In vitro assessment of the antimicrobial activity of tetracycline hydrochloride diluted in three different vehicles against Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum.

Background: The development and progression of periodontal diseases is a result of the dynamic interaction of microorganisms within their habitat, and changes in this habitat generate a dysbiotic state. Fusobacterium nucleatum and Prevotella intermedia are bridging microorganisms between the pioneer communities and other microorganisms responsible for periodontitis such as Porphyromonas gingivalis. Tetracycline hydrochloride (TTC-HCl) is commonly used as a coadjutant in periodontal treatment in the form of an antiseptic. However, there are no clear dilution or concentration protocols. Objective: This study aimed to evaluate the in vitro antimicrobial activity of TTC-HCl diluted in sterile water, saline solution, and 2% lidocaine with epinephrine 1:80,000 at concentration of 125, 250, and 500 mg, at three time points- 30, 60, and 120 s - on P. intermedia, F. nucleatum, and P. gingivalis using the Kelsey-Maurer technique. Materials and
Methods: The antimicrobial activity of TTC-HCl was evaluated at the proposed concentrations and times, dissolved in the different vehicles at pH 1.9 and 7.0, on F. nucleatum, P. intermedia, and P. gingivalis. The Kelsey-Maurer test was used to verify the presence or absence of colony-forming units. Each test was performed in triplicates with its respective viability controls.
Results: Inhibition of F. nucleatum, P. intermedia, and P. gingivalis was achieved with TTC-HCl at all concentrations, dissolved in distilled water, saline solution, and 2% lidocaine with epinephrine 1:80,000 for all times. Conclusions: The results show that TTC-HCl is a good antimicrobial alternative against F. nucleatum, P. intermedia, and P. gingivalis regardless of the vehicle in which it was dissolved, concentration, pH, or time used in this investigation. Copyright:

INTRODUCTION

It has been well-documented during recent years that microorganisms play an important role in the etiology of periodontal diseases.[123] Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum are considered periodontal pathogenic bacteria, although they have not just been isolated from lesions with periodontitis. They have also been found in gingivitis and periodontally healthy sites.[145] P. intermedia and F. nucleatum strains grow over a wide range of pH, P. intermedia at pH ranges of 5.0–7.0 and F. nucleatum 5.5–7.0 which is an indication of the great variability in pH fluctuation within the oral cavity environment.[6] Conversely, P. gingivalis grows at pH 6.0–7.5.[67]

Local administration of antimicrobials for periodontal disease has been a complement to conventional scaling and root planing therapy,[891011] particularly in patients with poor clinical response.[910111213] Studies using tetracycline–hydrochloride (TTC-HCl) have demonstrated antimicrobial activity against subgingival microbiota.[1415] Local forms of irrigation require characteristics such as substantivity on dentinal surfaces, which in the case of TTC-HCl, have been demonstrated to remain for up to ≥7 days in crevicular fluid. This is longer than other antimicrobials.[912161718] This single irrigation and doses cause TTC-HCl to act as an antiseptic.[19]

In addition to the antimicrobial effect of TTC, it has been associated with increased adhesion between crevicular glycoproteins and dentin because the tubules are exposed; stimulation, proliferation, and adhesion of fibroblasts;[1820] anticollagenase activity;[212223] and inhibition of osteoclast function[24] and neutrophil function.[25]

In vitro studies have also demonstrated the capacity of aqueous solutions of TTC-HCl to demineralize enamel and dentin surfaces, in turn delaying biofilm formation.[15] TTC has been applied with different vehicles such as petrolatum,[26] gels,[272829] polymer matrices,[3031] TTC fibers,[32333435] and microencapsulation systems.[36] Use in single irrigation applications[3738] or use of TTC paste for 3 min at a pH of 2-3 has been reported for decontamination of implant surfaces.[39]

The goal of this investigation was to evaluate the in vitro antimicrobial activity of TTC-HCl diluted in sterile water, saline solution, and 2% lidocaine with epinephrine 1:80,000 at pH 1.9 and 7.0 at concentrations of 125, 250, and 500 mg and at three time points (30, 60, and 120 s) on P. intermedia, F. nucleatum, and P. gingivalis using the Kelsey–Maurer technique.

MATERIALS AND METHODS

Three working dilutions of powder TTC-HCl (Genfar®) (solute) were prepared in three different vehicles. Dilutions of TTC-HCl were made in 4.5 ml of water, 0.9% saline solution (Baxter), and 2% lidocaine with epinephrine 1:80,000 (Newcaine 2% New Stetic S. A.) (solvent) at concentrations of 125, 250, and 500 mg. Nine milliliters of each concentration of the disinfectant was added to a sterile test tube for each microorganism tested. The original pH was 1.9. The same procedure was performed bringing the dilutions to pH 7.0 (pH adjusted with sodium hydroxide).

Culture medium

The culture medium used for the experimentation was Wilkins–Chalgren Agar (Oxoid™) supplemented with hemin 5 mg/ml (Sigma-Aldrich) and menadione 0.5 mg/ml (Sigma-Aldrich).[40]

Bacterial suspension

The microorganisms F. nucleatum (ATCC 25586), P. intermedia (ATCC 25611), and P. gingivalis (ATCC 33277) were inoculated and isolated on Wilkins–Chalgren Agar. A bacterial suspension was prepared in 10 ml of brain heart infusion (BHI) broth from the colonies obtained (Oxoid™) until a turbidity equivalent to McFarland scale 1 (OD: Optical density between 0.16 and 0.20 at a wavelength of 630 nm) was achieved.

Viability controls

Viability controls were employed to check the viability of the strains by counting colony-forming units (CFUs), which were tested against the disinfectant. Six serial base 10 dilutions were prepared with the bacterial suspension using BHI dilution broth. After the dilutions were prepared, 1 ml of the 10−6 dilution was inoculated at depth in 14 ml of Wilkins–Chalgren Agar; the mixture was then poured in Petri dishes and incubated at 37°C for 7 days under anaerobic conditions. CFUs were counted after 7 days, and the result was multiplied by the dilution factor 10−6, which corresponded to the actual bacterial inoculum.

Negative controls

Negative controls were conducted to exclude the bactericidal power of the diluent vehicles, namely water, 0.9% saline, and 2% lidocaine epinephrine 1:80,000 on the bacteria by counting CFUs. A volume of 4.5 ml of distilled water, 0.9% saline solution, and 2% lidocaine anesthetic with epinephrine 1:80,000 was placed in sterile test tubes, and 500 μl of the viable bacterial suspension at 1 on the McFarland scale and vortex homogenized for 10 s was added; 1 ml of each suspension was taken and inoculated on Wilkins–Chalgren Agar (Oxoid™) supplemented with 5 mg/ml hemin (Sigma-Aldrich) and 0.5 mg/ml menadione (Sigma-Aldrich) placed in Petri dishes and incubated at 37°C for 7 days under anaerobic conditions. This procedure was performed in triplicates.

Positive controls

Positive controls were used to compare the bactericidal power of the compounds assessed in this study with that of an antiseptic known as 0.12% chlorhexidine (Farpag) on bacteria by counting the CFUs. A volume of 4.5 ml of 0.12% chlorhexidine was placed in sterile tubes, 500 μl of the viable McFarland scale 1 bacterial suspension was added, and vortex homogenized for 10 s; 1 ml of each suspension was taken and inoculated in depth in 14 ml of Wilkins–Chalgren Agar (Oxoid ™); the mixture was then poured in Petri dishes and incubated at 37°C for 7 days under anaerobic conditions. This procedure was performed in triplicate.

Tetracycline hydrochloride effectiveness test: Kelsey–Maurer test

In sterile test tubes, 4.5 ml of each vehicle – distilled water, 0.9% saline solution or 2% lidocaine anesthetic with epinephrine 1:80,000 – was added to TTC-HCL (125, 250, and 500 mg) at pH 1.9 and pH 7.0; 500 μl of the bacterial suspension was added, homogenized in a vortex for 10 s, and allowed to act as a disinfectant at clinically viable times for 30, 60, and 120 s. The suspension was inoculated at each concentration and each time in the same way as for the viability controls. One milliliter of each suspension containing the bacteria was taken and inoculated at depth in Wilkins–Chalgren Agar supplemented with hemin 5 mg/ml (Sigma-Aldrich) and menadione 0.5 mg/ml (Sigma-Aldrich) at 45°C; the mixture then poured in Petri dishes. It was then incubated at 37°C under anaerobic conditions. This procedure was performed in triplicates.[40]

Reading the tests

The presence or absence of CFUs in the different vehicles was evaluated in water, 0.9% saline solution, and 2% lidocaine with epinephrine 1:80,000 at different concentrations of TTC (125, 250, and 500 mg) and at 30, 60, and 120 s. The same was performed for the different pH values and for the positive and negative controls. The microorganisms were re-inoculated and identified by MALDI-TOF to confirm the purity of the cultures.

Percentage of inhibition

CFUs from the 10−6 dilution of the viability control were counted. The number of colonies was multiplied by the dilution factor, which is equivalent to 100% of the bacterial inoculum. The number of colonies in the different vehicles, concentrations, times, and pH values used were counted. After this, a rule of three was used to determine the percentage of bacterial growth resistant to TTC-HCl.

Statistical analysis

Descriptive statistics were used for the statistical analysis; this included the use of a summary measure (percentages [%]), which expresses a number as a fraction of 100.

RESULTS

In the viability control, it was found that the bacterial inoculum of each ATCC strain was viable, and CFU counts were established on the McFarland scale. This inoculum was the one that came into contact with TTC-HCl. The negative controls (sterile distilled water, saline, and 2% lidocaine with epinephrine 1:80,000) had countless positive CFU growths, which effectively showed that dilution vehicles had no antimicrobial activity and the activity was provided by TTC-HCl at both acidic and neutral pH levels.

No CFUs were found at any of the concentrations, times, or pH values during the efficacy trial, resulting in 100% antimicrobial effectiveness [Table 1].

Table 1

Bactericidal activity of tetracycline hydrochloride (%) in Fusobacterium nucleatum, Prevotella intermedia, and Porphyromonas gingivalis

Concentration tetracycline HCl (mg)	Exposure time to tetracycline HCl
	30 s						60 s						120 s
	F. nucleatum		P. intermedia		P. gingivalis		F. nucleatum		P. intermedia		P. gingivalis		F. nucleatum		P. intermedia		P. gingivalis
	pH 1.9	pH 7.0	pH 1.9	pH 7.0	pH 1.9	pH 7.0	pH 1,9	pH 7.0	pH 1.9	pH 7.0	pH 1.9	pH 7.0	pH 1.9	pH 7.0	pH 1.9	pH 7.0	pH 1.9	pH 7.0
125	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100
250	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100
500	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100

Viability control: F. nucleatum 195×106 SD: ±8.33. P. intermedia 118×106 SD: ±21.63. P. gingivalis 2816×106 SD: ±13,89. HCl - Hydrochloride; F. nucleatum - Fusobacterium nucleatum; P. intermedia - Prevotella intermedia; P. gingivalis - Porphyromonas gingivalis; pH - Potential of hydrogen; SD - Standard deviation

DISCUSSION

This investigation is conducted to evaluate the antimicrobial activity of TTC-HCl dissolved in the most common vehicles that dentists have in their offices. The most common dilution liquids for this purpose mentioned in the literature are water and saline solution. Although anesthetics are not mentioned in the literature with TTC-HCl as a solvent in dentistry, they have been used in other fields.[4142] In addition, all dentists have wide access to it, many use it to dilute TTC-HCl, it is a sterile liquid, and it does not generate great cost. Dentists have to make this type of decision daily when choosing a vehicle to dilute TTC. Therefore, it is crucial to determine its activity and to help make a responsible and proven decision in an environment similar to that of the dental office setting adapted to the Kelsey–Maurer test which is based on real-time testing of the efficacy of an antiseptic or antimicrobial agent.[40]

This treatment is relevant considering that it is a common practice in so many countries around the world. It is based on applying a topic antibiotic to change the environment of the periodontal pocket and to enhance the treatment. Accordingly, it is important to prove the evidence and the real outcome with the bacteria's directly related.

The risks and/or benefits for patients should be considered in the systemic administration of antimicrobials, such as the adverse effects of antibiotics, and currently, antimicrobial resistance. Therefore, the evidence does not justify the indiscriminate use of systemic antimicrobials for all patients with periodontitis.[43] TTCs are used in periodontics for dentine conditioning, surface toxins such as endotoxin, and regeneration. Surface demineralization reveals the cementum collagen matrix, which induces fibroblast attachment and development by widening the orifices of the dentinal tubules, demonstrating antibacterial substantivity and residual antibacterial activity from 7 days to 4 weeks.[1644454647]

To date, no study has compared antimicrobial effects on the three periodontopathogens based on solvent, concentrations, pH, or time.

Antimicrobial therapy is performed in various periodontal procedures. The investigation carried out refers to the justification, adequate selection, dosage, and duration of antibiotic therapy to optimize its utility.[48] However, no difference in its action owing to dilution vehicles has been reported, with the ones chosen being the most common that dentists have available.

Local use of antibiotics has been explored for the control of subgingival microorganisms. One of the advantages of providing an antimicrobial locally, such as TTC-HCl, is that it can selectively alter bacterial growth so that mechanical therapy has a prolonged effect.[49] In addition to this, total drug used is low compared with systemic administration, and therefore, fewer side effects could be associated with these doses.[9384849]

The concentrations of TTC-HCl used in this investigation were selected based on those stated in the literature (100 mg/ml[163848] and 50 mg/ml.[50] The result of the antimicrobial activity found in this investigation is consistent with the antimicrobial effectiveness settled by Goodson, gaining Clinical attachment and an incremental reduction in bacterial counts, after the local intrasulcular delivery of TTC fiber therapy in adult periodontitis patients.[103251] TTC acts by inhibiting protein synthesis within the bacterial cell, and in vitro studies carried out to evaluate the susceptibility of antibiotics have demonstrated that most periodontal pathogenic bacteria, including P. intermedia, P. gingivalis, and F. nucleatum, are susceptible to this antibiotic. The lipophilic properties of TTCs explain its high penetration capacity in bacterial cellsHaga clic o pulse aquí para escribir texto.[5253] The antimicrobial activity of TTC can be compared with the use of chlorhexidine.[6754]

Antimicrobial effectiveness was determined at acidic and neutral pH, coinciding with the literature as the fermentation of sugars and low pH favor cariogenic bacteria but not periodontal pathogens.[55] It should be noted that the pH of TTC-HCl varies from 1.8 to 2.2, depending on whether it is diluted in water, an anesthetic, or saline solution, although fluctuations in pH in the oral cavity range between 5.5 and 7.0.[56] pH values of >6.0 or <4.5 increase the solubility of TTC significantly.[57] It can be suggested that pH affects the microorganisms used here in addition to the effect of TTC-HCl. The environment rich in proteolytic enzymes that causes gingivitis or periodontitis[5859] improves the growth of bacteria such as Fusobascterium and Prevotella. These microorganisms, in turn, increase the average pH to neutral values that can help generate an adequate environment[67] for the succession of inflammatory species that are more sensitive to acids such as P. gingivalis. This explains the susceptibility of these microorganisms[67] at acidic pH. However, the antimicrobial effect of TTC-HCl at basic pH can also be seen.

CONCLUSIONS

TTC-HCl has in vitro antimicrobial activity when diluted in sterile water, saline solution, and 2% lidocaine with epinephrine 1:80,000 at pH 1.9 and 7.0 and at concentrations of 125, 250, and 500 mg and at three time points–30, 60, and 120 s–on P. intermedia, F. nucleatum, and P. gingivalis.

Financial support and sponsorship

This project was funded by the Universidad Cooperativa de Colombia.

Conflicts of interest

There are no conflicts of interest.