecalis; sorbic acid molecules will enter the cells promoting that more sorbate ions, out- side the bacterial cell, are converted to sor- bic acid. To favor the existence of solubilized sorbic acid, looking to guarantee entrance to the bacterial cell, a co-solvent (ethanol) was added to the solutions tested. Besides being a co-solvent, ethanol is a solute that contributes to the increase in hyperosmo- larity of the solutions tested and can help killing bacteria per se. In this last regard ethanol has been evaluated as antimicrobial in both planktonic and biofilm-producing microorganisms. Using a solution contain- ing 25% of ethanol, Suchomel et al. found

a 96.5% reduction of live E. faecalis cells in planktonic form after 5 min (20). Nonethe- less when a 95% ethanol solution was tested against an E. faecalis biofilm, 34% of live cells were recorded 72 h after 5 min of ir- rigation (21). Similarly using a multispecies biofilm Duarte et al. reported 27% of live cells using 95% ethanol after 10 min of ir- rigation (22). In the present work, solution HS-A contained 19.2% of ethanol, while so- lution HS-B had 38.4%; both solutions were able to completely kill planktonic E. faeca- lis, even at 7 McFarland. Nevertheless, when using solution HS-C (which contained 19.2% of ethanol), live cells were found starting at 2 McFarland. Thus, we can establish that ethanol is not completely responsible of the antimicrobial effect of the solutions tested. But acts synergically with the hyperosmolar- ity of the solutions and the presence or sor- bic acid.

The ability to enter the cell and inhibit vital functions for survival was demonstrated in the present study by observing the antibac- terial effect shown by the solutions tested on bacterial cells both in planktonic form and in the form of biofilm. In the latter, SEM im- ages evidence modification of cellular mor- phology as the literature indicates; since the shocking effect on the cell causes water out- flow, thus reducing the cytoplasmic volume and inducing cell plasmolysis (23,24). This is why it was possible to observe deformed, concave, and flattened cells, not all of them but most. Based on the results of the micro- biological tests, we can conclude that solu- tion HS-B is effective at reducing bacterial load of a root canal with a biofilm of E. fae- calis. The hyperosmotic solutions elaborated on the basis of sodium chloride, potassium sorbate, and ethanol were effective at elimi- nating E. faecalis in planktonic form in the

0.5 to 7 McFarland range. In addition, solu- tion HS-B was effective at reducing bacterial load in infected canals with a mature biofilm of E. faecalis.

It seems unclear whether the bacterial reduction was caused by killing, detachment

of biofilm cells from the substrate, or both. The hyperosmotic solution appears to be able to disperse biofilms, based on some of the photomicrographs and it may also aid at cleaning the root canal system.

Along with the ability to provoke mor- phological changes consistent with hyperos- motic stress damage to bacterial cells. The contact of the hyperosmotic solution with the dentin surface does not cause structural changes, however further studies are needed to clarify the origin of some micro-cracks observed.

It can be concluded that the hyperos- motic solution (HS-B) showed considerable antibacterial capacity against E. faecalis in both planktonic and biofilm form; with the ability to generate changes in the morphol- ogy of bacterial cells consistent with hyper- osmotic shock. The need to conduct cytotox- icity tests is highlighted before it is possible to propose solution HS-B as irrigation agent or irrigation adjuvant in endodontics.

ACKNOWLEDGMENTS

Rojas Briones ME was recipient of a scholarship by the Consejo Nacional de Cien- cia y Tecnología (CONACYT), México. This study was conducted with funds from the Master in Endodontics program of the Sto- matology Faculty of the Autonomous Univer- sity of San Luis Potosí, San Luis Potosí, Méxi- co and the Doctorate of Biological Sciences of the Universidad Autónoma de Aguascali- entes, México.

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