Characterization of uropathogenic Escherichia coli phylogroups associated with antimicrobial resistance, virulence factor distribution, and virulence-related phenotypes.

In this study, we compared the characteristics of different uropathogenic Escherichia coli phylogroups. A total of 844 E. coli isolated from urine were enrolled and the antimicrobial susceptibility of E. coli to 22 antibiotics was determined by disk diffusion test. The distribution of phylogroups and 20 virulence factor genes was determined by PCR. Phenotypes associated with bacterial virulence, including motility, biofilm formation, and the production of curli and siderophore, were examined. Phylogroup B2 was dominant in our isolates (64.8%), followed by phylogroups D (8.6%), B1 (7.8%), F (6.0%), C (4.5%), A (3.1%), untypable (2.8%), E (1.8%), and clade I (0.5%). The prevalence of multidrug-resistant strains was highest in phylogroup C (86.8%), followed by E (80.0%), F (75.0%), and D (71.2%). Moreover, 23.5% of the phylogroup F E. coli were extensively drug-resistant. Phylogroup B2 E. coli had an average of the highest virulence factor genes (10.1 genes/isolate). Compared to phylogroup B2 E. coli, phylogroups F and clade I E. coli had higher motility while phylogroup C E. coli had lower motility. >60% of phylogroups A and C E. coli showed very low curli production. In contrast, 14%, 10%, and 7%, of E. coli in phylogroups F, B2, and E, produced a very high amount of curli, respectively. Surprisingly, phylogroup A E. coli showed the highest virulence to larvae, followed by phylogroups B2 and C. In summary, we first characterized and revealed that the antimicrobial resistance, virulence gene distribution, motility, and curli production, were associated with in E. coli phylogroups.

Plasmid-mediated quinolone resistance determinants in fluoroquinolone-nonsusceptible Escherichia coli isolated from patients with urinary tract infections in a university hospital, 2009-2010 and 2020.

OBJECTIVES: This study aimed to characterize the plasmid-mediated quinolone resistance (PMQR) in fluoroquinolone nonsusceptible E. coli (FQNSEC) isolated from patients with urinary tract infections (UTIs) in 2019-2010 and 2020. METHODS: A total of 844 E. coli isolates were collected from UTI patients at National Cheng Kung University Hospital. The antimicrobial susceptibility of E. coli isolates to 21 antibiotics was determined by disk diffusion tests. The distribution of phylogenetic groups, virulence factor, and PMQR genes was determined by PCR. Conjugation assays were performed to investigate the transferability of qnr genes from FQNSEC isolates to E. coli C600. RESULTS: We found 211 (41.9%) and 152 (44.7%) E. coli isolates were FQNSEC in 2009-2010 and 2020, respectively. Phylogenetic group B2 was dominant in FQNSEC isolates (52.34%), followed by group F (10.47%), group B1 (9.64%), and group D (9.64%). FQNSEC isolates were more resistant to 17 of 19 tested antimicrobial agents, compared to the fluoroquinolone susceptible E. coli. PMQR screening results showed 34, 22, and 10 FQNSEC isolates containing aac(6')-Ib-cr, qnr genes, and efflux pump genes (qepA or oqxAB), respectively. PMQR E. coli isolates were more nonsusceptible to gentamicin, amoxicillin, ampicillin/sulbactam, imipenem, cefazolin, cefuroxime, cefmetazole, ceftriaxone, ceftazidime, and cefepime compared to non-PMQR FQNSEC. Moreover, 16 of 22 qnr-carrying plasmids were transferrable to the recipient C600. CONCLUSION: Here, we reported the high prevalence of MDR- and XDR-E. coli in FQNSEC isolates. Moreover, qnr-carrying plasmids were highly transferable and led to the resistance to other classes of antibiotics in the transconjugants.

Potential of tailored amorphous multiporous calcium silicate glass for pulp capping regenerative endodontics-A preliminary assessment.

INTRODUCTION/OBJECTIVE: The tailored amorphous multi-porous (TAMP) material fabrication technology has led to a new class of bioactive materials possessing versatile characteristics. It has not been tested for dental applications. Thus, we aimed to assess its biocompatibility and ability to regenerate dental mineral tissue. METHODS: 30CaO-70SiO2 model TAMP discs were fabricated by a sol-gel method followed by in vitro biocompatibility testing with isolated human or mini-swine dental pulp stem cells (DPSCs). TAMP scaffolds were tested in vivo as a pulp exposure (pin-point, 1 mm, 2 mm, and entire pulp chamber roof) capping material in the molar teeth of mini-swine. RESULTS: The in vitro assays showed that DPSCs attached well onto the TAMP discs with comparable viability to those attached to culture plates. Pulp capping tests on mini-swine showed that after 4.5 months TAMP material was still present at the capping site, and mineral tissue (dentin bridge) had formed in all sizes of pulp exposure underneath the TAMP material. CONCLUSIONS: TAMP calcium silicate is biocompatible with both human and swine DPSCs in vitro and with pulp in vivo, it may help regenerate the dentin bridge after pulp exposure.

Pulp/Dentin Regeneration: It Should Be Complicated.

Stem cell-mediated regenerative endodontics has reached the human clinical trial phase; however, many issues still exist that prevent such technology to be a widely used clinical practice. These issues are not straightforward and are complicated. They should be because pulp regeneration is dealing with a small dead-end space. In addition, when regeneration is needed, the space is often heavily infected. The true standard of pulp regeneration should be everything except generation of some fibrous connective tissue and amorphous mineral deposit. As of now, we are still far short of reaching the standard of complete vascularized and innervated pulp regeneration with newly formed tubular dentin in all types of teeth. Thus, we need to go back to the bench and use established animal models or create new animal models to tackle those issues. This article will address several key issues including the possibility of pulp regeneration in small canals of molar teeth by enhancing the neovascularization, and whether the organized tubular dentin can be generated on the canal walls. Data from our semi-orthotopic tooth fragment mouse model have shown that complete pulp regeneration using dental pulp stem cells (DPSCs) in small canal has been inconsistent because of limited blood supply. This inconsistency is similar in our orthotopic miniature swine model, although in some cases vascularized pulp-like tissue can be formed throughout the canal space after DPSC transplantation. Furthermore, no tubular dentin was observed in the orthotopic pulp regeneration, despite the fact that DPSCs have the capacity to generate some tubular dentin-like structure in the hydroxyapatite/tricalcium phosphate-mediated ectopic pulp/dentin formation model in mice. Potential strategies to be tested to address these regeneration issues are discussed herein.

Effects of Restorative Materials on Dental Pulp Stem Cell Properties.

INTRODUCTION: Dental pulp stem cells (DPSCs) are multipotent progenitors for biotechnological practices, but the influences of existing restorations on their viability and differentiation are not well-known. This study was aimed to investigate in vivo and in vitro responses of DPSCs to restorative materials. METHODS: Class I cavities were prepared on molars scheduled to be extracted and then restored with a resin-based composite (RBC), a glass ionomer cement, or zinc oxide eugenol. Intact teeth were used as controls. Twelve molars in each group were extracted on day 7 or day 30 after restorations to assess the early or intermediate pulp responses and were then cut in half. One half was processed for histopathological analysis, and the other was used to isolate DPSCs for a colony-forming unit assay and real-time polymerase chain reaction for NANOG, OCT4, and CD44 expression. RESULTS: All restored teeth showed pulp damage at various levels, whereas mild to moderate inflammation persisted in the RBC group until day 30. The existence of DPSCs in the pulp cores of all groups was revealed based on CD44 immunoreactivity. Glass ionomer cement and zinc oxide eugenol did not affect the relative percentages of DPSCs in either early or intermediate stages, whereas RBCs reduced the percentage. The colony-forming units in all restoration groups were comparable with those in the control. Nevertheless, the restorations significantly enhanced OCT4 expression, especially in RBC/day 30. CONCLUSIONS: Dental restorations cause mild pulp damage but do not affect DPSC viability. RBC decreases DPSC densities but might increase the stemness of surviving DPSCs through an inflammation-stimulation process.

A Miniature Swine Model for Stem Cell-Based De Novo Regeneration of Dental Pulp and Dentin-Like Tissue.

The goal of this study was to establish mini-swine as a large animal model for stem cell-based pulp regeneration studies. Swine dental pulp stem cells (sDPSCs) were isolated from mini-swine and characterized in vitro. For in vivo studies, we first employed both ectopic and semi-orthotopic study models using severe combined immunodeficiency mice. One is hydroxyapatite-tricalcium phosphate (HA/TCP) model for pulp-dentin complex formation, and the other is tooth fragment model for complete pulp regeneration with new dentin depositing along the canal walls. We found that sDPSCs are similar to their human counterparts exhibiting mesenchymal stem cell characteristics with ability to form colony forming unit-fibroblastic and odontogenic differentiation potential. sDPSCs formed pulp-dentin complex in the HA/TCP model and showed pulp regeneration capacity in the tooth fragment model. We then tested orthotopic pulp regeneration on mini-swine including the use of multi-rooted teeth. Using autologous sDPSCs carried by hydrogel and transplanted into the mini-swine root canal space, we observed regeneration of vascularized pulp-like tissue with a layer of newly deposited dentin-like (rD) tissue or osteodentin along the canal walls. In some cases, dentin bridge-like structure was observed. Immunohistochemical analysis detected the expression of nestin, dentin sialophosphoprotein, dentin matrix protein 1, and bone sialoprotein in odontoblast-like cells lining against the produced rD. We also tested the use of allogeneic sDPSCs for the same procedures. Similar findings were observed in allogeneic transplantation. This study is the first to show an establishment of mini-swine as a suitable large animal model utilizing multi-rooted teeth for further cell-based pulp regeneration studies.

Treatment of Mature Permanent Teeth with Necrotic Pulps and Apical Periodontitis Using Regenerative Endodontic Procedures: A Case Series.

INTRODUCTION: Regenerative endodontic procedures (REPs) are usually used to treat human immature permanent teeth with necrotic pulps and/or apical periodontitis. Successful REPs result in the elimination of clinical signs/symptoms, the resolution of apical periodontitis, and, in some cases, thickening of the canal walls and/or continued root development with or without apical closure. REPs can restore the vitality of tissue in the canals of immature permanent teeth previously destroyed by infection or trauma. Vital tissue is inherited with immune defense mechanisms to protect itself from foreign invaders. Recently, REPs have also been used to successfully treat human mature permanent teeth with necrotic pulps and apical periodontitis. The purpose of this case series was to present the potential of using REPs for mature permanent teeth with necrotic pulps and apical periodontitis. METHODS: This case series consisted of 6 patients, 4 females and 2 males. The patients' ages ranged from 8-21 years old. Seven permanent teeth, 4 anterior and 3 molar teeth, with necrotic pulps and apical periodontitis were treated using REP. Radiographically, the root development of all teeth was almost completed except the apices of 2 molars, which showed slightly open. Complete chemomechanical debridement of the canals of the teeth was performed, and the canals were dressed with Metapaste (Meta Biomed Co, Ltd, Chungbuk, Korea) during treatment visits. Periapical bleeding into the canals was induced at the last treatment visit by placing a hand #20 or #25 K-file with the tip slightly bent through the apical foramina into the periapical tissues. A 3-mm thickness of mineral trioxide aggregate was placed into the coronal canals over semicoagulated blood. The access cavities were restored with either composite resin or amalgam. RESULTS: Follow-ups of the 7 teeth ranged from 8 to 26 months. The periapical lesions of 2 teeth were considered healed, and 5 teeth revealed healing. Clinical signs/symptoms were absent in all teeth at follow-up visits at different time points. None of the treated teeth responded to cold and electric pulp tests. CONCLUSIONS: This case series shows the potential of using REPs for mature teeth with necrotic pulp and apical periodontitis.

Establishing the pangolin mitochondrial D-loop sequences from the confiscated scales.

Pangolin scales are encountered in traditional East Asian medicines (TEAM) and the ever increasing demand for these scales has escalated the decline in the numbers of these mammals. The identification of protected pangolin species is necessary to enforce international and national legislation as well as assist with conservation measures. There is limited morphological feature on a pangolin scale thus requiring DNA analysis as a means of identification. We report on the isolation of DNA from pangolin scales and a strategy for obtaining the full length of the mitochondrial D-loop, being 1159 bp. Primer sets creating five overlapping amplicons were designed to amplify sections of this mitochondrial DNA locus. DNA from the blood stain of nineteen Formosan pangolins (Manis pentadactyla pentadactyla) along with 145 scale samples that were suspected to have come from pangolins, was amplified and sequenced; leading to a total of 91 D-loop sequences being obtained. The 19 Formosan pangolin sequences produced 5 haplotypes and 72 of the 145 seized scales provided useable sequence classified as a further 38 haplotypes. The D-loop sequences from those scales suspected to be from a pangolin had a higher similarity to any of the 19 samples taken from M. p. pentadactyla compared to a D-loop sequence from Manis tetradactyla (the only pangolin D-loop sequence in GenBank, NC_004027). These 43 haplotypes were used to establish a local database for the D-loop sequence of pangolins and add to the data of Manis sp. held on GenBank. The PCR amplification strategy development in this study could be used in forensic DNA identification of scales suspected to be from protected pangolin species.