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.

Effect of ferric and ferrous iron chelators on growth of Bacteroides fragilis under anaerobic conditions.

Growth of Bacteroides fragilis under anaerobic conditions in the presence of either haemin or protoporphyrin IX was inhibited by the ferrous iron chelator bipyridyl. The ferric-iron chelator desferrioxamine inhibited growth in the presence of protoporphyrin but not haemin, suggesting that even under anaerobic conditions Fe3+ is involved in uptake of non-haem iron, which is required in the absence of haemin. However, the ferric iron chelators 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and pyridoxal isonicotinoyl hydrazone (PIH) were only weakly inhibitory. Apotransferrin, which also binds Fe3+, inhibited growth, but this was not simply due to binding of iron in the medium, as under the reducing conditions present, transferrin was unable to bind iron. This study suggests that even under anaerobic conditions, uptake of non-haem iron by B. fragilis may involve conversion of Fe2+ to Fe3+.

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.

Intracanal medicaments: evaluation of the antibacterial effects of chlorhexidine, metronidazole, and calcium hydroxide associated with three vehicles.

The use of an intracanal medicament may be helpful in eliminating remaining bacteria that survived inside root canals after complete chemomechanical preparation. This study evaluated the antibacterial activities of medicaments that act by means of contact, and not by vapor release, against obligate and facultative anaerobic bacteria commonly found in endodontic infections. The medicaments used were 0.12% chlorhexidine gel; 10% metronidazole gel; calcium hydroxide plus distilled water, calcium hydroxide plus camphorated paramonochlorophenol (CPMC); and calcium hydroxide plus glycerin. An agar diffusion test was used, and the zones of bacterial inhibition around each medicament were recorded and compared. The results revealed that calcium hydroxide/CPMC paste was effective against all bacterial strains tested. Chlorhexidine was also inhibitory to all strains. It was about as effective as calcium hydroxide/CPMC paste against most of the strains. Metronidazole also caused inhibition of growth of all obligate anaerobes tested and was more effective than calcium hydroxide/CPMC against two strains. Calcium hydroxide mixed with distilled water or glycerin failed to show zones of bacterial inhibition, probably because of limitations of the agar diffusion test.

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.

Disinfection by calcium hydroxide pastes of dentinal tubules infected with two obligate and one facultative anaerobic bacteria.

Bovine dentine cylinders were experimentally infected with Actinomyces israelii, Fusobacterium nucleatum, or Enterococcus faecalis. The latter is a facultative anaerobic bacteria and the others are obligate anaerobes commonly found in endodontic infections. The infected specimens were exposed to pastes of calcium hydroxide mixed with saline solution or camphorated paramonochlorophenol for periods of 1 h, 1 day, and 1 week. The viability of bacteria after these exposure times was evaluated by specimen incubation in culture medium to compare the effectiveness of the pastes in disinfecting dentinal tubules. The calcium hydroxide/camphorated paramonochlorophenol paste effectively killed bacteria in the tubules after a 1-h period of exposure, except for E. faecalis that required one day of exposure. In contrast, the calcium hydroxide/saline paste was ineffective against E. faecalis and F. nucleatum even after 1 week of exposure. The results showed that camphorated paramonochlorophenol increased the antibacterial effects of calcium hydroxide.

A scanning electron microscopic evaluation of in vitro dentinal tubules penetration by selected anaerobic bacteria.

In vitro root canal dentinal tubule invasion by selected anaerobic bacteria commonly isolated from endodontic infections was evaluated. Dentinal cylinders obtained from bovine incisors were inoculated with bacteria, and microbial penetration into tubules was demonstrated by scanning electron microscopy. The results indicated that all bacterial strains tested were able to penetrate into dentinal tubules, but to different extents.

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).

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).

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).

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.

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.

Evaluation of the antibacterial activities of calcium hydroxide, chlorhexidine, and camphorated paramonochlorophenol as intracanal medicament. A clinical and laboratory study.

The antibacterial activities of camphorated paramonochlorophenol, chlorhexidine, and calcium hydroxide were compared using a clinical and laboratory evaluation. In the clinical experiment, root canals that yielded positive cultures a week after complete chemomechanical preparation and camphorated paramonochlorophenol dressing were medicated with one of the three substances tested. Postmedication samples were taken from the canal 1 week later. In the laboratory experiment, the agar diffusion test was used to evaluate the inhibitory activity of the medicaments against bacteria commonly found in endodontic infections. The results of the clinical evaluation showed that all medicaments were effective in reducing or eliminating the endodontic microbiota, as demonstrated by the incidence of negative cultures. There was no statistically significant difference among the medicaments tested. In the laboratory evaluation, camphorated paramonochlorophenol showed the largest zones of bacterial inhibition against all bacterial strains tested.

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.

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.

[In vitro susceptibility to a new antimicrobial agent (imipenem) of pathogens isolated from inpatients at various centers]. / Estudo da suscetibilidade "in vitro" a um novo antimicrobiano (imipenem) de patógenos isolados de pacientes hospitalares em vários centros.

Imipenem is a beta lactam antibiotic, a highly potent new carbapenem with broad antibacterial spectrum. To test the "in vitro" efficacy of this antimicrobial agent in pathogens more frequent in several Medical Centers in Brazil, susceptibility testing with 10 mcg imipenem disks and, or corresponding MIC were carried out with 1231 recent isolates of 41 different bacterial species, obtained mainly from hospitalized patients in 5 different medical centers of the cities of S. Paulo, Rio de Janeiro and Salvador. Our preliminary results with this antibiotic, in final phases of clinical and laboratorial experimentation in this country, are very promissing with, 96.79% of susceptibility of test isolates to 10 mcg imipenem disks and corresponding MIC correlation of up to 4 micrograms/ml. (92.31%). Of the 9 bacterial species more frequently isolated totaling 1108 (82%) of the 1230 test isolates, disk susceptibility was 99% (E. coli), 93% (Pseudomonas aeruginosas), 87% (Staphylococcus aureus), 100% (Klebsiella pneumoniae), 98% (Klebsiella sp), 97% (Proteus mirabiles), 94% (Enterobacter sp), 100% (Streptococcus faecalis) with good MIC correlation (up to 8 mcg/ml) and 100% for the anaerobic species Bacteroides sp (MIC up to 4 micrograms/ml). "In vitro" efficacy to hospital pathogens with high frequency of resistance to most antibiotics as Pseudomonas aeruginosa and to anaerobes notably Bacteroides sp is emphasized.

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.

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.

Bacteriology of chronic otitis media affecting children living in Rio de Janeiro.

A bacteriologic analysis was performed on the purulent exudates obtained from the middle ears of 45 children living in Rio de Janeiro with CSOM and spontaneous perforations of the ear drum. Anaerobic cultures showed anaerobic bacteria in association with aerobic or facultatively anaerobic bacteria, or both, in 30 (66.7%) specimens. Facultative anaerobic microorganisms occurred in 42 (93.3%) of the specimens analyzed, strict aerobes in 17 (37.8%), capnophilic in 6 (13.3%), and fungi in 4 (8.9%). The rate of different species of bacteria isolated per sample was 4.4:1.87 for anaerobic and 1.84 for facultative microorganisms. Multiple drug resistances in the aerobic and facultative bacteria were found, and tetracycline, erythromycin, and beta-lactam antibiotic resistances were accentuated in the anaerobic bacteria.

Antagonism among Bacteroides fragilis group strains isolated from middle ear exudates from patients with chronic suppurative otitis media.

The production and sensitivity of a bacteriocin-like substance in Bacteroides fragilis group strains isolated from middle ear exudates in children with suppurative otitis media were studied through antagonism assayed by the well method. The results of the crossed reactions showed that 10 strains (66.6%) were bacteriocinogenic, 9 were sensitive to at least 1 bacteriocin (60%), and none showed inhibitory activity against homologous strains. Different patterns of susceptibility to bacteriocin-like substances were observed among strains isolated from the same patient as well as different strains isolated from another patient. These findings indicate that bacteriocin typing of anaerobic bacteria isolated from middle ear exudates in children with otitis media might have use in epidemiologic studies.