Application of bioelectric effect to reduce the antibiotic resistance of subgingival plaque biofilm: An in vitro study.

CONTEXT: Biofilms are known for their antimicrobial resistance, and so is the subgingival plaque biofilm, the primary etiologic factor for periodontal infections. AIMS: The objective of this study is to investigate if the subgingival plaque biofilm resistance can be reduced using doxycycline in the presence of low-intensity electric field (bioelectric effect). SETTINGS AND
DESIGN: The study was an in vitro microbiological study.
MATERIALS AND METHODS: Subgingival plaque samples from chronic periodontitis patients were collected to grow subgingival plaque biofilms on hydroxyapatite disks. Hydroxyapatite disks with the plaque biofilms from each patient were divided into four groups: (i) No intervention - control, (ii) current alone - CU; (iii) doxycycline - AB, and (iv) combined treatment - CU + AB. After respective treatments, the disks were anaerobically incubated for 48 h, the biofilm was dispersed and subcultured and colony-forming unit/mL was estimated in all the four groups. STATISTICAL ANALYSIS: Statistical analysis was done using Mann-Whitney and Kruskal-Wallis tests for intergroup comparisons. T-test was done to assess the difference in current flow between the groups CU and CU + AB.
RESULTS: All the three treatment modalities showed antibacterial effect. Application of current alone resulted in reduced bacterial growth than control group. Doxycycline alone resulted in reduction in bacterial counts better than control and current alone groups. The combination treatment showed greatest inhibition of bacterial colonies.
CONCLUSION: The ability of doxycycline antibiotic in inhibiting plaque biofilm was significantly enhanced by application of a weak electric field (5 volts for 2 min).

INTRODUCTION

The subgingival plaque exists as a complex biofilm consisting of a multispecies consortium colonized on the surfaces of the oral cavity.[12] Periodontal disease is realized as a multispecies bacterial biofilm infection rather than an infection caused by a single pathogen.[34] Rams et al. studied the antibiotic susceptibility of selected subgingival periodontal pathogens from chronic periodontitis patients and reported resistance to antibiotics commonly used in periodontal practice.[5] The composition and organization of the biofilms limit diffusion of molecules, including antibiotics through the structure and into the biofilm. Other potential mechanisms implicated in biofilm resistance are the presence of antimicrobial destroying enzymes in the biofilm, quorum sensing and signaling systems, existence of altered growth rate (dormant bacteria), and exchange of genes.[67] By virtue of these properties, biofilm infections pose clinically challenging situations in their management and eradication.

Efficacy of mechanical plaque removal is limited and transient in the subgingival area.[89] Systemic and local antibiotic therapies are bound with problems of side effects, drug-resistant strains, and can be effective on planktonic bacteria with penetration barrier into biofilms.[10] The antibiotic concentrations required to inhibit or kill periodontal bacteria were earlier determined on planktonically grown bacteria in an in vitro environment of pure cultures and not in bacteria within a complex biofilm where antibiotic resistance is projected to be several magnitudes higher (1000–1500 times).[71112] However, such high doses are impractical as they dramatically increase the risk of side effects and contribute to the proliferation of multidrug-resistant strains.[1213]

It is prudent to focus on treatment modalities targeting the plaque biofilm rather than individual species to enhance the efficacy of existing antibiotics. Biofilm research has identified additional means to disrupt biofilms. Photodynamic therapy and ultrasound have been tried to target biofilm bacteria in periodontitis with conflicting results.[1415] Additional physicotherapeutic approaches like bioelectric effect (BE) was tried and tested to overcome antibiotic resistance effectively. Costerton et al. reported improved efficacy of antimicrobials when used along with a low-intensity electric fields simultaneously to the biofilm.[1617]

BE was shown to be a promising method to increase the efficacy of antibiotics on biofilms by applying electrical signals in combination with low doses of antibiotics. Following this principle, medical devices and implants are coated with controlled delivery of antibiotics to prevent biofilm infection.[18] Taken these observations together, the objective of this study is to evaluate if electrical current can enhance the efficacy of doxycycline against subgingival plaque biofilm bacteria.

MATERIALS AND METHODS

Inclusion and exclusion criteria

Participants suffering from chronic periodontitis between the ages of 30 and 65 years with no known systemic diseases, who had not received antibiotics within the previous 6 months and who were not taking medications that might influence the subgingival flora were included for the study. Chronic generalized periodontitis was characterized by ≥5 mm-probing depth and ≥3-mm attachment loss in at least two sites per quadrant. Pregnant women, lactating mothers, and patients who had received any surgical/nonsurgical periodontal therapy during the previous 6 months were excluded from the study.

Ten chronic generalized periodontitis patients were selected for saliva and subgingival plaque sample collection and ethical clearance was obtained from the Institutional Review Committee, Sri Sai College of Dental Surgery, Vikarabad. After obtaining the informed consent from the patient and the screening examination, Plaque Index (Sillness and Loe), Modified Gingival Index (Lobene), Sulcular Bleeding Index (Saxton), Probing pocket depths, gingival recession, and clinical attachment levels were recorded. A pilot study was done to ensure biofilm formation from the patient sample, to arrive at the time and growth conditions, to establish the electrical parameters, and to choose the appropriate antibiotic and its concentration before commencing the original study.

Blenkinsopp et al. and Costerton et al. reported that low-intensity electric fields (1.5–20 V/cm) can override the bacterial resistance when used along with the antibiotic. To apply the electric current, an electric power generator was fabricated to deliver a voltage within the range of 0–20 V. For electrical parameters, DC electric field is used in this study similar to most of the earlier studies.[161719] Using this range in our pilot study, the voltage and the time of application were arrived at by trial and error method with the voltage levels of 1 V, 5 V, 10 V, 15 V, and 20 V, each applied for 30 s, 60 s, 90 s, and 120 s, respectively. A time- and energy-dependent reduction in bacterial growth was observed with 1 V showing least effect and maximum effect with 20 V. These observations are in agreement with those of Kim et al.,[20] wherein they concluded that the total energy and not the type of electric signal is the key to determine the efficacy of BE treatment.

However, the optimum level that is well tolerated by the human body has to be arrived at, foreseeing the possible clinical applications of this concept. Hence, the safe tolerable voltage levels were tested on investigators first, and then, on 100 volunteers starting from 0 V and slowly increasing the voltage levels to a maximum of 8–10 V. A visual analog score (VAS) was designed based on their response and Dalziel's reports and used to assess the volunteered participants' response to varying levels of voltage applied.[21] To standardize, the electrodes were kept at mandibular first premolar for all the healthy volunteers. It was observed that most of the participants tolerated well up to 6–8 V, but started feeling tingling sensation. Hence, a safe well tolerable and comfortable 5 V was chosen to further carry out the study [Tables 1a and 1b] The VAS scores analysis showed that a voltage application of 5V for 2 min as the safe permissible limit, wherein the mean current flow of around or <1 mA.

Table 1a

Evaluation of patient perception on the application of current in the gingival sulcus

Table 1b

Analysis from visual analog score readings

Formation of subgingival plaque biofilm

In vitro subgingival plaque biofilm models were developed from subgingival plaque samples of chronic generalized periodontitis patients on sterile ceramic calcium hydroxyapatite disks (Clarkson Chromatography Products Inc., 213 South Main St. South Williamsport, PA 17702, USA) (HA) of 0.38” diameter × 0.06” thickness as described by Walker and Sedlacek [22] [Table 2]. The HA disks with biofilm from each patient were distributed into four groups as shown in the flow chart [Table 3];

Table 2

Flowchart showing subgingival plaque biofilm model formation

Table 3

Flow chart showing the distribution of hydroxyapatite disks per patient and the medium used in the respective groups

Control: No intervention

CU, current alone: Application of low-intensity electric field alone (5 V for 2 min)

AB, Antibiotic alone: Application of antibiotic (doxycycline 512 μg/ml)

CU + AB: Application of electric field (5 V for 2 min) and antibiotic (doxycycline 512 μg/ml).

HA disks of all the groups were incubated anaerobically for 48 h. Biofilm formation on the HA disk per patient was confirmed by scanning electron microscopy [Figures 1 and 2]. Plaque samples of chronic periodontitis patients were grown into biofilms and tested, each sample was replicated 3 times, and the average values of colony-forming unit (CFU)/mL were recorded.

Figure 1

Scanning electron microscopy picture of biofilm on HA disk (×2500)

Figure 2

Clumps of bacterial colonies (×5000)

Application of electric field to the biofilm

For electric field application, three-neck glass flasks were used. The two side necks were closed airtight with rubber septa (Sigma Aldrich, St. Louis, MO, USA). Immediately, a two-way glass rod was fitted on the center neck and air in the flask was removed using vacuum pump, thus ensuring vacuum in the flask [Figure 3]. Group II (CU alone) and Group IV (CU + AB) disks were transferred gently and quickly into sterile three-neck glass flasks in anaerobic workstation for the application of electric field along with their respective media. Two platinum electrodes were pierced through the rubber septa on the side necks and one was connected to the cathode and the other to anode. For the application of electric field, a regulated electric power supply (Future Tech Instruments, Secunderabad, India) was designed and fabricated to deliver electric voltage (DC) within the range of 0–20 V [Figure 4]. The amount of voltage applied; the time and the current flow could be observed from the digital readout displayed on the apparatus. Using this, DC, direct current voltage of 5V is applied for 2 min is applied the HA disks of CU and CU + AB groups. The mean current flowed was around 11 mA in both the groups. After current application, the flasks were anaerobically incubated for 48 h.

Figure 3

Three-neck glass flask with platinum wire electrodes on two-side openings closed airtight with rubber septa, two-way glass valve fitted at the center

Figure 4

0/20 DC regulated electric power supply

Subculture on blood agar

The biofilm disks were transferred to 1 ml of sterilized trypticase soy broth (TSB) in a test tube, gently sonicated to disrupt the biofilm matrix and disperse the bacterial growth at a low intensity to prevent exposure of the sample to atmospheric air. The bacterial dispersions were vortexed, serially diluted to a standard turbidity of 0.5 McF, and then plated on to blood agar (HiMedia) for anaerobic incubation of 48 h to calculate number of viable CFU/mL.

Statistical analysis

Statistical analysis was done for the CFUs/mL and current readings using SPSS software. Differences in CFUs obtained were tested using nonparametric tests to avoid the influence that high counts might have on the mean. Comparison of one group with all the other three was done with Kruskal–Wallis test. Mann–Whitney test was used to detect differences between two unpaired sets of observations. To check if doxycycline had any influence on the current flow, that is, between CU and CU + AB groups, statistical analysis was done using t-test for current readings in both the groups. The differences is considered statistically significant if P ≤ 0.05.

RESULTS

The clinical parameters of all the ten chronic periodontitis who donated saliva and plaque samples were presented in Table 4. The results in terms of log10 values of CFU/mL for each patient were tabulated [Table 5]. In all the patients, the control group showed high bacterial counts with a mean of 8.21 log10 CFU. Group CU had shown a reduction of 0.27 Log10 in the viable bacteria compared to control (P = 0.013). AB, antibiotic alone group, resulted in 3.44 log10 reduction in the CFU/mL compared to control group (P ≤ 0.001**). Combination treatment, CU + AB resulted in significantly high 4.6 log10 reduction of bacterial viable counts compared to control group (P ≤ 0.001**). The percentage difference in log10 values of CFU as compared to control group and the intergroup comparisons are presented in Figures 5 and 6, respectively.

Table 4

Mean age and clinical parameters of chronic periodontitis patients

Table 5

Mean logarithmic values (Log10) of colony-forming units in all the four groups

Figure 5

Percentage difference in log10 colony-forming unit/mL between the control group and test groups. CU – Current alone; AB – Antibiotic alone

Figure 6

Intergroup comparison of percentage difference in log10 colony-forming unit/mL. CU – Current alone; AB – Antibiotic alone

Current readings

The mean values of current in the two groups were calculated. The difference in the current flow in both the groups was not statistically significant at P ≥ 0.05, 95% confidence interval [Table 6].

Table 6

Mean values of current (milliamperes) that passed through the medium on application of 5V in both the groups

DISCUSSION

To study the BE, subgingival plaque biofilm model was required. Previous studies on BE were done on artificially created single-species biofilms [161723] which cannot simulate the situation in periodontitis, a multispecies disease. Several in vitro supragingival plaque models have been described in literature.[242526] However, only few studies have described the model for subgingival plaque biofilm, which is the actual culprit of periodontitis.[1922] In the present study, subgingival plaque biofilm model on hydroxyapatite disks was used.[1922] As per this model, biofilms formed from samples obtained from periodontally diseased patients were 69% similar in species and 57% similar in the proportions present. The biofilms grown for 48 h yielded heavy growth on culture, therefore to standardize, further maturation of biofilms was not required.

To apply the electric current, an electric power generator was fabricated to deliver a voltage within the range of 0–20 V. For electrical parameters, DC electric field is used in this study similar to most of the earlier studies.[161720] A time- and energy-dependent reduction in bacterial growth was observed with 1 V showing least effect and maximum effect with 20 V (Pilot Study). These observations are in agreement with those of Kim et al.,[20] wherein they concluded that the total energy and not the type of electric signal is the key to determine the efficacy of BE treatment. Caubet et al. reported that the synergy between direct current and antibiotic works better than radiofrequency current.[27]

Application of electric currents for therapeutic purpose is not new and was tried to drive chemotherapeutic molecules into solid tumors and in orthopedics for healing of bone fractures.[2829] Studies investigating possible tolerance levels of electric current to human body suggested that maximum harmless current that human body can perceive and sustain is around 5 mA.[2130] The parameters used in this study were cross-checked for in vivo application which ensured the voltage levels effective in vitro would be safe for in vivo use.

Previous studies on BE were done using aminoglycosides, ciprofloxacin, piperacillin, oxytetracycline on Pseudomonas aeruginosa or Klebsiella pneumoniae or Escherichia coli biofilms, etc. Lasserre et al. reported the application of BE on Porphyromonas gingivalis biofilm with 0.2% of chlorhexidine recently.[23] In the present study, doxycycline was used to investigate the BE on the biofilms grown from subgingival plaque samples of chronic periodontitis patients, which mimics closely to the clinical scenario. Earlier reports suggested that the maximum concentration of the drug released in the gingival crevicular fluid after application of locally applied doxycycline gel is around 20–25 mg/ml (after 15 min) and controlled release up to 16 μg/ml can be maintained for 10–12 days.[31323334] In this study, a concentration of 512 μg/ml doxycycline was used which 40 times lower than the maximum released drug levels with doxycycline gels. The results showed a reduction of 3.44 log10 in viable counts compared to control group. Antibiotic efficacy was further enhanced by 4.6 log10 reduction of viable counts with a transient exposure to mean electric current of 11 mA for 2 min, a proof of the concept of BE.

Electric currents can cause electrolysis of sodium chloride (NaCl). Rabinovitch and Stewart reported removal and inactivation of Staphylococcus epidermidis biofilm by the electrolysis products of chloride ions.[35] To avoid this, instead of metal salt solutions like sodium chloride, the medium itself was used as an electrolyte in this study. TSB plus distilled water in CU group and TSB plus doxycycline were the electrolytes in CU + AB group so that the reduction in bacterial count can be attributed totally to the treatment intervention. The mean amperage of current that flowed in both groups was 11.5 mA and 11.6 mA with doxycycline or with distilled water in electrolyte composition, respectively. Hence, doxycycline did not alter the current flow through the medium. For electrode selection, platinum (noble metal) electrodes were used in this study as corrosion was reported with the stainless steel electrode (cathode).[35]

Shirtliff et al., in their study, reported that the electrical current did not have any bactericidal action either alone or when coupled with free chlorine.[36] In the present study, application of current alone had shown 0.27 log10 reduction in CFU/mL compared to control group. This is in agreement with del Pozo et al. that current alone could have antibacterial effect, which was termed as electricidal effect.[37] Reported studies on BE applied low-intensity electric field for prolonged exposure time of 12–48 h, which seemed to be impractical in a clinical scenario. Lasserre et al. applied 1.5 mA and 10 mA DC currents for 10 min on P. gingivalis biofilm with 0.2% of chlorhexidine and stated a significant enhancement in chlorhexidine action with 10 mA.[23] In this study, it was observed that even a transient exposure of 2 min to electrical current of 5V (11 mA) can enhance the action of doxycycline on biofilm. However, the maturity of the biofilm, type of species, and the current parameters are slightly different in these studies to make a true comparison.

The results of this study showed a statistically significant difference in the number of viable bacterial counts (CFU/mL) when each group was compared with control group and when intragroup comparisons were made. The maximum reduction in the number of CFU/mL was observed in combination group CU + AB, followed by AB alone and CU alone.

Some suggested mechanisms for BE in the literature are as follows:

Electric current disrupts the charges in extracellular matrix, allowing penetration of antibiotics [15]

Electrochemical generation of potentiating oxidants [16]

Electroporation – The BE may depend largely on electrophoretic forces that allow antimicrobial agents to overcome diffusion barriers that would otherwise limit their access to their targets within bacterial cells [1516]

The BE could be related to electrolysis, with resultant increased delivery of oxygen to the biofilm, which might overcome biofilm biomass and cell wall barriers, as well as increase the metabolic activity and growth rate of the contained bacteria [38]

Increased convective transport due to contraction and expansion of the biofilms.[3940]

CONCLUSIONS AND LIMITATIONS

To conclude, the hypothesis was accepted, as the ability of antibiotic (doxycycline) in inhibiting plaque biofilm microbes was enhanced significantly by the application of low-intensity electric fields of 5V for 2 min. Moreover, electrical current alone at 5V for 2 min was effective in reducing bacterial counts significantly in biofilm.

However, this approach needs more in vitro studies to test antimicrobial susceptibility of periodontal pathogens in biofilm state with low-dose antibiotics and concomitant low-intensity electric current. Significant electric parameters, time, frequency of application, and optimal concentration of each antibiotic to achieve maximum effect on a specified biofilm need to be defined, following which, potential clinical applications in periodontitis can be addressed. Maturity of the biofilm could also be a variable to be addressed. As the biofilms become more mature, the resistance to antibiotics also may increase. While the effect of the electrical current alone or its complementary role is established in the present study, it is only an in vitro observation. One of the critical issues would be how to deliver the in vitro established voltage of electric current into the clinical realm. The electrical parameters or the device if any developed based on the parameters must be studied and tested in larger sample size before giving any conclusions. Observations from the pilot studies, however, showed patient acceptance and no side effects. Another issue to be resolved is the duration of the effectiveness of the bacterial inhibition thus achieved, that is, once the bacterial load is reduced, how long the effect would sustain and how often the retreatment has to be repeated.

One most important advantage with oral biofilms is the mode of delivery to oral cavity appears feasible unlike the deep-seated medical device-related biofilm infections. This type of treatment modality may be a novel approach to contain periodontopathic bacteria and with suitable refinements in technique and delivery system, it could be translated into a viable treatment option in the armory of a periodontist. Future clinical application of this regimen suggested was, to develop a small hand piece like a scaler that can disrupt the biofilm mechanically by the tip and deliver an antiseptic irrigation with low DCs that can target the biofilms present in the inaccessible areas, thereby optimizing the antimicrobial strategy. Another possible clinical application would be the use of these electric currents through a special tray designed for home care to improve supragingival plaque control during supportive periodontal therapy [23] or an intraoral irrigator like “Waterpik” that can be used for intrapocket irrigation in an electrical field.

The application of low-intensity electric field to disrupt the biofilm is on a similar analogy of LASER/photodynamic therapy in sterilization of gingival sulcus area. However, the application and local nature of this type of approach is similar to other locally delivered antimicrobial agents and may have the same limitations. To conclude, periodontitis cases who are refractory and resistant to regular and conventional treatment seem to lend themselves to this type of therapy. Since the antibiotic concentrations used along with the electric current are minimal, and yet effective on a biofilm, this approach may constitute a useful adjunct in the repertoire of treatment options.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.