Quantitative proteomic analysis in symptomatic and asymptomatic apical periodontitis.

AIM: To quantitatively and qualitatively compare the host proteomic profile in samples of symptomatic and asymptomatic apical periodontitis (AP) using nano-liquid chromatography-electron spray tandem mass spectrometry. METHODOLOGY: Samples were obtained from 18 patients with radiographically evident AP, divided into symptomatic and asymptomatic groups (nine per group) according to clinical characteristics. After sample collection, protein extraction, purification and quantification of the samples were performed, which were analysed by reverse-phase liquid chromatography coupled to mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in expression of proteins between the groups were calculated using the Monte Carlo algorithm, considering P < 0.05 for down-regulated proteins and 1 - P > 0.95 for up-regulated proteins. Proteins were identified with the embedded ion accounting algorithm in the software and a search of the Homo sapiens UniProt database. RESULTS: A total of 853 individual human proteins were identified. In the quantitative analysis, common proteins to both groups accounted for 143 proteins. Differences in expression between groups resulted in 51 up-regulated proteins (1 - P > 0.95) in the symptomatic group, including alpha-1-antitrypsin, protein S100-A8, myeloperoxidase, peroxiredoxin and lactotransferrin. This group also had 43 down-regulated proteins (P < 0.05), comprising immunoglobulin, neutrophil defensin, pyruvate kinase and alpha-enolase. The qualitative analysis considered only the exclusive proteins of each group. For the symptomatic group, 318 complete proteins and 29 fragments were identified, such as dedicator of cytokinesis protein, intersectin, prostaglandin, phospholipase DDHD2 and superoxide dismutase. For the asymptomatic group, 326 complete proteins and 37 fragments were identified, including azurocidin, C-reactive protein, collagen alpha, cathepsin, heat shock and laminin. CONCLUSIONS: Quantitative differences in the expression of common proteins in cases of symptomatic and asymptomatic AP were found, which were mostly related to host immune response in both groups. Exclusive proteins in the symptomatic group were mainly related to the host response to the presence of viruses in endodontic infections, oxidative stress and proteolytic enzymes. The results provide a basis for a better understanding of cellular and molecular pathways involved in AP, establishing specific proteomic profiles for symptomatic and asymptomatic conditions.

Biocompatibility, induction of mineralization and antimicrobial activity of experimental intracanal pastes based on glass and glass-ceramic materials.

AIM: To evaluate the biocompatibility, induction of mineralization and antimicrobial activity of experimental intracanal pastes based on two glass and glass-ceramic materials. Calcium hydroxide (Ca(OH)2 ) paste was used as the positive control. METHODOLOGY: The glass-ceramic powder [two-phased Biosilicate (BS-2P)] and F18 bioactive glass were mixed with distilled water (ratio 2 : 1), inserted in polyethylene tubes and implanted in the subcutaneous tissues of 16 rats. Empty tubes were used as negative control. After 7 and 30 days (n = 8), the rats were euthanized for haematoxylin-eosin, von Kossa, polarized light and osteopontin (OPN) immunolabeling analysis. Direct contact tests using a suspension of each paste were performed with Enterococcus faecalis planktonic cells to evaluate antimicrobial activity (24 h of contact), in a pilot study. The number of CFU mL-1 was calculated for each group. The antimicrobial analysis data were submitted to one-way anova and Tukey tests, whilst biocompatibility and immunohistochemical data were submitted to the Kruskal-Wallis and Dunn tests (P < 0.05). RESULTS: Most specimens of the control, BS-2P and Ca(OH)2 groups were associated with moderate inflammation seven days following implantation, whilst F18 was associated with moderate to severe inflammation, without differences amongst the groups (P > 0.05). At 30 days, most specimens of control, F18 and BS-2P groups had mild inflammation, whilst Ca(OH)2 had mild to moderate inflammation; however, no differences were determined amongst the groups (P > 0.05). The fibrous capsule was thick at 7 days, becoming thin at 30 days. All pastes induced von Kossa-positive structures and were birefringent to polarized light. At seven days, the BS-2P group had significantly more OPN immunolabeling compared to the control and Ca(OH)2 groups (P < 0.05). At 30 days, the F18 group had significantly more OPN immunolabeling compared to the control and Ca(OH)2 groups (P < 0.05). All pastes reduced the total number of E. faecalis; however, the reduction was only significant when comparing BS-2P and Ca(OH)2 groups to the control (P < 0.05). Only calcium hydroxide eliminated E. faecalis. CONCLUSIONS: Experimental BS-2P and F18 pastes were biocompatible, stimulated biomineralization and induced significant OPN immunolabeling compared to Ca(OH)2 . Only the BS-2P paste demonstrated antimicrobial activity comparable to Ca(OH)2 .

Antibiotic prescription for endodontic infections: a survey of Brazilian Endodontists.

AIM: To investigate antibiotic prescribing habits reported by Brazilian endodontists in specific clinical situations. METHODOLOGY: Brazilian endodontists (n = 13 853) were invited to answer an online questionnaire. The questionnaire consisted of two parts: the first part contained personal data such as age, gender, years of experience and location of endodontic practice; the second part included questions regarding their behaviour when prescribing antibiotics in dental practice. The subjects provided their registration number in the Regional Council of Dentistry (RCD) to prevent duplication of data. Data were collected and analysed by SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Chi-square and Fisher's exact tests were used to test the significance of possible associations (P < 0.05). RESULTS: From the 13 853 questionnaires, a total of 615 were answered (4.44%). The first-choice antibiotic was amoxicillin (81.5%), followed by amoxicillin + clavulanic acid (30.7%). For acute apical abscesses with intra- and extraoral diffuse swelling, fever and trismus, 90.1% reported they would prescribe antibiotics, whilst 88.1% reported they would prescribe antibiotics even without extraoral swelling, fever and trismus, and 20.5% would prescribe antibiotics in cases of chronic apical periodontitis, and sinus tract. The first-choice antibiotics varied by age of dentist (P < 0.001) and time elapsed since their endodontic graduation (P = 0.001). CONCLUSIONS: Many endodontists reported prescribing antibiotics in situations where they would not be indicated. Likewise, the general administration of antibiotics was longer in duration than necessary, reinforcing the need of continuous education regarding the use of antibiotics.

Microbiological profile and antimicrobial susceptibility pattern of infected root canals associated with periapical abscesses.

The aim of this investigation was to identify microorganisms from root canals with periapical abscesses and assess the susceptibility of specific anaerobic bacteria to selected antimicrobials and their ß-lactamase production. Sixty root canals were microbiologically investigated. The susceptibility of Anaerococcus prevotii, Fusobacterium necrophorum, F. nucleatum, Parvimonas micra, and Prevotella intermedia/nigrescens to antimicrobials was evaluated with the Etest, whereas ß-lactamase production was assessed with nitrocefin. A total of 287 different bacterial strains were recovered, including 201 strict anaerobes. The most frequently strict isolated anaerobes were A. prevotii, P. micra, and F. necrophorum. The selected bacteria were susceptible to all the tested antibiotics, except A. prevotii and Fusobacterium species to azithromycin and erythromycin, as well as A. prevotii and F. necrophorum to metronidazole. None of the microorganisms produced ß-lactamase. Gram-positive anaerobic bacteria predominated in the root canals with periapical abscesses. All microorganisms tested were susceptible to benzylpenicillin, amoxicillin, amoxicillin + clavulanate, cefaclor, and clindamycin, producing no ß-lactamase.

Bacterial examination of endodontic infections by clonal analysis in concert with denaturing high-performance liquid chromatography.

BACKGROUND/AIMS: The aim of this study was to examine the diversity of bacterial species in the infected root canals of teeth associated with endodontic abscesses by cloning and sequencing techniques in concert with denaturing high-performance liquid chromatography. METHODS: Samples collected from five infected root canals were subjected to polymerase chain reaction (PCR) with universal 16S ribosomal DNA primers. Products of these PCRs were cloned and sequenced. Denaturing high-performance liquid chromatography (DHPLC) was used as a screening method to reduce the number of clones necessary for DNA sequencing. RESULTS: All samples were positive for the presence of bacteria and a range of 7-13 different bacteria were found per root canal sample. In total, 48 different oral clones were detected among the five root canal samples. Olsenella profusa was the only species present in all samples. Porphyromonas gingivalis, Dialister pneumosintes, Dialister invisus, Lachnospiraceae oral clone, Staphylococcus aureus, Pseudoramibacter alactolyticus, Peptostreptococcus micros and Enterococcus faecalis were found in two of the five samples. The majority of the taxa were present in only one sample, for example Tannerella forsythia, Shuttleworthia satelles and Filifactor alocis. Some facultative anaerobes that are frequently isolated from endodontic infections such as E. faecalis, Streptococcus anginosus and Lactobacillus spp. were also found in this study. CONCLUSION: Clonal analysis of the microflora associated with endodontic infections revealed a wide diversity of oral species.

Incidence and antimicrobial susceptibility of Porphyromonas gingivalis isolated from mixed endodontic infections.

AIM: To investigate the prevalence of Porphyromonas gingivalis in root canals of infected teeth with periapical abscesses and to investigate the antimicrobial susceptibility of this species to some frequently prescribed antibiotics. METHODOLOGY: Samples were obtained from 70 root canals of abscessed teeth. Microbial sampling, isolation and bacterial identification were accomplished using appropriate culture methods for anaerobic species. The antimicrobial susceptibility of the 20 strains of P. gingivalis isolated was determined by using the E-test. The antimicrobial agents tested were amoxicillin, amoxicillin + clavulanate, azythromycin, benzylpenicillin, cephaclor, clindamycin, erythromycin, metronidazole and tetracycline. RESULTS: A total of 352 individual strains, belonging to 69 different species, were isolated. Eighty three percent of the strains were strict anaerobes and 47.5% of the isolated bacteria were Gram-negative. Porphyromonas gingivalis was found in 20 root canals and was most frequently found in symptomatic cases. Statistically, the presence of P. gingivalis was related to purulent exudates and pain on palpation (both P < 0.05). All P. gingivalis strains were sensitive to amoxicillin, amoxicillin + clavulanate, cephaclor, clindamycin, benzylpenicyllin, metronidazole and tetracycline. The lowest range of minimum inhibitory concentration (MIC) (0.026-0.125 microg mL(-1)) was observed against amoxicillin + clavulanate and clindamycin. The lowest MIC 90 was observed against clindamycin (0.064 microg mL(-1)). One strain was resistant to erythromycin and eight strains were resistant to azythromycin. CONCLUSION: Porphyromonas gingivalis pathogen is isolated with frequency from root canals of infected teeth with periapical abscesses. Amoxicillin, as well as amoxicillin-clavulanic acid and benzylpenicillin were effective against P. gingivalis.

Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens in endodontic lesions detected by culture and by PCR.

he aim of this study was to investigate the presence of four black-pigmented bacteria, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens, in endodontic infections by culture and polymerase chain reaction (PCR) analyses. Microbial samples were obtained from 50 teeth with untreated necrotic pulps (primary infection) and from 50 teeth with failing endodontic treatment (secondary infection). Microbiological strict anaerobic techniques were used for serial dilution, plating, incubation, and identification. For PCR detection, the samples were analyzed using species-specific primers of 16S rDNA and the downstream intergenic spacer region. Culture and PCR detected the test species in 13/100 and 50/100 of the study teeth, respectively. The organisms were cultured from 11/50 (22%) of primarily infected root canal samples and from 2/50 (4%) of secondary root canal samples. PCR detection identified the target species in 32/50 (64%) and 18/50 (36%) of primary and secondary infections, respectively. P. gingivalis was rarely isolated by culture methods (1%), but was the most frequently identified test species by PCR (38%). Similarly, P. endodontalis was not recovered by culture from any tooth studied, but was detected by PCR in 25% of the sampled teeth. PCR-based identification also showed higher detection rates of P. intermedia (33%) and P. nigrescens (22%) than culture (13%). In conclusion, P. gingivalis, P. endodontalis, P. intermedia, and P. nigrescens were identified more frequently in teeth with necrotic pulp than in teeth with failing endodontic treatment. Also, a higher frequency of black-pigmented species was detected by PCR than by culture.

Microbiological analysis of infected root canals from symptomatic and asymptomatic teeth with periapical periodontitis and the antimicrobial susceptibility of some isolated anaerobic bacteria.

The purpose of the present study was to investigate the correlation between the composition of the bacterial flora isolated from infected root canals of teeth with apical periodontitis with the presence of clinical signs and symptoms, and to test the antibiotic susceptibility of five anaerobic bacteria mostly commonly found in the root canals of symptomatic teeth against various substances using the E-test. Microbial samples were taken from 48 root canals, 29 symptomatic and 19 asymptomatic, using adequate techniques. A total of 218 cultivable isolates were recovered from 48 different microbial species and 19 different genera. Root canals from symptomatic teeth harbored more obligate anaerobes and a bigger number of bacterial species than the asymptomatic teeth. More than 70% of the bacterial isolates were strict anaerobes. Statistical analysis used a Pearson Chi-squared test or a one-sided Fisher's Exact test as appropriate. Suggested relationships were found between specific microorganisms, especially gram-negative anaerobes, and the presence of spontaneous or previous pain, tenderness to percussion, pain on palpation and swelling amoxicillin, amoxicillin + clavulanate and cephaclor were effective against all the strains tested. The lowest susceptibility rate was presented by Prevotella intermedia/nigrescens against Penicillin G. Our results suggested that specific bacteria are associated with endodontic symptoms of infected teeth with periapical periodontitis and the majority of the anaerobic bacterial species tested were susceptible to all antibiotics studied.