RESUMO
INTRODUCTION: A significant portion of the bacteria taking part of the microbiome associated with apical periodontitis still remain to be cultivated and phenotypically characterized. This molecular study evaluated the prevalence of selected as-yet-uncultivated and difficult-to-culture bacterial taxa in infected root canals and their susceptibility to chemomechanical procedures. METHODS: Root canals of single-rooted teeth with apical periodontitis were prepared using rotary nickel-titanium instruments and 2.5% sodium hypochlorite as the irrigant. DNA extracts from samples taken before (S1) and after (S2) chemomechanical preparation were surveyed for the presence of 7 as-yet-uncultivated phylotypes and 1 difficult-to-culture species using end-point polymerase chain reaction. Samples were also subjected to quantitative analysis of total bacteria and levels of the 2 most prevalent taxa. RESULTS: Bacteroidaceae sp. HOT-272 (24%) and Fretibacterium fastidiosum (20%) were the most prevalent taxa in S1. Their mean counts in S1 were 8.25 × 10(3) and 2.13 × 10(3) rRNA gene copies, corresponding to 0.18% and 0.55% of the total bacteria. Chemomechanical debridement promoted a highly statistically significant reduction in total bacterial counts (P < .001), but 64% of the canals were still positive for bacterial presence. Of the target taxa, only Bacteroidaceae sp. HOT-272 and F. fastidiosum were detected in S2 (each one in 1 sample). The reduction in counts of both taxa was also highly significant (P < .001). CONCLUSIONS: Findings confirmed that several as-yet-uncultivated and difficult-to-grow bacterial taxa can participate in the microbiome associated with apical periodontitis. Two of them were found in relatively high prevalence but rarely as a dominant species. Chemomechanical procedures were highly effective in completely eliminating these taxa or at least substantially reducing their numbers.
Assuntos
Bactérias/classificação , Periodontite Periapical/microbiologia , Preparo de Canal Radicular/métodos , Carga Bacteriana , Técnicas Bacteriológicas , Bacteroidaceae/classificação , Bacteroidaceae/isolamento & purificação , DNA Bacteriano/análise , Ligas Dentárias/química , Cavidade Pulpar/microbiologia , Necrose da Polpa Dentária/microbiologia , Desenho de Equipamento , Bactérias Anaeróbias Gram-Negativas/classificação , Bactérias Anaeróbias Gram-Negativas/isolamento & purificação , Humanos , Megasphaera/classificação , Megasphaera/isolamento & purificação , Microbiota , Níquel/química , Fenótipo , Reação em Cadeia da Polimerase/métodos , Irrigantes do Canal Radicular/uso terapêutico , Hipoclorito de Sódio/uso terapêutico , Titânio/químicaRESUMO
OBJECTIVE: The aim of this study was to detect the prevalence of selected bacterial species in intraoral sites of patients with chronic periodontitis (CP) using multiplex polymerase chain reaction (PCR). METHODOLOGY: Samples were collected from the tongue dorsum, buccal mucosa, supragingival and subgingival plaque and saliva of 30 patients with untreated CP. Multiplex PCR was used to determine prevalence rates, which were then compared using a chi-square test. Significance level was set at p<0.05. Mean and standard deviation values were used to evaluate variations in prevalence according to site. RESULTS: The prevalence of S. mutans was 70% in saliva; 60% in samples collected from the tongue dorsum; 50% in samples collected from the buccal mucosa; 56.5% in the supragingival plaque; and 53.5% in the subgingival plaque. The prevalence of E. faecalis ranged from 3.5% to 13.5% in all intraoral microenvironment. The highest prevalence of P. gingivalis was found in subgingival plaque (53.5%), and of P. intermedia in supragingival plaque (33.5%), subgingival plaque (30%) and tongue dorsum (33.5%). The prevalence of bacteria did not vary significantly among the intraoral sites. CONCLUSIONS: All studied bacteria were identified in intraoral sites. S. mutans, P. gingivalis and P. intermedia had high prevalence rates, but the prevalence of E. faecalis was low. Multiplex PCR proved to be an adequate method for epidemiological studies.
Assuntos
Bacteroidaceae/classificação , Periodontite Crônica/microbiologia , Lactobacillales/classificação , Reação em Cadeia da Polimerase/métodos , Adulto , Bochecha/microbiologia , Periodontite Crônica/classificação , Placa Dentária/microbiologia , Enterococcus faecalis/isolamento & purificação , Feminino , Gengiva/microbiologia , Hemorragia Gengival/classificação , Humanos , Masculino , Mucosa Bucal/microbiologia , Bolsa Periodontal/classificação , Porphyromonas gingivalis/isolamento & purificação , Prevotella intermedia/isolamento & purificação , Saliva/microbiologia , Streptococcus mutans/isolamento & purificação , Língua/microbiologiaRESUMO
OBJECTIVE: The aim of this study was to detect the prevalence of selected bacterial species in intraoral sites of patients with chronic periodontitis (CP) using multiplex polymerase chain reaction (PCR). METHODOLOGY: Samples were collected from the tongue dorsum, buccal mucosa, supragingival and subgingival plaque and saliva of 30 patients with untreated CP. Multiplex PCR was used to determine prevalence rates, which were then compared using a chi-square test. Significance level was set at p<0.05. Mean and standard deviation values were used to evaluate variations in prevalence according to site. RESULTS: The prevalence of S. mutans was 70 percent in saliva; 60 percent in samples collected from the tongue dorsum; 50 percent in samples collected from the buccal mucosa; 56.5 percent in the supragingival plaque; and 53.5 percent in the subgingival plaque. The prevalence of E. faecalis ranged from 3.5 percent to 13.5 percent in all intraoral microenvironment. The highest prevalence of P. gingivalis was found in subgingival plaque (53.5 percent), and of P. intermedia in supragingival plaque (33.5 percent), subgingival plaque (30 percent) and tongue dorsum (33.5 percent). The prevalence of bacteria did not vary significantly among the intraoral sites. CONCLUSIONS: All studied bacteria were identified in intraoral sites. S. mutans, P. gingivalis and P. intermedia had high prevalence rates, but the prevalence of E. faecalis was low. Multiplex PCR proved to be an adequate method for epidemiological studies.
Assuntos
Adulto , Feminino , Humanos , Masculino , Bacteroidaceae/classificação , Periodontite Crônica/microbiologia , Lactobacillales/classificação , Reação em Cadeia da Polimerase/métodos , Bochecha/microbiologia , Periodontite Crônica/classificação , Placa Dentária/microbiologia , Enterococcus faecalis/isolamento & purificação , Gengiva/microbiologia , Hemorragia Gengival/classificação , Mucosa Bucal/microbiologia , Bolsa Periodontal/classificação , Porphyromonas gingivalis/isolamento & purificação , Prevotella intermedia/isolamento & purificação , Saliva/microbiologia , Streptococcus mutans/isolamento & purificação , Língua/microbiologiaRESUMO
OBJECTIVES: This paper reviews the principles of polymerase chain reaction (PCR) methodology, its application in identification of endodontic pathogens and the perspectives regarding the knowledge to be reached with the use of this highly sensitive, specific and accurate methodology as a microbial identification test. DATA SOURCES: Studies published in the medical, dental and biological literature. STUDY SELECTION: Evaluation of published epidemiological studies examining the endodontic microbiota through PCR methodology. CONCLUSIONS: PCR technology has enabled the detection of bacterial species that are difficult or even impossible to culture as well as cultivable bacterial strains showing a phenotypically divergent or convergent behaviour. Moreover, PCR is more rapid, much more sensitive, and more accurate when compared with culture. Its use in endodontics to investigate the microbiota associated with infected root canals has expanded the knowledge on the bacteria involved in the pathogenesis of periradicular diseases. For instance, Tannerella forsythensis (formerly Bacteroides forsythus), Treponema denticola, other Treponema species, Dialister pneumosintes, and Prevotella tannerae were detected in infected root canals for the first time and in high prevalence when using PCR analysis. The diversity of endodontic microbiota has been demonstrated by studies using PCR amplification, cloning and sequencing of the PCR products. Moreover, other fastidious bacterial species, such as Porphyromonas endodontalis, Porphyromonas gingivalis and some Eubacterium spp., have been reported in endodontic infections at a higher prevalence than those reported by culture procedures.