ORCID Profile
0000-0001-5569-7755
Current Organisation
University of South Australia
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Gene Expression (incl. Microarray and other genome-wide approaches) | Veterinary Microbiology (excl. Virology) | Characterisation of Biological Macromolecules | Medicinal and Biomolecular Chemistry | Veterinary Epidemiology | Biologically Active Molecules | Physical Chemistry (Incl. Structural) | Veterinary Sciences not elsewhere classified | Veterinary Sciences | Structural Chemistry and Spectroscopy | Structural Biology (incl. Macromolecular Modelling) |
Expanding Knowledge in the Agricultural and Veterinary Sciences | Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Chemical Sciences | Livestock Product Traceability and Quality Assurance | Expanding Knowledge in the Medical and Health Sciences | Food Safety
Publisher: Elsevier BV
Date: 11-2018
DOI: 10.1016/J.EJMECH.2018.09.053
Abstract: Antibiotic resistance among clinically significant bacterial pathogens is becoming a prevalent threat to public health, and new antibacterial agents with novel mechanisms of action hence are in an urgent need. Utilizing computational docking method and structure-based optimization strategy, we rationally designed and synthesized two series of isoxazol-3-yl- and isoxazol-5-yl-containing benzamide derivatives that targeted the bacterial cell ision protein FtsZ. Evaluation of their activity against a panel of Gram-positive and -negative pathogens revealed that compounds B14 and B16 that possessed the isoxazol-5-yl group showed strong antibacterial activity against various testing strains, including methicillin-resistant Staphylococcus aureus and penicillin-resistant S. aureus. Further molecular biological studies and docking analyses proved that the compound functioned as an effective inhibitor to alter the dynamics of FtsZ self-polymerization via a stimulatory mechanism, which finally terminated the cell ision and caused cell death. Taken together, these results could suggest a promising chemotype for development of new FtsZ-targeting bactericidal agent.
Publisher: Wiley
Date: 16-09-2014
DOI: 10.1002/MBO3.212
Publisher: Springer Science and Business Media LLC
Date: 27-12-2019
DOI: 10.1038/S41598-019-56248-7
Abstract: There is increasing demand for safe and effective sanitizers for irrigation water disinfection to prevent transmission of foodborne pathogens to fresh produce. Here we compared the efficacy of pH-neutral electrolyzed oxidizing water (EOW), sodium hypochlorite (NaClO) and chlorine dioxide (ClO 2 ) against single and mixed populations of E. coli , Listeria and Salmonella under a range of pH and organic matter content. EOW treatment of the mixed bacterial suspension resulted in a dose-dependent ( mg/L free chlorine), rapid ( min) and effective (4–6 Log 10 ) reduction of the microbial load in water devoid of organic matter under the range of pH conditions tested (pH, 6.0, 7.0, 8.4 and 9.2). The efficacy of EOW containing 5 mg/L free chlorine was unaffected by increasing organic matter, and compared favourably with equivalent concentrations of NaClO and ClO 2 . EOW at 20 mg/L free chlorine was more effective than NaClO and ClO 2 in reducing bacterial populations in the presence of high (20–100 mg/L) dissolved organic carbon, and no regrowth or metabolic activity was observed for EOW-treated bacteria at this concentration upon reculturing in rich media. Thus, EOW is as effective or more effective than other common chlorine-based sanitizers for pathogen reduction in contaminated water. EOW’s other characteristics, such as neutral pH and ease of handling, indicate its suitability for fresh produce sanitation.
Publisher: Elsevier BV
Date: 02-2017
DOI: 10.1016/J.BMCL.2017.01.042
Abstract: Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chlor henicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rif icin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps.
Publisher: Elsevier BV
Date: 02-2016
DOI: 10.1016/J.EJMECH.2016.10.065
Abstract: Novel series of novel 3-O-arylalkylcarbamoyl descladinosylazithromycin derivatives with the 2'-O-acetyl and 11,12-cyclic carbonate groups, the 11,12-cyclic carbonate group and the 11-O-arylalkylcarbamoyl side chain, and 2'-O-arylalkylcarbamoyl descladinosylazithromycin with the 11,12-cyclic carbonate group were designed, synthesized and evaluated for their antibacterial activity using broth microdilution method. The results showed that the majority of the target compounds showed moderate to favorable activity against six kinds of susceptible strains and almost all of them displayed significantly improved activity compared with references against three erythromycin-resistant strains of S. pneumoniae B1 expressing the ermB gene, S. pneumoniae AB11 expressing the ermB and mefA genes, and S. pyogenes R1. In particular, compound 6h exhibited the most potent activity against susceptible B. subtilis ATCC9372 (0.5 μg/mL), penicillin-resistant S. epidermidis (0.125 μg/mL), and erythromycin-resistant S. pneumoniae B1 (1 μg/mL) and S. pneumoniae AB11 (1 μg/mL), which were 2-, 2-, 256-, 256-fold better activity than azithromycin, respectively. Additionally, compounds 6f (0.5 μg/mL) and 6g (0.25 μg/mL) were the most active against S. pneumoniae A22072, which were 8- and 16-fold better activity than azithromycin (4 μg/mL). As for erythromycin-resistant S. pyogenes R1, compound 5a presented the most excellent activity (8 μg/mL), showing 32- and 32-fold higher activity than azithromycin (256 μg/mL) and clarithromycin (256 μg/mL).
Publisher: Elsevier BV
Date: 2018
DOI: 10.1016/J.EJMECH.2017.11.102
Abstract: A novel series of 4-substituted 2-naphthamide derivatives were designed, synthesized and evaluated for their biological activity. In particular, the ability of the compounds to potentiate the action of antibiotics, to inhibit Nile Red efflux and to target AcrB specifically was investigated. The results indicated that most of the 4-substituted 2-naphthamide derivatives were able to synergize with the antibiotics tested, and inhibit Nile Red efflux by AcrB in the resistant phenotype. Subsequent exclusion of compounds with off target effects such as outer- or inner membrane permeabilization identified compounds 7c, 7g, 12c, 12i and 13g as efflux pump inhibitors (EPIs). Particularly, compounds 7c, 7g and 12i were found to be the most potent EPIs, which synergized with the two substrates tested at lower concentrations than that of parent A3, demonstrating an improvement in potency as compared to A3. Additionally, when the outer membrane of E. coli was permeabilized, compound 12c displayed a huge increase in efficacy and was able to synergize with erythromycin at a concentration that was 16 times lower than that of the parent A3. Hence we were able to design and synthesize compounds that displayed significant increase in efficacy as EPIs against AcrB.
Publisher: MDPI AG
Date: 22-09-2023
DOI: 10.3390/D15101028
Publisher: Frontiers Media SA
Date: 04-08-2020
Publisher: Portland Press Ltd.
Date: 15-04-2002
DOI: 10.1042/0264-6021:3630243
Abstract: The molecular mass of the galactose-H(+) symport protein GalP, as its histidine-tagged derivative GalP(His)(6), has been determined by electrospray MS (ESI-MS) with an error of <0.02%. One methionine residue, predicted to be present from the DNA sequence, was deduced to be absent. This is a significant advance on the estimation of the molecular masses of membrane-transport proteins by SDS/PAGE, where there is a consistent under-estimation of the true molecular mass due to anomalous electrophoretic migration. Addition of a size-exclusion chromatography step after Ni(2+)-nitrilotriacetate affinity purification was essential to obtain GalP(His)(6) suitable for ESI-MS. Controlled trypsin, trypsin+chymotrypsin and CNBr digestion of the protein yielded peptide fragments suitable for ESI-MS and tandem MS analysis, and accurate mass determination of the derived fragments resulted in identification of 82% of the GalP(His)(6) protein. Tandem MS analysis of selected peptides then afforded 49% of the actual amino acid sequence of the protein the absence of the N-terminal methionine was confirmed. Matrix-assisted laser-desorption ionization MS allowed identification of one peptide that was not detected by ESI-MS. All the protein eptide mass and sequence determinations were in accord with the predictions of amino acid sequence deduced from the DNA sequence of the galP gene. [ring-2-(13)C]Histidine was incorporated into GalP(His)(6) in vivo, and ESI-MS analysis enabled the measurement of a high (80%) and specific incorporation of label into the histidine residues in the protein. MS could also be used to confirm the labelling of the protein by (15)NH(3) (93% enrichment) and [(19)F]tryptophan (83% enrichment). Such MS measurements will serve in the future analysis of the structures of membrane-transport proteins by NMR, and of their topology by indirect techniques.
Publisher: Elsevier BV
Date: 02-2021
Publisher: Frontiers Media SA
Date: 21-06-2021
DOI: 10.3389/FMICB.2021.597735
Abstract: Klebsiella pneumoniae is a Gram-negative pathogen that has become a worldwide concern due to the emergence of multidrug-resistant isolates responsible for various invasive infectious diseases. Biofilm formation constitutes a major virulence factor for K. pneumoniae and relies on the expression of fimbrial adhesins and aggregation of bacterial cells on biotic or abiotic surfaces in a coordinated manner. During biofilm aggregation, bacterial cells communicate with each other through inter- or intra-species interactions mediated by signallng molecules, called autoinducers, in a mechanism known as quorum sensing (QS). In most Gram-negative bacteria, intra-species communication typically involves the LuxI/LuxR system: LuxI synthase produces N -acyl homoserine lactones (AHLs) as autoinducers and the LuxR transcription factor is their cognate receptor. However, K. pneumoniae does not produce AHL but encodes SdiA, an orphan LuxR-type receptor that responds to exogenous AHL molecules produced by other bacterial species. While SdiA regulates several cellular processes and the expression of virulence factors in many pathogens, the role of this regulator in K. pneumoniae remains unknown. In this study, we describe the characterization of sdiA mutant strain of K. pneumoniae . The sdiA mutant strain has increased biofilm formation, which correlates with the increased expression of type 1 fimbriae, thus revealing a repressive role of SdiA in fimbriae expression and bacterial cell adherence and aggregation. On the other hand, SdiA acts as a transcriptional activator of cell ision machinery assembly in the septum, since cells lacking SdiA regulator exhibited a filamentary shape rather than the typical rod shape. We also show that K. pneumoniae cells lacking SdiA regulator present constant production of QS autoinducers at maximum levels, suggesting a putative role for SdiA in the regulation of AI-2 production. Taken together, our results demonstrate that SdiA regulates cell ision and the expression of virulence factors such as fimbriae expression, biofilm formation, and production of QS autoinducers in K. pneumoniae .
Publisher: MDPI AG
Date: 16-06-2021
DOI: 10.3390/ANTIBIOTICS10060727
Abstract: Our recent focus on the “lost antibiotic” unguinol and related nidulin-family fungal natural products identified two semisynthetic derivatives, benzguinols A and B, with unexpected in vitro activity against Staphylococcus aureus isolates either susceptible or resistant to methicillin. Here, we show further activity of the benzguinols against methicillin-resistant isolates of the animal pathogen Staphylococcus pseudintermedius, with minimum inhibitory concentration (MIC) ranging 0.5–1 μg/mL. When combined with sub-inhibitory concentrations of colistin, the benzguinols demonstrated synergy against Gram-negative reference strains of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (MICs of 1–2 μg/mL in the presence of colistin), whereas the benzguinols alone had no activity. Administration of three intraperitoneal (IP) doses of 20 mg/kg benzguinol A or B to mice did not result in any obvious adverse clinical or pathological evidence of acute toxicity. Importantly, mice that received three 20 mg/kg IP doses of benzguinol A or B at 4 h intervals exhibited significantly reduced bacterial loads and longer survival times than vehicle-only treated mice in a bioluminescent S. aureus murine sepsis challenge model. We conclude that the benzguinols are potential candidates for further development for specific treatment of serious bacterial infections as both stand-alone antibiotics and in combination with existing antibiotic classes.
Publisher: Wiley
Date: 22-07-2005
Abstract: The ATP binding cassette (ABC) transporter LmrA from the bacterium Lactococcus lactis is a homolog of the human multidrug resistance P-glycoprotein (ABCB1), the activity of which impairs the efficacy of chemotherapy. In a previous study, LmrA was shown to mediate ethidium efflux by an ATP-dependent proton-ethidium symport reaction in which the carboxylate E314 is critical. The functional importance of this key residue for ABC proteins was suggested by its conservation in a wider family of related transporters however, the structural basis of its role was not apparent. Here, we have used homology modeling to define the structural environment of E314. The residue is nested in a hydrophobic environment that probably elevates its pKa, accounting for the pH dependency of drug efflux that we report in this work. Functional analyses of wild-type and mutant proteins in cells and proteoliposomes support our proposal for the mechanistic role of E314 in proton-coupled ethidium transport. As the carboxylate is known to participate in proton translocation by secondary-active transporters, our observations suggest that this substituent can play a similar role in the activity of ABC transporters.
Publisher: Elsevier BV
Date: 06-2003
Publisher: Portland Press Ltd.
Date: 13-08-2010
DOI: 10.1042/BJ20091860
Abstract: The MexAB–OprM drug efflux pump is central to multidrug resistance of Pseudomonas aeruginosa. The ability of the tripartite protein to confer drug resistance on the pathogen is crucially dependent on the presence of all three proteins of the complex. However, the role of each protein in the formation of the intact functional complex is not well understood. One of the key questions relates to the (in)ability of MexB to act independently of its cognitive partners, MexA and OprM. In the present study, we have demonstrated that, in the absence of MexA and OprM, MexB can: (i) recruit AcrA and TolC from Escherichia coli to form a functional drug-efflux complex (ii) transport the toxic compound ethidium bromide in a Gram-positive organism where the periplasmic space and outer membrane are absent and (iii) catalyse transmembrane chemical proton gradient (ΔpH)-dependent drug transport when purified and reconstituted into proteoliposomes. Our results represent the first evidence of drug transport by an isolated RND (resistance–nodulation–cell ision)-type multidrug transporter, and provide a basis for further studies into the energetics of RND-type transporters and their assembly into multiprotein complexes.
Publisher: Elsevier BV
Date: 09-2021
Publisher: Public Library of Science (PLoS)
Date: 16-07-2018
Publisher: Elsevier BV
Date: 02-2021
Publisher: Elsevier BV
Date: 12-2017
Abstract: The bacterial species and specific spoilage organisms associated with the Southern Australian King George Whiting (KGW) and Tasmanian Atlantic Salmon (TAS), and the efficacy of a HOCl-containing water-based sanitization product (Electro-Chemically Activated Solution, by ECAS4) in extending the shelf life of KGW and TAS fillets were evaluated. Fillets were washed with an ECAS4 solution containing either 45 ppm or 150 ppm of free chlorine and bacterial species enumerated on selective and non-selective media, followed by identification of pure isolates by 16 S rRNA gene sequencing. The dominant spoilage microbiota in KGW and TAS fillets stored at 4 ± 1 °C were Pseudomonas spp. and Shewanella spp. At either concentration, ECAS4 significantly reduced total bacterial load and specific spoilage organisms on KGW and TAS fillets (approx. 1-2 log colony-forming units) during storage and significantly extended the shelf life of the fillets by 2 and 4 days, respectively. The significant increase in shelf life and quality of fillets was corroborated by raw and cooked sensory evaluation. ECAS4 sanitization could have a significant impact on the overall food industry, translating into health and economic benefits through reduction of food spoilage bacteria and potentially, foodborne pathogens without many of the disadvantages of currently approved biocides.
Publisher: Portland Press Ltd.
Date: 04-2016
DOI: 10.1042/BSR20160046
Abstract: Iron is essential for the survival and virulence of pathogenic bacteria. The FeoB transporter allows the bacterial cell to acquire ferrous iron from its environment, making it an excellent drug target in intractable pathogens. The protein consists of an N-terminal GTP-binding domain and a C-terminal membrane domain. Despite the availability of X-ray crystal structures of the N-terminal domain, many aspects of the structure and function of FeoB remain unclear, such as the structure of the membrane domain, the oligomeric state of the protein, the molecular mechanism of iron transport, and how this is coupled to GTP hydrolysis at the N-terminal domain. In the present study, we describe the first homology model of FeoB. Due to the lack of sequence homology between FeoB and other transporters, the structures of four different proteins were used as templates to generate the homology model of full-length FeoB, which predicts a trimeric structure. We confirmed this trimeric structure by both blue-native-PAGE (BN-PAGE) and AFM. According to our model, the membrane domain of the trimeric protein forms a central pore lined by highly conserved cysteine residues. This pore aligns with a central pore in the N-terminal GTPase domain (G-domain) lined by aspartate residues. Biochemical analysis of FeoB from Pseudomonas aeruginosa further reveals a putative iron sensor domain that could connect GTP binding/hydrolysis to the opening of the pore. These results indicate that FeoB might not act as a transporter, but rather as a GTP-gated channel.
Publisher: Portland Press Ltd.
Date: 10-2005
DOI: 10.1042/BST20051008
Publisher: Elsevier BV
Date: 12-2020
Publisher: Frontiers Media SA
Date: 29-08-2022
DOI: 10.3389/FMICB.2022.967949
Abstract: Acinetobacter baumannii is a pathogen with high intrinsic antimicrobial resistance while multidrug resistant (MDR) and extensively drug resistant (XDR) strains of this pathogen are emerging. Treatment options for infections by these strains are very limited, hence new therapies are urgently needed. The bacterial cell ision protein, FtsZ, is a promising drug target for the development of novel antimicrobial agents. We have previously reported limited activity of cinnamaldehyde analogs against Escherichia coli . In this study, we have determined the antimicrobial activity of six cinnamaldehyde analogs for antimicrobial activity against A. baumannii . Microscopic analysis was performed to determine if the compounds inhibit cell ision. The on-target effect of the compounds was assessed by analyzing their effect on polymerization and on the GTPase activity of purified FtsZ from A. baumannii . In silico docking was used to assess the binding of cinnamaldehyde analogs. Finally, in vivo and in vitro safety assays were performed. All six compounds displayed antibacterial activity against the critical priority pathogen A. baumannii , with 4-bromophenyl-substituted 4 displaying the most potent antimicrobial activity (MIC 32 μg/mL). Bioactivity was significantly increased in the presence of an efflux pump inhibitor for A. baumannii ATCC 19606 (up to 32-fold) and significantly, for extensively drug resistant UW 5075 (greater than 4-fold), suggesting that efflux contributes to the intrinsic resistance of A. baumannii against these agents. The compounds inhibited cell ision in A. baumannii as observed by the elongated phenotype and targeted the FtsZ protein as seen from the inhibition of polymerization and GTPase activity. In silico docking predicted that the compounds bind in the interdomain cleft adjacent to the H7 core helix. Di-chlorinated 6 was devoid of hemolytic activity and cytotoxicity against mammalian cells in vitro , as well as adverse activity in a Caenorhabditis elegans nematode model in vivo . Together, these findings present halogenated analogs 4 and 6 as promising candidates for further development as antimicrobial agents aimed at combating A. baumannii . This is also the first report of FtsZ-targeting compounds with activity against an XDR A. baumannii strain.
Publisher: Informa UK Limited
Date: 2004
DOI: 10.1080/09687860400003941
Abstract: NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1'-(13)C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.
Publisher: Portland Press Ltd.
Date: 28-02-2017
DOI: 10.1042/EBC20160053
Abstract: The crisis of antimicrobial resistance (AMR) is one of the most serious issues facing us today. The scale of the problem is illustrated by the recent commitment of Heads of State at the UN to coordinate efforts to curb the spread of AMR infections. In this review, we explore the biochemistry behind the headlines of a few stories that were recently published in the public media. We focus on ex les from three different issues related to AMR: (i) hospital-acquired infections, (ii) the spread of resistance through animals and/or the environment and (iii) the role of antimicrobial soaps and other products containing disinfectants in the dissemination of AMR. Although these stories stem from three very different settings, the underlying message in all of them is the same: there is a direct relationship between the use of antimicrobials and the development of resistance. In addition, one type of antimicrobial could select for cross-resistance to another type and/or for multidrug resistance. Therefore, we argue the case for increased stewardship to not only cover clinical use of antibiotics, but also the use of antimicrobials in agriculture and stewardship of our crucially important biocides such as chlorhexidine.
Publisher: Portland Press Ltd.
Date: 07-01-2005
DOI: 10.1042/BJ20040791
Abstract: The human BCRP (breast cancer resistance protein, also known as ABCG2) is an ABC (ATP-binding cassette) transporter that extrudes various anticancer drugs from cells, causing multidrug resistance. To study the molecular determinants of drug specificity of BCRP in more detail, we have expressed wild-type BCRP (BCRP-R) and the drug-selected cancer cell line-associated R482G (Arg482→Gly) mutant BCRP (BCRP-G) in Lactococcus lactis. Drug resistance and the rate of drug efflux in BCRP-expressing cells were proportional to the expression level of the protein and affected by the R482G mutation, pointing to a direct role of BCRP in drug transport in L. lactis. In agreement with observations in mammalian cells, the BCRP-R-mediated transport of the cationic substrates rhodamine 123 and tetramethylrosamine was significantly decreased compared with the activity of BCRP-G. In addition, BCRP-R showed an enhanced interaction with the anionic anticancer drug methotrexate when compared with BCRP-G, suggesting that structure/substrate specificity relationships in BCRP, as observed in eukaryotic expression systems, are maintained in prokaryotic L. lactis. Interestingly, BCRP-R exhibited a previously unestablished ability to transport antibiotics, unconjugated sterols and primary bile acids in L. lactis, for which the R482G mutation was not critical. Since Arg482 is predicted to be present in the intracellular domain of BCRP, close to transmembrane segment 3, our results point to a role of this residue in electrostatic interactions with charged substrates including rhodamine 123 and methotrexate. Since unconjugated sterols are neutral molecules and bile acids and many antibiotics are engaged in protonation/deprotonation equilibria at physiological pH, our observations may point either to a lack of interaction between Arg482 and neutral or neutralized moieties in these substrates during transport or to the interaction of these substrates with regions in BCRP not including Arg482.
Publisher: Frontiers Media SA
Date: 25-04-2019
Publisher: Microbiology Society
Date: 17-12-2021
Abstract: Carbapenems are potent broad-spectrum β-lactam antibiotics reserved for the treatment of serious infections caused by multidrug-resistant bacteria such as Pseudomonas aeruginosa . The surge in P. aeruginosa resistant to carbapenems is an urgent threat, as very few treatment options remain. Resistance to carbapenems is predominantly due to the presence of carbapenemase enzymes. The assessment of 147 P . aeruginosa isolates revealed that 32 isolates were carbapenem non-wild-type. These isolates were screened for carbapenem resistance genes using PCR. One isolate from wastewater contained the Adelaide imipenemase gene ( bla AIM-1 ) and was compared phenotypically with a highly carbapenem-resistant clinical isolate containing the bla AIM-1 gene. A further investigation of wastewater s les from various local healthcare and non-healthcare sources as well as river water, using probe-based qPCR, revealed the presence of the bla AIM-1 gene in all the s les analysed. The widespread occurrence of bla AIM-1 throughout Adelaide hinted at the possibility of more generally extensive spread of this gene than originally thought. A blast search revealed the presence of the bla AIM-1 gene in Asia, North America and Europe. To elucidate the identity of the organism(s) carrying the bla AIM-1 gene, shotgun metagenomic sequencing was conducted on three wastewater s les from different locations. Comparison of these nucleotide sequences with a whole-genome sequence of a P. aeruginosa isolate revealed that, unlike the genetic environment and arrangement in P. aeruginosa , the bla AIM-1 gene was not carried as part of any mobile genetic elements. A phylogenetic tree constructed with the deduced amino acid sequences of AIM-1 suggested that the potential origin of the bla AIM-1 gene in P. aeruginosa might be the non-pathogenic environmental organism, Pseudoxanthomonas mexicana .
Publisher: Elsevier BV
Date: 05-2021
Publisher: Oxford University Press (OUP)
Date: 29-05-2012
DOI: 10.1111/J.1574-6968.2012.02594.X
Abstract: Drug efflux pumps such as MexAB-OprM from Pseudomonas aeruginosa confer resistance to a wide range of chemically different compounds. Within the tripartite assembly, the inner membrane protein MexB is mainly responsible for substrate recognition. Recently, considerable advances have been made in elucidating the drug efflux pathway through the large periplasmic domains of resistance-nodulation- ision (RND) transporters. However, little is known about the role of amino acids in other parts of the protein. We have investigated the role of two conserved phenylalanine residues that are aligned around the cytoplasmic side of the central cavity of MexB. The two conserved phenylalanine residues have been mutated to alanine residues (FAFA MexB). The interaction of the wild-type and mutant proteins with a variety of drugs from different classes was investigated by assays of cytotoxicity and drug transport. The FAFA mutation affected the efflux of compounds that have targets inside the cell, but antibiotics that act on cell wall synthesis and membrane probes were unaffected. Combined, our results indicate the presence of a hitherto unidentified cytoplasmic-binding site in RND drug transporters and enhance our understanding of the molecular mechanisms that govern drug resistance in Gram-negative pathogens.
Publisher: Elsevier BV
Date: 2020
Publisher: MDPI AG
Date: 23-09-2022
DOI: 10.3390/ANTIBIOTICS11101301
Abstract: Multidrug-resistant (MDR) Gram-negative pathogens, especially Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Enterobacter spp., are recognized by the World Health Organization as the most critical priority pathogens in urgent need of drug development. In this study, the in vitro antimicrobial activity of robenidine analogues NCL259 and NCL265 was tested against key human and animal Gram-negative clinical isolates and reference strains. NCL259 and NCL265 demonstrated moderate antimicrobial activity against these Gram-negative priority pathogens with NCL265 consistently more active, achieving lower minimum inhibitory concentrations (MICs) in the range of 2–16 µg/mL. When used in combination with sub-inhibitory concentrations of polymyxin B to permeabilize the outer membrane, NCL259 and NCL265 elicited a synergistic or additive activity against the reference strains tested, reducing the MIC of NCL259 by 8- to 256- fold and the MIC of NCL265 by 4- to 256- fold. A small minority of Klebsiella spp. isolates (three) were resistant to both NCL259 and NCL265 with MICs 256 µg/mL. This resistance was completely reversed in the presence of the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PAβN) to yield MIC values of 8–16 µg/mL and 2–4 µg/mL for NCL259 and NCL256, respectively. When NCL259 and NCL265 were tested against wild-type E. coli isolate BW 25113 and its isogenic multidrug efflux pump subunit AcrB deletion mutant (∆AcrB), the MIC of both compounds against the mutant ∆AcrB isolate was reduced 16-fold compared to the wild-type parent, indicating a significant role for the AcrAB-TolC efflux pump from Enterobacterales in imparting resistance to these robenidine analogues. In vitro cytotoxicity testing revealed that NCL259 and NCL265 had much higher levels of toxicity to a range of human cell lines compared to the parent robenidine, thus precluding their further development as novel antibiotics against Gram-negative pathogens.
Publisher: Springer Science and Business Media LLC
Date: 12-2003
DOI: 10.1038/NATURE02173
Publisher: MDPI AG
Date: 05-12-2020
DOI: 10.3390/ANTIBIOTICS9120873
Abstract: The bacterial cell ision protein, FtsZ, has been identified as a target for antimicrobial development. Derivatives of 3-methoxybenzamide have shown promising activities as FtsZ inhibitors in Gram-positive bacteria. We sought to characterise the activity of five difluorobenzamide derivatives with non-heterocyclic substituents attached through the 3-oxygen. These compounds exhibited antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA), with an isopentyloxy-substituted compound showing modest activity against vancomycin resistant Enterococcus faecium (VRE). The compounds were able to reverse resistance to oxacillin in highly resistant clinical MRSA strains at concentrations far below their MICs. Three of the compounds inhibited an Escherichia coli strain lacking the AcrAB components of a drug efflux pump, which suggests the lack of Gram-negative activity can partly be attributed to efflux. The compounds inhibited cell ision by targeting S. aureus FtsZ, producing a dose-dependent increase in GTPase rate which increased the rate of FtsZ polymerization and stabilized the FtsZ polymers. These compounds did not affect the polymerization of mammalian tubulin and did not display haemolytic activity or cytotoxicity. These derivatives are therefore promising compounds for further development as antimicrobial agents or as resistance breakers to re-sensitive MRSA to beta-lactam antibiotics.
Publisher: American Society for Microbiology
Date: 17-03-2022
DOI: 10.1128/MRA.00890-21
Abstract: Here, we present the completely closed genome sequence of Pasteurella multocida 17BRD-035, a bovine respiratory disease (BRD) pathogen from Queensland, Australia, with genes that confer resistance to β-lactams, tilmicosin, and tetracycline. It consists of a single 2,624,884-bp chromosome and an average GC content of 40.23% and belongs to the newly described Rural Industries Research and Development Corporation (RIRDC) sequence type 394.
Publisher: MDPI AG
Date: 17-06-2021
DOI: 10.3390/MICROORGANISMS9061322
Abstract: Bovine respiratory disease (BRD) causes high morbidity and mortality in beef cattle worldwide. Antimicrobial resistance (AMR) monitoring of BRD pathogens is critical to promote appropriate antimicrobial stewardship in veterinary medicine for optimal treatment and control. Here, the susceptibility of Mannheimia haemolytica and Pasteurella multicoda isolates obtained from BRD clinical cases (deep lung swabs at post-mortem) among feedlots in four Australian states (2014–2019) was determined for 19 antimicrobial agents. The M. haemolytica isolates were pan-susceptible to all tested agents apart from a single macrolide-resistant isolate (1/88 1.1%) from New South Wales (NSW). Much higher frequencies of P. multocida isolates were resistant to tetracycline (18/140 12.9%), tilmicosin (19/140 13.6%), tulathromycin/gamithromycin (17/140 12.1%), and icillin enicillin (6/140 4.6%). Five P. multocida isolates (3.6%), all obtained from NSW in 2019, exhibited dual resistance to macrolides and tetracycline, and a further two Queensland isolates from 2019 (1.4%) exhibited a multidrug-resistant phenotype to icillin enicillin, tetracycline, and tilmicosin. Random- lified polymorphic DNA (RAPD) typing identified a high degree of genetic homogeneity among the M. haemolytica isolates, whereas P. multocida isolates were more heterogeneous. Illumina whole genome sequencing identified the genes msr(E) and mph(E)encoding macrolide resistance, tet(R)-tet(H) or tet(Y) encoding tetracycline resistance, and blaROB-1 encoding icillin enicillin resistance in all isolates exhibiting a corresponding resistant phenotype. The exception was the tilmicosin-resistant, tulathromycin/gamithromycin-susceptible phenotype identified in two Queensland isolates, the genetic basis of which could not be determined. These results confirm the first emergence of AMR in M. haemolytica and P. multocida from BRD cases in Australia, which should be closely monitored.
Publisher: Springer Science and Business Media LLC
Date: 05-07-2009
DOI: 10.1038/NMETH.1347
Publisher: Informa UK Limited
Date: 19-12-2020
Publisher: Elsevier BV
Date: 04-2006
DOI: 10.1016/J.TIPS.2006.02.008
Abstract: ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many in idual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.
Publisher: Elsevier BV
Date: 03-2023
Publisher: Elsevier BV
Date: 04-2019
DOI: 10.1016/J.BMCL.2019.02.003
Abstract: A series of novel 5-methoxy-2,3-naphthalimide derivatives were designed, synthesized and evaluated for their biological activities. In particular, the ability of the compounds to synergize with antimicrobials, to inhibit Nile Red efflux, and to target AcrB was assayed. The results showed that the most of the tested compounds more sensitized the Escherichia coli BW25113 to the antibiotics than the parent compounds 7c and 15, which were able to inhibit Nile Red efflux. Significantly, compound A5 possessed the most potent antibacterial synergizing activity in combination with levofloxacin by 4 times and 16 times at the concentration of 8 and 16 µg/mL, respectively, whilst A5 could effectively abolish Nile Red efflux at 100 μM. Additionally, target effect of A5 was confirmed in the outer- or inner membrane permeabilization assays. Therefore, A5 is an excellent lead compound for further structural optimization.
Publisher: Frontiers Media SA
Date: 28-04-2015
Publisher: Elsevier BV
Date: 09-2021
Publisher: MDPI AG
Date: 23-03-2022
DOI: 10.3390/ANTIBIOTICS11040429
Abstract: Antimicrobial-resistant bacterial infections are a major and costly public health concern [...]
Publisher: Elsevier BV
Date: 06-2018
DOI: 10.1016/J.BMCL.2018.04.015
Abstract: A novel series of 5-methyl-2-phenylphenanthridium derivatives were displayed outstanding activity against a panel of antibiotic-sensitive and -resistant bacteria strains compared with their precursor sanguinarine, ciprofloxacin and oxacillin sodium. Compounds 7 l, 7m and 7n were found to display the most effective activity against five sensitive strains (0.06-2 μg/mL) and three resistant strains (0.25-4 μg/mL). The kinetic profiles indicated that compound 7l possessed the strongest bactericidal effect on S. aureus ATCC25923, with the MBC value of 16 μg/mL. The cell morphology and the FtsZ polymerization assays indicated that these compounds inhibited the bacterial proliferation by interfering the function of bacterial FtsZ. The SARs showed that all the 4-methyl-substituted 5-methyl-2-phenylphenanthridium subseries could be further investigated as the FtsZ-targeting antibacterial agents.
Publisher: Elsevier BV
Date: 06-2021
Publisher: Elsevier BV
Date: 02-2017
DOI: 10.1016/J.BMCL.2016.12.081
Abstract: Novel series of 3-substituted 2,6-difluorobenzamide derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their in vitro antibacterial activity against various phenotype of Gram-positive and Gram-negative bacteria, and their cell ision inhibitory activity against three representative strains. As a result, 3-chloroalkoxy derivative 7, 3-bromoalkoxy derivative 12 and 3-alkyloxy derivative 17 were found to exhibit the best antibacterial activity against Bacillus subtilis with MICs of 0.25-1μg/mL, and good activity (MIC<10μg/mL) against both susceptible and resistant Staphylococcus aureus. Additionally, all the three compounds displayed potent cell ision inhibitory activity with MIC values of below 1μg/mL against Bacillus subtilis and Staphylococcus aureus.
Publisher: Wiley
Date: 05-2022
Publisher: Portland Press Ltd.
Date: 28-02-2017
DOI: 10.1042/EBC20160063
Abstract: Gram-negative bacteria are responsible for a large proportion of antimicrobial-resistant infections in humans and animals. Among this class of bacteria are also some of the most successful environmental organisms. Part of this success is their adaptability to a variety of different niches, their intrinsic resistance to antimicrobial drugs and their ability to rapidly acquire resistance mechanisms. These mechanisms of resistance are not exclusive and the interplay of several mechanisms causes high levels of resistance. In this review, we explore the molecular mechanisms underlying resistance in Gram-negative organisms and how these different mechanisms enable them to survive many different stress conditions.
Publisher: MDPI AG
Date: 24-10-2020
DOI: 10.3390/MICROORGANISMS8111647
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen displaying high intrinsic antimicrobial resistance and the ability to thrive in different ecological environments. In this study, the ability of P. aeruginosa to develop simultaneous resistance to multiple antibiotics and disinfectants in different natural niches were investigated using strains collected from clinical s les, veterinary s les, and wastewater. The correlation between biocide and antimicrobial resistance was determined by employing principal component analysis. Molecular mechanisms linking biocide and antimicrobial resistance were interrogated by determining gene expression using RT-qPCR and identifying a potential genetic determinant for co- and cross-resistance using whole-genome sequencing. A subpopulation of P. aeruginosa isolates belonging to three sequence types was resistant against the common preservative benzalkonium chloride and showed cross-resistance to fluoroquinolones, cephalosporins, aminoglycosides, and multidrug resistance. Of these, the epidemiological high-risk ST235 clone was the most abundant. The overexpression of the MexAB-OprM drug efflux pump resulting from amino acid mutations in regulators MexR, NalC, or NalD was the major contributing factor for cross-resistance that could be reversed by an efflux pump inhibitor. This is the first comparison of antibiotic-biocide cross-resistance in s les isolated from different ecological niches and serves as a confirmation of laboratory-based studies on biocide adapted isolates. The isolates from wastewater had a higher incidence of multidrug resistance and biocide-antibiotic cross-resistance than those from clinical and veterinary settings.
Publisher: Elsevier BV
Date: 2016
DOI: 10.1016/J.SCITOTENV.2016.10.083
Abstract: Four mercury (Hg) contaminated soils with different pH (7.6, 8.5, 4.2 and 7.02) and total organic carbon contents (2.1, 2.2, 4 and 0.9%) were subjected to bioremediation utilizing a Hg volatilizing bacterial strain Sphingobium SA2 and nutrient amendment. In a field with ~280mg/kgHg, 60% of Hg was removed by bio-augmentation in 7days, and the removal was improved when nutrients were added. Whereas in artificially spiked soils, with ~100mg/kgHg, removal due to bio-augmentation was 33 to 48% in 14days. In the field contaminated soil, nutrient amendment alone without bio-augmentation removed 50% of Hg in 28days. Nutrient amendment also had an impact on Hg remediation in the spiked soils, but the best results were obtained when the strain and nutrients both were applied. The development of longer root lengths from lettuce and cucumber seeds grown in the remediated soils confirmed that the soil quality improved after bioremediation. This study clearly demonstrates the potential of Hg-reducing bacteria in remediation of Hg-contaminated soils. However, it is desirable to trap the volatilized Hg for enhanced bioremediation.
Publisher: Elsevier BV
Date: 03-2004
Publisher: Elsevier BV
Date: 04-1999
Abstract: The heat-stable protease from Chryseobacterium indologenes Ix9a was purified to homogeneity using immobilized metal affinity chromatography. The enzyme was characterized as a metalloprotease with an approximate relative molecular mass of 24,000, a pH optimum of 6.5, and a high temperature optimum (50 degrees C). The metal chelator EDTA and the Zn2+-specific chelator 1,10-phenanthroline were identified as inhibitors and atomic absorption analysis showed that the enzyme contained Ca2+ and Zn2+. The activity of the apoenzyme could be restored with Ca2+, Zn2+, Mg2+, and Co2+. Phosphoramidon and Gly-d-Phe did not inhibit Chryseobacterium indologenes Ix9a protease. Heat inactivation did not follow first order kinetics, but showed biphasic inactivation curves. The protease has a Km of 0.813 microg. ml-1 for casein as substrate. Amino acid analysis showed that the protease contains a high amount of small amino acids like glycine, alanine, and serine, but a low concentration of methionine and no cysteine at all. Electrospray mass spectrometry of proteolysis fragments formed when insulin B chain was hydrolyzed showed cleavage at the amino terminal of leucine, tyrosine, and phenylalanine. A hydrophobic amino acid at the carboxyl donating side seems to increase the rate of reaction.
Publisher: Elsevier BV
Date: 08-2023
Publisher: Elsevier BV
Date: 08-2017
DOI: 10.1016/J.BMCL.2017.06.005
Abstract: 5-Methylphenanthridium derivatives were designed, synthesized and evaluated for their in vitro antibacterial activity and cell ision inhibitory activity against various Gram-positive and -negative bacteria. Among them, compounds 5A2, 5B1, 5B2, 5B3, 5C1 and 5C2 displayed the best on-target antibacterial activity with an MIC value of 4µg/mL against B. subtilis ATCC9372 and S. pyogenes PS, showing over 2-fold better activity than sanguinarine. The SARs showed that the 5-methylphenanthridium derivatives with the alkyl side chains at the 2-postion, especially the straight alkyl side chains exerted better on-target antibacterial activity.
Publisher: Bentham Science Publishers Ltd.
Date: 24-03-2016
DOI: 10.2174/1389450116666151001103948
Abstract: The increasing multi-drug resistance has become a major threat to the public health. Overexpression of multidrug efflux pumps is one of the major mechanisms of drug resistance in bacteria. Since active efflux of antibacterial agents plays a significant role in mediating drug resistance in bacteria, the inhibition of efflux pumps appears to be a promising strategy to restore antibacterial potency. In recent years, in order to address this grave problem of multiple drug resistance mediated by efflux pump, a large number of efflux pump inhibitors have been discovered and tested, including natural products, antibiotics and synthetic molecules. This review mainly describes recent achievements in the search for new molecules that are able to inhibit efflux pumps in both Gram-positive and Gram-negative bacteria, in particular emphasis on natural and synthetic inhibitors of the NorA efflux pump in Staphylococcus aureus, MexAB-OprM efflux pump in Pseudomonas aeruginosa, and AcrAB-TolC efflux pump in Enterobacteriaceae, giving special attention to their mechanisms of action, structure-activity relationships and synergetic effect with clinically available antibiotics.
Publisher: American Chemical Society (ACS)
Date: 20-02-2023
Publisher: Springer US
Date: 27-11-2020
DOI: 10.1007/978-1-0716-0163-1_7
Abstract: Antimicrobial resistance (AMR) is rapidly becoming one of the great healthcare challenges. A common mechanism employed by pathogenic bacteria to avoid the action of certain antibiotics is to overexpress efflux pumps that can extrude these drugs from the cell rendering them ineffective. Small molecule inhibitors that target bacterial efflux pumps provide a route toward reversing AMR. Here, we describe the application of surface plasmon resonance (SPR) technology to characterize protein:small molecule interactions between the inner membrane protein AcrB subunit of the Escherichia coli AcrA-AcrB-TolC efflux pump and its substrates and novel inhibitors. The SPR assay provides quantitative data about the kinetics of binding that can help guide the development of new chemotherapies to combat AMR.
Publisher: Portland Press Ltd.
Date: 04-2019
DOI: 10.1042/BSR20180474
Abstract: The speed at which bacteria develop antimicrobial resistance far outpace drug discovery and development efforts resulting in untreatable infections. The World Health Organisation recently released a list of pathogens in urgent need for the development of new antimicrobials. The organisms that are listed as the most critical priority are all Gram-negative bacteria resistant to the carbapenem class of antibiotics. Carbapenem resistance in these organisms is typified by intrinsic resistance due to the expression of antibiotic efflux pumps and the permeability barrier presented by the outer membrane, as well as by acquired resistance due to the acquisition of enzymes able to degrade β-lactam antibiotics. In this perspective article we argue the case for reversing resistance by targeting these resistance mechanisms – to increase our arsenal of available antibiotics and drastically reduce antibiotic discovery times – as the most effective way to combat antimicrobial resistance in these high priority pathogens.
Publisher: Elsevier BV
Date: 09-2003
Publisher: Portland Press Ltd.
Date: 12-1999
DOI: 10.1042/BST0270893
Publisher: Elsevier BV
Date: 10-2019
DOI: 10.1016/J.WATRES.2018.05.055
Abstract: Many pathogens can enter into a viable but nonculturable (VBNC) state in response to harsh environmental stresses. Bacteria in this state can retain certain features of viable cells, such as cellular integrity, metabolic activity, or virulence and may present health risks associated with drinking water. In this study, we investigated the ability of chlorination and chloramination, which are widely used methods to disinfect drinking water, to induce Escherichia coli into a VBNC state. After treatment with chlorine and chloramine at concentrations of 1, 2, 3, and 4 mg/L, the counts of culturable E. coli cells decreased from 10
Publisher: Elsevier BV
Date: 2020
DOI: 10.1016/J.EJMECH.2019.111910
Abstract: Drug efflux pumps confer multidrug resistance to dangerous bacterial pathogens which makes these proteins promising drug targets. Herein, we present initial chemical optimization and structure-activity relationship (SAR) data around a previously described efflux pump inhibitor, nordihydroguaretic acid (NDGA). Four series of novel NDGA analogues that target Escherichia coli AcrB were designed, synthesized and evaluated for their ability to potentiate the activity of antibiotics, to inhibit AcrB-mediated substrate efflux and reduce off-target activity. Nine novel structures were identified that increased the efficacy of a panel of antibiotics, inhibited drug efflux and reduced permeabilization of the bacterial outer and inner membranes. Among them, WA7, WB11 and WD6 possessing broad-spectrum antimicrobial sensitization activity were identified as NDGA analogues with favorable properties as potential AcrB inhibitors, demonstrating moderate improvement in potency as compared to NDGA. In particular, WD6 was the most broadly active analogue improving the activity of all four classes of antibacterials tested.
Publisher: Elsevier BV
Date: 12-2020
Publisher: Elsevier BV
Date: 09-2003
DOI: 10.1016/S0924-8579(03)00212-7
Abstract: The crystallization of MsbA, an ATP-binding cassette (ABC) transporter involved in the transport of Lipid A in Escherichia coli, provided a fascinating glimpse into the high-resolution structure of an ABC transporter at 4.8 A. The E. coli crystal structure of MsbA reveals a dimer. Although the structure of the MsbA monomer is consistent with the biochemistry of ABC transporters, including the human multidrug resistance P-glycoprotein, the interface between the monomers in the MsbA dimer may not reflect the biologically relevant interface. We considered the interface in a two-armed MsbA dimer, named spiral. Our findings indicate that (i) the spiral MsbA dimer may have biological relevance for ABC transporters that interact with lipophilic substrates, and (ii) the dimer interface observed in the crystal structure of E. coli MsbA represents a crystallization artefact. A comparison of the spiral MsbA dimer with the recently published structure of MsbA in Vibrio cholera is also described.
Publisher: American Society for Microbiology
Date: 03-10-2023
Publisher: Wiley
Date: 15-02-2008
Publisher: American Chemical Society (ACS)
Date: 27-10-2023
Publisher: Elsevier BV
Date: 06-2018
DOI: 10.1016/J.VETMIC.2018.04.004
Abstract: Methicillin-resistant coagulase-negative staphylococci (MRCoNS) have recently emerged as a significant cause of bovine mastitis worldwide. Here we describe the isolation of MRCoNS from cases of bovine mastitis from a single dairy farm in Australia. Fourteen CoNS isolates were identified as MRCoNS on the basis of having an oxacillin MIC of ≥0.5 μg/mL. The isolates were speciated as S. chromogenes (n = 1) S. fleurettii (n = 1), S. haemolyticus (n = 2), S. sciuri (n = 5), S. simulans (n = 1) S. succinus (n = 2) and S. xylosus (n = 2). Five of the isolates (S. fleuretti, S. haemolyticus S. sciuri and two S. succinus) were mecA-positive. We also detected a previously described S. sciuri mecA homolog in four oxacillin-resistant S. sciuri isolates. The remainder of the putative MRCoNS did not contain any mecA-related resistance determinants in their genomes. Comparative genomic analysis of three previously published S. sciuri isolates, from humans, a squirrel and a cereal crop (rice), and a representative isolate from our study demonstrated clustering and a high degree of genetic homogeneity (>95%), suggesting S. sciuri has low host specificity. In conclusion, CoNS, in particular S. sciuri, may act as a reservoir for SCCmec elements that can easily be spread between different host species by direct cross-infection.
Publisher: Elsevier BV
Date: 04-2017
DOI: 10.1016/J.BBAMEM.2017.08.024
Abstract: Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in other processes such as virulence and biofilm formation. Hence efflux pump inhibition, as a means to reverse antimicrobial resistance in clinically relevant pathogens, has gained increased momentum over the past two decades. Significant advances in the structural and functional analysis of AcrB have informed the selection of efflux pump inhibitors (EPIs). However, an accurate method to determine the kinetics of efflux pump inhibition was lacking. In this study we standardised and optimised surface plasmon resonance (SPR) to probe the binding kinetics of substrates and inhibitors to AcrB. The SPR method was also combined with a fluorescence drug binding method by which affinity of two fluorescent AcrB substrates were determined using the same conditions and controls as for SPR. Comparison of the results from the fluorescent assay to those of the SPR assay showed excellent correlation and provided validation for the methods and conditions used for SPR. The kinetic parameters of substrate (doxorubicin, novobiocin and minocycline) binding to AcrB were subsequently determined. Lastly, the kinetics of inhibition of AcrB were probed for two established inhibitors (phenylalanine arginyl β-naphthylamide and 1-1-naphthylmethyl-piperazine) and three novel EPIs: 4-isobutoxy-2-naphthamide (A2), 4-isopentyloxy-2-naphthamide (A3) and 4-benzyloxy-2-naphthamide (A9) have also been probed. The kinetic data obtained could be correlated with inhibitor efficacy and mechanism of action. This study is the first step in the quantitative analysis of the kinetics of inhibition of the clinically important RND-class of multidrug efflux pumps and will allow the design of improved and more potent inhibitors of drug efflux pumps. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.
Publisher: Elsevier BV
Date: 07-2022
DOI: 10.1016/J.VETMIC.2022.109460
Abstract: Histophilus somni is a prevalent commensal organism of the upper respiratory tract of cattle and a major causative agent of bovine respiratory disease (BRD) and other syndromes including myocarditis and infectious thromboembolic meningoencephalitis. This study investigated the antimicrobial susceptibility and phylogenetic relationships of H. somni isolates obtained from lung, heart, and other tissues at post-mortem as well as nasal mucosa swabs from cases of BRD in Australian feedlots (2004-2019). Broth microdilution Minimal Inhibitory Concentration (MIC) assays were determined for 19 antimicrobials using three different media (CLSI approved Veterinary Fastidious Medium [VFM], Mueller-Hinton fastidious broth medium supplemented with yeast extract [MHF-Y] and Columbia Broth [CB] supplemented with 5% lysed horse blood). For all antimicrobials, MICs obtained using CB medium were identical or within 1 dilution step of the MICs obtained for VFM and MHF-Y media. Therefore, CB may be a suitable medium for H. somni antimicrobial susceptibility testing similar to MHF-Y medium. None of the 70 Australian H. somni isolates exhibited resistance to antimicrobials with CLSI breakpoints including those commonly used in the treatment of BRD in Australia (first-line tetracyclines [chlortetracycline and oxytetracycline], second-line macrolides [tulathromycin], and third-line extended-spectrum cephalosporin [ceftiofur]). Whole-genome sequence analysis of 65 H. somni isolates for genomic single nucleotide polymorphism differences identified four phylogenetic clusters, each containing isolates from different Australian states, feedlots and tissue sources that clustered together. These findings demonstrate limited genetic ersity and the absence of significant antimicrobial resistance among Australian isolates of H. somni isolated from feedlot cattle.
Publisher: Research Square Platform LLC
Date: 17-05-2023
DOI: 10.21203/RS.3.RS-2670219/V1
Abstract: The increasing rate of carbapenem-resistant bacteria within healthcare environments is an issue of great concern that needs urgent attention. This resistance is driven by metallo-β-lactamases (MBLs), which can catalyse the hydrolysis of almost all clinically available β-lactams and are resistant to all the available β-lactamase inhibitors. In this study, a novel MBL was identified in a multidrug resistant isolate of the opportunistic pathogen, Chryseobacterium indologenes . Sequence analysis predicted this MBL (CIM-1) to be a lipoprotein with an atypical lipobox. Characterization of CIM-1 revealed it to be a membrane-associated, high-affinity carbapenemase with a broad spectrum of activity that includes all cephalosporins and carbapenems. This is the first report of the identification and characterisation of an MBL with an atypical lipobox and only the second lipidated MBL to be characterised after critically important NDM-1. We also identified more lipidated MBLs with non-canonical lipoboxes highlighting the necessity in further investigation of lipidated MBLs.
Publisher: Springer Science and Business Media LLC
Date: 20-04-2014
DOI: 10.1038/NATURE13205
Publisher: Oxford University Press (OUP)
Date: 21-05-2019
DOI: 10.1111/JAM.14298
Abstract: The antimicrobial activity of cinnamon essential oil and cinnamaldehyde against bacterial and fungal pathogens associated with canine otitis externa, as well as the effect of their combination with EDTA were investigated. Antimicrobial susceptibility testing was performed using the broth microdilution method while spot-plating technique was used to determine their bactericidal activity. Time-kill kinetics and checkerboard assays were performed to confirm the bactericidal activity and combination effects of the compounds. Cinnamon oil and cinnamaldehyde exhibited antimicrobial activity against Gram-positive and Gram-negative pathogens, as well as Malassezia pachydermatis. Synergistic interaction was shown when EDTA (672 μg ml Cinnamon essential oil and cinnamaldehyde, either used alone or in combination with EDTA, were effective against the causative micro-organisms of canine otitis externa. The data suggest that cinnamaldehyde could be a promising antimicrobial agent against canine otitis externa. This study shows that cinnamon essential oil and cinnamaldehyde, especially the latter, could be used in combination with EDTA as novel treatment for sensitive and resistant bacterial and fungal pathogens involved in canine otitis externa.
Publisher: Informa UK Limited
Date: 10-2021
DOI: 10.1080/14786419.2021.1983570
Abstract: Bioactivity-guided fraction of an extract of
Publisher: American Society for Microbiology
Date: 15-03-2018
DOI: 10.1128/JVI.01774-17
Abstract: The innate immune response plays a pivotal role during human cytomegalovirus (HCMV) primary infection. Indeed, HCMV infection of primary fibroblasts rapidly triggers strong induction of type I interferons (IFN-I), accompanied by proinflammatory cytokine release. Here, we show that primary human foreskin fibroblasts (HFFs) infected with a mutant HCMV TB40/E strain unable to express UL83-encoded pp65 (v65Stop) produce significantly higher IFN-β levels than HFFs infected with the wild-type TB40/E strain or the pp65 revertant (v65Rev), suggesting that the tegument protein pp65 may d en IFN-β production. To clarify the mechanisms through which pp65 inhibits IFN-β production, we analyzed the activation of the cGAS/STING/IRF3 axis in HFFs infected with either the wild type, the revertant v65Rev, or the pp65-deficient mutant v65Stop. We found that pp65 selectively binds to cGAS and prevents its interaction with STING, thus inactivating the signaling pathway through the cGAS/STING/IRF3 axis. Consistently, addition of exogenous cGAMP to v65Rev-infected cells triggered the production of IFN-β levels similar to those observed with v65Stop-infected cells, confirming that pp65 inactivation of IFN-β production occurs at the cGAS level. Notably, within the first 24 h of HCMV infection, STING undergoes proteasome degradation independently of the presence or absence of pp65. Collectively, our data provide mechanistic insights into the interplay between HCMV pp65 and cGAS, leading to subsequent immune evasion by this prominent DNA virus. IMPORTANCE Primary human foreskin fibroblasts (HFFs) produce type I IFN (IFN-I) when infected with HCMV. However, we observed significantly higher IFN-β levels when HFFs were infected with HCMV that was unable to express UL83-encoded pp65 (v65Stop), suggesting that pp65 (pUL83) may constitute a viral evasion factor. This study demonstrates that the HCMV tegument protein pp65 inhibits IFN-β production by binding and inactivating cGAS early during infection. In addition, this inhibitory activity specifically targets cGAS, since it can be bypassed via the addition of exogenous cGAMP, even in the presence of pp65. Notably, STING proteasome-mediated degradation was observed in both the presence and absence of pp65. Collectively, our data underscore the important role of the tegument protein pp65 as a critical molecular hub in HCMV's evasion strategy against the innate immune response.
Publisher: MDPI AG
Date: 10-08-2021
DOI: 10.3390/MICROORGANISMS9081697
Abstract: One approach to combat the increasing incidence of multidrug-resistant (MDR) bacterial pathogens involves repurposing existing compounds with known safety and development pathways as new antibacterial classes with potentially novel mechanisms of action. Here, triclabendazole (TCBZ), a drug originally developed to treat Fasciola hepatica (liver fluke) in sheep and cattle, and later in humans, was evaluated as an antibacterial alone or in combination with sub-inhibitory concentrations of polymyxin B (PMB) against clinical isolates and reference strains of key Gram-positive and Gram-negative bacteria. We show for the first time that in vitro, TCBZ selectively kills methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius at a minimum inhibitory concentration (MIC) range of 2–4 µg/mL, and vancomycin-resistant enterococci at a MIC range of 4–8 µg/mL. TCBZ also inhibited key Gram-negative bacteria in the presence of sub-inhibitory concentrations of PMB, returning MIC90 values of 1 µg/mL for Escherichia coli, 8 µg/mL for Klebsiella pneumoniae, 2 µg/mL for Acinetobacter baumannii and 4 µg/mL for Pseudomonasaeruginosa. Interestingly, TCBZ was found to be bacteriostatic against intracellular S. aureus but bactericidal against intracellular S. pseudintermedius. Additionally, TCBZ’s favourable pharmacokinetic (PK) and pharmacodynamic (PD) profile was further explored by in vivo safety and efficacy studies using a bioluminescent mouse model of S. aureus sepsis. We show that repeated four-hourly oral treatment of mice with 50 mg/kg TCBZ after systemic S. aureus challenge resulted in a significant reduction in S. aureus populations in the blood to 18 h post-infection (compared to untreated mice) but did not clear the bacterial infection from the bloodstream, consistent with in vivo bacteriostatic activity. These results indicate that additional pharmaceutical development of TCBZ may enhance its PK/PD, allowing it to be an appropriate candidate for the treatment of serious MDR bacterial pathogens.
Publisher: S. Karger AG
Date: 2007
DOI: 10.1159/000099641
Abstract: LmrP is a secondary active multidrug transporter from i Lactococcus lactis /i . The protein belongs to the major facilitator superfamily and utilizes the electrochemical proton gradient (inside negative and alkaline) to extrude a wide range of lipophilic cations from the cell. Previous work has indicated that ethidium, a monovalent cationic substrate, is exported by LmrP by electrogenic antiport with two (or more) protons. This observation raised the question whether these protons are translocated sequentially along the same pathway, or through different routes. To address this question, we constructed a 3-D homology model of LmrP based on the high-resolution structure of the glycerol-3P/Pi antiporter GlpT from i Escherichia coli /i , and we tested by mutagenesis the possible proton conduction points suggested by this model. Similar to the template, LmrP is predicted to contain an internal cavity formed at the interface between the two halves of the transporter. On the surface of this cavity lie two clusters of polar, aromatic and carboxylate residues with potentially important function in proton shuttling. Cluster 1 in the C-terminal half contains D235 and E327 in immediate proximity of each other, and is located near the apex of the cavity. Cluster 2 in the N-terminal half contains D142. Analyses of LmrP mutants containing charge-conservative or carboxyl-to-amide replacements at positions 142, 235 and 327 suggest that D142 is part of a dedicated proton translocation pathway in the ethidium translocation reaction. In contrast, D235 and E327 are part of an independent pathway, in which D235 interacts with protons. E327 appears to modulate the pKa of D235 and plays a role in the interaction with ethidium. These results are consistent with the proposal that major facilitator superfamily proteins consist of two membrane domains, one of which is involved in substrate binding and the other in ion coupling, and they indicate that there are two proton conduction pathways at play in the transport mechanism.
Publisher: Elsevier BV
Date: 09-2014
DOI: 10.1016/J.PEP.2014.06.012
Abstract: The FeoB Fe(II) transporter from the drug resistant pathogen, Pseudomonas aeruginosa is essential for ferrous iron transport and is implicated in virulence and biofilm development. Hence it is an attractive target for the development of new anti-infective drugs. FeoB is an intriguing protein that consists of a cytosolic N-terminal GTPase domain and an integral membrane domain which most likely acts as ferrous iron permease. Characterisation of FeoB is critical for developing therapeutics aimed at inhibiting this protein. However, structural and functional analysis of FeoB is h ered by the lack of high yield homogenously pure protein which is monodisperse, stable and active in solution. Here we describe the optimised procedure for the recombinant expression of FeoB from P. aeruginosa and provide an evaluation of the most favourable purification, pH and detergent conditions. The functional reconstitution of FeoB in liposomes is also described. This represents the first detailed procedure for obtaining a pure, active and stable FeoB solution in milligram quantities which would be amenable to biochemical, biophysical and structural studies.
Publisher: American Society for Microbiology
Date: 15-09-2005
DOI: 10.1128/JB.187.18.6363-6369.2005
Abstract: MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis , our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis . MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with K i values of 16 and 11 μM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a K i of 57 μM, in a similar range as the K i for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbA-mediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005).
Publisher: Elsevier BV
Date: 03-2020
Publisher: Elsevier BV
Date: 03-2018
DOI: 10.1016/J.BMCL.2018.02.001
Abstract: 3-Methoxybenzamide (3-MBA) derivatives have been identified as novel class of potent antibacterial agents targeting the bacterial cell ision protein FtsZ. As one of isosteres for the amide group, 1,2,3-triazole can mimic the topological and electronic features of the amide, which has gained increasing attention in drug discovery. Based on these considerations, we prepared a series of 1H-1,2,3-triazole-containing 3-MBA analogues via isosteric replacement of the terminal amide with triazole, which had increased antibacterial activity. This study demonstrated the possibility of developing the 1H-1,2,3-triazole group as a terminal amide-mimetic element which was capable of both keeping and modulating amide-related bioactivity. Surprisingly, a different action mode of these new 1H-1,2,3-triazole-containing analogues was observed, which could open new opportunities for the development of antibacterial agents.
Publisher: Royal Society of Chemistry (RSC)
Date: 2000
DOI: 10.1039/A809409H
Publisher: American Society for Microbiology
Date: 18-10-2023
DOI: 10.1128/AAC.00424-23
Publisher: Frontiers Media SA
Date: 30-11-2022
DOI: 10.3389/FCIMB.2022.1063407
Abstract: The spread of artemisinin resistant Plasmodium falciparum parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites via two mechanisms: ‘delayed death’ by inhibiting the bacterium-like ribosomes of the apicoplast, and ‘quick-killing’ that kills rapidly across the entire blood stage development. Here, 22 azithromycin analogues were explored for delayed death and quick-killing activities against P. falciparum (the most virulent human malaria) and P. knowlesi (a monkey parasite that frequently infects humans). Seventeen analogues showed improved quick-killing against both Plasmodium species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for P. falciparum and P. knowlesi , respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of P. falciparum parasites (& hrs treatment) and were & -fold more selective against P. falciparum than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite Toxoplasma gondii and the gram-positive bacterium Streptococcus pneumoniae did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment. The azithromycin analogues characterised in this study expand the structural ersity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.
Publisher: Wiley
Date: 28-09-2016
DOI: 10.1111/CBDD.12658
Abstract: Novel series of 3‐ O ‐arylalkylbenzamide and 3‐ O ‐arylalkyl‐2,6‐difluorobenzamide derivatives were synthesized and evaluated for their on‐target activity and antibacterial activity. The results indicated that the 3‐ O ‐arylalkyl‐2,6‐difluorobenzamide derivatives possessed much better on‐target activity and antibacterial activity than the 3‐ O ‐arylalkylbenzamide derivatives. Among them, 3‐ O ‐chlorobenzyl derivative 36 was the most effective in antibacterial activity (0.5, 4, and 8 μ g/mL) against Bacillus subtilis ATCC 9372, methicillin‐resistant Staphylococcus aureus ATCC 29213, and penicillin‐resistant Staphylococcus aureus PR , while 3‐ O ‐methylbenzyl derivative 41 only exhibited the most potent activity (2 μ g/mL) against Staphylococcus aureus ATCC 25923.
Publisher: Elsevier BV
Date: 02-2008
DOI: 10.1016/J.BCP.2007.10.022
Abstract: LmrA is an ATP-binding cassette (ABC) multidrug transporter from Lactococcus lactis, and is a structural homologue of the human multidrug resistance P-glycoprotein (ABCB1), the overexpression of which is associated with multidrug resistance in tumours. We recently observed that a truncated version of LmrA lacking the nucleotide-binding domain mediates a proton motive force-dependent ethidium transport reaction by catalyzing proton-ethidium symport. This finding raised the question whether proton motive force-dependent transport can also be observed for other drugs, and whether this reaction is also relevant for full-length LmrA. Furthermore, the observations on LmrA-MD raised the question whether ATP-dependent transport by LmrA in intact cells could be due to the activity of independent ABC transporters that might become upregulated in the lactococcal cells due to the overexpression of LmrA the recently identified ABC multidrug transporter LmrCD was put forward as a possible candidate. Here, we investigated the energy coupling to the transport of the hiphilic dye Hoechst 33342 in proteoliposomes containing purified LmrA. For this purpose, LmrA was obtained from lactococcal cells lacking the genomic lmrA and lmrCD genes, in which LmrA was expressed from a plasmid. To separate ATP-dependence from proton motive force-dependence, we also used mutant LmrA proteins, which were affected in their ability to hydrolyse ATP. Our studies in proteoliposomes demonstrate that LmrA can catalyze Hoechst 33342 transport independent of auxiliary proteins, in an ATP-dependent fashion and a transmembrane chemical proton gradient (interior acidic)-dependent fashion.
Publisher: Elsevier BV
Date: 07-2019
DOI: 10.1016/J.MICPATH.2019.05.016
Abstract: Stone formation and catheter blockage are major complications of Proteus UTIs. In this study, we investigated the ability of allicin to inhibit P. mirabilis-induced struvite crystallization and catheter blockage using a synthetic bladder model. Struvite crystallization inhibition study was carried out using P. mirabilis lysate as urease enzyme source in synthetic urine (SU). Struvite productions were monitored by phase contrast light microscopy and measurements of pH, Mg
Publisher: MDPI AG
Date: 17-03-2021
DOI: 10.3390/ANTIBIOTICS10030307
Abstract: In this study, we optimized and compared different transmission electron microscopy (TEM) methods to visualize changes to Gram-negative bacterial morphology induced by treatment with a robenidine analogue (NCL195) and colistin combination. Aldehyde-fixed bacterial cells (untreated, treated with colistin or NCL195 + colistin) were prepared using conventional TEM methods and compared with ultrathin Tokuyasu cryo-sections. The results of this study indicate superiority of ultrathin cryo-sections in visualizing the membrane ultrastructure of Escherichia coli and Pseudomonas aeruginosa, with a clear delineation of the outer and inner membrane as well as the peptidoglycan layer. We suggest that the use of ultrathin cryo-sectioning can be used to better visualize and understand drug interaction mechanisms on the bacterial cell membrane.
Publisher: MDPI AG
Date: 15-04-2022
DOI: 10.3390/MICROORGANISMS10040825
Abstract: The authors wish to make the following corrections to this paper [...]
Publisher: MDPI AG
Date: 06-01-2022
DOI: 10.3390/ANTIBIOTICS11010065
Abstract: In this study, we investigated the potential of an analogue of robenidine (NCL179) to expand its chemical ersity for the treatment of multidrug-resistant (MDR) bacterial infections. We show that NCL179 exhibits potent bactericidal activity, returning minimum inhibitory concentration/minimum bactericidal concentrations (MICs/MBCs) of 1–2 µg/mL against methicillin-resistant Staphylococcus aureus, MICs/MBCs of 1–2 µg/mL against methicillin-resistant S. pseudintermedius and MICs/MBCs of 2–4 µg/mL against vancomycin-resistant enterococci. NCL179 showed synergistic activity against clinical isolates and reference strains of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa in the presence of sub-inhibitory concentrations of colistin, whereas NCL179 alone had no activity. Mice given oral NCL179 at 10 mg/kg and 50 mg/kg (4 × doses, 4 h apart) showed no adverse clinical effects and no observable histological effects in any of the organs examined. In a bioluminescent S. aureus sepsis challenge model, mice that received four oral doses of NCL179 at 50 mg/kg at 4 h intervals exhibited significantly reduced bacterial loads, longer survival times and higher overall survival rates than the vehicle-only treated mice. These results support NCL179 as a valid candidate for further development to treat MDR bacterial infections as a stand-alone antibiotic or in combination with existing antibiotic classes.
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2021
End Date: 05-2023
Amount: $860,365.00
Funder: Australian Research Council
View Funded ActivityStart Date: 06-2015
End Date: 12-2016
Amount: $860,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2017
End Date: 07-2018
Amount: $510,000.00
Funder: Australian Research Council
View Funded Activity