Melbourne Dental School - Research Publications
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ItemMultifunctional Antimicrobial Polypeptide-Selenium Nanoparticles Combat Drug-Resistant Bacteria(AMER CHEMICAL SOC, 2020-12-16)Antibiotic-resistant bacteria are a severe threat to human health. The World Health Organization's Global Antimicrobial Surveillance System has revealed widespread occurrence of antibiotic resistance among half a million patients across 22 countries, with Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae being the most common resistant species. Antimicrobial nanoparticles are emerging as a promising alternative to antibiotics in the fight against antimicrobial resistance. In this work, selenium nanoparticles coated with the antimicrobial polypeptide, ε-poly-l-lysine, (Se NP-ε-PL) were synthesized and their antibacterial activity and cytotoxicity were investigated. Se NP-ε-PL exhibited significantly greater antibacterial activity against all eight bacterial species tested, including Gram-positive, Gram-negative, and drug-resistant strains, than their individual components, Se NP and ε-PL. The nanoparticles showed no toxicity toward human dermal fibroblasts at the minimum inhibitory concentrations, demonstrating a therapeutic window. Furthermore, unlike the conventional antibiotic kanamycin, Se NP-ε-PL did not readily induce resistance in E. coli or S. aureus. Specifically, S. aureus began to develop resistance to kanamycin from ∼44 generations, whereas it took ∼132 generations for resistance to develop to Se NP-ε-PL. Startlingly, E. coli was not able to develop resistance to the nanoparticles over ∼300 generations. These results indicate that the multifunctional approach of combining Se NP with ε-PL to form Se NP-ε-PL is a highly efficacious new strategy with wide-spectrum antibacterial activity, low cytotoxicity, and significant delays in development of resistance.
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ItemComplementation in trans of Porphyromonas gingivalis Lipopolysaccharide Biosynthetic Mutants Demonstrates Lipopolysaccharide Exchange(American Society for Microbiology, 2021-04-21)Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host. IMPORTANCE: Porphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.
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ItemIADR 100 Years On: Driving Science, Engagement, and Globalization Post COVID-19(SAGE PUBLICATIONS INC, 2021-12)
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ItemTaxonomy of Oral Bacteria(ELSEVIER ACADEMIC PRESS INC, 2018-01-01)The oral cavity is a collection of diverse microenvironments, each inhabited by a community of microorganisms, the majority of which are bacteria and their phages. Given the appropriate conditions, some of these bacteria can cause destruction of the teeth or their supporting hard and soft tissues. For over 300 years microbiologists have been characterising these microbial communities, in both oral health and disease. In this chapter, we take the reader on a journey through time as we discuss the various methods that have been utilised in the characterisation of the bacteria calling the oral cavity home, and how the use of these methods has informed our understanding of oral bacterial communities and the diversity of their members.
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ItemAdolescent temperament dimensions as stable prospective risk and protective factors for salivary C-reactive protein(WILEY, 2018-02)OBJECTIVE: Temperament has associations with later physical health outcomes, yet there is a dearth of research exploring the connection between temperament and mechanisms that have known associations with these health outcomes. Recent research has delineated a connection between personality and inflammation during adulthood, but this association has not yet been studied in adolescent samples. DESIGN: We investigated whether stable adolescent temperament (averaged over two years), specifically effortful control and negative emotionality, provided a more robust prediction of inflammation as measured by salivary C-reactive protein (sCRP), than depressive symptoms. METHODS: Temperament and depressive symptoms were measured in a sample of sixty-three adolescents (37 males) when they were approximately 12 years old (mean age = 12.30, SD = 0.69) and again when they were approximately 14 years old (mean age = 14.84, SD = 0.49). Levels of sCRP were determined approximately 7 months later (mean = 6.77, SD = 2.99) when participants were approximately 15 years old (mean age = 15.49, SD = 0.49). RESULTS: Regression analyses revealed that effortful control (EC) was significantly associated with lower sCRP levels, while higher negative emotionality (NE) was significantly associated with higher sCRP levels. Furthermore, these associations were larger than those for depressive symptoms and were differentially impacted by the addition of covariates. Implications for the role of stable risk and protective factors in inflammatory processes are discussed. CONCLUSIONS: These findings are the first to show associations between adolescent temperament and inflammation. Furthermore, these findings extend previous personality research to temperamental research in a younger sample of adolescents. Statement of contribution What is already known? There is a large extant literature on the association between depressive symptoms and inflammation. There is a smaller extant literature on the association between personality and inflammation. No studies have examined how adolescent temperament traits may relate to inflammation. What does this study add? Longitudinal data collection over the course of 3 years in an adolescent sample. Addresses the question of whether temperament factors relate to inflammation. Temperament provides a more robust predictor of later inflammation than depressive symptoms.
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ItemA panel of microRNAs can be used to determine oral squamous cell carcinoma(WILEY, 2017-11)BACKGROUND: Subjective histopathology is currently used to diagnose oral squamous cell carcinoma (OSCC). We tested if abundances of a panel of microRNA could be an objective OSCC indicator. METHOD: Literature review enabled identification of 10 microRNAs associated with oral and head and neck malignancies. We extracted RNA from formalin-fixed paraffin-embedded biopsies; 20 each with OSCC, dysplasia, or histologically normal epithelium (HNE) and 10 with oral lichen planus (OLP). Relative abundances of microRNAs in HNE and OSCC were determined using reverse transcription and then real-time PCR with global mean normalization. MicroRNAs differentially expressed (test microRNA, T-miR) and non-differentially expressed (normalization microRNA, N-miR) were identified. The raw microRNA Cq data were incorporated in a developed algorithm that output a T-miR expression value (T-miREV) score. Raw Cq data from HNE, OSCC, dysplasia, and OLP samples were then used to test the algorithm scoring and OSCC classification. RESULTS: Four test and normalization microRNAs were identified. Algorithm output of T-mirEV >1 or <-1 indicated high and low OSCC probability score, respectively, and gave 88.9% sensitivity, 100% specificity, and 93.5% accuracy. Grouping high and intermediate T-mirEV scores (T-miREV ≥-1) resulted in sensitivity of 90%, specificity of 65%, and accuracy of 77.5% in OSCC classification. All 20 dysplasias and eight of 10 OLP had T-miREV ≥-1 indicating intermediate to high probability of malignant changes. CONCLUSION: A microRNA panel combined with our algorithm can identify tissue with probable oncogenic changes. IMPACT: The developed algorithm serves as a baseline for prospective trials, which may result in potential clinical utility.
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ItemRole of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the Joint EFP/ORCA workshop on the boundaries between caries and periodontal disease(WILEY, 2017-03)BACKGROUND AND AIMS: The scope of this working group was to review (1) ecological interactions at the dental biofilm in health and disease, (2) the role of microbial communities in the pathogenesis of periodontitis and caries, and (3) the innate host response in caries and periodontal diseases. RESULTS AND CONCLUSIONS: A health-associated biofilm includes genera such as Neisseria, Streptococcus, Actinomyces, Veillonella and Granulicatella. Microorganisms associated with both caries and periodontal diseases are metabolically highly specialized and organized as multispecies microbial biofilms. Progression of these diseases involves multiple microbial interactions driven by different stressors. In caries, the exposure of dental biofilms to dietary sugars and their fermentation to organic acids results in increasing proportions of acidogenic and aciduric species. In gingivitis, plaque accumulation at the gingival margin leads to inflammation and increasing proportions of proteolytic and often obligately anaerobic species. The natural mucosal barriers and saliva are the main innate defence mechanisms against soft tissue bacterial invasion. Similarly, enamel and dentin are important hard tissue barriers to the caries process. Given that the present state of knowledge suggests that the aetiologies of caries and periodontal diseases are mutually independent, the elements of innate immunity that appear to contribute to resistance to both are somewhat coincidental.
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ItemOral swirl samples - a robust source of microRNA protected by extracellular vesicles(WILEY, 2017-04)BACKGROUND: MicroRNAs are small non-coding RNAs which are dysregulated in disease states, such as oral cancer. Extracellular vesicles, a potential source of microRNA, are found in saliva. OBJECTIVE: To demonstrate that a quantifiable amount of microRNA can be isolated from oral swirl samples. Additionally, we hypothesized that extracellular vesicles may protect contained microRNA from degradation in these samples. METHOD: A polyethylene glycol-based precipitation was used for extracellular vesicle enrichment of oral swirl samples. Comparison was made between samples treated with and without RNase. Further, samples from three subjects were exposed to a range of conditions over 7 days and assessed for presence of microRNA by reverse-transcription quantitative PCR. Extracellular vesicles from samples were identified under transmission electron microscopy. RESULTS: An adequate quantity of microRNA for qPCR analysis was extractable from samples despite exposure to conditions under which degradation of RNA would be expected. CONCLUSION: A technique was developed to isolate an adequate quantity of microRNA for analysis from oral swirl samples. Extracellular vesicle-associated microRNA may be protected from degradation. This technique moves towards chairside application of translational microRNA research in the field of oral cancer prognostics.
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ItemC-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide(WILEY-V C H VERLAG GMBH, 2017-01-05)Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL-1 ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.
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ItemCandida virulence and ethanol-derived acetaldehyde production in oral cancer and non-cancer subjects(WILEY, 2016-11)OBJECTIVES: To compare biofilm-forming ability, hydrolytic enzymes and ethanol-derived acetaldehyde production of oral Candida isolated from the patients with oral cancer and matched non-oral cancer. MATERIAL AND METHODS: Fungal biofilms were grown in RPMI-1640 medium, and biofilm mass and biofilm activity were assessed using crystal violet staining and XTT salt reduction assays, respectively. Phospholipase, proteinase, and esterase production were measured using agar plate method, while fungal acetaldehyde production was assessed via gas chromatography. RESULTS: Candida isolated from patients with oral cancer demonstrated significantly higher biofilm mass (P = 0.031), biofilm metabolic activity (P < 0.001), phospholipase (P = 0.002), and proteinase (P = 0.0159) activity than isolates from patients with non-oral cancer. High ethanol-derived acetaldehyde-producing Candida were more prevalent in patients with oral cancer than non-oral cancer (P = 0.01). In univariate regression analysis, high biofilm mass (P = 0.03) and biofilm metabolic activity (P < 0.001), high phospholipase (P = 0.003), and acetaldehyde production ability (0.01) were significant risk factors for oral cancer; while in the multivariate regression analysis, high biofilm activity (0.01) and phospholipase (P = 0.01) were significantly positive influencing factors on oral cancer. CONCLUSION: These data suggest a significant positive association between the ability of Candida isolates to form biofilms, to produce hydrolytic enzymes, and to metabolize alcohol to acetaldehyde with their ability to promote oral cancer development.