Comparative analysis of endodontic pathogens using checkerboard hybridization in relation to culture.

BACKGROUND/AIM: The purpose of this study was to detect bacterial species and to quantify the total number of bacteria from samples of infected root canals before (S1) and after chemo-mechanical preparation using 2% chlorhexidine (CHX) gel as auxiliary chemical substance (S2) and after 7 days of intracanal dressing (S3) to compare microbial changes. METHOD: Twenty-four teeth were selected for this study. Chemo-mechanical preparation was performed using 2% CHX gel, then three different intracanal medicaments [M1: Ca(OH)(2) paste; M2: 2% CHX gel; and M3: Ca(OH)(2) paste plus 2% CHX gel] were used for 7 days. Checkerboard DNA-DNA hybridization was performed to detect 40 bacterial species. Aerobic and anaerobic culture techniques were used to determine the bacterial community by counting the colony-forming units (CFU). RESULTS: The species most frequently identified by checkerboard in S1 were: Fusobacterium nucleatum ssp. polymorphum, Treponema socranskii ssp. socranskii, Parvimonas micra and Enterococcus faecalis. In S2 and S3 a total of eight different species were identified; and only one of them was gram-positive (E. faecalis). Microorganisms were not identified after use of M2 for 7 days. The quantification obtained on agar plates ranged from 4 x 10(5) to 2.6 x 10(6) CFU/ml in S1, mean CFU was reduced by 99.96% in S2, and there was no statistical difference between the CFU in S2 and S3. CONCLUSION: The antibacterial effect of the mechanical preparation supplemented by the use of an antibacterial auxiliary substance greatly reduced the microorganisms in the main root canal.

Effect of root canal procedures on endotoxins and endodontic pathogens.

BACKGROUND/AIMS: The purpose of this study was to determine the amount of endotoxin (lipopolysaccharide) and cultivable bacteria in human necrotic root canals before (S1) and after chemo-mechanical preparation using chlorhexidine (CHX) gel as auxiliary chemical substance (S2), and after 7 days of intracanal dressing (S3) in order to evaluate the anti-endotoxin and antimicrobial effects of endodontic procedures. METHOD: Twenty-four teeth were selected for the present study. Chemo-mechanical preparation was performed using 2% CHX gel and three different intracanal medicaments [CaOH2 paste; 2% CHX gel; and CaOH2 + 2% CHX gel]. A quantitative chromogenic Limulus amoebocyte lysate assay was used to measure the amount of endotoxin. Aerobic and anaerobic techniques were used to isolate and identify bacteria, and to determine the bacterial reduction by counting colony-forming units (CFU). RESULTS: Endotoxins and bacteria were present in 100% of the initial samples, with endotoxin concentration ranging from 62.93 to 214.56 UE/ml and CFU ranging from 4 x 10(5) to 2.6 x 10(6). After chemo-mechanical preparation a mean endotoxin reduction of 44.4% was found. Eight (33.3%) root canals were still positive by culture analysis with a mean reduction of bacteria (CFU) of 99.96%. After 7 days of intracanal dressing, endotoxin concentration decreased by only 1.4% compared with S2, and residual bacteria were recovered by culture analysis in 13 cases (54.1%). No significant difference was found among different intracanal medicaments. CONCLUSION: Relatively high values of endotoxin were still present in the root canal after chemo-mechanical preparation although the majority of bacteria were eliminated. No improvement was achieved by 7 days of intracanal dressing.

Quantification and characterization of Synergistes in endodontic infections.

INTRODUCTION: Bacterial species belonging to the poorly characterized division Synergistes have recently been reported in endodontic infections, and therefore may be part of the etiology of periradicular diseases. The objective of this study was to characterize and quantify the predominant Synergistes phylotypes in infected root canals. METHODS: We analyzed 32 necrotic teeth, each from a different patient, with radiographic evidence of apical periodontitis and with primary endodontic infections. RESULTS: Using real-time quantitative polymerase chain reaction based on Synergistes-specific primers, seven of the 32 cases were found to be positive. Comparative sequence analysis showed that each of the seven samples was infected by one numerically dominant phylotype. Diversity among phylotypes was such that they could be grouped into three major evolutionary branches within the Synergistes division. The size of the total Synergistes population ranged from 4.5 x 10(4) to 1.5 x 10(6) 16S rRNA gene copies, and the median proportion accounted for 0.79% of the total bacterial community. For comparison, we also quantified such recognized endodontic pathogens as Prevotella intermedia, Porphyromonas gingivalis, and Treponema. The first two species were found in five and nine cases, respectively, with a median proportion below 0.01%, while Treponema was found in 18 cases with a median proportion of 1.48%. CONCLUSION: Thus, the prevalence and quantity of Synergistes was clearly within the range of the other analyzed pathogens, suggesting their clinical relevance in endodontic infections. Furthermore, the diversity of Synergistes found at the diseased sites designates infected root canals as an important human ecosystem providing several unique micro-niches for this novel group of bacteria.

Microarrays complement culture methods for identification of bacteria in endodontic infections.

The aim of this study was to investigate the microbial composition of necrotic root canals using culture methods and microarray technology. Twenty uniradicular teeth with radiographic evidence of periapical bone loss and with no previous endodontic treatment were selected for this study. For molecular diagnosis a DNA chip with 20 different species-specific, 16S-rDNA-directed catcher probes was used. The microorganisms most frequently detected by the DNA chip were: Micromonas micros, Fusobacterium nucleatum ssp., Tannerella forsythia, Treponema denticola, Veillonella parvula, Eubacterium nodatum, Porphyromonas gingivalis, Actinomyces odontolyticus, and Streptococcus constellatus. As expected, additional important bacterial taxa were found by culture analysis, but microorganisms such as T. forsythia and T. denticola could not be identified. In conclusion, microarrays may provide significant additional information regarding the endodontic microbiota by detecting bacterial species that are otherwise difficult or impossible to culture.