Microbial profile comparisons of saliva, pooled and site-specific subgingival samples in periodontitis patients

RESEARCH ARTICLE Microbial profile comparisons of saliva, pooled and site-specific subgingival samples in periodontitis patients Daniel Belstrøm1*, Maria Lynn Sembler-Møller2, Maria Anastasia Grande1, Nikolai Kirkby3, Sean Liam Cotton4, Bruce J. Paster4,5, Palle Holmstrup1 1 Section for Periodontology, Microbiology, and Community Dentistry, Department of Odontology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark, 2 Section for Oral Medicine, Department of Odontology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark, 3 Department of Medical Microbiology, Copenhagen University Hospital, Copenhagen, Denmark, 4 The Forsyth Institute, Cambridge, MA, United States of America, 5 Department of Oral Medicine, Infection & Immunity, Harvard School of Dental Medicine, Boston, MA, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * Abstract Objectives OPEN ACCESS Citation: Belstrøm D, Sembler-Møller ML, Grande MA, Kirkby N, Cotton SL, Paster BJ, et al. (2017) Microbial profile comparisons of saliva, pooled and site-specific subgingival samples in periodontitis patients. PLoS ONE 12(8): e0182992. https://doi. org/10.1371/journal.pone.0182992 Editor: Özlem Yilmaz, Medical University of South Carolina, UNITED STATES Received: June 20, 2017 Accepted: July 27, 2017 The purpose of this study was to compare microbial profiles of saliva, pooled and site-specific subgingival samples in patients with periodontitis. We tested the hypotheses that saliva can be an alternative to pooled subgingival samples, when screening for presence of periopathogens. Design Site specific subgingival plaque samples (n = 54), pooled subgingival plaque samples (n = 18) and stimulated saliva samples (n = 18) were collected from 18 patients with generalized chronic periodontitis. Subgingival and salivary microbiotas were characterized by means of HOMINGS (Human Oral Microbe Identification using Next Generation Sequencing) and microbial community profiles were compared using Spearman rank correlation coefficient. Published: August 11, 2017 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Results Pronounced intraindividual differences were recorded in site-specific microbial profiles, and site-specific information was in general not reflected by pooled subgingival samples. Presence of Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, Filifactor alocis, Tannerella forsythia and Parvimona micra in site-specific subgingival samples were detected in saliva with an AUC of 0.79 (sensitivity: 0.61, specificity: 0.94), compared to an AUC of 0.76 (sensitivity: 0.56, specificity: 0.94) in pooled subgingival samples. Funding: This study was supported financially by the Danish Dental Association. Conclusions Competing interests: The authors have declared that no competing interests exist. Site-specific presence of periodontal pathogens was detected with comparable accuracy in stimulated saliva samples and pooled subgingival plaque samples. Consequently, saliva PLOS ONE | https://doi.org/10.1371/journal.pone.0182992 August 11, 2017 1 / 11 Salivary and subgingival microbiotas may be a reasonable surrogate for pooled subgingival samples when screening for presence of periopathogens. Future large-scale studies are needed to confirm findings from this study. Introduction The oral cavity is harbored by a complex and diverse microbiota, which comprises more than 700 different predominant bacterial species [1]. The oral microbiota shows a high degree of site-specific characteristics [2], which is shaped by ecological conditions and perturbations in different oral locations [3;4]. A symbiotic relationship between the resident oral microbiota and the host is essential for oral homeostasis, while alteration of the subgingival microbiota is critically involved in development of periodontitis [5]. Subgingival colonization by specific species, including the red complex bacterial species, i.e., Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola has been reported to be strongly associated with progression of periodontitis as determined by using DNA-DNA checkerboard analysis [6]. The development of contemporary molecular methods has expanded the list of potential periodontal pathogens, including Parvimonas micra and Filifactor alocis [7–10]. Analysis of subgingival plaque is considered the gold standard when studying periodontitis-associated microbial profiles. Ideally, analysis of single-site should be performed, but for practical and economic reasons, pooled subgingival plaque samples have often been employed [11]. However, it is well-known that detailed site-specific information might get lost when pooled samples are employed for microbial analysis [12]. Collection of subgingival plaque samples can be a tedious procedure, which can only be performed by trained dental professionals. On the other hand, saliva is easily obtained, even at home by the patients themselves [13;14]. Thus, saliva has been suggested an alternative to local microbial samples for analysis of periodontitis-associated biomarkers [15]. Salivary presence and relative abundance of red complex periodontal pathogens associates with periodontitis [16;17], and several studies have reported a positive correlation between presence of specific periopathogens in pooled subgingival samples and saliva samples [18–21]. However, to the best of our knowledge a direct comparison on the efficacy of using saliva samples as an alternative to pooled subgingival samples for screening of site-specific presence of periopathogens has not been performed. Thus, the purpose of this study was two-fold. 1: to characterize intra-individual variations in site-specific subgingival samples. 2: to compare the efficacy of using saliva samples and pooled subgingival samples for screening of site-specific presence of specific periopathogens. We tested the hypotheses that saliva can be an alternative to pooled subgingival samples, when screening for presence of periopathogens. Materials and methods Study design Using data from a previous study, a sample size of n = 18 was estimated based on a power calculation with a difference in mean value of Shannon index of 0.1 as primary outcome (α = 0.05, β = 0.20, E = 0.10, S(Δ) = 0.15) [22]. Therefore 18 patients (11 male and 7 females, mean age: 54 yrs.) attending Copenhagen University School of Dentistry for periodontitis treatment were included in this study (Table 1). Patients were screened for eligibility based on full-mouth recordings of PLOS ONE | https://doi.org/10.1371/journal.pone (...truncated)