Effect of Probiotic Lactobacillus salivarius on Peri-Implantitis Pathogenic Bacteria: An In Vitro Study

Background Varied treatment modalities have been described in the past for the management of peri-implant diseases but the evidence of the use of probiotics for the treatment of peri-implantitis is limited. The aim of this study was to determine the antagonistic growth effects of Lactobacillus salivarius on the growth of peri-implantitis pathogens. Material and method Anin vitroassessment of probiotic L. salivarius on peri-implantitis pathogens was done using the serial tube dilution method. Minimum inhibitory concentration was calculated for five subgingival pathogens namely Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Streptococcus salivarius, and Staphylococcus aureus. Minimum inhibitory concentration (MIC) is defined as the lowermost concentration of any drug that prevents the noticeable growth of the test organism. In vitro assessment to determine the MIC is necessary for an in vivo application. The MIC value will also help to find the drug’s accurate dosage. Results Peri-implantitis pathogens were cultured from individuals diagnosed with peri-implantitis. Except for A. actinomycetemcomitans, all other pathogens were susceptible to the probiotic. S. salivarius had the lowest MIC (0.8mg/mL). Conclusion The MIC value for pathogens will help to determine the effective mode and form of probiotic that can be used for the treatment of peri-implantitis.


Introduction
The term "Probiotic" is used to classify those substances that are released by one organism to enhance the growth of another [1], and is coined after the Greek word bio-tikos which means "for life". They are considered beneficial for the health when taken in adequate amounts [2]. Since its initial use, several authors have found a positive correlation between probiotics and gut health, oral health, halitosis, dental caries, and oral candidiasis [3][4][5]. Periodontal inflammation has also benefited from the use of probiotics. Various Lactobacilli strains have been studied in the past. Lactobacillus acidophilus strain was found to be beneficial when used in patients having gingivitis, periodontitis, and pregnancy-induced gingivitis [6]. Lactobacillus brevis and Lactobacillus reuteri have also been shown to improve gingival bleeding in individuals [7,8]. Lactobacillus salivarius was found to reduce the gingival probing depth and also reduce the periodontal pathogens in dental plaque [9]. The successful results of probiotics in periodontal therapy have given rise to exploring their beneficial role in peri-implant diseases. Peri-implant mucositis and periimplantitis constitute peri-implant diseases. Peri-implant mucositis is an inflammatory condition without bone loss and is limited to the surrounding tissues of the implant. On the contrary, peri-implantitis is associated with the loss of supporting bone [10]. Peri-implantitis microflora is more complex, consisting of mainly anaerobic gram-negative bacteria. When compared to periodontitis, tissue destruction was significantly higher among individuals with peri-implantitis. Although various treatment options are available, limited evidence is present on the usage of probiotics for the treatment of peri-implant diseases [11,12]. Thus, this study was carried out to evaluate the in vitro effect of probiotic L. salivarius on periimplantitis pathogens. The aim of this study was to demonstrate the antagonistic growth effects of probiotic L. salivarius on growth suppression of peri-implantitis pathogens, such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Streptococcus salivarius, and Staphylococcus aureus, using the serial tube dilution method.

Materials And Methods
This study was carried out at M.A. Rangoonwala College of Dental Sciences and Research Centre, Pune. Ethical clearance was obtained from the institutional ethical committee (Reference number: MCES/EC/Perio.PhD/349-A/2016). Patients reporting to the department of periodontology and diagnosed with peri-implantitis were recruited for this study and selected if they complied with the selection criteria. The inclusion criteria included patients who were 18 years or older with a minimum of one implant having peri-implantitis. Peri-implantitis was diagnosed if the probing depth around the implant was ≥ 4 mm, bleeding on probing, loss of supporting bone when viewed on radiographs, and with no implant mobility. Patients consuming tobacco, with systemic diseases, and/or under medication for systemic health were excluded. All subjects provided appropriate informed consent according to the guidelines of the Helsinki Declaration.
After the patients were selected for the study, scaling was done to remove supra-gingival plaque. Subgingival plaque samples were then collected to obtain the peri-implantitis pathogens. Sterile paper point (30no.) was used for the same. After keeping it in the peri-implant pocket for 30 seconds, it was transferred instantly into a sterile Eppendorf Tube® (Eppendorf Corp., Hamburg, Germany). A transport medium containing thioglycollate broth, 2 ml (0.4% agar, 0.15% thioglycollate buffered saline) was used for the same. Once the sample was collected, it was sent to the microbiological lab for further investigation. Each collected sample was incubated under aerobic and anaerobic conditions to identify five peri-implant pathogens: P. gingivalis, A. actinomycetemcomitans, P. intermedia, S. salivaruis, and S. aureus. To support the growth of the bacteria, various culture media were used. Aerobes: Blood Agar and MacConkey Agar, Anaerobes: Blood Agar, and Bacteroides Bile Esculin (BBE) Agar.
L. Salivarius was procured from Agharkar Research Institute, Pune, and was cultured according to their recommendation. Rogosa agar (selective medium) was used to culture L. Salivarius, with an incubation period of three to four days at 37°C. Bergey's Manual® of Systematic Bacteriology (Springer: New York) was taken as a reference for identification of the colonies based on their colony, biochemical, and morphological characteristics. Minimum inhibitory concentration (MIC) by means of the serial tube dilution method was employed to measure the effect of probiotic L. Salivarius [13]. For initial preparation, 20μl of lactobacilli strain was mixed with the 380μl of Thioglycollate broth to make a volume of 400μl. The first dilution was prepared by adding 200μl from this tube into a separate test tube containing 200μl of Thioglycollate broth. This was termed as 10−1 dilution. To make the next dilution, 200μl was added from this 10-1 to a test tube containing 200μl of Thioglycollate broth. This was termed as 10-2 dilution. Similarly, a total of nine dilutions were prepared. Culture suspensions of the five peri-implantitis pathogens were made by adding 5μl from their maintained stock cultures into 2ml of Thioglycollate broth. From this, 200μl was added into each serially diluted tube. All these tubes were then incubated in an anaerobic jar for 48-72 hours at 37°C. The presence of any turbidity indicated the growth of the organism. The tube that contained the least concentration of the lactobacilli strain with no turbidity was regarded as the MIC for that particular microorganism.

Results
Subgingival plaque samples were collected to obtain five different pathogens namely: S. aureus, S. salivaris, P. intermedia, A. actinomycetemcomitans, and P. gingivalis. These pathogens were tested against probiotic L. Salivarius to determine the MIC values ( Table 1).

Discussion
In peri-implantitis, there is progressive destruction of the hard and soft tissues surrounding the implant [14]. Peri-implantitis is known as a multi-factorial disease with numerous risk factors for the same. Various pathogens are also associated with the progression of peri-implantitis. In the study by Persson et al., the pathogens found to be associated with peri-implantitis were Treponema denticola, Tannerella forsythia, Streptococcus mitis, Streptococcus intermedius, S. aureus, P. gingivalis, Haemophilus influenzae, Helicobacter pylori, Campylobacter rectus, A. actinomycetemcomitans [15].
Progression of peri-implantitis leads to excessive loss of supporting tissues causing implant failure or loss of the implant. The treatment modalities of peri-implantitis vary from non-surgical therapy, surgical therapy, and local and systemic antimicrobial therapy. Yet, some patients do not show any response to any of the treatments [16]. Also, antibiotic resistance limits the use of antibiotics in these cases [17,18]. Therefore, probiotics as an adjunct therapy in the management of peri-implantitis is studied in recent years. The use of probiotics in treating caries, halitosis, gingival and periodontal diseases have been reported in the past, but very little literature is available on the use of probiotics in treating peri-implantitis.
For this study, five pathogens were selected that were known to be present predominantly in periimplantitis sites (P. gingivalis, A. actinomycetemcomitans, P. intermedia, S. salivarius, and S. aureus). These pathogens were obtained from the patients who were diagnosed with peri-implantitis. They were then tested against the antagonistic effect of L. salivarius.
L. salivarius is amongst the major species in human saliva [19,20]. Their property includes the production of organic acids from the fermentation of carbohydrates, thereby interfering with the growth of other neighboring microorganisms [21]. Because of this antagonistic property they can be used to combat the spread of infection and improve the host immunity. Authors have suggested their beneficial role when used to treat periodontal and peri-implant diseases [19]. Thus, for this study, L. salivarius was used to test its suppressive effect against the peri-implantitis pathogens.
Based on the susceptibility of the microorganisms, the MIC can be low or high. The MIC is defined as the lowermost concentration of a drug that, after overnight incubation, will suppress the growth of an organism [13]. In this present study, S. salivarius had the lowest MIC (0.8mg/mL) whereas P. gingivalis and P. intermedia had higher MIC (50mg/mL). Similarly, previous authors have also reported in vitro effect of L. salivarius on P. gingivalis, P. intermedia, and Prevotella nigrescens [22]. L. salivarius has also been shown to decrease pocket probing depth and plaque index in people diagnosed with periodontal disease [23,24]. L. salivarius and Lactobacillus fermentum and their concentrated fermentative broth inhibited the growth of P. gingivalis, Streptococcus sanguis, and Streptococcus mutans [25]. A. actinomycetemcomitans was not found to be susceptible to L. salivarius in this present study. Thus, to identify the precise value of MIC for A. actinomycetemcomitans, additional dilutions of >100 mg/mL are required.