Biomedical subjects

J F Siqueira

Publications and source records attributed to J F Siqueira.

At least 19 recordsLinked to original sources

PubMed2006

Identification of Enterococcus faecalis in root-filled teeth with or without periradicular lesions by culture-dependent and-independent approaches.

Enterococcus faecalis is the most commonly found species in root-filled teeth evincing recalcitrant periradicular lesions and as a consequence, a role in causation of endodontic treatment failure has been suggested. The purpose of this study was to evaluate the prevalence of this bacterial species in root-filled teeth with or without periradicular lesions. Identification of E. faecalis was carried out by polymerase chain reaction (PCR) or conventional culture procedures. Overall, E. faecalis was detected by species-specific 16S rRNA gene-based PCR in 40/50 teeth (80%), while culture revealed the occurrence of this species in 8/50 teeth (16%). PCR was significantly more effective than culture in detecting this bacterial species (p < 0.001). Of 27 root-filled teeth with no periradicular lesions, E. faecalis was found in 22 cases (81.5%) by PCR and in five cases (18.5%) by culture. Of 23 root-filled teeth with periradicular lesions, E. faecalis was identified in 18 cases (78%) by PCR and in three cases (13%) by culture. Regardless of the identification technique used, no significant difference was observed when comparing the occurrence of E. faecalis in root-filled teeth with and without periradicular lesions (p > 0.05). Although these findings apparently put into question the status of E. faecalis as the main species causing endodontic treatment failure, other related factors still need to be clarified before this assumption turns into certainty.

Adult↗

PubMed2006

Culture-independent detection of Eikenella corrodens and Veillonella parvula in primary endodontic infections.

Eikenella corrodens and Veillonella parvula are normal cultivable inhabitants of the oral cavity but their presence in endodontic infections has not been as common as it could be anticipated. This might have been because of shortcomings of culture techniques when it comes to bacterial isolation or identification. The present study intended to survey samples from primary endodontic infections for the presence of E. corrodens and V. parvula using a culture-independent 16S rRNA gene-based nested PCR protocol. Genomic DNA was isolated directly from samples taken from different forms of periradicular lesions, and the presence of E. corrodens and V. parvula was determined by nested PCR. Specificity for each primer pair was confirmed by sequence analysis of PCR products from positive clinical samples. V. parvula and E. corrodens were, respectively, detected in 33% and 14% of the root canals associated with chronic apical periodontitis. Both V. parvula and E. corrodens were found in 10% of the cases diagnosed as acute apical periodontitis. V. parvula and E. corrodens were detected in 21% and 26% of the samples from acute apical abscesses, respectively. In general, species-specific nPCR allowed the detection of V. parvula in 24% and E. corrodens in 18% of the samples taken from primary endodontic infections. Findings confirmed that V. parvula and E. corrodens can take part in the microbiota of primary endodontic infections, but in prevalence values somewhat higher when compared to most of the previous culture studies that had reported recovery of these species.

Acute Disease↗

PubMed2006

Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections.

The purpose of the present study was to use terminal restriction fragment length polymorphism analysis and the 16S rRNA gene clone library to investigate the diversity of the microbiota associated with asymptomatic and symptomatic endodontic infections and to compare the bacterial community structure in these two clinical conditions. Samples were taken from asymptomatic endodontic infections associated with chronic periradicular lesions and from symptomatic infections clinically diagnosed as acute abscesses. 16S rRNA genes from DNA isolated from clinical samples were used to construct clone libraries or were subjected to terminal restriction fragment length polymorphism analysis. Sequence analysis of 186 clones revealed 42 taxa; 23 (55%) were uncultivated phylotypes, of which seven were unique to endodontic infections. Clone sequencing and terminal restriction fragment length polymorphism analysis revealed that the most commonly detected taxa were Fusobacterium nucleatum (including terminal restriction fragment types 1 and 2), Peptostreptococcus micros/Peptostreptococcus sp. oral clone AJ062/BS044/FG014, Prevotella species, Dialister species, Mogibacterium species, Lachnospiraceae oral clone 55A-34, Filifactor alocis, Megasphaera sp. oral clone CS025/BS073, and Veillonella sp. oral clone BP1-85/Veillonella dispar/V. parvula. Bacteroides-like sp. oral clone X083/Bacteroidales oral clone MCE7_20 and Dialister sp. oral clone BS016/MCE7_134 were detected only in asymptomatic teeth. On the other hand, F. nucleatum terminal restriction fragment type 2, Prevotella intermedia, Dialister pneumosintes, and some phylotypes were exclusively detected in symptomatic samples. Bacterial profiles of symptomatic endodontic infections generated by terminal restriction fragment length polymorphism analysis were clearly different from those of asymptomatic infections. Overall, the average number of terminal restriction fragments in symptomatic samples was significantly larger than in asymptomatic samples. Molecular analysis of the microbiota associated with symptomatic or asymptomatic endodontic infections indicates that the endodontic bacterial diversity is greater than previously described by culture methods and that the structure of the microbiota differ significantly between asymptomatic and symptomatic infections.

Adolescent↗

PubMed2005

Searching for Archaea in infections of endodontic origin.

Archaea is a highly diverse group of prokaryotes, whose members have been traditionally recognized as extremophiles. Recently, some of these microorganisms have also been found to thrive in nonextreme environments, including the human body. Methanogenic archaea have been detected in samples from subgingival plaque associated with periodontal disease and a pathogenetic role is suspected. The purpose of this study was to survey samples taken from different types of endodontic infections for the presence of archaea. Samples were taken from untreated and treated root canals associated with asymptomatic chronic periradicular lesions as well as from cases diagnosed as acute periradicular abscesses. Overall, 96 samples were obtained. DNA from samples was extracted by using two different protocols and used as template for polymerase chain reaction amplification using oligonucleotide universal primers for the domains Archaea or Bacteria. Samples were also checked for the presence of spirochetes by making use of a group-specific primer. While bacteria were present in all samples, no case yielded archaeal DNA. Spirochetes occurred in a high number of cases. Our findings suggested that members of the Archaea domain are not members of the microbiota present in different types of endodontic infections and thereby may not be implicated in the etiology of apical periodontitis.

Archaea↗

PubMed2005

Exploiting molecular methods to explore endodontic infections: Part 1--current molecular technologies for microbiological diagnosis.

Endodontic infections have been traditionally studied by culture-dependent methods. However, as with other areas of clinical microbiology, culture-based investigations are plagued by significant problems, including the probable involvement of viable but uncultivable micro-organisms with disease causation and inaccurate microbial identification. Innumerous molecular technologies have been used for microbiological diagnosis in clinical microbiology, but only recently some of these techniques have been applied in endodontic microbiology research. This paper intended to review the main molecular methods that have been used or have the potential to be used in the study of endodontic infections. Moreover, advantages and limitations of current molecular techniques when compared to conventional methods for microbial identification are also discussed.

Bacterial Typing Techniques↗

PubMed2005

Exploiting molecular methods to explore endodontic infections: Part 2--Redefining the endodontic microbiota.

The second part of this review discusses the application of molecular methods in endodontic microbiology research for a comprehensive characterization of the microbiota associated with different types of endodontic infections. Despite their recent introduction in endodontic research, molecular methods have already given a significant contribution to the understanding of endodontic infections and the future holds the perspective of a still better refinement of the knowledge about these infections. Molecular methods have revealed a higher complexity of the endodontic microbiota than previously reported by cultivation approaches. In addition to detecting some cultivable species in increased prevalence, molecular methods have also expanded the list of putative endodontic pathogens by inclusion of some fastidious bacterial species or even uncultivated bacteria that have never been previously found in endodontic infections.

DNA Probes↗

PubMed2005

Occurrence of two newly named oral treponemes - Treponema parvum and Treponema putidum - in primary endodontic infections.

BACKGROUND/AIMS: Recent evidence from molecular genetic studies has revealed that oral Treponema species are involved in infections of endodontic origin. This study assessed the occurrence of two newly named oral treponemes - Treponema parvum and Treponema putidum - in primary endodontic infections using a culture-independent identification technique. METHODS: Genomic DNA was isolated directly from clinical samples, and a 16S rRNA gene-based nested polymerase chain reaction (PCR) assay was used to determine the presence of T. parvum and T. putidum. Species-specific primer pairs were developed by aligning closely related 16S rRNA gene sequences. The specificity for each primer pair was validated by running PCR against a panel of oral bacteria and by sequence analysis of PCR products from positive clinical samples. RESULTS: T. parvum was detected in 52% of the root canals associated with chronic apical periodontitis, in 20% of the cases diagnosed as acute apical periodontitis, and in no abscessed case. In general, T. parvum was detected in 26% of the samples from primary endodontic infections. T. putidum was found in only one case of acute apical periodontitis (2% of the total number of cases investigated). CONCLUSIONS: The devised nested PCR protocol was able to identify both T. parvum and T. putidum directly in clinical samples and demonstrated that these two treponemes can take part in endodontic infections.

DNA, Bacterial↗

PubMed2005

Species-directed 16S rRNA gene nested PCR detection of Olsenella species in association with endodontic diseases.

AIMS: To investigate the occurrence of Olsenella uli and Olsenella profusa in samples taken from infections of endodontic origin using a devised 16S RNA gene-based nested PCR protocol. METHODS AND RESULTS: DNA extracted from clinical samples was initially amplified using universal 16S rRNA gene primers followed by a second round of amplification using the first PCR products to detect a specific fragment of either O. uli or O. profusa 16S rRNA gene. Olsenella uli was detected in 27% of samples from teeth with asymptomatic periradicular lesions and in 20% of pus samples taken from symptomatic (abscess) lesions. Olsenella profusa was detected in 7% of asymptomatic teeth and in 13% of symptomatic teeth. A new Olsenella phylotype was also detected. CONCLUSIONS: Olsenella species can take part in the microbiota associated with infections of the root canal of human teeth and a role in the pathogenesis of periradicular diseases is suspected. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study demonstrates that molecular genetic methods keep expanding the list of candidate endodontic pathogens to include Olsenella species.

Actinobacteria↗

PubMed2005

Uncultivated phylotypes and newly named species associated with primary and persistent endodontic infections.

Endodontic infections have been traditionally studied by culture methods, but recent reports showing that over 50% of the oral microbiota is still uncultivable (B. J. Paster et al., J. Bacteriol. 183:3770-3783, 2001) raise the possibility that many endodontic pathogens remain unknown. This study intended to investigate the prevalence of several uncultivated oral phylotypes, as well as newly named species in primary or persistent endodontic infections associated with chronic periradicular diseases. Samples were taken from the root canals of 21 untreated teeth and 22 root-filled teeth, all of them with radiographic evidence of periradicular bone destruction. Genomic DNA was isolated directly from each sample, and 16S rRNA gene-based nested or heminested PCR assays were used to determine the presence of 13 species or phylotypes of bacteria. Species-specific primers had already been validated in the literature or were developed by aligning closely related 16S rRNA gene sequences. Species specificity for each primer pair was confirmed by running PCRs against a panel of several oral bacteria and by sequencing DNA from representative positive samples. All species or phylotypes were detected in at least one case of primary infections. The most prevalent species or phylotypes found in primary infections were Dialister invisus (81%), Synergistes oral clone BA121 (33%), and Olsenella uli (33%). Of the target bacteria, only these three species were detected in persistent infections. Detection of uncultivated phylotypes and newly named species in infected root canals suggests that there are previously unrecognized bacteria that may play a role in the pathogenesis of periradicular diseases.

Actinobacteria↗

PubMed2005

Novel bacterial phylotypes in endodontic infections.

Although molecular studies have revealed potential oral pathogens among the phyla Spirochaetes and Deferribacteres, their occurrence in endodontic infections has not been consistently investigated. In this study, we devised a nested PCR-DGGE approach to survey samples from infected root canals for the presence of members of these two phyla, and to examine their diversity. The primers used also amplified DNA from Atopobium species. Eight of 10 cases showed bands representative of the target bacterial groups. DGGE profiles revealed a mean number of 6.5 intense and faint bands. No single band occurred in all profiles. Sequences from intense bands excised from the gel showed similarities to species/phylotypes of all target groups--Flexistipes species (Deferribacteres phylum), uncharacterized spirochetes, and Atopobium species. Analysis of these data indicates that uncultivated Spirochaetes and Deferribacteres phylotypes are frequent members of the endodontic microbiota and may be potential pathogens involved with the etiology of periradicular diseases.

Actinobacteria↗

PubMed2004

Geographical differences in bacteria detected in endodontic infections using polymerase chain reaction.

The polymerase chain reaction (PCR) is an innovative nucleic acid-based assay that has the highest sensitivity of any microbiological technique for the detection of bacteria. The purpose of this study was to use PCR to detect the presence of specific species of bacteria in samples collected from two geographical locations. Microbial samples from abscesses of endodontic origin were collected from patients in Portland, Oregon, and Rio de Janeiro, Brazil. PCRs with species-specific oligonucleotide primers for the 16S ribosomal RNA gene were used for detection of the bacteria after DNA extraction from each clinical sample. Statistical analysis revealed that there was a significant difference in detection of the bacteria between the two geographical locations for Prevotella intermedia, Prevotella nigrescens, Prevotella tannerae, Fusobacterium nucleatum, and Porphyromonas gingivalis, but not for Porphyromonas endodontalis, Fusobacterium necrophorum, and Enterococcus faecalis. These results suggest that differences in bacteria detected or cultured in studies can be associated with geographical location.

Adult↗

PubMed2004

Treponema species associated with abscesses of endodontic origin.

Spirochetes have been frequently observed in abscesses of endodontic origin, but they have rarely been identified. This study sought to investigate the prevalence of eight oral treponemes in acute periradicular abscesses using a species-specific nested polymerase chain reaction assay. Purulent exudate was collected by aspiration from 19 cases diagnosed as acute periradicular abscesses and DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first polymerase chain reaction products to detect a specific fragment of the 16S rDNA of each Treponema species. The species-specific nPCR assay used in this study allowed the detection of Treponema denticola in 79%(15 of 19), Treponema socranskii in 26%(5 of 19), Treponema pectinovorum in 21% (4 of 19), Treponema amylovorum in 16% (3 of 19), and Treponema medium in 5% (1 of 19) of the cases. Spirochetal DNA was found in 89% of the cases (17 of 19). The number of Treponema species per case ranged from 1 to 3 (mean, 1.5). Treponema vincentii, Treponema lecithinolyticum and Treponema maltophilum were not detected in any pus sample. The present data lend support to the assertion that Treponema species, particularly T. denticola and T. socranskii, may be involved in the pathogenesis of acute periradicular abscesses.

Adolescent↗

PubMed2004

Investigation of bacterial communities associated with asymptomatic and symptomatic endodontic infections by denaturing gradient gel electrophoresis fingerprinting approach.

The purpose of the present study was to investigate the bacterial communities associated with asymptomatic and symptomatic endodontic infections and to compare denaturing gradient gel electrophoresis (DGGE) fingerprinting patterns of these two clinical conditions. The root canal microbiota of teeth associated with asymptomatic or symptomatic periradicular lesions was profiled by the PCR-DGGE method and then compared, taking into consideration the banding patterns. Bacteria were present in all examined cases. Comparative analysis of the two clinical conditions revealed bands that were common to both symptomatic and asymptomatic cases, but most DGGE bands appeared to be unique for each clinical condition. No single band occurred in all profiles. The mean number of bands detected in the 16S rDNA community profiles were 12.1 +/- 9.4 (range 2-29) for symptomatic samples and 6.7 +/- 2.7 (range 2-11) for asymptomatic ones. Clustering methods and principal component analysis of DGGE banding pattern placed the samples according to the presence or absence of symptoms. Four intense bands that were excised from the gel and sequenced showed similarities to species of the Campylobacter genus (found in 5/12 asymptomatic and in 3/11 symptomatic cases), Fusobacterium genus (4/11 symptomatic cases), Acinetobacter genus (5/12 asymptomatic cases), and Enterobacteriaceae family (11/12 asymptomatic and 2/11 symptomatic cases). The profiles of the predominant bacterial community appeared to be unique for each individual. These findings confirm that endodontic infections are polymicrobial and showed that there are significant differences in the predominant bacterial composition between asymptomatic and symptomatic cases.

Acinetobacter↗

PubMed2003

Detection of Filifactor alocis in endodontic infections associated with different forms of periradicular diseases.

Recent molecular studies have expanded the list of suspected endodontic pathogens. The aim of this study was to evaluate the occurrence of Filifactor alocis in primary endodontic infections associated with different forms of periradicular diseases. Identification by nested polymerase chain reaction was performed in root canal samples from teeth associated with either asymptomatic periradicular lesions or acute apical periodontitis. Samples were also taken by aspiration of purulent exudate associated with acute apical abscesses. DNA extracted from the samples was initially amplified using universal 16S rDNA primers followed by a second round of amplification using the first PCR products to detect a specific fragment of F. alocis 16S rDNA. F. alocis was detected in 12/21 (57.1%) root canal samples from teeth showing asymptomatic periradicular lesions and in 3/10 (30%) samples taken from root canals associated with acute apical periodontitis. This species occurred in 8/19 (42.1%) pus aspirates obtained from abscessed teeth. In general, F. alocis was detected in 23/50 (46%) samples taken from endodontic infections. These findings suggest that F. alocis is involved in the etiology of different forms of periradicular diseases and has the potential to be an endodontic pathogen.

Adolescent↗

PubMed2003

Oral treponemes in primary root canal infections as detected by nested PCR.

AIM: To investigate the prevalences of four Treponema species in primary root canal infections using a nested PCR assay. METHODOLOGY: Samples were obtained from 32 infected root canals. Twenty-two cases showed chronic asymptomatic periradicular lesions and 10 symptomatic cases were diagnosed as acute apical periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect a specific fragment of the 16S rDNA of each Treponema denticola, T. socranskii, T. vincentii and T. pectinovorum. RESULTS: Bacteria were present in all cases sampled. T. denticola was detected in 77.3% of the asymptomatic cases, T. socranskii in 40.9%, T. vincentii in 18.2% and T. pectinovorum in 13.6%. In the cases diagnosed as acute apical periodontitis, T. denticola was detected in 80%, whilst T. socranskii and T. vincentii were detected in 40% and 10% of cases, respectively. No symptomatic case yielded T. pectinovorum. In general, nested PCR detected T. denticola in 78.1% of the cases, T. socranskii in 40.6%, T. vincentii in 15.6% and T. pectinovorum in 9.4%. At least one of the four Treponema species was found in 84.4% of the cases examined. CONCLUSIONS: The species T. denticola was detected in a large number of the cases examined: the prevalence of T. socranskii was also relatively high. The species T. vincentii and T. pectinovorum were also found, but in a smaller number of cases. Based on these data, the recognized pathogenicity of these microorganisms and their involvement with other oral diseases, they should be included in the restricted set of putative endodontic pathogens.

Adolescent↗

PubMed2003

Campylobacter gracilis and Campylobacter rectus in primary endodontic infections.

AIM: A species-specific nested polymerase chain reaction (PCR) assay was used to investigate the occurrence of Campylobacter gracilis and C. rectus in primary root canal infections. METHODOLOGY: Samples were collected from 57 single-rooted teeth with carious lesions, necrotic pulps and radiographic evidence of periradicular disease. Twenty-eight cases were diagnosed as chronic asymptomatic periradicular lesions, 12 cases as acute apical periodontitis, and 17 cases as acute periradicular abscess. DNA was extracted from the samples and initially amplified using universal 16S rDNA primers. A second round of amplification using the first PCR products was performed to specifically detect C. gracilis or C. rectus in the samples. RESULTS: Campylobacter gracilis and C. rectus were, respectively, detected in 21.4 (6 of 28) and 30% (6 of 20) of the root canals associated with chronic asymptomatic periradicular lesions. Campylobacter gracilis was found in 16.7% (2 of 12) of the cases diagnosed as acute apical periodontitis, whilst C. rectus was found in 33.3% (two of six cases). In the abscessed cases, C. gracilis and C. rectus were detected in 23.5 (4 of 17) and 11.8% (2 of 17) of the cases, respectively. No association of these species with clinical symptoms was observed (P > 0.01) In general, species-specific nPCR allowed the detection of C. gracilis in 21.1% (12 of 57) and C. rectus in 23.3% (10 of 43)of the samples taken from primary endodontic infections. CONCLUSIONS: Findings confirmed the assertion that both C. gracilis and C. rectus participate in infections of endodontic origin and suggest a pathogenetic role with regard to periradicular diseases.

Adolescent↗

PubMed2002

Clonality of Fusobacterium nucleatum in root canal infections.

Fusobacterium nucleatum is a gram-negative non-spore-forming, non-motile, obligate anaerobic rod that is normally isolated from the oral cavity. Several studies have reported a significant heterogeneity within the F. nucleatum species. The aim of the present study was to analyze the clonal diversity of F. nucleatum strains isolated from intracanal infections and to evaluate the presence of Enterobacterial Repetitive Intergenic Consensus (ERIC)-like sequences in the genome of F. nucleatum. Samples were collected from 13 single-root teeth from adult patients, all having carious lesions, necrotic pulps and radiographic evidence of periradicular bone loss. F. nucleatum was isolated from two different patients (subjects 5 and 7) by culture. Amplification of 19 colonies from subject 5 and 15 colonies from subject 7 using ERIC primers resulted in four clonal types, two per subject. An intense amplicon of approximately 700 bp was generated by ERIC-PCR for all F. nucleatum isolates and F. nucleatum ssp. polymorphum ATCC 10953. The amplification reaction using primer 1254 confirmed the results obtained with the ERIC primer. Our findings indicate that DNA fingerprints provided by ERIC- and Arbitrarily Primed (AP)-PCR may constitute a powerful tool for investigating F. nucleatum clonal diversity.

Adult↗

PubMed2002

Direct amplification of rRNA gene sequences for identification of selected oral pathogens in root canal infections.

AIM: The purpose of this study was to assess the prevalence of selected oral pathogens in root canal infections and their relationship with symptoms using a highly sensitive technique, the polymerase chain reaction. METHODOLOGY: Samples were obtained from 91 infected teeth associated with periradicular lesions, including cases of acute periradicular abscesses. DNA was extracted from the samples and analysed for the presence of target microbial species using a PCR-based identification assay. RESULTS: All samples were positive for the presence of bacteria. Streptococcus anginosus group was detected in 16.7%, Fusobacterium nucleatum in 14.3%, and Bacteroides forsythus in 7.1% of the abscess samples. No pus sample yielded Actinomyces israelii, Actinobacillus actinomycetemcomitans or fungal species. In general, B. forsythus was found in 20% of the cases (16 of 80), S. anginosus in 12% (6 of 50), F. nucleatum in 10% (6 of 60) and A. israelii in 5% (two of 40). A. actinomycetemcomitans was not detected in any case. Fungi were present in only one of 50 cases (2%). There was no correlation between the species and symptoms. CONCLUSIONS: Direct molecular approaches appear to be a valuable tool for the rapid and reliable diagnosis of infectious diseases, as well as for research purposes. There was no correlation between target microbial species and symptoms.

Actinomyces↗