Antimicrobial effect of chlorhexidine on Aggregatibacter actinomycetemcomitans biofilms associated with peri-implantitis

Background. This study aimed to assessthe antimicrobial effect of chlorhexidine (CHX) on Aggregatibacter actinomycetemcomitans biofilms isolated from subgingival plaque of peri-implantitis lesions. Methods. Thirteen patients requiring peri-implantitis treatment were consecutively selected and their subgingival biofilm was collected by inserting fine sterile paper points into peri-implant pockets for 15 seconds. A. actinomycetemcomitans was isolated from the subgingival biofilm and cultured. In this study, the standard strain of A. actinomycetemcomitans served as the positive control group and a blank disc impregnated with water served as the negative control; 0.1 mL of the bacterial suspension was cultured on specific culture medium and blank discs (6 mm in diameter) impregnated with 0.2%CHX mouthrinse (Behsa Pharmaceutical Co.) and negative control discs were placed on two sides of the bacterial culture plate. The size of growth inhibition zone was measured by a blinded independent observer in millimetres. Results. According to the results of disc diffusion test, the mean diameter of growth inhibition zone of A. actinomycetemcomitans around discs impregnated with CHX was larger in both standard (positive control) and biofilm samples of A. actinomycetemcomitans compared to the negative control group (blank disc) (P<0.001). Conclusion. Use of0.2% CHX mouthwash had antibacterial effects on A. actinomycetemcomitans species isolated from peri-implantitis sites.


Introduction
eri-implantitis is defined as a destructive inflammatory process of the peri-implant soft and hard tissues, often resulting in loss of supporting bone structure beyond biological bone remodelling. 1,2 Reports are variable regarding the incidence and prevalence of peri-implantitis and a prevalence of 6.61% over a 9-14-year period, 3 23% during a 10-year period 4 and 36.6% during an average of 8.4 years of loading. 5 The specific role of bacteria in peri-implantitis was argued recently. Periodontal pathogens such as A. actinomycetemcomitans, Porphyromonas gingivalis, Porphyromonas intermedia, Tannerella forsythia and Treponema denticola have been isolated from peri-implantitis pockets. 6 At present, association of no single microorganism has been confirmed with implant failure but a shift has been noticed from a predominately gram-positive non-motile, aerobic and facultative anaerobic microorganisms towards gram-negative, motile, anaerobic bacteria. 7 The available clinical protocols for prevention and treatment of peri-implantitis are variable and include non-surgical and surgical approaches but because of incomplete removal of biofilm due to difficult accessibility, screw-shaped design of the implant and the rough implant surface, the predictability of nonsurgical treatment, particularly mechanical debridement, has been questioned 8,9 and additional treatments such as laser therapy, use of antibiotics and/or antiseptics such as chlorhexidine, stannous fluoride, hydrogen peroxide and 35% phosphoric acid gel have been suggested recently. 10 Chlorhexidine (CHX) has been the gold standard oral antiseptic for plaque control for the past 2 decades with no possibility of systemic toxicity, microbial infection or supra-infection. 11 A recent review of the literature also revealed that rinsing with CHX and saline solution was efficient to decontaminate implants with sandblasted/acid-etched and titanium plasma-sprayed surfaces. 10 Considering the predominant role of CHX as the leading oral antiseptic in surgical and non-surgical treatment of peri-implantitis and gap of published information on the susceptibility of A. actinomycetemcomitans biofilm to oral antiseptics and mouthwashes, this study sought to assess the antimicrobial effect of CHX on A. actinomycetemcomitans biofilm isolated from subgingival plaque of peri-implantitis lesions.

Methods
The study protocol was approved by the Ethics Committee of Tehran University of Medical Sciences number 4827and written informed consent was obtained from the participants.

Patient selection
The participants were selected among patients seeking peri-implantitis treatment in the Department of Periodontics, Tehran University of Medical Sciences. The inclusion and exclusion criteria are presented in Figures 1 and 2. Presence of periimplant mucositisis often characterized by bleeding on probing and/or suppuration and is usually associated with probing depths ≥4 mm with any degree of detectable bone loss following initial bone remodelling after implant placement.

Biofilm extraction
The diseased sites were isolated with cotton rolls and subgingival biofilms were obtained using fine sterile paper points (#30) by placing them in peri-implant pockets for 15 seconds. The paper points were immediately placed in a small glass containing thioglycollate broth (Fluid Thioglycollate Medium) and transferred to a laboratory within 30 minutes.

Transferring the samples
In this study, 1 mL of thioglycollate was used as the transfer medium and immersed in boiling water bath for 10 minutes before sampling. Therefore, oxygen was removed from the medium. The bacteria were separated from the paper points by vortexing for 30 seconds. The samples were then cultured in specific A. actinomycetemcomitans culture medium (Brucella agar enriched with lysed sheep blood, vitamin K1, fetal bovine serum, bacitracin, vancomycin and hemin) by a standard loop and incubated in anaerobic conditions in anaerobic jars containing pack-gas and catalyst (which produced CO 2 ) at 37°C for 48 hours.
After colony growth, star-shaped colonies were sampled. The presence of gram-negative A. actinomycetemcomitans was detected by gram staining and microscopic study. In addition, to identify A. actinomycetemcomitans biochemical diagnostic tests (oxidase, catalase and glucose tests) were performed onthesamples.
Finally, samples which were assessed in this procedure were frozen and stored at -70°C. In order to evaluate the effect of CHX on bacteria, frozen A. actinomycetemcomitans was placed at room temperature to thaw. Then, 0.1 mL volume of bacterial suspension was diluted in broth to an optical density of 0.5 McFarland concentration. Finally, 50 μL of this suspension was added to specific A. actinomycetemcomitans medium (as mentioned before) and incubated at 37°C in the presence of 5% CO 2 for 24 hours.

Disc diffusion method
After 24 hours of broth culture (0.5 McFarland), A. actinomycetemcomitans bacteria were aseptically subcultured and evenly spread on blood agar plates using a sterile swab. Three to five minutes were allowed for the culture medium to adhere to the plate. Next, blank discs (6 mm in diameter) impregnated with 0.2% CHX mouthrinse (Behsa Pharmaceutical Co.) and negative control discs (not impregnated with0.2% CHX) were placed on the bacterial culture plate using sterile forceps, and were finally incubated in an anaerobic jar at 37°C for 48 hours. The growth inhibition zone was measured as the distance from the edge of the disc to the edge of bacterial colonies by a blinded independent observer in millimetres ( Figure 3).
In this study, we used two control groups: one was the standard strain of A. actinomycetemcomitans as the positive control group toassess the efficacy of CHX disc and the other one was a blank disc impregnated with water, free of any active agent against A. actinomycetemcomitans as the negative control group.
In order to find differences between the two groups, the diameters of growth inhibition zones were analysed by Student's t-test. Statistical significance was set at P < 0.05. SPSS19 (SPSS Inc., Chicago, IL, USA) was used for data analysis.

Results
A total of 13 patients (4 males and 9 females) with a mean age of 43.85 years and an average probing depth of 6.85 mm were evaluated as shown in Table  1. Six samples were collected from the maxilla (46.2%) and seven from the mandible (53.8%).
According to the results of disc diffusion test (Table 2), the mean diameter of A. actinomycetemcomitans growth inhibition zone around discs impregnated with CHX was significantly larger in both standard (positive control) and biofilm samples of A. actinomycetemcomitans compared to the negative control group(blank disc), and was 0 after 48 hours of incubation (P<0.001). Negative controls did not demonstrate any zone of growth inhibition.
Comparison of the mean diameter of A. actinomycetemcomitans growth inhibition zone around discs impregnated with CHX in standard groups (in 4 groups) and those obtained from patients with periimplantitis revealed a significant difference (P < 0.001).

Discussion
Although data on the susceptibility of A. actinomycetemcomitans and oral biofilm to oral antiseptics are scarce, some papers have reported that the use of antibiotics and/or antiseptics such as CHX, stannous fluoride, hydrogen peroxide and 35% phosphoric acid gel along with mechanical and nonsurgical treatments was effective for the treatment of periimplantitis lesions. 10 The results of this study indicated that CHX had asignificant effect on biofilms collected from subgingival plaque of patients with peri-implantitis in comparison with no use of CHX. These findings are consistent with those of previous studies. 12,13 Chlorhexidine with its broad-spectrum grampositive and gram-negative antibacterial activity is known as the gold standard of oral antiseptics. 14 Literature review revealed no similar studies on the primary antibacterial effect of oral antiseptics on A. actinomycetemcomitans biofilm associated with periimplantitis. However, CHX proved to have a predictable efficacy in decreasing the bacterial load on titanium surfaces among other antiseptics such as sodium hypochlorite, hydrogen peroxide, essential oils and citric acid, and this may suggest optimal efficacy against peri-implantitis. 15 The mechanism of action of CHX in eliminating the bacteria is related to cationic molecules attached to negatively charged bacterial cell surface and consequent leakage and destruction of the cell wall. 16,17 Since CHX has no selective ability for destroying bacterial and nonbacterial proteins, mechanical cleaning of implant surface prior to applying CHX is recommended. 18,19 One of the main advantages of CHX is its prolonged activity but this effect may induce negative side effects such as potential cytotoxicity against cell lines; thus to minimize the possibility of side effects of CHX, rinsing the implant surface would be beneficial to prevent deleterious effects on implant surfaces. 20 A recent review of the literature also revealed that rinsing with CHX and saline solution was suita-ble to decontaminate implants with sandblasted/acidetched and titanium plasma-sprayed surfaces. 10 Finally, considering the limitations of this in vitro study it can be concluded that CHX has significant effects on A. actinomycetemcomitans biofilm extracted from subgingival plaque of patients with periimplantit is lesions. Although the results of this study are relatively encouraging, further in vitro, ex vivo and animal studies as well as randomized clinical trials are needed to introduce the optimal protocol of using CHX in the treatment of peri-implantitis and achieve greater suppression of anaerobic bacteria on the implant surfaces.

Conclusion
Within the limitations of this study, the results showed that 0.2% CHX mouthwash had significant antibacterial effects on A. actinomycetemcomitans species isolated from subgingival plaque of periimplantitis patients.

Ethics approval
The study protocol was approved by the Ethics Committee of Tehran University of Medical Sciences number 4827and written informed consent was obtained from all the participants.