The Main Bacterial Communities Identified in the Sites Affected by Periimplantitis: A Systematic Review

(1) Background: Periimplantitis is an infectious condition that affects the periimplant tissue and is of bacterial etiology. However, to date, the exact bacterial flora involved in its occurrence is not known. The aim of this literature review was to summarize the articles published on this topic and to identify the main bacterial species isolated in periimplantitis. (2) Methods: The articles published in three databases were researched: Pubmed, Embase and Web of Science using Prisma guides and combinations of MeSH terms. We selected 25 items from the 980 found by applying the inclusion and exclusion criteria. (3) Results: We quantified the results of the 25 studies included in this review. In general, the most commonly identified bacterial species were Gram-negative anaerobic species, as Prevotella, Streptococcus, Fusobacterium and Treponema. (4) Conclusion: The most frequent bacteria in the periimplantitis sites identified in this review are Gram-negative anaerobic species, also involved in the pathogenesis of the periodontal disease.


Introduction
The loss of dental units due to increased life expectancy, aging, lack of regular checkups at the dentist, poor oral hygiene or various accidents leads to the installation of edentulousness. Once edentation appears, the functionality of the dento-maxillary apparatus, the homeostasis of the alveolar bone, as well as the balance of the digestive and nervous system are affected by the psychological and masticatory implications of tooth loss.
In recent years, the concern among dental practitioners for finding prosthetic rehabilitation solutions as functional as possible and for preserving the remaining dental structures has led to the widespread use of dental implants as a treatment solution of edentulous patients. There are several types of biomaterials from which dental implants can be made, the most used being titanium and titanium alloys, materials with high biocompatibility, high success rates over long periods of follow-up and good mechanical properties [1]. Although titanium implants are widely used, they also have drawbacks such as the gray color that can be visible in the frontal area with thin gingival tissue, which is why more aesthetic alternatives have been found. One of these is the use of Zirconia for dental implants, a material that has aesthetic properties as translucency, the color similar to natural teeth and biocompatibility with the surrounding soft and hard tissues [1][2][3]. The mechanical properties of Zirconia are encouraging, as it has high flexural strength, increased resistance to fracture and corrosion, as well as a low thermal conductivity [3]. Regarding osseointegration, most studies show that the two materials have similar properties [3,4]. Colonization plaque accumulation and gingival bleeding due to neglect of oral hygiene, lack of regular visits to the dental office, as well as by decreasing the healing capacity of the tissues and affecting the body's response to treatment [14,16,17]. Patients with periodontal disease or periimplantitis have a higher risk of developing complications associated with COVID-19, studies suggesting that lesions in the periodontal and periimplant pockets are an entry point for the virus into the general circulation [15,16]. Certain constituents of plaque in the periimplant sulcus, such as Treponema denticola, Porphyromonas gingivalis or Candida, release proteases that degrade the basement membrane facilitating viral and bacterial invasion [15]. Periodontal and periimplant pockets, rich in aggressive pathogens, can also be a starting point for microorganisms that can be aspirated by people infected with SARS-CoV-2, leading to various complications [16].
The information in the literature about the microbiota in periimplantitis is inaccurate and it is not specified exactly which bacteria are associated with periimplantitis. Recent data suggests that periimplantitis is a polymicrobial anaerobic infection that does not fully correspond to the severity of the disease [12].
The bacterial community located in the oral cavity is diverse, and information on microorganisms involved in the development of periimplant infectious diseases as well as standardized treatment protocols for these diseases are limited. This literature review aimed to highlight the main bacterial colonies isolated from periimplant sites affected by periimplantitis by synthesizing information published on this topic in specialized studies.

Search Strategy
The methodological design of this study is in line with the PRISMA 2020 criteria and guidelines [18]. The protocol of the review was registered within the International Prospective Register of Systematic Reviews (PROSPERO) under registration number CRD42022335476. In this study, we address the following question: "Which are the most common bacterial species in periimplantitis?" To answer this question, a systematic literature search was carried out in 3 databases: PubMed, Embase, and Web of Science. The search strategy consisted of different combinations of MeSH keywords: "periimplantitis", "bacteria", "biofilm", "microorganisms", "microbiota", "dental implant": (bacteria) AND (periimplantitis), (biofilm) AND (periimplantitis), (microorganisms) AND (periimplantitis), (microbiota) AND (periimplantitis), (biofilm) AND (dental implant), ((bacteria) AND (dental implant)) AND (periimplantitis), (microbiome) AND (periimplantitis), ("Bacterial strains and periimplantitis" or "biofilms and periimplantitis" or "bacterial cultures and periimplantitis" or "types of bacteria and periimplantitis"), the filters applied being: Clinical Study, Clinical Trial, Randomized Controlled Trial, Other Animals, Humans, in the last 10 years. Two researchers independently performed the database literature search, and subsequently the results were confronted.

Inclusion and Exclusion Criteria
The inclusion criteria were the articles published in the three databases mentioned above, in the last ten years, between January 2012 until March 2022. We selected studies performed on patients with dental implants suffering from periimplantitis and those in which samples of bacterial plaque were collected from periimplant sulcus affected by periimplantitis, analyzed and provided results on the microbial flora involved in periimplantitis.
Among the exclusion criteria were: articles published in a language other than English or French, articles other than those mentioned above, such as systematic reviews or metaanalyzes, experimental or in vitro studies.

Study Selection and Data Collection
Initially, the titles and abstracts of the selected articles were checked for the relevance of the topic, in relation to the proposed research. Subsequently, a full text analysis of all eligible articles was performed based on the inclusion and exclusion criteria. All articles that met the eligibility criteria were selected, and a standardized document was used to collect information on authors, year of publication, bacterial isolation methods and bacteria identified from these studies. Initially, 980 items from the 3 databases were selected according to the search terms mentioned above. Subsequently, after the elimination of duplicates, 201 articles remained, which were analyzed in detail based on inclusion and exclusion criteria. Finally, 25 articles were selected that met the eligibility criteria and introduced in this review. The selected articles were analyzed and classified according to the methods of isolation and identification of bacterial species. To collect data from selected articles, a table was created that included information on the authors of the articles, the year of publication, the study design, population characteristics, the isolation techniques and microbiological methods of bacterial identification, as well as the main bacterial species identified in each study ( Table 1).

Bibliographic Documentation and Selection of Articles
The process of searching and selecting the articles included in this literature review according to the PRISMA requirements is presented in Figure 1. Initially, 980 articles were selected from the three databases: PubMed, Embase and Web of Science that matched the search terms. Out of these, 779 duplicates were identified and removed. The remaining 201 articles were checked, 11 of them being removed because they were not available in full text, another 17 being literature reviews and 19 being in vitro studies. Other elimination criteria were the evaluation of specific bacteria, the lack of quantitative analysis of bacterial species or the absence of periimplantitis. Finally, after a detailed analysis, 25 articles that analyze the bacterial flora involved in periimplantitis were included in this study (Figure 1). Table 1 summarizes the most relevant information in the included studies, such as title, authors, year of publication, study design, population characteristics, methods of isolation and bacterial identification, and the main bacterial species identified. Out of a total of 25 articles, six of them used classical bacterial culture methods to identify microorganisms involved in the development of periimplantitis and 17 used modern molecular biology techniques, DNA-DNA hybridization or RNA sequencing, and two of them used combined bacterial culture and DNA hybridization techniques.

Clinical and Microbiological Characteristics of the Included Studies
Quality assessment of the included studies in this review performed using NOS Scale emphasized that 16 studies were of high quality, eight studies were of moderate quality and one study was of low quality. The results are summarized in Table 2. The average NOS score for the quality assessment of the included studies in this review was 6.6.  Table 1 summarizes the most relevant information in the included studies, such as title, authors, year of publication, study design, population characteristics, methods of isolation and bacterial identification, and the main bacterial species identified. Out of a total of 25 articles, six of them used classical bacterial culture methods to identify microorganisms involved in the development of periimplantitis and 17 used modern molecular biology techniques, DNA-DNA hybridization or RNA sequencing, and two of them used combined bacterial culture and DNA hybridization techniques.

Clinical and Microbiological Characteristics of the Included Studies
Quality assessment of the included studies in this review performed using NOS Scale emphasized that 16 studies were of high quality, eight studies were of moderate quality and one study was of low quality. The results are summarized in Table 2. The average NOS score for the quality assessment of the included studies in this review was 6.6.
A classification of the microorganisms identified in the 25 studies included in this review was made, and the frequency with which each microorganism was found in them was subsequently quantified. In addition, a separate classification was performed on A classification of the microorganisms identified in the 25 studies included in this review was made, and the frequency with which each microorganism was found in them was subsequently quantified. In addition, a separate classification was performed on studies that used the bacterial culture technique to identify microorganisms, analyzing the differences between the two classifications.
In general, the most commonly identified bacterial species were Prevotella, and Streptococcus, Gram-negative anaerobic species. Prevotella was found in 17 of the 25 studies, so with a frequency of 68%. In this bacterial species, Prevotella intermedia was isolated in 7 of the 17 studies (41.17%), Prevotella nigrescens in 4 of 17 (23.52%), Prevotella fusca, Prevotella multiformis and Prevotella denticola, in one of the 17 articles (5.88%).
Streptococcus was found in 17 of the 25 studies, so with a frequency of 68%. The following bacterial species were identified from the genus Streptococcus The genera Fusobacterium were isolated in 16 out of the 25 studies, so with a frequency of 64%. Of the genus Fusobacterium, Fusobacterium nucleatum was isolated in 8 of 16 cases, respectively, 50%.
The genus Neisseria, identified in 9 of the 25 studies (36%), the species Neisseria flavescens being found in 2 of the 9 studies (22.22%).
Regarding the species identified by microbial cultures, 6 of the 25 studies that used this method to isolate bacteria from periimplant sites were identified. The most common were the genera Veillonella, Prevotella and Fusobacterium, with a frequency of 3 out of 6 studies, respectively, 50%. Prevotella intermedia was identified in 1 of 3 studies (33.33%), Prevotella oralis in 1 of 3 (33.33%) and Fusobacterium nucleatum in 2 of 3 studies (66.66%).

Discussion
The complications related to dental implants are mainly inflammatory lesions of the bone and soft tissues surrounding the implants and their restorative components, induced by the accumulation of bacterial biofilm [8,44]. Periimplantitis is defined as an inflammatory disease of the mucosa surrounding an implant accompanied by progressive loss of periimplant support bone [44]. It is generally perceived that after implant insertion and initial loading, during the healing process, between 0.5 and 2 mm of crestal bone height is lost. Any radiographic evidence of additional bone loss suggests a periimplant condition [8]. Periimplantitis has an infectious etiology, and the diversity of bacteria in the oral cavity is increased. However, information on the microbiota involved in periimplantitis is limited and it is unclear whether there is a specific group of bacteria that may be associated with periimplantitis. Through this review, we wanted to summarize the results obtained in specialized studies, in order to identify the main pathogens involved in the development of periimplantitis. The main species identified in most studies included in this literature review are Gram-negative anaerobic bacteria such as those in the red complex, a highly virulent complex containing particularly aggressive species as Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. These bacteria are also correlated with the loss of the gingivo-periodontal junction in periodontal diseases. At the same time, bacteria from the orange complex, Prevotella intermedia, Fusobacterium nucleatum, Campylobacter, Streptococcus constellatus, Peptostreptococcus and Eubacterium were identified in most studies in this review. These bacteria have the ability to make connections and enhance the destructive effect of bacteria in the red complex by promoting their colonization. These findings are consistent with other studies published in the literature [9,11], which also show that the main components of the bacterial community are Gram-negative anaerobic bacteria, as confirmed in this study. Perimucositis and periimplantitis have similar characteristics to gingivitis and parodontitis, that occur around natural dentition. These diseases have multifactorial etiologies and are strongly correlated with bacterial biofilm, especially with Gram-negative anaerobic bacteria [11]. Numerous studies have found the presence of a high number of periodontopathogenic bacteria of the red complex and those in the orange complex in the sites affected by periimplantitis and have highlighted the correlations between the two conditions and how they influence each other when coexisting within the same oral cavity [9,10,12,13,45].
In addition to Gram-negative anaerobic bacteria and those in the red and orange complexes, a high number of studies included in this review discovered Gram-negativeaerobic species such as Neisseria, yellow and purple complex species: Streptococcus, Veillonella, and Gram-positive anaerobic species: Filifactor, Parvimonas micra.
Therefore, the most common bacteria in the periimplantitis sites identified in this review are Gram-negative anaerobic species, also involved in the pathogenesis of the periodontal disease. This aspect is particularly important to establish new preventive measures and new therapeutic protocols, in order to reduce the incidence of this condition among patients.
The treatment of periimplant diseases is complex, the management of these diseases being difficult to achieve. The study of the bacterial flora involved in periimplantitis and the knowledge of the microorganisms incriminated in the pathogenesis of this disease can be a starting point in the research of new therapeutic means in order to achieve a personalized treatment targeted on the main periimplant pathogens. New preventive measures may also be developed in order to reduce the incidence of these conditions in patients or to prevent recurrence after treatment. All the studies included in this review were assessed for risk of bias with NOS Scale Tool [42], independently by two authors. Divergences in the assessment were solved by discussion and by re-evaluating the article. According to NOS Scale there are three groups of risk of bias: low risk of bias (7-9 NOS scores), high risk of bias (4-6 NOS scores) and very high risk of bias (0-3 NOS scores) [46]. The majority of the studies included in this review are at low risk of bias and only one study is considered at very high risk of bias.
The main limitation of this review is the relatively heterogeneity of the studies included in this research. The included articles have different study design, different methods of identifying the clusters of bacteria involved in periimplantitis and the studies are performed on different types of implants. Additionally, the results of counting bacteria are reported in different measurements units. Analysis of the differences in bacterial colonization regarding the type, material and surface design of the implants was impossible because this information was not available in all the articles included in this review.

Conclusions
Summarizing the results obtained in this review, we can state that there is a correlation between the germs involved in the pathogenesis of periodontal diseases and those that cause periimplant diseases. The bacteria identified in most of the analyzed studies: Prevotella, Streptococcus, Fusobacterium, Treponema, Tannerella, and Porphyromonas gingivalis are mostly anaerobic bacteria, pathogens with high virulence, also involved in the development of periodontal diseases, which require special treatment protocols.