Australian Infectious Diseases Research Center

St. Lucia, Australia

Australian Infectious Diseases Research Center

St. Lucia, Australia
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Lee K.T.,ARC Technology | Coffey J.W.,ARC Technology | Robinson K.J.,ARC Technology | Muller D.A.,ARC Technology | And 11 more authors.
Langmuir | Year: 2017

Immunoassays are ubiquitous across research and clinical laboratories, yet little attention is paid to the effect of the substrate material on the assay performance characteristics. Given the emerging interest in wearable immunoassay formats, investigations into substrate materials that provide an optimal mix of mechanical and bioanalytical properties are paramount. In the course of our research in developing wearable immunoassays which can penetrate skin to selectively capture disease antigens from the underlying blood vessels, we recently identified significant differences in immunoassay performance between gold and polycarbonate surfaces, even with a consistent surface modification procedure. We observed significant differences in PEG density, antibody immobilization, and nonspecific adsorption between the two substrates. Despite a higher PEG density formed on gold-coated surfaces than on aminefunctionalized polycarbonate, the latter revealed a higher immobilized capture antibody density and lower nonspecific adsorption, leading to improved signal-to-noise ratios and assay sensitivities. The major conclusion from this study is that in designing wearable bioassays or biosensors, the design and its effect on the antifouling polymer layer can significantly affect the assay performance in terms of analytical specificity and sensitivity. (Figure Presented). © 2016 American Chemical Society.


Totsika M.,Australian Infectious Diseases Research Center | Totsika M.,University of Queensland | Upton M.,University of Manchester | Beatson S.A.,Australian Infectious Diseases Research Center | And 4 more authors.
Journal of Infectious Diseases | Year: 2013

Background. Escherichia coli O25b:H4-ST131 represents a predominant clone of multidrug-resistant uropathogens currently circulating worldwide in hospitals and the community. Urinary tract infections (UTIs) caused by E. coli ST131 are typically associated with limited treatment options and are often recurrent.Methods. Using established mouse models of acute and chronic UTI, we mapped the pathogenic trajectory of the reference E. coli ST131 UTI isolate, strain EC958.Results. We demonstrated that E. coli EC958 can invade bladder epithelial cells and form intracellular bacterial communities early during acute UTI. Moreover, E. coli EC958 persisted in the bladder and established chronic UTI. Prophylactic antibiotic administration failed to prevent E. coli EC958-mediated UTI. However, 1 oral dose of a small-molecular-weight compound that inhibits FimH, the type 1 fimbriae adhesin, significantly reduced bacterial colonization of the bladder and prevented acute UTI. Treatment of chronically infected mice with the same FimH inhibitor lowered their bladder bacterial burden by >1000-fold.Conclusions. In this study, we provide novel insight into the pathogenic mechanisms used by the globally disseminated E. coli ST131 clone during acute and chronic UTI and establish the potential of FimH inhibitors as an alternative treatment against multidrug-resistant E. coli. © 2013 The Author.


Wailan A.M.,University of Queensland | Sidjabat H.E.,University of Queensland | Yam W.K.,University of Queensland | Yam W.K.,Agency for Science, Technology and Research Singapore | And 21 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2016

blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1, and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A, and ISCR1 may have been involved in the acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones, but different regions carrying blaNDM are found in different locations. Tn3-derived inverted-repeat transposable elements (TIME) appear to have been involved in the acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterization of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements, and plasmids and provide insights into the possible routes for transmission of blaNDM genes among species of the Enterobacteriaceae family. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


Lee K.T.,University of Queensland | Lee K.T.,ARC Technology | Muller D.A.,University of Queensland | Muller D.A.,Australian Infectious Diseases Research Center | And 13 more authors.
Analytical Chemistry | Year: 2014

Herein we demonstrate the use of a wearable device that can selectively capture two distinct circulating protein biomarkers (recombinant P. falciparum rPfHRP2 and total IgG) from the intradermal fluid of live mice in situ, for subsequent detection in vitro. The device comprises a microprojection array that, when applied to the skin, penetrates the outer skin layers to interface directly with intradermal fluid. Because of the complexity of the biological fluid being sampled, we investigated the effects of solution conditions on the attachment of capture antibodies, to optimize the assay detection limit both in vitro and on live mice. For detection of the target antigen diluted in 20% serum, immobilization conditions favoring high antibody surface density (low pH, low ionic strength) resulted in 100-fold greater sensitivity in comparison to standard conditions, yielding a detection limit equivalent to the plate enzyme-linked immunosorbent assay (ELISA). We also show that blocking the device surface to reduce nonspecific adsorption of target analyte and host proteins does not significantly change sensitivity. After injecting mice with rPfHRP2 via the tail vein, we compared analyte levels in both plasma and skin biopsies (cross-sectional area same as the microprojection array), observing that skin samples contained the equivalent of ∼8 μL of analyte-containing plasma. We then applied the arrays to mice, showing that surfaces coated with a high density of antibodies captured a significant amount of the rPfHRP2 target while the standard surface showed no capture in comparison to the negative control. Next, we applied a triplex device to both control and rPfHRP2-treated mice, simultaneously capturing rPfHRP2 and total IgG (as a positive control for skin penetration) in comparison to a negative control device. We conclude that such devices can be used to capture clinically relevant, circulating protein biomarkers of infectious disease via the skin, with potential applications as a minimally invasive and lab-free biomarker detection platform. © 2014 American Chemical Society.


Corrie S.R.,University of Queensland | Corrie S.R.,Australian Infectious Diseases Research Center | Corrie S.R.,ARC Technology | Coffey J.W.,University of Queensland | And 8 more authors.
Analyst | Year: 2015

Biosensors are being developed to provide rapid, quantitative, diagnostic information to clinicians in order to help guide patient treatment, without the need for centralised laboratory assays. The success of glucose monitoring is a key example of where technology innovation has met a clinical need at multiple levels-from the pathology laboratory all the way to the patient's home. However, few other biosensor devices are currently in routine use. Here we review the challenges and opportunities regarding the integration of biosensor techniques into body fluid sampling approaches, with emphasis on the point-of-care setting. © 2015 The Royal Society of Chemistry.


Yeow B.,University of Queensland | Coffey J.W.,University of Queensland | Muller D.A.,University of Queensland | Muller D.A.,Australian Infectious Diseases Research Center | And 5 more authors.
Analytical Chemistry | Year: 2013

Herein, we report the fabrication, characterization, and testing of a polymer microprojection array, for the direct and selective capture of circulating biomarkers from the skin of live mice. First, we modified polycarbonate wafers using an electrophilic aromatic substitution reaction with nitric acid to insert aromatic nitro-groups into the benzene rings, followed by treatment with sodium borohydride to reduce the nitro-groups to primary amines. Initial characterization by ultraviolet-visible (UV-vis) spectroscopy suggested that increasing acid concentration led to increased depth of material modification and that this was associated with decreased surface hardness and slight changes in surface roughness. Chemical analysis with X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance fourier transform infrared (ATR-FT-IR) spectroscopy showed nitrogen species present at the surface for all acid concentrations used, but subsurface nitrogen species were only observed at acid concentrations >35%. The nitrogen species were identified as a mixture of nitro, imine, and amine groups, and following reduction, there was sufficient amounts of primary amine groups for covalent attachment of a polyethylene glycol antifouling layer and protein capture probes, as determined by colorimetric and radiometric assays. Finally, the modification scheme was applied to polycarbonate microprojection arrays, and we show that these devices achieve flank skin penetration depths and biomarker yields comparable with our previously reported gold-coated silicon arrays, with very low nonspecific binding even in 10% mouse serum (in vitro) or directly in mouse skin (in vivo). This study is the first demonstration showing the potential utility of polymer microprojections in immunodiagnostics applications. © 2013 American Chemical Society.


Arras S.D.M.,Australian Infectious Diseases Research Center | Arras S.D.M.,University of Queensland | Chua S.M.H.,Australian Infectious Diseases Research Center | Chua S.M.H.,University of Queensland | And 8 more authors.
PLoS ONE | Year: 2016

Low rates of homologous integration have hindered molecular genetic studies in Cryptococcus neoformans over the past 20 years, and new tools that facilitate genome manipulation in this important pathogen are greatly needed. To this end, we have investigated the use of a Class 2 CRISPR system in C. neoformans (formerly C. neoformans var. grubii). We first expressed a derivative of the Streptococcus pyogenes Cas9 nuclease in C. neoformans, and showed that it has no effect on growth, production of virulence factors in vitro, or virulence in a murine inhalation model. In proof of principle experiments, we tested the CAS9 construct in combination with multiple self-cleaving guide RNAs targeting the wellcharacterized phosphoribosylaminoamidazole carboxylase-encoding ADE2 gene. Utilizing combinations of transient and stable expression of our constructs, we revealed that functionality of our CRISPR constructs in C. neoformans is dependent upon the CAS9 construct being stably integrated into the genome, whilst transient expression of the guide RNA is sufficient to enhance rates of homologous recombination in the CAS9 genetic background. Given that the presence of the CRISPR nuclease does not influence virulence in a murine inhalation model, we have successfully demonstrated that this system is compatible with studies of C. neoformans pathogenesis and represents a powerful tool that can be exploited by researchers in the field. © 2016 Arras et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Wailan A.M.,University of Queensland | Sartor A.L.,Queensland Government | Zowawi H.M.,University of Queensland | Zowawi H.M.,King Saud bin Abdulaziz University for Health Sciences | And 6 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2015

The carbapenem resistance determinant blaNDM-1 has been found in various Gram-negative bacteria and upon different plasmid replicon types (Inc). Here, we present four patients within two hospitals in Pakistan harboring between two and four NDM-1- producing Gram-negative bacilli of different species coresident in their stool samples. We characterize the blaNDM-1 genetic contexts of these 11 NDM-1-producing Gram-negative bacilli in addition to other antimicrobial resistance mechanisms, plasmid replicon profiles, and sequence types (STs) in order to understand the underlying acquisition mechanisms of carbapenem resistance within these bacteria. Two common plasmid types (IncN2 and IncA/C) were identified to carry blaNDM-1 among the six different bacterial species isolated from the four patients. Two of these strains were novel Citrobacter freundii ST 20 and ST 21. The same IncN2-type blaNDM-1 genetic context was found in all four patients and within four different species. The IncA/C-type blaNDM-1 genetic context was found in two different species and in two of the four patients. Combining genetic context characterization with other molecular epidemiology methods, we were able to establish the molecular epidemiological links between genetically unrelated bacterial species by linking their acquisition of an IncN2 or IncA/C plasmid carrying blaNDM-1 for carbapenem resistance. By combining plasmid characterization and in-depth genetic context assessment, this analysis highlights the importance of plasmids in antimicrobial resistance. It also provides a novel approach for investigating the underlying mechanisms of blaNDM-1-related spread between bacterial species and genera via plasmids. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


PubMed | Australian Infectious Diseases Research Center
Type: | Journal: Langmuir : the ACS journal of surfaces and colloids | Year: 2016

Immunoassays are ubiquitous across research and clinical laboratories, yet little attention is paid to the effect of the substrate material on the assay performance characteristics. Given the emerging interest in wearable immunoassay formats, investigations into substrate materials that provide an optimal mix of mechanical and bioanalytical properties are paramount. In the course of our research in developing wearable immunoassays which can penetrate skin to selectively capture disease antigens from the underlying blood vessels, we recently identified significant differences in immunoassay performance between gold and polycarbonate surfaces, even with a consistent surface modification procedure. We observed significant differences in PEG density, antibody immobilization, and nonspecific adsorption between the two substrates. Despite a higher PEG density formed on gold-coated surfaces than on amine-functionalized polycarbonate, the latter revealed a higher immobilized capture antibody density and lower nonspecific adsorption, leading to improved signal-to-noise ratios and assay sensitivities. The major conclusion from this study is that in designing wearable bioassays or biosensors, the design and its effect on the antifouling polymer layer can significantly affect the assay performance in terms of analytical specificity and sensitivity.


PubMed | Australian Infectious Diseases Research Center, Institute of Environmental Science and Research, University of Queensland, Royal Brisbane and Womens Hospital and University of New South Wales
Type: Journal Article | Journal: Antimicrobial agents and chemotherapy | Year: 2016

blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1, and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A, and ISCR1 may have been involved in the acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones, but different regions carrying blaNDM are found in different locations. Tn3-derived inverted-repeat transposable elements (TIME) appear to have been involved in the acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterization of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements, and plasmids and provide insights into the possible routes for transmission of blaNDM genes among species of the Enterobacteriaceae family.

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