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Background: Molecular taxonomic assignments in oral microbial communities have been made using probe-matching approaches, but never compared to those obtained by more readily accepted tree-based approaches. Objective: To compare community composition profiles obtained from a probe-matching approach (HOMINGS) to those from a closed-ended tree-based approach (QIIME using the eHOMD database). Design: HOMINGS and QIIME were used for parallel analysis of ten mock community samples, and of 119 supragingival plaque samples from ecologically unique sites (sound tooth surfaces in healthy subjects, sound tooth surfaces in patients with primary Sjögren’s Syndrome, and carious lesions in Sjögren’s Syndrome patients). Linear discriminant analysis Effective Size (LEfSe) was used to identify discriminating taxa among the natural plaque samples. Results: Community composition profiles of all samples were congruent between the two analysis aproaches. Alpha and beta diversity of the natural plaque communities were likewise similar. Communities from pSS patients and those from individuals with normal salivary flow differed in alpha and beta diversity. Both classification approaches yielded differences in composition predicted for samples from these subject cohorts, and discriminating taxa were similar between approaches. Conclusions: A direct comparison demonstrates that HOMINGS is largely equivalent to the tree-based approach as implemented here.

Esta pesquisa comparou o perfil e diversidade da composição bacteriana do biofilme supragengival coletado em diferentes condições bucais de sujeitos carie ativos, carie inativos e livres de caries. Dezesseis indivíduos (13-76; med=23.5 anos), selecionados na Faculdade de Odontologia da Universidade Federal do Rio Grande do Sul, foram alocados em três grupos: cárie ativos (CA) (n=7); cárie inativos (CI) (n=3); e, sem experiência de cárie (CF) (n=6). Vinte e uma amostras de placa supragengival (“pool”) foi obtida dos sujeitos CA a partir de três condições de saúde dental: LNCA: lesão não cavitada ativa (n=7); LNCI: lesão não cavitada inativa (n=7); S: superfície saudável. O biofilme supragengival foi coletado depois de 12 horas sem escovação dental, armazenado em solução estabilizadora de RNA, concentrado e congelado a -80oC. O RNA total foi extraído utilizando Lisozima e o kit UltraClean® Microbial RNA Isolation (MO-BIO). As bibliotecas genômicas foram preparadas com o protocolo True Seq® Sample Preparation Guide, Low Sample (LS) Protocol Illumina (Illumina, Inc., San Diego, CA) e sequenciadas no sequenciador Illumina HiSeq 3000, resultando em bilhões de 2x150 pares de base. As sequencias foram enviadas para o servidor MG-RAST (Metagenomics Analysis Server) para as analises de bioinformática. Sequência de alta qualidade (3,542,190) foram agrupadas em unidades taxonômicas operacionais (OTUs; 97% identidade; SILVA SSU), representando 915 gêneros independentes pertencentes a 29 filos, quatro considerados com elevada abudancia (Actinobacteria, Firmicutes, Bacteroidetes and Fusobacteria). Todos os sujeitos compartilharam 4493 OTUs (123 gêneros), indicando a presença de um microbioma central da placa dental. A analise de alfa diversidade revelou uma menor diversidade microbiana para os sítios saudáveis dos sujeitos carie ativos versus os dos sem carie (CA-S vs CF-S), e para os sujeitos carie ativos versus os sujeitos sem carie (CA vs CF). Os generos dominantes incluiram Actinomyces, Corynebacterium, Capnocytophaga, Leptotrichia, Veillonella, Prevotella, Streptococcus, Eubacterium e Neisseria. Veillonella e Leptotrichia foram correlacionados positivamente com caries, e Prevotella com saúde. Corynebacterium e Capnocytophaga estavam mais próximos do que Actinomyces, mas aguparam-se juntos formando um importante “cluster” com elevada abundância tanto em saude como em doença. A análise da Ordenação por Escalonamento Métrico Multidimensional mostra que os sítios nos sujeitos carie-ativos (CA-LNCA, CA-LNCI e CA-S) estão mais próximos uns dos outros do que os sujeitos CI-LNCI ou dos sujeitos CF. Nós observamos um alto nível de diversidade da fração ativa da comunidade bacteriana (abordagem baseada em RNA) que está relacionada com o elevado número de organismos detectados no biofilme supragengival e não devido a espécies mortas ou inativas, destacando que a cárie dentária é uma doença polimicrobiana, onde consórcios microbianos multiespécie são metabolicamente ativos nas lesões. As comunidades bacterianas supragengivais apresentam uma semelhança intrapessoal, mas a diversidade interpessoal e a composição bacteriana diferente revelam que o status de atividade de cárie do indivíduo é mais importante do que os sitios. This research compared the bacterial composition profile and diversity from supragingival biofilm collected in different oral conditions from active- and inactive-caries subjects, and from caries-free subjects. Sixteen individuals (13-76; med=23.5 years-old), selected at the Faculty of Dentistry, Federal University of Rio Grande do Sul, were allocated in three groups: cariesactive (CA) (n=7); caries inactive (CI) (n=3); and, caries-free (CF) (n=6). Twenty-one supragingival plaque samples (pools) were obtained from CA subjects from three dental health conditions: ANCL: active non-cavitated lesions (n=7); INCL: inactive non-cavitated lesions (n=7); S: sound dental surfaces (n=7). The supragingival biofilm was collected after 12 hours without toothbrushing, stored in RNA stabilization solution, pelleted and frozen in -80oC. The total RNA extraction was made using Lysozyme and UltraClean® Microbial RNA Isolation kit (MO-BIO). The genomic libraries were prepared using True Seq® Sample Preparation Guide, Low Sample (LS) Protocol Illumina (Illumina, Inc., San Diego, CA), and sequenced by Illumina HiSeq 3000, resulting in billions of 2x150 base pairs. The sequences were uploaded into sever MG-RAST (Metagenomics Analysis Server) for bioinformatics analysis. High-quality sequences (3,542,190) were clustered into genus operational taxonomic units (OTUs; 97% identity; SILVA SSU), representing 915 independent genera belonging to 29 phyla, four considered higher abundant (Actinobacteria, Firmicutes, Bacteroidetes and Fusobacteria). All subjects shared 4493 OTUs (123 genera), indicating the presence of a core plaque microbiome. The α diversity analysis showed less bacterial diversity in sound sites from caries active compared to caries-free (CA-S vs CF-S), and for caries active subjects versus caries-free subjects (CA vs CF). The dominant genera included Actinomyces, Corynebacterium, Capnocytophaga, Leptotrichia, Veillonella, Prevotella, Streptococcus, Eubacterium, and Neisseria. Veillonella and Leptotrichia were positively related with caries, and Prevotella with health. Corynebacterium and Capnocytophaga were closer than Actinomyces but toghether formed an important cluster with high abundance in health and disease. The Metric Multidimensional Scaling Ordination analysis shows that sites from active subjects (CAANCL, CA-INCL and CA-S) are closer to each other than CI-INCL subjects or CF-S subjects. We found a high level of diversity in the active fraction of the bacterial community (RNAbased approach) that is related with the high number of organisms detected in supragingival biofilm and not due to dead or inactive species, highlighting that dental caries is a polymicrobial disease, where multispecies microbial consortia are metabolically active in the lesions. Supragingival bacterial communities presents an intrapersonal similarity, but interpersonal diversity and different bacterial composition reveals that the subject’s caries activity status matters more than sites.

Background Urease is an enzyme produced by plaque bacteria hydrolysing urea from saliva and gingival exudate into ammonia in order to regulate the pH in the dental biofilm. The aim of this study was to assess the urease activity among oral bacterial species by using the rapid urease test (RUT) in a micro-plate format and to examine whether this test could be used for measuring the urease activity in site-specific supragingival dental plaque samples ex vivo. Methods The RUT test is based on 2% urea in peptone broth solution and with phenol red at pH 6.0. Oral bacterial species were tested for their urease activity using 100 μl of RUT test solution in the well of a micro-plate to which a 1 μl amount of cells collected after growth on blood agar plates or in broth, were added. The color change was determined after 15, 30 min, and 1 and 2 h. The reaction was graded in a 4-graded scale (none, weak, medium, strong). Ex vivo evaluation of dental plaque urease activity was tested in supragingival 1 μl plaque samples collected from 4 interproximal sites of front teeth and molars in 18 adult volunteers. The color reaction was read after 1 h in room temperature and scored as in the in vitro test. Results The strongest activity was registered for Staphylococcus epidermidis, Helicobacter pylori, Campylobacter ureolyticus and some strains of Haemophilus parainfluenzae, while known ureolytic species such as Streptococcus salivarius and Actinomyces naeslundii showed a weaker, variable and strain-dependent activity. Temperature had minor influence on the RUT reaction. The interproximal supragingival dental plaque between the lower central incisors (site 31/41) showed significantly higher scores compared to between the upper central incisors (site 11/21), between the upper left first molar and second premolar (site 26/25) and between the lower right second premolar and molar (site 45/46). Conclusion The rapid urease test (RUT) in a micro-plate format can be used as a simple and rapid method to test urease activity in bacterial strains in vitro and as a chair-side method for testing urease activity in site-specific supragingival plaque samples ex vivo.

Aim: The purpose of the present study was to investigate in periodontitis patients the relationship between the number of bacteria in the saliva and the amount of de novo plaque formation before and after treatment.Methods: At baseline, before any treatment was provided, patients rinsed with 10 ml sterile saline. After professional tooth cleaning the patients were instructed to abolish all tooth cleaning procedures for the subsequent 24 h. After this period, the rinsing procedure was repeated and the amount of de novo plaque was assessed. Three months after the initial periodontal therapy was completed the experiment was repeated. The microbiological evaluation of the rinsing samples was carried out by means of phase contrast microscopy and anaerobic culturing.Results: After treatment the amount of de novo plaque was less compared to before treatment, 0.40 and 0.65 respectively. Both before and after treatment more de novo plaque was present at sites with inflammation than at healthy sites. In order to evaluate the contribution of the numbers of salivary bacteria to the amount of de novo plaque formation an analysis was carried out for healthy sites. This analysis included only healthy sites as determined before treatment and the same sites after treatment. The results showed a significant reduction in the de novo plaque formation after treatment (0.49 before and 0.22 after treatment). Phase contrast microscopic evaluation showed that the number of bacteria in the rinsing samples after treatment was less than before treatment. After treatment also a reduction was found in the prevalence of Prevotella intermedia, Tannerella forsythensis and Peptostreptococcus micros in the rinsing samples.Conclusion: The present study confirms the observation in the literature that the periodontal condition is of major importance in the rate of de novo plaque formation. In addition, the results suggest that the number of bacteria in the saliva may play a role.