Bacteremia after supragingival scaling and dental extraction: Culture and molecular analyses.

OBJECTIVE: To study the incidence and magnitude of bacteremia after dental extraction and supragingival scaling. SUBJECTS AND METHODS: Blood samples were taken before and 5 and 30 min after dental extraction and supragingival scaling from individuals at high (n = 44) or negligible risk (n = 51) for infective endocarditis. The former received prophylactic antibiotic therapy. Samples were subjected to aerobic and anaerobic culture and quantitative real-time polymerase chain reaction to determine the incidence of bacteremia and total bacterial levels. RESULTS: Patients who did not receive prophylactic antibiotic therapy had a higher incidence of positive blood cultures (30% 5 min after extraction) than patients who received prophylactic antibiotic therapy (0% 5 min after extraction; p < .01). Molecular analysis did not reveal significant differences in the incidence or magnitude of bacteremia between the two patient groups either 5 or 30 min after each of the procedures evaluated. Extraction was associated with higher incidence of bacteremia than supragingival scaling by blood culture (p = .03) and molecular analysis (p = .05). CONCLUSIONS: Molecular methods revealed that dental extraction and supragingival scaling were associated with similar incidence of bacteremia in groups receiving or not prophylactic antibiotic therapy. However, blood culture revealed that antibiotic therapy reduced viable cultivable bacteria in the bloodstream in the extraction group.

Cytokine production in response to endodontic infection in germ-free mice.

This study evaluated the cytokine profiles (type 1 or type 2) that are triggered by and modulate endodontic periapical infections in the root canal system of germ-free mice. Microorganisms isolated from two patients with pulpal necrosis were inoculated into two groups of experimental animals: group I (Gemella morbillorum) and group II (Bifidobacterium adolescentis, Fusobacterium nucleatum and Clostridium butyricum). In vitro, G. morbillorum induced type 1 cytokine synthesis, while the modulation processed in vivo seemed to have the opposite effect, with a reduction in the basal levels of IL-12 and IFN-gamma, IL-4-independent down-modulation. In vitro, microorganisms from group II, in poly-infection, induced a reduction of type 1 cytokine levels from day 10 to day 20, which seemed to be modulated via IL-4. In vivo, however, a predominance of the immune response to one species over the others occurred.

Actinomyces species, streptococci, and Enterococcus faecalis in primary root canal infections.

The purpose of this study was to evaluate the prevalence of Actinomyces species, streptococci, and Enterococcus faecalis in primary root canal infections by using a molecular genetic method. Samples were obtained from 53 infected teeth, of which 27 cases were diagnosed as acute periradicular abscesses. DNA was extracted to evaluate the occurrence of 13 bacterial species by using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Polymerase chain reaction using an ubiquitous bacterial primer was undertaken to check the presence of bacterial DNA in clinical samples. All root canal samples contained bacteria as demonstrated by polymerase chain reaction. The checkerboard DNA-DNA hybridization assay allowed the detection of streptococci in 22.6% of the samples, Actinomyces species in 9.4%, and E. faecalis in 7.5%. The most prevalent species were members of the Streptococcus anginosus group. With regard to the asymptomatic lesions, the most prevalent species were S. intermedius (11.5% of the cases), E. faecalis (11.5%), and S. anginosus (7.7%). S. constellatus was the most prevalent species in pus samples (25.9% of the cases). The other most prevalent species in abscessed teeth were A. gerencseriae (14.8%), S. gordonii (11.1%), S. intermedius (11.1%), A. israelii (7.4%), S. anginosus (7.4%), and S. sanguis (7.4%). S. constellatus was the only species positively associated with acute periradicular abscess (p < 0.01).

Prevalence of mutans streptococci in 93 members from six Brazilian families.

Several studies report that mutans streptococci (MS) are closely associated with caries in humans and that there is a correlation between the number of carious lesions and the levels of MS in the saliva of children and adults. The presence of MS in the saliva of 93 members of six Brazilian families with at least 3 generations was investigated. Samples of whole unstimulated saliva were collected and diluted. Aliquots of 50 microliters of each suspension were dropped onto SB20 agar and incubated in a candle jar at 37 degrees C for 72 h. Colonies resembling MS were counted, collected, seeded in thioglycoilate medium and subjected to biochemical typing. Mutans streptococci were isolated from 80 subjects (86.0%) and the counts ranged from 3.0 x 10(2) (log 2.477) to 1.6 x 10(8) (log 8.204) CFU/ml of saliva. All of the 73 adults were colonized by MS, but the bacteria were detected in only 7 (35.0%) of the 20 children evaluated. Streptococcus mutans occurred in 78 subjects (97.5%), and 51 (63.7%) were monocolonized. S. sobrinus occurred in 29 individuals (36.3%) and 2 (2.5%) were monocolonized. Twenty-seven (33.8%) subjects were multicolonized with S. mutans and S. sobrinus. This study showed a high prevalence (86.0%) of mutans streptococci in the saliva of members of the studied families, which suggests the risk of intrafamilial transmission.

Implantation of bacteria from human pulpal necrosis and translocation from root canals in gnotobiotic mice.

The aim of this study was to determine whether microorganisms recovered from infected human root canals were able to survive and translocate to a local lymph node when experimentally inoculated into the root canal system of germ-free mice. The microorganisms isolated from two patients with pulpal necrosis were inoculated in two groups of experimental animals; group I (Gemella morbillorum) and group II (Bifidobacterium adolescentis, Fusobacterium nucleatum, and Clostridium butyricum). G. morbillorum showed the highest frequency of colonization and translocation to the draining lymph node. In group II only F. nucleatum and C. butyricum colonized and translocated when inoculated in tri-association. When the bacteria from group II were inoculated in monoinfection all three species colonized the root canal of germ-free mice and translocated to the draining lymph node, but with different frequencies. We conclude that selective mechanisms occur in which some bacterial species are fit to survive, multiply, and translocate in the germ-free mouse model.

Antifungal effects of endodontic medicaments.

The eradication of root canal infection is paramount in endodontic treatment. Because fungi are involved in some types of root canal infections, the purpose of this study was to investigate the antifungal effects of several medicaments against the following selected fungal species: Candida albicans, Candida glabrata, Candida guilliermondii, Candida parapsilosis, and Saccharomyces cerevisiae. The agar diffusion test was the method used. Calcium sulphate or zinc oxide in glycerin showed no inhibitory effects towards any selected fungal species. The pastes of calcium sulphate or calcium hydroxide in camphorated paramonochlorophenol (CPMC)/glycerin showed the most pronounced antifungal effects. Calcium hydroxide in glycerin or chlorhexidine, and chlorhexidine in a detergent also showed antifungal activity, which was, however, much lower than the pastes of calcium sulphate or calcium hydroxide in CPMC/glycerin. Strategies using medicaments that have antifungal effects may assist in the successful management of persistent or secondary endodontic infections caused by fungi.

Effects of solutions used in infants' oral hygiene on biofilms and oral microorganisms.

The purpose of this work was to evaluate the effects of oral hygiene solutions used for infants on biofilms formed in vitro from infants' saliva and dental plaque: ATCC reference strains A. viscosus; C. albicans; L. casei; S. mitis; S. mutans; S. oralis; S. sanguis; S. sobrinus and clinically isolated microorganisms (saliva) C. albicans, S. mitis, S. mutans, S. oralis, S. sanguis and S. sobrinus. After exposure of the oral biofilms to H2O2 diluted 1/4 to 1/16; and NaF 0.02 percent, concentrated and diluted 1/2, for 1 and 3 minutes, the viable count of microorganisms, compared to the controls was significantly reduced (p < 0.05). They also showed a significant antimicrobial effect for all the microorganisms evaluated, when compared to the control (p < 0.05). Exposure to sodium bicarbonate solution and a camomile solution, for 1 and 3 minutes, was not significantly lethal to oral biofilms nor to any microorganism evaluated, regardless of whether they were concentrated or diluted. We do not recommend the use of H2O2 but suggest using the camomile solution and NaF 0.02 percent in a rational manner for cleaning the infant's mouth.

Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA.

The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.

Checkerboard DNA-DNA hybridization analysis of endodontic infections.

OBJECTIVE: The purpose of this investigation was to examine the microbiota of infected root canals by using a molecular genetic method. STUDY DESIGN: The presence and levels of 42 bacterial species were determined in 28 root canal samples by using whole genomic DNA probes and checkerboard DNA-DNA hybridization. To confirm the presence of bacterial DNA in clinical samples, a polymerase chain reaction with an ubiquitous bacterial primer was undertaken. RESULTS: The results of the checkerboard DNA-DNA hybridization analysis showed that 22 of the 42 DNA probes tested were reactive with 1 or more samples. The number of bacterial species in the root canal samples ranged from 1 to 17 (mean, 4.7). Seventeen of the 28 root canal samples were positive for at least 1 DNA probe. The most prevalent species found were as follows: Bacteroides forsythus (39. 3% of the cases); Haemophilus aphrophilus (25%); Corynebacterium matruchotii (21.4%); Porphyromonas gingivalis (17.9%); and Treponema denticola (17.9%). CONCLUSIONS: The microbiologic data of the present investigation indicated that molecular genetic methods can provide significant additional knowledge regarding the endodontic microbiota by detecting bacterial species that are difficult or impossible to culture. In addition, our findings support the current concept that endodontic infections are mixed infections of polymicrobial etiology.

Prevalence of Brachyspira species isolated from diarrhoeic pigs in Brazil.

Pathogenic intestinal spirochaetes of pigs include Brachyspira (formerly Serpulina) hyodysenteriae, the cause of swine dysentery, and Brachyspira pilosicoli, the cause of porcine colonic spirochetosis (PCS). The purpose of this study was to assess the relative importance of Brachyspira species in diarrhoeal disease of growing pigs on farms in southern Brazil. The intensity and pattern of haemolysis, the production of indole and the hydrolysis of hippurate by reference and field porcine intestinal spirochaetes were compared with 16S-ribosomal RNA (mRNA)- and 23S-rRNA-based polymerase chain reaction assays for the identification of B hyodysenteriae and B pilosicoli. Between July and October 1998, 206 rectal swabs were taken from pigs on 17 farms with a history of diarrhoea developing within 30 days after they had been moved from nursery to growing facilities. Of 49 beta-haemolytic spirochaetes that were cultured, 29 (59.2 per cent) were grown in pure culture for phenotypic and genotypic characterisation, leaving 20 untyped. Of the 29 typed isolates, eight isolates obtained from six farms were identified as B hyodysenteriae, and 15 isolates obtained from seven other farms were identified as B pilosicoli; the remaining six isolates were identified as weakly beta-haemolytic commensal spirochaetes. There was complete agreement between the results of the phenotypic and genotypic analyses.

Effects of three different infant dentifrices on biofilms and oral microorganisms.

The purpose of this work was to evaluate the effects of infant dentifrices: A--with lactoperoxidase, glucose oxidase and lactoferrin; B--with 1100 ppm of NaF and sodium lauryl sulfate; C--with extract of calendula. The dentifrices were test on biofilms formed in vitro from saliva and dental plaque of infants, using reference strains A. viscosus (ATCC 43146); C. albicans (ATCC 51501); L. casei (ATCC 4646); S. mitis (ATCC 49456); S. mutans (ATCC 25175); S. oralis (ATCC 35037); S. sanguis (ATCC 10586); S. sobrinus (ATCC 27609) and isolated clinically microorganisms C. albicans, S. mitis, S. mutans, S. oralis, S. sanguis, S. sobrinus and Lactobacillus sp. Twenty infants were chosen, who were beginning treatment at the Infants Clinic of the Pediatric Dentistry Department, Federal University of Rio de Janeiro. A pool of unstimulated saliva and a pool of dental plaque were collected from which biofilms were produced. Supernatants from each dentifrice were prepared and concentrated and diluted solutions of the dentifrices and a control sterile diluent were tested against the biofilms produced, for 1 and 3 minutes, and against the microorganisms. The results were statistically analyzed by the ANOVA and Tukey Test. After the exposure of the biofilms produced both from saliva and from dental plaque, to the dentifrice B concentrated and 1/2, for 1 and 3 minutes, the viable microorganisms count (CFU/ml), compared to the controls, was significantly reduced (p < 0.05). However, exposure to the dentifrices A and C concentrated and dentifrice B 1/4 and 1/8, for 1 and 3 minutes, was not significantly lethal to the biofilms. The dentifrices A and C, either concentrated or diluted (1/2 to 1/128) and the dentifrice B in the dilutions 1/16 to 1/128 did not have an antimicrobial effect on any microorganism evaluated. For all the microorganisms evaluated, the dentifrice B concentrated and in the 1/2 dilution showed a significant antimicrobial effect, when compared with the control (p < 0.05).

Mechanical reduction of the bacterial population in the root canal by three instrumentation techniques.

The in vitro reduction of the bacterial population in the root canal by the mechanical action of instrumentation and irrigation was evaluated. Root canals inoculated with a Enterococcus faecalis suspension were instrumented using hand Nitiflex files, Greater Taper (GT) files, and Profile 0.06 taper Series 29 rotary instruments. Irrigation was performed using sterile saline solution. Root canals were sampled before and after instrumentation. In the group of the Nitiflex files, samples were also taken after each file size. After serial dilution, samples were plated onto Mitis-Salivarius agar, and the colony forming units grown were counted. All techniques and instruments tested were able to reduce significantly the number of bacterial cells in the root canal. Instrumentation to a Nitiflex #30 was significantly more effective than GT files. There were no significant differences when comparing the effects of the Profile instrument #5 with either the GT files or the Nitiflex #30. Enlargement to a Nitiflex #40 was significantly more effective in eliminating bacteria when compared with the other techniques and instruments tested (p < 0.05). The results of this study showed that the instrumentation and irrigation can mechanically remove more than 90% of bacterial cells from the root canal.

Coronal leakage of two root canal sealers containing calcium hydroxide after exposure to human saliva.

This in vitro study evaluated the coronal leakage of human saliva into root canals filled by lateral condensation of gutta-percha using two root canal sealers containing calcium hydroxide. Obturated canals with gutta-percha and either Sealapex of Sealer 26 were mounted in an apparatus and then exposed to saliva. The number of days required for total recontamination of the root canal was recorded. Evaluation was conducted for 60 days. Thirty-five percent of the specimens of the Sealer 26 group showed entire recontamination at 60 days. Eighty percent of the root canals obturated with Sealapex showed complete recontamination at 60 days. Sealer 26 presented significantly less leakage than Sealapex (p < 0.01).

Effectiveness of four chemical solutions in eliminating Bacillus subtilis spores on gutta-percha cones.

Gutta-percha cones should be free of pathogenic micro-organisms before being used for root canal filling. This study was carried out to evaluate the effectiveness of four chemical agents in eliminating Bacillus subtilis spores from gutta-percha cones. The solutions tested were 5.25% sodium hypochlorite, 2% glutaraldehyde, 2% chlorhexidine digluconate, and 70% ethyl alcohol. The gutta-percha cones coated with spores were placed into contact with the chemical agents for 1, 3, 5 and 10 min. The results showed that 5.25% sodium hypochlorite was effective in destroying the spores after 1 min of contact. Glutaraldehyde, chlorhexidine and ethyl alcohol did not decontaminate the gutta-percha cones even after 10 min of contact.

Antibacterial effects of endodontic irrigants on black-pigmented gram-negative anaerobes and facultative bacteria.

The antibacterial effect of endodontic irrigants was evaluated against four black-pigmented Gram-negative anaerobes and four facultative anaerobic bacteria by means of the agar diffusion test. All solutions used were inhibitory against all bacterial strains tested. A 4% NaOCl solution provided the largest average zone of bacterial inhibition of this study that was significantly superior when compared with the other solutions, except 2.5% NaOCl (p < 0.05). Based on the averages of the diameters of the zones of bacterial growth inhibition, the antibacterial effects of the solutions could be ranked from strongest to weakest as follows: 4% NaOCl; 2.5% NaOCl; 2% chlorhexidine; 0.2% chlorhexidine, EDTA, and citric acid; and 0.5% NaOCl.

Recontamination of coronally unsealed root canals medicated with camphorated paramonochlorophenol or calcium hydroxide pastes after saliva challenge.

This in vitro study evaluated the ability of some medications to prevent recontamination of coronally unsealed root canals by bacteria from saliva. The medications tested were camphorated paramonochlorophenol (CPMC) applied in cotton pellets in the pulp chamber; calcium hydroxide/saline solution paste filling the root canal; and calcium hydroxide/CPMC/glycerin paste also filling the root canal. Medicated canals were exposed to saliva, and the number of days required for total recontamination to occur was recorded. Canals medicated with CPMC in cotton pellets were thoroughly recontaminated within an average of 6.9 days. Canals filled with calcium hydroxide/saline solution and calcium hydroxide/CPMC/glycerin showed entire recontamination within an average of 14.7 and 16.5 days, respectively. Calcium hydroxide pastes were significantly more effective than CPMC (p < 0.05).

Pathogenicity of facultative and obligate anaerobic bacteria in monoculture and combined with either Prevotella intermedia or Prevotella nigrescens.

The pathogenicity of obligate and facultative anaerobic bacteria commonly found in endodontic infections was tested using a mouse model. The capacity of inducing abscesses was evaluated seven days after subcutaneous injection of the bacteria in pure culture and in combinations with either Prevotella intermedia or Prevotella nigrescens. Nine of the fifteen bacterial strains tested were pathogenic in pure culture. No statistically significant differences were detected between these strains in pure culture and in mixtures with either P. intermedia or P. nigrescens. Synergism between the bacterial strains was only apparent when associating Porphyromonas endodontalis with P. intermedia or P. nigrescens. Histopathological examination of tissue sections from induced abscesses revealed an acute inflammatory reaction, dominated by polymorphonuclear leukocytes. Sections from the control group using sterile medium showed no evidence of inflammatory reaction.

Influence of different vehicles on the antibacterial effects of calcium hydroxide.

This study evaluated the influence of three different vehicles on the antibacterial activity of calcium hydroxide against four bacterial species commonly found in endodontic infections. For this purpose, a broth dilution test using 24-well cell culture plates was performed. Results showed that all pastes were effective in killing the bacteria tested, but at different times. The calcium hydroxide/camphorated paramonochlorophenol/glycerin paste was the most effective against the four bacterial strains tested.

Evaluation of the effectiveness of sodium hypochlorite used with three irrigation methods in the elimination of Enterococcus faecalis from the root canal, in vitro.

The effectiveness of 4.0% sodium hypochlorite (NaOCl) used with three irrigation methods in the elimination of Enterococcus faecalis from the root canal was tested in vitro. Root canals contaminated with E. faecalis were treated as follows: (i) irrigation with 2 mL of NaOCl solution and agitation with hand files; (ii) irrigation with 2 mL of NaOCl solution and ultrasonic agitation; (iii) irrigation with NaOCl alternated with hydrogen peroxide. Contaminated canals irrigated with sterile saline solution served as the control. Paper points used to sample bacteria from the root canals were transferred to tubes containing 5 mL of brain heart infusion (BHI) broth. Tubes were incubated and the appearance of broth turbidity was indicative of bacteria remaining in the root canal. There were no statistically significant differences between the experimental groups. However, NaOCl applied by the three methods tested, was significantly more effective than the saline solution (control group) in disinfecting the root canal.