MICROresist-PI: Metagenomic snapshot of the microbiome and resistome in peri-implantitis: pilot study

AIM
The MICROresist-PI project intends to provide new experimental knowledge on oral microbial communities from peri-implantitis-affected dental implants in comparison with the microbiota found in healthy implants, considering inter-patient and intra-patient variation. A pilot study on the microbiome and resistome of such communities will be carried out using metagenomics sequencing in order to pave the way to the full characterization of microbial dysbiosis in peri-implantitis (PI). The ultimate and future goal will be to find novel biomarkers that could assist in the diagnosis and preventive treatment design of PI.
THE PROBLEM
Dental implants have been used to treat edentulism routinely in dental practice. It is estimated that the current yearly increase of dental implants’ placements is about 14% with projections of reaching up to 23% in the year 2026 [1]. However, with the increasing use of dental implants, peri-implant diseases are also becoming more prevalent, and their clear definition and pathogenesis understanding are crucial for implementing adequate treatment. Such biological
complications affecting dental implants are inflammatory conditions associated with a microbial challenge. Peri-implantitis (PI) is the major peri-implant complication and can lead to dental implant loss [2]. More in-depth studies are still needed to fully disclose the microbial dysbiotic state in PI. A shotgun metagenomic sequencing analysis used to profile taxonomic composition and functional potential of microbial communities, including bacteria, archaea, viruses and protozoa, has been hardly applied to analyze the full microbiome in PI. In fact, most studies done on the microbiome of PI have focused on the bacteriome, using 16S rRNA gene sequencing [3,4]. Additionally, to date, there is little or no insight into the oral resistome [5]. Getting more understanding of particular microbiomes, such as those of saliva and subgingival biofilm, and
into their resistomes in both health and disease states can provide valuable data to improve diagnostic and treatment strategies. THE STRATEGY, METHODOLOGY AND KNOW-HOW
The project execution will rely on Egas Moniz Dental Clinic (EMDC), a national reference dental hospital for the treatment of PI, and on a team with large experience in Dentistry, Peri-Implantology, Microbiology, Molecular Biology, and Biostatistics. Moreover, the Research Unit involved, the Interdisciplinary Research Centre Egas Moniz (CiiEM), is committed to foster research and innovation to improve health and disease treatment.
Upon Egas Moniz Ethics Committee approval, patients with healthy implants (Group HI) and patients with co-occurrence of diagnosed PI-affected implants and healthy implants (Group PI) will be recruited from EMDC throughout a 9-month period, according to a set of pre-established inclusion and exclusion criteria. At least n = 20 patients will be enrolled in each group. Data and clinical information will be collected from participants. Then, several samples will be collected as follows: 1) one saliva sample will be collected from each patient on both groups; 2) one biofilm sample will be collected from a healthy implant site of each patient included in Group HI; and 3) two biofilm samples, one from a healthy implant site and one from a PI site, will be collected from each patient belonging to Group PI. Afterwards, DNA will be extracted from all samples. DNA samples (n=20) with enough concentration and quality, from each type of sample within the same patient in each group will be obtained and used in shotgun metagenomic sequencing analysis. This technique will provide taxonomic profiling (diversity and abundance)
as well as insights on the spectrum of function genes, namely antibiotic resistance genes (ARGs) on each sample. Considering the multidimensional nature of the gathered and obtained data, a multivariate analysis approach will be performed. Furthermore, we will also look for correlations between the patient's clinical data and the associated microbiome or resistome.
EXPECTED OUTCOMES AND IMPACT
The structure and role of polymicrobial communities in the pathology and progression of PI are still not well known. A number of questions remain to be addressed. For instance, what are the detailed differences in genomic architecture between microbial biofilms from patients with PI and those with healthy implants? How do changes in microbial composition drive the pathogenesis of PI? Is the saliva microbiome modulated by the PI microbiome within the same
patient? Does the resistome of microbial communities from patients with PI differ in terms of composition or abundance from the resistome found within the microbiome of patients with healthy implants? The MICROresist-PI project entails a pilot study involving next-generation sequencing technology that will generate data crucial to shed light on the above questions.