ORIGINAL RESEARCH published: 08 October 2020 doi: 10.3389/fcimb.2020.583761 Oral and Fecal Microbiome in Molar-Incisor Pattern Periodontitis Pâmela Pontes Penas Amado 1, Dione Kawamoto 1, Emmanuel Albuquerque-Souza 2, Diego Castillo Franco 3,4, Luciana Saraiva 2, Renato Corrêa Viana Casarin 5, Anna Carolina Ratto Tempestini Horliana 6 and Marcia Pinto Alves Mayer 1,2* 1 Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil, 2 Division of Periodontology, Department of Stomatology, School of Dentistry, University of São Paulo, São Paulo, Brazil, 3 Department of Biological Oceanography, Oceanographic Institute, University of São Paulo, São Paulo, Brazil, 4 Institute of Environmental Sciences, Faculty of Biology, Jagiellonian University, Kraków, Poland, 5 Department of Prosthodontics and Periodontics, Piracicaba Dental School, State University of Campinas, São Paulo, Brazil, 6 Biophotonics Applied to Health Sciences, University Nove de Julho, São Paulo, Brazil In order to improve our understanding on the microbial complexity associated with Edited by: Georgios N. Belibasakis, Grade C/molar-incisor pattern periodontitis (GC/MIP), we surveyed the oral and fecal Karolinska Institutet (KI), Sweden microbiomes of GC/MIP and compared to non-affected individuals (Control). Seven Reviewed by: Afro-descendants with GC/MIP and seven age/race/gender-matched controls were Anders Johansson, evaluated. Biofilms from supra/subgingival sites (OB) and feces were collected and Umeå University, Sweden Oumkeltoum Ennibi, submitted to 16S rRNA sequencing. Aggregatibacter actinomycetemcomitans (Aa) JP2 Mohammed V University in Rabat, clone genotyping and salivary nitrite levels were determined. Supragingival biofilm of Morocco GC/MIP presented greater abundance of opportunistic bacteria. Selenomonas was *Correspondence: Marcia Pinto Alves Mayer increased in subgingival healthy sites of GC/MIP compared to Control. Synergistetes [email protected] and Spirochaetae were more abundant whereas Actinobacteria was reduced in OB of GC/MIP compared to controls. Aa abundance was 50 times higher in periodontal sites Specialty section: This article was submitted to with PD≥ 4 mm of GC/MIP than in controls. GC/MIP oral microbiome was characterized Microbiome in Health and Disease, by a reduction in commensals such as Kingella, Granulicatella, Haemophilus, Bergeyella, a section of the journal and Streptococcus and enrichment in periodontopathogens, especially Aa and sulfate Frontiers in Cellular and Infection Microbiology reducing Deltaproteobacteria. The oral microbiome of the Aa JP2-like+ patient was Received: 15 July 2020 phylogenetically distant from other GC/MIP individuals. GC/MIP presented a higher Accepted: 09 September 2020 abundance of sulfidogenic bacteria in the feces, such as Desulfovibrio fairfieldensis, Published: 08 October 2020 Erysipelothrix tonsillarum, and Peptostreptococcus anaerobius than controls. These Citation: Amado PPP, Kawamoto D, preliminary data show that the dysbiosis of the microbiome in Afro-descendants with Albuquerque-Souza E, Franco DC, GC/MIP was not restricted to affected sites, but was also observed in supragingival and Saraiva L, Casarin RCV, subgingival healthy sites, as well as in the feces. The understanding on differences of the Horliana ACRT and Mayer MPA (2020) Oral and Fecal Microbiome in microbiome between healthy and GC/MIP patients will help in developing strategies to Molar-Incisor Pattern Periodontitis. improve and monitor periodontal treatment. Front. Cell. Infect. Microbiol. 10:583761. Keywords: dental plaque, aggressive periodontitis, oral microbiome, fecal microbiome, human microbiome, 16S doi: 10.3389/fcimb.2020.583761 rRNA sequencing, Aggregatibacter actinomycetemcomitans, dysbiosis Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 October 2020 | Volume 10 | Article 583761 Amado et al. Periodontitis-Associated Oral and Fecal Microbiome INTRODUCTION MATERIALS AND METHODS Periodontitis is a multifactorial inflammatory disease that Cross-Sectional Study Individuals affects periodontal tissues in response to a dysbiotic microbial Afro-descendants with periodontitis Stage III, Grade C/MIP community (Hajishengallis, 2015). The most common forms of (GC/MIP), aged between 18 and 25 years, and age/race/gender- periodontitis observed in clinical practice were recently included matched non-affected individuals (Control) were selected at the in a single large category (Caton et al., 2018; Papapanou et al., School of Dentistry of the University of São Paulo (FOUSP), after 2018), and the previous localized aggressive periodontitis is now protocol approval by Research Ethics Committee at Biomedical classified as molar/incisor pattern periodontitis (MIP) (Tonetti Sciences Institute of University of São Paulo (number: 1.821.309). et al., 2018). Although MIP shares some general features with Those who agreed to participate in the study signed an informed other forms of periodontitis, its classification as a distinct disease consent form. entity is still under discussion (Fine et al., 2018, 2019). GC/MIP cases were diagnosed as follows: interdental clinical MIP, as the name suggests, affects incisors and first molars attachment loss (CAL) ≥5mm at the site of greatest loss; of adolescents and young adults associated to minimal plaque radiographic alveolar bone loss extending at least to middle third and rapid rate of progression (Fine et al., 2019). Genetic of the root; % bone loss/age >1.0; early onset disease (18–25 characteristics and high incidence of MIP in members of the same years), angular alveolar bone defects, and MIP (Tonetti et al., family support a strong familial aggregation (Meng et al., 2007). 2018). The inclusion criteria for the Control were as follows: lack Despite its rare prevalence worldwide (0.1–2%), adolescents of of sites with PD >3 mm (assuming no pseudo pockets); bleeding African and Middle Eastern descent present a 10-fold higher on probing (BoP) ≤20% (Joss et al., 1994); no caries or extensive risk for MIP than other populations (Fine et al., 2019). MIP is restoration; and at least 28 permanent teeth. commonly associated with the putative pathogen Aggregatibacter Exclusion criteria were previous subgingival periodontal actinomycetemcomitans (Aa) (Slots et al., 1980; Zambon et al., therapy (scaling and root planning and/or periodontal 1983), and the highly virulent strains belonging to the JP2 surgical therapy); use of medications that could affect the clone are associated with disease progression in young Afro- periodontium, such as corticosteroids or antibiotic treatment descendants (Bueno et al., 1998; Haubek et al., 2008; Höglund in the previous 6 months and/or mouthwashes containing Åberg et al., 2014; Ennibi et al., 2019). antimicrobials; systemic diseases that could affect the progression Currently, it is well-known that periodontitis is induced of periodontitis (e.g., diabetes and immunological disorders); by the activity of the entire microbial community in the pregnant or lactating; and smokers. subgingival biofilm of affected sites, characterized by higher amounts of pathogens, and decreased proportion of commensal Clinical Assessment microorganisms (Faveri et al., 2009; van Essche et al., 2013) and BoP was evaluated based on the presence (1) or absence (0) of not by a single pathogen (Fine et al., 2013a). However, there are bleeding up to 30 s after probing; probing depth (PD) measured no reports on the composition of complex bacteria communities as the distance (in millimeters) from the free gingival margin to associated with MIP using next generation sequencing methods. the bottom of the pocket; gingival recession (GR) measured as the Moreover, periodontitis has been associated to several distance from the cementum-enamel junction to the free gingival systemic diseases and inflammatory disorders of the margin; CAL was measured as PD plus GR. When there was no gastrointestinal tract (Kumar, 2017), although a closer look GR, CAL was determined as the distance from the cementum- at this two-way relationship is still necessary. Hypothetically, enamel junction to the bottom of the pocket. All parameters were the dysbiotic oral microbiota not only induces a periodontal obtained at six sites per tooth (mesiobuccal, buccal, distobuccal, hyperinflammatory response, but it would be a source of distolingual, lingual and mesiolingual), except for third molars persistent systemic inflammation, serving as inoculum using a North Carolina probe (Hu-Friedy, Chicago, USA). Mean for the gut. Indeed, the oral administration of Aa and and standard deviation for PD and CAL were calculated by full- Porphyromonas gingivalis in mice causes alterations in the mouth, as well as number of sites with PD ≥4 mm and PD fecal microbiota (Arimatsu et al., 2014; Komazaki et al., ≥6 mm. Measurements reproducibility was calculated by intra- 2017). In addition, the fecal microbiome of individuals class correlation coefficient for PD (ICC = 0.86) and CAL (ICC affected by more common forms of periodontitis showed = 0.85) in two separate examinations. increased levels of Firmicutes, Proteobacteria, Verrucomicrobia and Euryarchaeota, and decreased levels of Bacteroidetes Samples Collection compared to healthy individuals (Lourenςo et al., 2018). All biofilm samples were collected from one site per quadrant However, a link between the oral and the fecal microbiomes in in the molar-incisor region using sterile periodontal curettes and patients with unusual periodontitis