Melbourne Dental School - Research Publications

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    Real-time, label-free detection and identification of bacteria through non-invasive optical imaging.
    Qazi, F ; Verma, R ; Redmond, CE ; Khalid, A ; O'Brien-Simpson, NM ; Tomljenovic-Hanic, S (Elsevier BV, 2024)
    Currently, traditional and newer molecular and mass spectrometry techniques of identifying bacteria from biological samples requires lengthy sample preparation, growth and labelling/staining assays. Thus, there is a pressing clinical need for an adjunct method that accurately identifies bacteria in real time. Here we report on the evaluation of confocal microscopy for the identification of clinically important and multi-drug resistant (MDR) bacteria in real time, using their intrinsic fluorescence features, i.e., emission spectra and fluorescence lifetime. The results demonstrate that difference in emission spectra and fluorescence lifetimes can be used as a fingerprint for identification of 12 bacterial species and MDR strains in real-time. Photostability or time-traces of bacteria demonstrated that these parameters could be used for tracking and recording without a need for labelling. Further, dilution experiments demonstrated that using intrinsic fluorescence S. aureus, Klebsiella pneumoniae and Escherichia coli bacteria can be detected and identified at clinically relevant concentrations as low as 2 × 102 CFU/mL. This non-invasive, non-labelling optical methodology may serve as the basis for development of a device that would quickly and accurately identify bacteria in biological samples. Thus, this intrinsic fluorescence technique would provide clinicians information, within minutes from sampling, to base accurate and specific treatments for patients.
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    Engineering highly effective antimicrobial selenium nanoparticles through control of particle size
    Huang, T ; Holden, JA ; Heath, DE ; O'Brien-Simpson, NM ; O'Connor, AJ (Royal Society of Chemistry, 2019-08-21)
    The overuse of antibiotics has induced the rapid development of antibiotic resistance in bacteria. As a result, antibiotic efficacy has become limited, and infection with multidrug-resistant bacteria is considered to be one of the largest global human health threats. Consequently, new, effective and safe antimicrobial agents need to be developed urgently. One promising candidate to address this requirement is selenium nanoparticles (Se NPs), which are made from the essential dietary trace element Se and have antimicrobial activity against Gram-positive bacteria. The size of nanomaterials can strongly affect their biophysical properties and functions; however, the effects of the size of Se NPs on their antibacterial efficacy has not been systematically investigated. Therefore, in this work, spherical Se NPs ranging from 43 to 205 nm in diameter were fabricated, and their mammalian cytotoxicity and antibacterial activity as a function of their size were systematically studied. The antibacterial activity of the Se NPs was shown to be strongly size dependent, with 81 nm Se NPs showing the maximal growth inhibition and killing effect of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). The Se NPs were shown to have multi-modal mechanisms of action that depended on their size, including depleting internal ATP, inducing ROS production, and disrupting membrane potential. All the Se NPs were non-toxic towards mammalian cells up to 25 μg mL−1. Furthermore, the MIC value for the 81 nm particles produced in this research is 16 ± 7 μg mL−1, significantly lower than previously reported MIC values for Se NPs. This data illustrates that Se NP size is a facile yet critical and previously underappreciated parameter that can be tailored for maximal antimicrobial efficacy. We have identified that using Se NPs with a size of 81 nm and concentration of 10 μg mL−1 shows promise as a safe and efficient way to kill S. aureus without damaging mammalian cells.
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    Antimicrobial nanoparticle coatings for medical implants: Design challenges and prospects
    Li, X ; Huang, T ; Heath, DE ; O'Brien-Simpson, NM ; O'Connor, AJ (AMER INST PHYSICS, 2020-11)
    Microbial colonization, infection, and biofilm formation are major complications in the use of implants and are the predominant risk factors in implant failure. Although aseptic surgery and the administration of antimicrobial drugs may reduce the risk of infection, the systemic use of antibiotics can lead to a lack of efficacy, an increase in the risk of tissue toxicity, and the development of drug-resistant infections. To reduce implant-related infections, antimicrobial materials are increasingly being investigated and applied to implant surfaces using various methods depending on the agents and their microbicidal mechanisms. Through the development of biomaterials and nanotechnology, antimicrobial nanoparticles are becoming promising candidates for implant coatings, as their multifactorial antimicrobial mechanisms combat microbial adherence, viability, and biofilm formation. Despite their antimicrobial promise, the application of nanoparticles onto implant surfaces while retaining their antimicrobial potency faces many challenges. Herein, we review the potential and challenges associated with the design and implementation of antimicrobial nanoparticle coatings for the medical implant industry, particularly focusing on manufacturing considerations, sterilization, long-term stability, protein fouling, regulation, and safety, with a view to providing researchers the necessary tools to aid the translation of materials from the bench to the clinic.
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    Multifunctional Antimicrobial Polypeptide-Selenium Nanoparticles Combat Drug-Resistant Bacteria
    Huang, T ; Holden, JA ; Reynolds, EC ; Heath, DE ; O'Brien-Simpson, NM ; O'Connor, AJ (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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    Using inorganic nanoparticles to fight fungal infections in the antimicrobial resistant era
    Huang, T ; Li, X ; Maier, M ; O'Brien-Simpson, NM ; Heath, DE ; O'Connor, AJ (ELSEVIER SCI LTD, 2023-03-01)
    Fungal infections pose a serious threat to human health and livelihoods. The number and variety of clinically approved antifungal drugs is very limited, and the emergence and rapid spread of resistance to these drugs means the impact of fungal infections will increase in the future unless alternatives are found. Despite the significance and major challenges associated with fungal infections, this topic receives significantly less attention than bacterial infections. A major challenge in the development of fungi-specific drugs is that both fungi and mammalian cells are eukaryotic and have significant overlap in their cellular machinery. This lack of fungi-specific drug targets makes human cells vulnerable to toxic side effects from many antifungal agents. Furthermore, antifungal drug resistance necessitates higher doses of the drugs, leading to significant human toxicity. There is an urgent need for new antifungal agents, specifically those that can limit the emergence of new resistant species. Non-drug nanomaterials have primarily been explored as antibacterial agents in recent years; however, they are also a promising source of new antifungal candidates. Thus, this article reviews current research on the use of inorganic nanoparticles as antifungal agents. We also highlight challenges facing antifungal nanoparticles and discuss possible future research opportunities in this field. STATEMENT OF SIGNIFICANCE: Fungal infections pose a growing threat to human health and livelihood. The rapid spread of resistance to current antifungal drugs has led to an urgent need to develop alternative antifungals. Nanoparticles have many properties that could make them useful antimycotic agents. To the authors' knowledge, there is no published review so far that has comprehensively summarized the current development status of antifungal inorganic nanomaterials, so we decided to fill this gap. In this review, we discussed the state-of-the-art research on antifungal inorganic nanoparticles including metal, metal oxide, transition-metal dichalcogenides, and inorganic non-metallic particle systems. Future directions for the design of inorganic nanoparticles with higher antifungal efficacy and lower toxicity are described as a guide for further development in this important area.
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    One step antimicrobial coatings for medical device applications based on low fouling polymers containing selenium nanoparticles
    Li, F ; Huang, T ; Pasic, P ; Easton, CD ; Voelcker, NH ; Heath, DE ; O'Brien-Simpson, NM ; O'Connor, AJ ; Thissen, H (Elsevier, 2023-07-01)
    All indwelling and implantable medical devices are associated with a risk of infection, and antimicrobial technologies that can provide effective protection against pathogen colonization and biofilm formation over the lifetime of these devices are urgently required. Here, strategies that combine multiple layers of defense have emerged as particularly promising. We have combined a copolymer coating based on 2-hydroxypropyl acrylamide and N-benzophenone acrylamide with novel, optimally sized antimicrobial selenium nanoparticles (Se NPs). The photoreactive polymer allowed the crosslinking and covalent anchoring of the coating in a single step, and the exceptionally low attachment of bacteria was demonstrated. Our results also demonstrated that the incorporation of the antimicrobial Se NPs provides the coating with an additional bactericidal functionality towards the Gram-positive bacteria S. aureus and E. faecalis, which are widely recognized as the most prevalent pathogens linked to medical device-associated infections and more broadly nosocomial infections. The multiple layers of defense provided effective inhibition of the growth of both bacteria strains in areas where the coating had been removed, as well as in the supernatant. Moreover, our results demonstrate the feasibility to modulate the release of Se NPs from the coating by tailoring coating parameters such as the nanoparticle to polymer ratio. Our cytotoxicity study further confirmed the superior biocompatibility of Se NPs compared to the well-established silver nanoparticles over a broad concentration range. Our multifunctional coating approach is expected to be translated into medical device applications due to its ease of manufacture and effective antimicrobial protection.
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    Detection and identification of amino acids and proteins using their intrinsic fluorescence in the visible light spectrum
    Verma, R ; Pyreddy, S ; Redmond, CE ; Qazi, F ; Khalid, A ; O'Brien-Simpson, NM ; Shukla, R ; Tomljenovic-Hanic, S (ELSEVIER, 2023-11-22)
    The detection and identification of biomolecules are essential in the modern era of medical diagnostics. Several approaches have been established, but they have significant limitations such as laborious and time-consuming sample preparation, analysis, and the need to use external probes which provide adequate but not desired levels of accuracy and sensitivity. Herein, we have explored successfully a non-invasive technique to detect and identifybiomolecules such as amino acids and proteins by utilizing their intrinsic fluorescence. The developed confocal microscopy method revealed high and photostable emission counts of these biomolecules including amino acids (tryptophan, phenylalanine, tyrosine, proline, histidine, cysteine, aspartic acid, asparagine, isoleucine, lysine, glutamic acid, arginine) and proteins (HSA, BSA) when they are excited with a green laser. The fluorescence lifetime of the samples enabled the identification and distinction of known and blind samples of biomolecules from each other. The developed optical technique is straightforward, non-destructive and does not require laborious labeling to identify specific proteins, and may serve as the basis for the development of a device that would quickly and accurately identify proteins at an amino acid level. Therefore, this approach would open an avenue for precise detection in imaging and at the same time increases our understanding of chemical dynamics at the molecular level.
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    Star-Peptide Polymers are Multi-Drug-Resistant Gram-Positive Bacteria Killers
    Li, W ; Hadjigol, S ; Mazo, AR ; Holden, J ; Lenzo, J ; Shirbin, SJ ; Barlow, A ; Shabani, S ; Huang, T ; Reynolds, EC ; Qiao, GG ; O'Brien-Simpson, NM (AMER CHEMICAL SOC, 2022-06-08)
    Antibiotic resistance in bacteria, especially Gram-positive bacteria like Staphylococcus aureus, is gaining considerable momentum worldwide and unless checked will pose a global health crisis. With few new antibiotics coming on the market, there is a need for novel antimicrobial materials that target and kill multi-drug-resistant (MDR) Gram-positive pathogens like methicillin-resistant Staphylococcus aureus (MRSA). In this study, using a novel mixed-bacteria antimicrobial assay, we show that the star-peptide polymers preferentially target and kill Gram-positive pathogens including MRSA. A major effect on the activity of the star-peptide polymer was structure, with an eight-armed structure inducing the greatest bactericidal activity. The different star-peptide polymer structures were found to induce different mechanisms of bacterial death both in vitro and in vivo. These results highlight the potential utility of peptide/polymers to fabricate materials for therapeutic development against MDR Gram-positive bacterial infections.
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    Systematic comparison of activity and mechanism of antimicrobial peptides against nosocomial pathogens
    Lin, B ; Hung, A ; Li, R ; Barlow, A ; Singleton, W ; Matthyssen, T ; Sani, M-AD ; Hossain, MA ; Wade, J ; O'Brien-Simpson, NM ; Li, W (ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER, 2022-03-05)
    The World Health Organisation has deemed several multi-drug resistant (MDR) nosocomial bacterial pathogens to be of significant threat to human health. A stark increase in morbidity, mortality and the burden to healthcare systems around the world can be attributed to the development of resistance in these bacteria. Accordingly, alternative antimicrobial agents have been sought as an attractive means to combat MDR pathogens, with one such example being antimicrobial peptides (AMPs). Given the reported activity of AMPs, including Pardaxin, MSI-78, dermaseptin-PC (DMPC) and Cecropin B, it is important to understand their activities and modes of action against bacteria for further AMP design. In this study, we compared these AMPs against a panel of nosocomial bacterial pathogens, followed by detailed mechanistic studies. It was found that Pardaxin (1-22) and MSI-78 (4-20) displayed the most pronounced antimicrobial activity against the tested bacteria. The mechanistic studies by membrane permeability and molecular dynamics simulation further confirmed the strong membrane interaction and structure of Pardaxin (1-22) and MSI-78 (4-20), which contributed to their potent activity. This study demonstrated a structure and activity guidance for further design of Pardaxin (1-22) and MSI-78 (4-20) as therapeutics against MDR pathogens. The different effects of DMPC (1-19) and Cecropin B (1-21) on membrane integrity and phospholipid membrane interactions provided critical information for the rational design of next-generation analogues with specificity against either Gram-negative or Gram-positive bacteria.
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    The overview of antimicrobial peptide-coated implants against oral bacterial infections Special Issue: Emerging Investigators
    Sun, Z ; Ma, L ; Sun, X ; Sloan, AJ ; O'Brien-Simpson, NM ; Li, W (WILEY, 2023-06)
    Abstract Dental implants are the most common therapeutic approach for resolving tooth loss and damage. Despite technical advances in treatment, implant failure rates can be as high as 23% with the major cause of peri‐implantitis: a multi‐species bacterial infection. With an annual growth rate in implant placements of 8.78% per annum, implant failure caused by bacterial infection is a significant oral and general health issue. The rise in antibiotic resistance in oral bacteria further adds pressure to implant failure; thus, there is a need for adjunctive therapy to improve implant outcomes. Due to the broad spectrum of activity and a low risk of inducing bacterial resistance, peptide antibiotics are emerging as a promising implant coating material to reduce/prevent peri‐implantitis and improve dental implant success rates. In this review, we summarised the current strategies of coating antimicrobial peptides (AMPs) onto dental implant material surfaces with multi‐functional properties to enhance osteoblast growth and prevent bacterial infections. This review compared the recent reported literature on dental implant coating with AMPs, which will provide an overview of the current dental implant coating strategies using AMPs and insights for future clinical applications.