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Characterization of the Oral Microbiome of Medically Controlled Type-2 2 Diabetes Patients

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Characterization of the Oral Microbiome of Medically Controlled Type-2 2 Diabetes Patients bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.031070; this version posted April 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Characterization of the oral microbiome of medically controlled type-2 2 diabetes patients 3 4 Ana Almeida-Santosa,b, Daniela Martins-Mendesc,d,e,f, Magdalena Gayà-Vidala*, Lucía 5 Pérez-Pardala*, Albano Beja-Pereiraa,b,g,#,* 6 7 aCentro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO-UP), InBIO, 8 Universidade do Porto, Rua Padre Armando Quintas,4485-661 Vairão, Portugal. 9 bDGAOT, Faculty of Sciences, University of Porto, Porto, Portugal. 10 cInternal Medicine Department, Centro Hospitalar de Vila Nova de Gaia/Espinho EPE, 11 Vila Nova de Gaia, Portugal. 12 dDiabetic Foot Clinic, Endocrinology, Diabetes and Metabolism Department, Centro 13 Hospitalar de Vila Nova de Gaia/Espinho EPE, Vila Nova de Gaia, Portugal. 14 eDepartment of Biomedicine, Faculty of Medicine, University of Porto, Porto, Portugal. 15 fi3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal. 16 gSustainable Agrifood Production Research Centre (GreenUPorto), Faculty of Sciences, 17 University of Porto, Porto, Portugal 18 19 Running Head: Oral Microbiome of Type-2 Diabetes Patients 20 21 22 23 # Corresponding address: [email protected] 24 *Magdalena Gayà-Vidal, Lucía Pérez-Pardal, and Albano Beja-Pereira contributed 25 equally to this work. Author order was determined on the basis of seniority. 26 27 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.031070; this version posted April 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 28 Abstract 29 Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease that is becoming a 30 significant global health care problem. Several studies have shown that people with 31 diabetes are more susceptible to oral problems, such as periodontitis and, although the 32 causes are still inconclusive, the oral microbiota seems to be an important factor in this 33 interaction. This study aimed to characterize the oral microbiome of a sample representing 34 T2DM patients from Portugal and exploit potential associations between some 35 microorganisms and variables like teeth brushing, smoking habits, and nutrient intake. 36 By sequencing the hypervariable regions V3-V4 of the 16S rRNA gene in 50 individuals 37 belonging to a group of diabetes patients and a control group, we found a total of 233 38 taxa, from which only 65% were shared between both groups. No differences were found 39 in terms of alpha and beta diversity between groups or habits categories. Also, there were 40 no significant differences in the oral microbiome profiles of control and diabetes patients. 41 Only the class Synergistia and the genus TG5, which are related to periodontitis, were 42 statistically more frequent in the control group. This finding can be justified by the fact 43 that these diabetic patients usually have their oral health under close medical control than 44 an average healthy person, which in this study was represented by the control group. 45 46 IMPORTANCE: Diabetes has become a significant global health care issue as its 47 incidence continues to increase exponentially, with type 2 diabetes being responsible for 48 more than 90% of these cases. Portugal is one of the countries with a higher prevalence 49 of diabetes in Europe. It has been reported that diabetic people have an increased risk of 50 developing several health problems such as oral infections mostly caused by opportunistic 51 pathogens. Some studies have pointed out a relationship between diabetes and oral 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.031070; this version posted April 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 52 microbiome. Therefore, the characterization of the microbial ecosystem of the mouth in 53 reference groups is crucial to provide information to tackle oral health pathogen-borne 54 conditions. In this study, we provide the first characterization of the oral microbiome of 55 type 2 diabetes mellitus patients from Portugal, and therefore, contributing new data and 56 knowledge to elucidate the relationship between diabetes and the oral microbiome. 57 58 59 60 61 62 63 Keywords: oral microbiome, type 2 diabetes mellitus, 16S rRNA gene sequencing, 64 microbiota, Next-Generation Sequencing, Portugal 65 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.031070; this version posted April 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 66 Introduction 67 Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by chronic 68 hyperglycemia caused by defects in insulin secretion, which can contribute to the 69 development of resistance to its action (1, 2). T2DM is becoming more common, also in 70 children and adolescents, and therefore, represents a significant global health care 71 problem (3). 72 As more studies are focused on the impact of T2DM on the medium-term patients’ health, 73 a growing number of studies have been reporting a close association between diabetes 74 and susceptibility for some oral illnesses, such as periodontitis (2), derived from the 75 deregulation of the oral microbiota equilibrium that increments the establishment of 76 pathogenic organisms, which leads to the presence of oral pathogenic bacteria, causing 77 the deregulation of the oral microbiota equilibrium, and vice-versa. 78 The oral microbiota is among the most diverse and dynamic ecosystems of the human 79 body, in which it has been identified more than 700 species of bacteria (4). Bacterial phyla 80 Firmicutes, Actinobacteria, Fusobacteria, Proteobacteria, and Bacteroidetes dominate 81 the oral microbiota, accounting for 80-95 % of the total species (5). These 82 microorganisms normally harmoniously co-exist with their host due to coevolution, 83 however, behavioral factors such as poor oral hygiene and diet, debilitated immune 84 systems, genetics, medication and, certain diseases can lead to a dysbiotic oral ecosystem 85 (6). This microbial imbalance is normally associated with an overgrowth of pathogenic 86 microorganisms, which can lead to more susceptibility to oral illness (7, 8). 87 The oral microbiota plays an important role in the relationship between periodontitis and 88 diabetes (9) since it influences glycemic control (10). Certain bacteria such as 89 Porphyromonas gingivalis, one of the main strains of periodontal disease, releases a 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.031070; this version posted April 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 90 lipopolysaccharide (LPS), which triggers numerous pro-inflammatory cytokines that are 91 responsible for periodontal tissue destruction as well as increases insulin resistance (1, 92 11, 12). However, other taxa could be related to T2DM. For example, J. Long et al. (4) 93 compared the oral microbiome profiles from African Americans subjects with T2DM 94 with non-diabetic obese individuals and non-diabetics with normal-weight and found a 95 higher abundance of taxa in the phylum Actinobacteria, which were associated with a 96 lower diabetes risk; whereas Actinomycetaceae, Bifidobacteriaceae, Coriobacteriaceae, 97 Corynebacteriaceae, and Micrococcaceae families were less abundant among diabetic 98 subjects compared to normal-weight controls. Additionally, another study found a 99 decrease in the biological and phylogenetic oral microbiome diversity in diabetics in 100 comparison to non-diabetics from South Arabia, evidence that was related to an increase 101 in the pathogenic content in the diabetic’s oral microbiome (13). 102 Presently, only two studies on the relationship between type 2 Diabetes Mellitus and the 103 oral microbiome were made on European populations (14, 15). However, one of them 104 was focused on the subgingival microbiome (14), and the other one on obese T2DM (15), 105 and both of them considered a limited sample size (n < 20). Since the oral microbiome is 106 an important factor for the maintenance of human health, more studies are needed to better 107 understand the relationship between diabetes and oral microbiome. 108 In this study, we characterized the oral microbiome of a group of medically controlled 109 type 2 diabetes patients from Portugal and compared them to a control group, by using 110 amplicon sequencing of the hypervariable V3-V4 regions of the 16s rRNA from saliva 111 samples of 50 individuals. 112 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.031070; this version posted April 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 113 Results 114 Sequencing data and taxonomic assignment 115 A total of 12,754,645 raw reads were obtained with a mean of 255,092.9 reads per sample 116 (ranging from 147 to 1,085,170 reads per sample).
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