Mulla et al. BMC Oral Health (2021) 21:408 https://doi.org/10.1186/s12903-021-01762-2 RESEARCH Open Access In vitro assessment of the efect of probiotic lactobacillus reuteri on peri-implantitis microfora Munaz Mulla1, Mushir Mulla2, Shashikanth Hegde1* and Ajit V. Koshy3 Abstract Background: Probiotics afect both the development and stability of microbiota by altering the colonization of pathogens and thus helps in stimulating the immune system of the individual. The aim of the present study is to assess the efect of probiotics on peri-implantitis microfora, by determining the minimum inhibitory concentration (MIC) of Lactobacillus reuteri, that can be efectively administered as an antimicrobial agent on specifc peri-implantitis pathogens. Hence, this study will be helpful in fnding the MIC of L. Reuteri that can be efectively administered as an antimicrobial agent on specifc peri-implantitis pathogens. Methods: This experimental research was conducted on patients visiting the periodontology department in M. A. Rangoonwala college of dental sciences and research centre. Sub-gingival plaque samples were collected from peri- implantitis patients to identify various peri-implantitis microorganisms. The identifed microorganisms were compared to each other and Chi-Square test was used to calculate statistical signifcance. The isolated microorganisms were subjected to the efect of probiotic Lactobacillus reuteri in-vitro. Minimum inhibitory concentration (MIC) was assessed using serial dilution method. Results: The research results showed the presence of Porphyromonas gingivalis, Aggregatibacter actinomycetem- comitans, Prevotella intermedia, Streptococcus salivaris and Staphylococcus aureus in the subgingival samples from peri-implantitis patients. Statistically, signifcantly higher proportion of samples had Porphyromonas gingivalis. When subjected to the efect of L. reuteri, all the microorganisms were afected by L.reuteri except Aggregatibacter actinomycetemcomitans. Conclusion: This study provides the various MIC value for each isolated pathogen against L.reuteri. The authors recommend to avoid using standard guidelines for probiotic dose in the treatment of peri-implant infections as the antimicrobial profle is diferent for each periodontal pathogen. Keywords: Peri-implantitis, Probiotics, Lactobacillus reuteri, Minimum inhibitory concentration (MIC) Background any natural tooth, gum diseases can also afect the dental Several treatment options are available to replace miss- implants leading to peri-implantitis. ing teeth. But over the years, placing dental implants are Peri-implantitis is an infammatory process, which has considered as the best treatment modality [1]. Just like an efect on the tissues surrounding an osseo-integrated implant, leading to the loss of supporting bone [2]. Every *Correspondence: [email protected] afected implant is always a threat to the durability of 1 Department of Periodontology, Yenepoya Dental College, University the associated prosthetic replacement. Peri-implant dis- road, Deralakatte, Mangalore, Karnataka 575018, India eases are common fnding following implant therapy as Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mulla et al. BMC Oral Health (2021) 21:408 Page 2 of 7 peri-implant mucositis is seen among 30.7% of all dental other than toothbrush and dentifrice, undergone peri- implants, and peri-implantitis is seen among 9.6% of all odontal therapy in the last 6 months, undergone peri- the dental implants [3–6]. Evaluation of the literature has implantitis treatment during the last 6 months. Written shown that the microbiota related to peri-implantitis is informed consents were obtained from all participants more complex when compared to a healthy peri-implant based on the guidelines of the Declaration of Helsinki. conditions. Te microbiota consists mostly of anaerobic Tis study was approved by the Institutional Ethics gram-negative bacteria [7]. Even in dental implants, a Committee of M.A.Rangoonwala college of dental sci- cause-efect relationship has been identifed between the ences and research Centre, Pune. accumulation of bacterial plaque and the development To avoid the inter-examiner bias, only one princi- of infammatory changes in the soft tissues surround- pal investigator was employed to collect the samples. ing oral implants. Tus, procedures are now aimed to Tirty-fve patient samples were collected based on prevent and treat any such peri-implantitis through dif- the selection criteria. Microbial samples were collected ferent methods. Tese include mechanical debridement, from the peri-implantitis pockets at the following surgical therapy with or without regenerative procedures, visit. Prior to sampling, the supra-gingival plaque was local or systemic antibiotics. removed with a sterile curette. Cotton rolls were used Various new strategies are also introduced to manage to isolate the site along with air syringe to gently dry this infection such as by recreating the healthy micro- the area. Subgingival samples were obtained by insert- bial environment by the use of non-pathogenic micro- ing #30 sterilized paper points into the deepest prob- organisms. Tis helps to stimulate the host immunity ing point and kept for 30 s. Te paper points were then by inhibiting the growth of pathogenic microorganisms. removed and immediately placed in a sterile Eppendorf One of the strategies is the use of Probiotics to manage tube prepared with transportation medium of 2 ml peri-implantitis. Probiotics are live microorganisms that of Tioglycollate broth (0.4% agar, 0.15% Tioglycol- are administered in sufcient amounts to produce a ben- late bufered saline). Immediately after the collection efcial efect on the host animal [8]. Minimum inhibi- of samples, they were transferred in an ice box to the tory concentration (MIC) is the lowest concentration of microbiological lab. Each sample was made 2 sets, one a drug that inhibits the visible growth of test organism. of which was kept to incubate under aerobic condition In vitro detection of MIC of a drug against pathogens will and the other under anaerobic condition. act as a guideline for its in vivo application. MIC scores Te sample was mixed thoroughly and 5 µl aliquots are usually used to identify an efective dose of the drug were inoculated using a sterile loop onto petri plates of against the pathogen [9]. Blood Agar & MacConkey’s Agar for the Aerobes and Te aim of the present study is to assess the efect of Kanamycin Blood Agar & Bacteroides Bile Esculin (BBE) probiotics on peri-implantitis microfora, by determining Agar for Anaerobes. the minimum inhibitory concentration (MIC) of Lacto- For Aerobic, plates were kept in an incubator for 24 h bacillus reuteri. Tus, this study will be helpful in fnding at 37 °C. For Anaerobic, plates were kept in Gas pack the MIC of L. Reuteri that can be efectively administered anaerobic jar for 5–6 days at 37 °C. After incubation as an antimicrobial agent on specifc peri-implantitis multiple distinguished colonies were observed on the pathogens. plates, which were further sub cultured individually to obtain pure colonies of each. Te pure colonies were then Methods identifed using colony, morphological and biochemi- Patients with peri-implantitis were recruited from cal characteristics as per the standard Bergey’s Manual the periodontology department of M.A.Rangoonwala of Systematic Bacteriology. Chi-Square test was used to college of dental sciences and research Centre, Pune calculate statistical signifcance between the identifed from June, 2016 to July, 2019. Te participants for organisms. the research were selected based on the selection cri- Te periodontal organism which were isolated was teria. Te inclusion criteria were: Age-18 years and then subjected to the efect of probiotic Lactobacillus above, patients presenting at least one implant with reuteri in-vitro. L.reuteri was cultured in Rogosa Agar- peri-implantitis (an implant with a probing depth (Selective medium), incubated at 37 °C for 3–4 days. Serial tube dilution technique was followed in this of ≥ 4 mm), signs of peri-implantitis (loss of supporting bone as estimated on radiographs, bleeding on prob- study to detect the MIC due to its ability to determine ing or suppuration), no implant mobility. Te exclusion antimicrobial activity along with its MIC and is based on criteria were: Consumption of any form of tobacco, the guidelines