• A clinical evaluation of the ability of finishing files to supplement the removal of bacteria and endotoxin from primarily infected root canals (Part I- Initial evaluation)

      Kim, Eunice; Martinho, Frederico C. (2020)
      Aim: The aim of this study was to evaluate the ability of XP-endo Finisher (XPF) to supplement the removal of bacteria and endotoxin from primary infected root canals after instrumentation. Methodology: This randomized and blinded controlled trial included eight subjects. Instrumentation was performed using Vortex Blue or XP-endo Shaper, followed by supplemental instrumentation with XPF. All canals were irrigated with 2.5% sodium hypochlorite. Bacterial and endotoxin samples were taken using sterile paper points. Samples were collected before and after instrumentation and after XPF. Results: Bacteria was present in all root canals. After XPF, bacterial mean was reduced from 255 ± 311.82 CFU/mL to 2.5 ± 7.07 CFU/mL (p= .056). Endotoxin was detected in all root canals by the LAL method (KQCL test). After XPF, endotoxin mean was significantly reduced from .85 ± .26 EU/mL to .03 ± .01 EU/mL (p= .00004). Conclusion: The findings of this study showed that the supplemental use of the XP-endo Finisher after root canal instrumentation was effective in significantly reducing endotoxin but not bacteria present in primary endodontic infections.
    • Bacterial Adhesion to Various Implant Surfaces

      Khatra, Navpreet Kaur; Masri, Radi, 1975- (2018)
      Dental implants provide a major course of treatment for patients who are partially or completely edentulous. These implants are biocompatible metal anchors that are surgically positioned in the jawbone to support the prostheses where natural teeth are missing. However, the use of dental implants has some disadvantages, which can result in complications. Once the clean implant surface is exposed in the oral cavity, it is immediately coated with salivary pellicle and subsequently colonized by oral microbial species. In fact, microbial adhesion and accumulation on implants are considered to play major roles in the pathogenesis of peri-implant mucositis and peri-implantitis. The physico- chemical characteristics of specific material surface are known to significantly influence the bacterial adhesion process. Therefore, the surface characteristics of dental implants have been refined and restructured over a period of time to improve the interaction of implants with host cells and tissues. Hence, investigating the microbiological aspects related to implant surfaces will provide important insights relevant to expectations of treatment outcome. To that end, in this study, we aimed to comparatively evaluate microbial adherence and accumulation on five different types of implants. Specimens were provided by Dental Implant Systems, Biodenta Group and implant surfaces included: .3- .5 μm anodized surface, 1 μm BST surface, .8- 1.0 μm anodized surface, 1.6μm SLA surface and .3- .4 μm machined surface. To assess microbial adherence, the cariogenic bacterial species Streptococcus mutans and the bacterial pathogen Staphylococcus aureus were studied. Results from these studies were analyzed using analysis of variance (ANOVA) and significant differences were further analyzed by Tukey’s Honestly Significant Difference (HSD) test. Pearson’s r was also used to evaluate the association between surface roughness and bacterial accumulation. The present study has demonstrated that not only surface roughness but other physicochemical properties such as surface charge, energy, wettability and biological factors such as host immune response and oral hygiene influence bacterial adhesion and accumulation around implant surfaces.
    • The Effect of Residual Bacteria on Dental Pulp Regeneration

      Nosrat, Ali; Fouad, Ashraf F. (2015)
      Tissue regeneration requires an interaction of stem cells and growth factors in a bioactive scaffold. This study utilized the ferret canine as an in situ animal model to investigate a clinically applicable tissue engineering approach for dentin-pulp regeneration. On the other hand, ideal root canal disinfection for dental pulp regeneration is a challenge. There is no study available to address the effect of residual bacteria on the outcome of dental pulp regeneration in previously infected root canals. Therefore, the aim of this study was two-fold: 1) To determine histologically, the efficacy of delivering stem cells within a bioactive scaffold directly into the root canal space compared with the traditional revascularization method and 2) to determine the effect of residual bacteria on the histological and radiographic outcomes of dental pulp regeneration procedures. Periapical lesions were induced in 24 canine teeth of 6 ferrets. Dental pulp stem cells were isolated, characterized, encapsulated in a hydrogel scaffold, and injected in half of the experimental teeth. The other half was treated using the traditional endodontic protocol with a blood clot scaffold. After an evaluation period of 3 months, the animals were sacrificed and block sections were processed for radiographic, histological and histo-bacteriological analyses. Sections were evaluated for the presence/absence of an odontoblast layer, dentin associated mineralized tissue (DAMT), bony islands, intra-canal and periapical inflammation, and bacteria. Data were analyzed using Fisher exact test and one-way analysis of variance (ANOVA) with the p≤0.05. Results of the study showed that there was no significant difference between the traditional and the tissue engineering groups in terms of presence and amount of DAMT and bony islands (p>0.05). Presence of residual bacteria was associated with lack of radiographic growth (p<0.0001) and with presence of intra-canal and periapical inflammation (p<0.05). There was significantly higher amounts of DAMT formed in teeth with no residual bacteria compared to teeth with bacteria (p<0.0001). The results of this study showed that residual bacteria play a critical role in the outcome of regenerative endodontic treatments.
    • Evaluation of Factors Related to Long-term Healing in Endodontic Treatment

      Orgel, Adam William; Fouad, Ashraf F. (2012)
      Introduction: This study sought to determine the effects of residual root canal bacteria and treatment factors on endodontic treatment outcomes up to 6 years post-operatively using traditional radiography (PA radiographs) and limited cone beam computed tomography (CBCT). Methods: Root canal samples were obtained from 50 patients with pulp necrosis and a periapical lesion, following two-visit treatment using contemporary chemomechanical preparation techniques. PCR, with broad range 16S rDNA bacterial primers, was performed followed by cloning and sequencing on pre-obturation specimens. Periapical and CBCT radiographs were taken at 10 months to 6 years post-treatment. Images were scored by two blinded, calibrated endodontists using the conventional periapical index (PAI) and a CBCT index. Statistical analysis was performed using bivariate and multivariate regression analysis for treatment factors. Kaplan-Meier survival analysis and Pearson's regression analysis was used for year-over-year changes. Results: 41 patients were included in the final data analysis. Recall decreased from 98% at 10-17 months to 51% at 33+ months. The percentage of patients healed at 10-17 months, 18-32 months and 33+ months and overall was 65%, 63%, and 66% respectively. Factors influencing outcomes were presence of bacteria at time of obturation (56% when present, 81% absent), primary versus persistent disease (71% vs. 39%), tooth type (85% anterior,23% molar), obturation to the radiographic apex (100% to the apex, 56% not to the apex), and larger master apical file sizes (73%MAF>45 vs. 36% MAF<45).. Short-term outcomes had a strong positive relationship with final outcome (Pearson's, r2=0.56, p=<0.01). Conclusions: Bacterial DNA presence at the time of obturation adversely affects short-term and intermediate-term endodontic treatment outcomes, but this effect is not found in the long-term. Various patient factors such as tooth type and treatment factors such as master apical file size influence outcomes. Short-term treatment outcomes are good predictors of long-term outcomes.

      Lahanda Wadu, Vijitha de Silva; Parrish, Nicole (2016)
      Antimicrobial resistance is an emerging threat. The Chesapeake Bay and upper watershed were previously found to harbor AR in multiple bacteria during the summer months. During the winter of 2013, we conducted a pilot study to survey bacterial diversity and resistance in isolates from the Chesapeake Bay and upper watershed. Water samples were collected from nine sites on the Chesapeake Bay and upper watershed. All bacterial isolates were identified to the species level using MALDI-ToF MS. Antimicrobial susceptibility testing was performed for all Gram-negative isolates using Etest and 14 antibiotics. A total of 35 bacterial isolates were recovered from all sites, including 8 Gram-positive and 27 Gram-negative organisms. Resistance to chloramphenicol and imipenem exists in the Chesapeake Bay and upper watershed. Further studies are needed to better understand the antibiotic resistance identified in this study and potential sources of antibiotic exposure and transmission.
    • The Rickettsia Ankyrin Repeat Protein 2 is a type IV secreted effector and co-localizes with markers of the endoplasmic reticulum in mammalian cells

      Lehman, Stephanie Shae; Azad, Abdu F.; 0000-0002-4568-3082 (2018)
      Spotted fever and typhus group Rickettsia species are neglected zoonotic, vector-borne bacteria that cause fatal infections and are distributed worldwide. These pathogens efficiently invade host endothelial cells, rapidly escape the phagosome, and establish an "intracytosolic niche," where they grow to high numbers before cell lysis. Despite more than two decades of research, the mechanisms of rickettsial pathogenesis and cytosolic survival remain poorly understood. A candidate virulence factor, Rickettsia ankyrin repeat protein 2 (RARP-2), is largely conserved in pathogenic strains, absent in non-pathogenic strains, and shows increased expression in the mammalian stage of infection. Based on these observations, we hypothesized that RARP-2 functions to enhance pathogenesis during rickettsial infection in mammals. This study demonstrates that RARP-2 is a cytosolically localized effector that is secreted by the type IV secretion system (T4SS) of Rickettsia species from both the typhus and spotted fever groups in order to modulate host pathways. Analysis of homolog distribution and domains of RARP-2 in 43 Rickettsia spp. allowed for the detailed characterization of the C-terminal ankyrin repeats, an N-terminal putative cysteine protease motif, and a C-terminal secretion signal characterized by disorder and flexibility. R. typhi RARP-2 is maximally expressed during the cytosolic phase of infection, is secreted via the T4SS, and overexpression results in the formation of cytosolic vesicular structures. The virulent R. rickettsii str. Sheila Smith (SS) RARP-2 homolog was observed to have 7 more Ank repeats than the avirulent str. Iowa-RARP-2. While both SS- and Iowa-RARP-2 homologs are secreted by the T4SS, only expression of SS-RARP-2 in the avirulent Iowa strain induced a lytic plaque phenotype characteristic of virulent strains. SS-RARP-2 also induced the formation of vesicular like structures in the host cytoplasm that were composed of ER membranes. Mutation of the putative protease active site of SS-RARP-2 abolished the lytic plaque phenotype but did not eliminate association with host membranes. Characterizing RARP-2 and its cognate secretion system marks a significant advance in our understanding of rickettsial biology during the cytosolic stage of infection, and adds to the collective knowledge of how Ank domains are manipulated by obligate intracellular bacteria to regulate host cells.
    • Structural and molecular interactions of quaternary ammonium compounds with bacteria

      Kim, Sang-Nyun; Nauman, Robert K. (1995)
      The molecular interaction of quaternary ammonium compounds (QAC) with bacterial cells was investigated using various techniques to obtain the knowledge on what kinds of damage are done to the cell that results in cell death. Electron microscopy and flow cytometry were used to evaluate direct effects of didecyldimethyl ammonium chloride (DDAC) to Pseudomonas aeruginosa cells. The characteristic effects of DDAC observed on the bacterial structure were the formation and pinching off of blebs from the outer membrane and the formation of multilamellar structures within the nucleoid area. This was followed by coagulation of the entire nucleoid region and cytoplasm. The morphological changes observed in the intact gram-negative bacterium probably accounted for the bactericidal action of DDAC. An extensive theoretical discussion was provided to explain the observed effects of DDAC on the bacterial envelopes and cytoplasm. A flow cytometric approach was evaluated to determine its use for assessing antibacterial activity of a variety of QAC on P. aeruginosa ATCC 15442. LIVE/DEAD BacLight{dollar}\rm \sp{lcub}TM{rcub}{dollar} viability kit and the SYTOX{dollar}\rm \sp{lcub}TM{rcub}{dollar} kit were used as vital fluorescent stains. The DDAC had the greatest activity by converting the most number of bacteria in the population from green to red, followed by tetradecyl benzalkonium chlorides (BKC), hexadecyl BKC and dodecyl BKC. The flow cytometer provided an excellent means to assess the antibacterial activity of individual QAC. In order to decipher the interaction of DDAC with E. coli lipids, a steady-state and time-resolved emission spectra of 2-dimethylamino-6-lauroyinaphthalene (Laurdan), and steady-state anisotropy decay of 1,6-diphenyl-1,3,5,-hexatriene (DPH) were performed. Finally, {dollar}\sp{lcub}31{rcub}{dollar}P NMR spectroscopy allowed for convenient and qualitative measurements of the effects of DDAC on lipid polymorphism which seems relevant in the DDAC effects on membrane perturbation and possibly bactericidal activity. A schematic model for DDAC-membrane interactions that takes into account Laurdan spectral data, and turbidimetric data in conjunction with {dollar}\sp{lcub}31{rcub}{dollar}P NMR results was presented, which attempts to explain the different modes of action of DDAC on lipid supramolecular structures with respect to molar % of DDAC to lipids.
    • The Role of Type I and III Interferons in the Pathogenesis of Bordetella pertussis Infection and Disease.

      Ardanuy, Jeremy; Carbonetti, Nicholas H. (2020)
      Bordetella pertussis is a Gram-negative bacterial pathogen that infects human respiratory tracts and is the causative agent for the disease pertussis, otherwise known as whooping cough. In 2012, there were 48,277 reported cases in the United States, the most since 1955. Symptoms build up to severe paroxysmal coughing, often for 10+ weeks after onset. For infants, pertussis can be fatal due to complications including pulmonary hypertension, pneumothorax, high-level circulating lymphocytosis, and pneumonia. In adults, the disease is severe too, with long-lasting cough, lung damage, and serious symptoms resulting in hospitalization. A difficulty in treatment/prevention of pertussis is a suboptimal vaccine that confers waning immunity, and a lack of effective treatments available. Antibiotics are administered to patients to prevent transmission, but usually don’t change the clinical course of disease. Host directed therapeutics treating pertussis could benefit individuals with severe cough and save the lives of infected infants. Using RNA-sequencing transcriptomics we investigated lung gene expression responses to Bordetella pertussis infection in adult mice, revealing that type I and III interferon (IFN) responses and signaling pathways may play an important role in promoting inflammatory responses. In B. pertussis infected mice, lung type I/III IFN responses correlated with increased proinflammatory cytokine expression and lung inflammatory pathology. In mutant mouse models with increased type I IFN signaling, B. pertussis exacerbated lung inflammatory pathology, whereas knockout mice with deficiencies in type I/III IFN signaling had reduced lung inflammation compared to wild-type mice. In direct contrast, infant mice didn’t upregulate type I/III IFNs in response to B. pertussis infection and were protected from lethal infection by increased type I IFN signaling, indicating age dependent effects of type I/III IFN signaling during B. pertussis infection. The induction of type I/III IFNs was found to be MyD88 dependent, and TLR9 and STING were identified as DNA sensing pattern recognition receptors required for type I/III IFN responses, as well as for typical levels of lung inflammatory pathology. This observation, coupled with results showing DNase treatment of B. pertussis-infected mice causing reduced lung pathology, indicated a DNA dependent induction of type I/III IFNs, making these targets for therapeutic intervention.