scholarly journals Identification and Localization of Extraradicular Biofilm-Forming Bacteria Associated with Refractory Endodontic Pathogens

2005 ◽  
Vol 71 (12) ◽  
pp. 8738-8743 ◽  
Author(s):  
Nobuo Noguchi ◽  
Yuichiro Noiri ◽  
Masahiro Narimatsu ◽  
Shigeyuki Ebisu
Keyword(s):  
Biofilm Formation ◽  
Bacterial Species ◽  
Fusobacterium Nucleatum ◽  
Immunohistochemical Localization ◽  
Rrna Gene ◽  
Root Tips ◽  
Bacterial Dna ◽  
Gene Assay ◽  
Periapical Periodontitis ◽  
Root Surfaces

ABSTRACT Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.

scholarly journals Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization

2009 ◽  
Vol 58 (10) ◽  
pp. 1359-1366 ◽  
Author(s):  
Ali Al-Ahmad ◽  
Marie Follo ◽  
Ann-Carina Selzer ◽  
Elmar Hellwig ◽  
Matthias Hannig ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Bacterial Colonization ◽  
Bacterial Species ◽  
Fusobacterium Nucleatum ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Actinomyces Naeslundii ◽  
Oral Biofilms

Oral biofilms are one of the greatest challenges in dental research. The present study aimed to investigate initial bacterial colonization of enamel surfaces in situ using fluorescence in situ hybridization (FISH) over a 12 h period. For this purpose, bovine enamel slabs were fixed on buccal sites of individual splints worn by six subjects for 2, 6 and 12 h to allow biofilm formation. Specimens were processed for FISH and evaluated with confocal laser-scanning microscopy, using probes for eubacteria, Streptococcus species, Veillonella species, Fusobacterium nucleatum and Actinomyces naeslundii. The number of adherent bacteria increased with time and all tested bacterial species were detected in the biofilm formed in situ. The general percentage composition of the eubacteria did not change over the investigated period, but the number of streptococci, the most frequently detected species, increased significantly with time (2 h: 17.7±13.8 %; 6 h: 20.0±16.6 %; 12 h: 24.7±16.1 %). However, ≤1 % of the surface was covered with bacteria after 12 h of biofilm formation in situ. In conclusion, FISH is an appropriate method for quantifying initial biofilm formation in situ, and the proportion of streptococci increases during the first 12 h of bacterial adherence.

scholarly journals Sensitive Identification of Bacterial DNA in Clinical Specimens by Broad-Range 16S rRNA Gene Enrichment

2020 ◽  
Vol 58 (12) ◽  
Author(s):  
Sara Rassoulian Barrett ◽  
Noah G. Hoffman ◽  
Christopher Rosenthal ◽  
Andrew Bryan ◽  
Desiree A. Marshall ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nucleic Acids ◽  
Bacterial Species ◽  
False Negative ◽  
Case Series ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Bacterial Dna ◽  
Clinical Specimens

ABSTRACT The broad-range detection and identification of bacterial DNA from clinical specimens are a foundational approach in the practice of molecular microbiology. However, there are circumstances under which conventional testing may yield false-negative or otherwise uninterpretable results, including the presence of multiple bacterial templates or degraded nucleic acids. Here, we describe an alternative, next-generation sequencing approach for the broad range detection of bacterial DNA using broad-range 16S rRNA gene hybrid capture (“16S Capture”). The method is able to deconvolute multiple bacterial species present in a specimen, is compatible with highly fragmented templates, and can be readily implemented when the overwhelming majority of nucleic acids in a specimen derive from the human host. We find that this approach is sensitive to detecting as few as 17 Staphylococcus aureus genomes from a background of 100 ng of human DNA, providing 19- to 189-fold greater sensitivity for identifying bacterial sequences than standard shotgun metagenomic sequencing, and is able to successfully recover organisms from across the eubacterial tree of life. Application of 16S Capture to a proof-of-principle case series demonstrated its ability to identify bacterial species that were consistent with histological evidence of infection, even when diagnosis could not be established using conventional broad range bacterial detection assays. 16S Capture provides a novel means for the efficient and sensitive detection of bacteria embedded in human tissues and for specimens containing highly fragmented template DNA.

Presence of Bacteria in Spontaneous Achilles Tendon Ruptures

2017 ◽  
Vol 45 (9) ◽  
pp. 2061-2067 ◽  
Author(s):  
Christer G. Rolf ◽  
Sai-Chuen Fu ◽  
Chelsea Hopkins ◽  
Ju Luan ◽  
Margaret Ip ◽  
...  
Keyword(s):  
Achilles Tendon ◽  
Acl Reconstruction ◽  
Bacterial Infections ◽  
Bacterial Species ◽  
Hamstring Tendon ◽  
Universal Primers ◽  
Rrna Gene ◽  
Low Grade ◽  
Bacterial Dna ◽  
Normal Tendon

Background: The structural pathology of Achilles tendon (AT) ruptures resembles tendinopathy, but the causes remain unknown. Recently, a number of diseases were found to be attributed to bacterial infections, resulting in low-grade inflammation and progressive matrix disturbance. The authors speculate that spontaneous AT ruptures may also be influenced by the presence of bacteria. Hypothesis: Bacteria are present in ruptured ATs but not in healthy tendons. Study Design: Cross-sectional study; Level of evidence, 3. Methods: Patients with spontaneous AT ruptures and patients undergoing anterior cruciate ligament (ACL) reconstruction were recruited for this study. During AT surgical repair, excised tendinopathic tissue was collected, and healthy tendon samples were obtained as controls from hamstring tendon grafts used in ACL reconstruction. Half of every sample was reserved for DNA extraction and the other half for histology. Polymerase chain reaction (PCR) was conducted using 16S rRNA gene universal primers, and the PCR products were sequenced for the identification of bacterial species. A histological examination was performed to compare tendinopathic changes in the case and control samples. Results: Five of 20 AT rupture samples were positive for the presence of bacterial DNA, while none of the 23 hamstring tendon samples were positive. Sterile operating and experimental conditions and tests on samples, controlling for harvesting and processing procedures, ruled out the chance of postoperative bacterial contamination. The species identified predominantly belonged to the Staphylococcus genus. AT rupture samples exhibited histopathological features characteristic of tendinopathy, and most healthy hamstring tendon samples displayed normal tendon features. There were no apparent differences in histopathology between the bacterial DNA–positive and bacterial DNA–negative AT rupture samples. Conclusion: The authors have demonstrated the presence of bacterial DNA in ruptured AT samples. It may suggest the potential involvement of bacteria in spontaneous AT ruptures.

scholarly journals Patterns of Oral Microbiota in Patients with Apical Periodontitis

2021 ◽  
Vol 10 (12) ◽  
pp. 2707
Author(s):  
Izabela Korona-Glowniak ◽  
Dominika Piatek ◽  
Emilia Fornal ◽  
Anna Lukowiak ◽  
Yuriy Gerasymchuk ◽  
...  
Keyword(s):  
Root Canal ◽  
Secondary Infection ◽  
Bacterial Species ◽  
Keystone Species ◽  
Pcr Amplification ◽  
Permanent Teeth ◽  
Rrna Gene ◽  
Oral Microbiota ◽  
Root Canals ◽  
Periapical Periodontitis

In this study, microbial diversity of the root canal microbiota related to different endodontic infections was investigated. In total, 45 patients with endo–perio lesions (8 patients), chronic periapical periodontitis (29 patients) and pulp necrosis (8 patients) were recruited. In 19 (42.2%) patients there was secondary infection of root canals. Microbial specimens were collected from root canals of non-vital teeth with or without changes in periapical area visible in X-ray. Then, oral microbiota were detected and identified using the culture method and real-time PCR amplification primers and hydrolysis-probe detection with the 16S rRNA gene as the target. Overall, 1434 species/genes from 41 different genera of 90 various microbial species were retrieved. Of the major reported phyla, Firmicutes (62.9%), Actinobacteria (14.0%), Bacteroidetes (12.1%), Proteobacteria (9.1%) and Fusobacteria (4.2%) were detected. Of the bacterial species, 54.6% were strict anaerobes. Corynebacterium matruchotii (p = 0.039) was present significantly more frequently in chronic periapical periodontitis. Moreover, the higher values of Decayed, Missing and Filled Permanent Teeth index were positively correlated with relative abundance of Actinomyces spp. (p = 0.042), Lactobacillus spp. (p = 0.006), Propionibacterium spp. (p = 0.024) and Rothia spp. (p = 0.002). The multivariate analyses revealed differences in total root canal samples, where components that affected grouping of root samples into four main categories were identified. Anaerobic Gram-negative bacteria predominated in root canals of teeth with pulp necrosis and periapical lesions. Facultative anaerobic Gram-positive bacteria predominated in canals with secondary infections. All detected members of mixed population groups that might serve as keystone species contributed to the entire community in its clinical relevance.

scholarly journals Structural changes in the oral microbiome of the adolescent patients with moderate or severe dental fluorosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Qian Wang ◽  
Xuelan Chen ◽  
Huan Hu ◽  
Xiaoyuan Wei ◽  
Xiaofan Wang ◽  
...  
Keyword(s):  
Community Structure ◽  
Microbial Community ◽  
Microbial Community Structure ◽  
Structural Changes ◽  
Dental Fluorosis ◽  
Bacterial Species ◽  
Fusobacterium Nucleatum ◽  
Oral Microbiome ◽  
Rrna Gene ◽  
Oral Diseases

AbstractDental fluorosis is a very prevalent endemic disease. Although oral microbiome has been reported to correlate with different oral diseases, there appears to be an absence of research recognizing any relationship between the severity of dental fluorosis and the oral microbiome. To this end, we investigated the changes in oral microbial community structure and identified bacterial species associated with moderate and severe dental fluorosis. Salivary samples of 42 individuals, assigned into Healthy (N = 9), Mild (N = 14) and Moderate/Severe (M&S, N = 19), were investigated using the V4 region of 16S rRNA gene. The oral microbial community structure based on Bray Curtis and Weighted Unifrac were significantly changed in the M&S group compared with both of Healthy and Mild. As the predominant phyla, Firmicutes and Bacteroidetes showed variation in the relative abundance among groups. The Firmicutes/Bacteroidetes (F/B) ratio was significantly higher in the M&S group. LEfSe analysis was used to identify differentially represented taxa at the species level. Several genera such as Streptococcus mitis, Gemella parahaemolysans, Lactococcus lactis, and Fusobacterium nucleatum, were significantly more abundant in patients with moderate/severe dental fluorosis, while Prevotella melaninogenica and Schaalia odontolytica were enriched in the Healthy group. In conclusion, our study indicates oral microbiome shift in patients with moderate/severe dental fluorosis. We identified several differentially represented bacterial species enriched in moderate and severe fluorosis. Findings from this study suggests that the roles of these bacteria in oral health and related diseases warrant more consideration in patients with moderate and severe fluorosis.

scholarly journals Effects of Sodium Tripolyphosphate on Oral Commensal and Pathogenic Bacteria

2019 ◽  
Vol 68 (2) ◽  
pp. 263-268 ◽  
Author(s):  
JI-HOI MOON ◽  
MI HEE NOH ◽  
EUN-YOUNG JANG ◽  
SEOK BIN YANG ◽  
SANG WOOK KANG ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Sodium Tripolyphosphate ◽  
Bacterial Species ◽  
Fusobacterium Nucleatum ◽  
Food Additive ◽  
Prevotella Intermedia ◽  
Commensal Bacterium ◽  
Total Growth ◽  
Opportunistic Pathogenic

Polyphosphate (polyP) is a food additive with antimicrobial activity. Here we evaluated the effects of sodium tripolyphosphate (polyP3, Na5P3O10) on four major oral bacterial species, in both single- and mixed-culture. PolyP3 inhibited three opportunistic pathogenic species: Fusobacterium nucleatum, Prevotella intermedia, and Porphyromonas gingivalis. On the contrary, a commensal bacterium Streptococcus gordonii was relatively less susceptible to polyP3 than the pathogens. When all bacterial species were co-cultured, polyP3 (≥ 0.09%) significantly reduced their total growth and biofilm formation, among which the three pathogenic bacteria were selectively inhibited. Collectively, polyP3 may be an alternative antibacterial agent to control oral pathogenic bacteria.

CPP-ACP Promotes SnF2 Efficacy in a Polymicrobial Caries Model

2018 ◽  
Vol 98 (2) ◽  
pp. 218-224 ◽  
Author(s):  
S.G. Dashper ◽  
P. Shen ◽  
C.P.C. Sim ◽  
S.W. Liu ◽  
C.A. Butler ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Fusobacterium Nucleatum ◽  
Rrna Gene ◽  
Enamel Demineralization ◽  
Actinomyces Naeslundii ◽  
Phosphate Ions ◽  
Human Enamel ◽  
Caries Model ◽  
Casein Phosphopeptide ◽  
Bioavailable Calcium

Dental caries is associated with plaque dysbiosis, leading to an increase in the proportions of acidogenic and aciduric bacteria at the expense of alkali-generating commensal species. Stannous fluoride (SnF2) slows the progression of caries by remineralization of early lesions but has also been suggested to inhibit glycolysis of aciduric bacteria. Casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) promotes fluoride remineralization by acting as a salivary biomimetic that releases bioavailable calcium and phosphate ions, and the peptide complex has also been suggested to modify plaque composition. We developed a polymicrobial biofilm model of caries using 6 bacterial species representative of supragingival plaque that were cultured on sound human enamel and pulsed with sucrose 4 times a day to produce a high cariogenic challenge. We used this model to explore the mechanisms of action of SnF2 and CPP-ACP. Bacterial species in the biofilms were enumerated with 16S rRNA gene sequence analyses, and mineral loss and lesion formation were determined in the enamel directly under the polymicrobial biofilms via transverse microradiography. The model tested the twice-daily addition of SnF2, CPP-ACP, or both. SnF2 treatment reduced demineralization by 50% and had a slight effect on the composition of the polymicrobial biofilm. CPP-ACP treatment caused a similar inhibition of enamel demineralization (50%), a decrease in Actinomyces naeslundii and Lactobacillus casei abundance, and an increase in Streptococcus sanguinis and Fusobacterium nucleatum abundance in the polymicrobial biofilm. A combination of SnF2 and CPP-ACP resulted in a greater suppression of the acidogenic and aciduric bacteria and a significant 72% inhibition of enamel demineralization.

scholarly journals In Vitro Evaluation of the Effect of Oral Probiotic Weissella cibaria on the Formation of Multi-Species Oral Biofilms on Dental Implant Surfaces

2021 ◽  
Vol 9 (12) ◽  
pp. 2482
Author(s):  
Mi-Sun Kang ◽  
Geun-Yeong Park
Keyword(s):  
Biofilm Formation ◽  
Quantitative Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Microscopic Analysis ◽  
Fusobacterium Nucleatum ◽  
Titanium Implant ◽  
Culture Methods ◽  
Weissella Cibaria ◽  
Actinomyces Naeslundii

Oral probiotics are beneficial bacteria that can help prevent periodontal disease. However, little is known about the effects of oral probiotics on the formation of implant biofilms. This study aimed to evaluate the effects of oral probiotics Weissella cibaria CMU and CMS1 in an in vitro complex biofilm model on titanium implant surfaces. First, it was identified through colony biofilm assay that W. cibaria CMU and CMS1 inhibit the formation of multi-species biofilms formed by eight types of bacteria. Two types of saliva-coated titanium discs inoculated with early (Streptococcus gordonii, Streptococcus oralis, Streptococcus sanguinis, Actinomyces naeslundii, and Veillonella parvula), secondary (Fusobacterium nucleatum and Prevotella intermedia), and late (Porphyromonas gingivalis) colonizers were treated with the oral probiotics and then incubated anaerobically for three days. The effects of oral probiotics on titanium disc biofilm formation were analyzed using culture methods, quantitative polymerase chain reaction (qPCR), and microscopic analysis. Both probiotics significantly inhibited the formation of biofilm, and all eight bacterial species were significantly reduced. The effectiveness of both probiotic strains was confirmed by all the methods used. Oral probiotics may have dramatically reduced the biofilm formation of secondary colonizers that act as bridges, thus inhibiting biofilm formation on the titanium surface. Our results suggest that the probiotic W. cibaria offers new possibilities for the prevention of peri-implant mucositis.

scholarly journals Two-Step PCR-Based Assay for Identification of Bacterial Etiology of Otitis Media with Effusion in Infected Lebanese Children

1998 ◽  
Vol 36 (5) ◽  
pp. 1185-1188 ◽  
Author(s):  
Ghassan M. Matar ◽  
Nada Sidani ◽  
Michel Fayad ◽  
Usamah Hadi
Keyword(s):  
Otitis Media ◽  
Otitis Media With Effusion ◽  
Bacterial Species ◽  
Cost Effective ◽  
Universal Primers ◽  
Rrna Gene ◽  
Specific Primer ◽  
Specific Primers ◽  
Bacterial Dna ◽  
Species Specific

We developed and evaluated a two-step PCR-based assay with universal primers and genus- or species-specific primers for the detection of the most prevalent bacterial etiologies of otitis media with effusion (OME) in children from Lebanese hospitals. These etiologies included Haemophilus, Streptococcus, and Moraxella (Branhamella)catarrhalis, which were detected in middle-ear effusion (MEE) samples taken from children with OME. A total of 47 MEE samples were aspirated from 36 patients during insertion of a tympanostomy tube performed particularly for OME. The duration of effusion in all patients was ≥2 months. DNA was extracted from MEE samples, and PCR was initially done with DNA extracts by using the universal primers RW01 and DG74, which flank an ∼370-bp fragment found in the 16S rRNA gene of all bacterial species. For the identification of specific bacteria, we used in three separate reaction mixtures the following genus- or species-specific primers: (i) aHaemophilus-specific probe (probe RDR125) as a primer along with DG74, (ii) a Streptococcus-specific primer (primer STR1; designed by us) along with DG74, and (iii) an M. catarrhalis-specific primer pair (primer pair MCA1-MCA2). Thirty-five MEE samples (74.5%) gave the expected 370-bp band, indicating the presence of bacterial DNA in the tested samples. Of the 35 PCR-positive samples tested, 33 (94.3%) were positive forHaemophilus, 3 (8.6%) were positive forStreptococcus, and 10 (28.6%) were positive for M. catarrhalis. Ten samples (28.6%) exhibited a mixed infection and were positive for both Haemophilus and M. catarrhalis. Culture was simultaneously performed for all 47 MEE samples. Ten of the 47 MEE samples (21.3%) exhibited bacterial growth. These 10 were PCR positive for bacterial DNA. The remaining 25 PCR-positive samples were negative by culture, thus showing about 53% discordance between PCR results and those of culture. The PCR assay proved to be more sensitive than culture, more rapid, less cumbersome, and more cost-effective than the available PCR-Southern hybridization-based assays.

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