Publications by authors named "Mark A Schembri"

184 Publications

Short-term Oral Steroids Significantly Improves Chronic Rhinosinusitis Without Nasal Polyps.

Laryngoscope 2021 Mar 4. Epub 2021 Mar 4.

Adelaide School of Medicine, The University of Adelaide, Adelaide, South Australia, Australia.

Objectives/hypothesis: The efficacy of short-term oral corticosteroids in chronic rhinosinusitis without nasal polyps (CRSsNP) is unknown. The aim of this controlled study was to assess the immediate and long-term outcomes from a short course of a commonly used oral corticosteroid, prednisolone, in well-defined CRSsNP patients.

Study Design: Prospective, observational controlled study.

Methods: A prospective-controlled study of CRSsNP patients treated with prednisolone at 0.5 mg/kg tapered over 10 days and non-prednisolone treated CRSsNP patients (controls) and follow-up at 2, 6, and 12 months. Baseline and follow-up SinoNasal Outcome Test (SNOT)-22, nasal endoscopy (Lund-Kennedy), and sinus CT scan scores (Lund-Mackay) were compared.

Results: At 2 months, there was a significant improvement in the SNOT-22, nasal endoscopy, and sinus CT scan scores in the prednisolone group (P < .0001) compared with controls (p = ns, Mann-Whitney U test). 52.5% of prednisolone-treated CRSsNP patients had improved symptoms and did not require sinus surgery at 12 months compared with 14.3% of controls (P < .001). Side-effects were reported in 8.9% of prednisolone-treated patients. Patients who benefited from prednisolone had a median symptom duration of 7.25 (99% confidence, upper limit of 11) months compared with 18 months in those requiring surgery.

Conclusions: Short-term oral prednisolone significantly improved all three clinical measures of disease in CRSsNP patients and avoided surgical intervention in 52.5% patients in the first 12 months. Patients with symptoms for less than 11 months were most likely to benefit. The side-effects of oral steroids require careful consideration and further studies are needed to ascertain appropriate dosage and treatment duration.

Level Of Evidence: 3 Laryngoscope, 2021.
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http://dx.doi.org/10.1002/lary.29495DOI Listing
March 2021

Discovery of Bacterial Fimbria-Glycan Interactions Using Whole-Cell Recombinant Escherichia coli Expression.

mBio 2021 02 23;12(1). Epub 2021 Feb 23.

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia

Chaperone-usher (CU) fimbriae are the most abundant Gram-negative bacterial fimbriae, with 38 distinct CU fimbria types described in alone. Some CU fimbriae have been well characterized and bind to specific glycan targets to confer tissue tropism. For example, type 1 fimbriae bind to α-d-mannosylated glycoproteins such as uroplakins in the bladder via their tip-located FimH adhesin, leading to colonization and invasion of the bladder epithelium. Despite this, the receptor-binding affinity of many other CU fimbria types remains poorly characterized. Here, we used a recombinant strain expressing different CU fimbriae, in conjunction with glycan array analysis comprising >300 glycans, to dissect CU fimbria receptor specificity. We initially validated the approach by demonstrating the purified FimH lectin-binding domain and recombinant expressing type 1 fimbriae bound to a similar set of glycans. This technique was then used to map the glycan binding affinity of six additional CU fimbriae, namely, P, F1C, Yqi, Mat/Ecp, K88, and K99 fimbriae. The binding affinity was determined using whole-bacterial-cell surface plasmon resonance. This work describes new information in fimbrial specificity and a rapid and scalable system to define novel adhesin-glycan interactions that underpin bacterial colonization and disease. Understanding the tropism of pathogens for host and tissue requires a complete understanding of the host receptors targeted by fimbrial adhesins. Furthermore, blocking adhesion is a promising strategy to counter increasing antibiotic resistance and is enabled by the identification of host receptors. Here, we use a defined heterologous expression system to identify glycan receptors for six chaperone-usher fimbriae and identify novel receptors that are consistent with their known function. The same system was used to measure the kinetics of binding to the identified glycan, wherein bacterial cells were immobilized onto a biosensor chip and the interactions with glycans were quantified by surface plasmon resonance. This novel, dual-level analysis, where screening for the repertoire of glycan binding and the hierarchy of affinity of the identified ligands is determined directly from a natively expressed fimbrial structure on the bacterial cell surface, is superior in both throughput and biological relevance.
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http://dx.doi.org/10.1128/mBio.03664-20DOI Listing
February 2021

Genomic surveillance, characterization and intervention of a polymicrobial multidrug-resistant outbreak in critical care.

Microb Genom 2021 Mar 18;7(3). Epub 2021 Feb 18.

Pathology Queensland, Central Laboratory, Brisbane, QLD, Australia.

Infections caused by carbapenem-resistant (CR-Ab) have become increasingly prevalent in clinical settings and often result in significant morbidity and mortality due to their multidrug resistance (MDR). Here we present an integrated whole-genome sequencing (WGS) response to a persistent CR-Ab outbreak in a Brisbane hospital between 2016-2018.. and isolates were sequenced using the Illumina platform primarily to establish isolate relationships based on core-genome SNPs, MLST and antimicrobial resistance gene profiles. Representative isolates were selected for PacBio sequencing. Environmental metagenomic sequencing with Illumina was used to detect persistence of the outbreak strain in the hospital. In response to a suspected polymicrobial outbreak between May to August of 2016, 28 CR-Ab (and 21 other MDR Gram-negative bacilli) were collected from Intensive Care Unit and Burns Unit patients and sent for WGS with a 7 day turn-around time in clinical reporting. All CR-Ab were sequence type (ST)1050 (Pasteur ST2) and within 10 SNPs apart, indicative of an ongoing outbreak, and distinct from historical CR-Ab isolates from the same hospital. Possible transmission routes between patients were identified on the basis of CR-Ab and SNP profiles. Continued WGS surveillance between 2016 to 2018 enabled suspected outbreak cases to be refuted, but a resurgence of the outbreak CR-Ab mid-2018 in the Burns Unit prompted additional screening. Environmental metagenomic sequencing identified the hospital plumbing as a potential source. Replacement of the plumbing and routine drain maintenance resulted in rapid resolution of the secondary outbreak and significant risk reduction with no discernable transmission in the Burns Unit since. We implemented a comprehensive WGS and metagenomics investigation that resolved a persistent CR-Ab outbreak in a critical care setting.
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http://dx.doi.org/10.1099/mgen.0.000530DOI Listing
March 2021

Characterization of DtrJ as an IncC plasmid conjugative DNA transfer component.

Mol Microbiol 2021 Feb 10. Epub 2021 Feb 10.

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia.

Incompatibility group C (IncC) plasmids are large (50-400 kb), broad host range plasmids that drive the spread of genes conferring resistance to all classes of antibiotics, most notably the bla gene that confers resistance to last-line carbapenems and the mcr-3 gene that confers resistance to colistin. Several recent studies have improved our understanding of the basic biological mechanisms driving the success of IncC, in particular the identification of multiple novel IncC conjugation genes by transposon directed insertion-site sequencing. Here, one of these genes, dtrJ, was examined in further detail. The dtrJ gene is located in the DNA transfer locus on the IncC backbone, and quantitative reverse-transcriptase PCR analysis revealed it is transcribed in the same operon as the DNA transfer genes traI and traD (encoding the relaxase and coupling protein, respectively) and activated by the AcaDC regulatory complex. We confirmed that DtrJ is not required for pilus biogenesis or mate pair formation. Instead, DtrJ localizes to the membrane, where it interacts with the coupling protein TraD and functions as an IncC DNA transfer protein. Overall, this work has defined the role of DtrJ in DNA transfer of IncC plasmids during conjugation.
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http://dx.doi.org/10.1111/mmi.14697DOI Listing
February 2021

Repurposing a neurodegenerative disease drug to treat Gram-negative antibiotic-resistant bacterial sepsis.

Sci Transl Med 2020 11;12(570)

School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, Queensland 4072, Australia.

The emergence of polymyxin resistance in carbapenem-resistant and extended-spectrum β-lactamase (ESBL)-producing bacteria is a critical threat to human health, and alternative treatment strategies are urgently required. We investigated the ability of the hydroxyquinoline analog ionophore PBT2 to restore antibiotic sensitivity in polymyxin-resistant, ESBL-producing, carbapenem-resistant Gram-negative human pathogens. PBT2 resensitized , , , and to last-resort polymyxin class antibiotics, including the less toxic next-generation polymyxin derivative FADDI-287, in vitro. We were unable to select for mutants resistant to PBT2 + FADDI-287 in polymyxin-resistant containing a plasmid-borne gene or carrying a chromosomal mutation. Using a highly invasive strain engineered for polymyxin resistance through mutation, we successfully demonstrated the efficacy of PBT2 + polymyxin (colistin or FADDI-287) for the treatment of Gram-negative sepsis in immunocompetent mice. In comparison to polymyxin alone, the combination of PBT2 + polymyxin improved survival and reduced bacterial dissemination to the lungs and spleen of infected mice. These data present a treatment modality to break antibiotic resistance in high-priority polymyxin-resistant Gram-negative pathogens.
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http://dx.doi.org/10.1126/scitranslmed.abb3791DOI Listing
November 2020

Companion Animals Are Spillover Hosts of the Multidrug-Resistant Human Extraintestinal Pandemic Clones ST131 and ST1193.

Front Microbiol 2020 2;11:1968. Epub 2020 Sep 2.

Australian Centre for Antimicrobial Resistance Ecology, School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA, Australia.

sequence types 131 (ST131) and 1193 are multidrug-resistant extraintestinal pathogens that have recently spread epidemically among humans and are occasionally isolated from companion animals. This study characterized a nationwide collection of fluoroquinolone-resistant (FQ ) isolates from extraintestinal infections in Australian cats and dogs. For this, 59 cat and dog FQ clinical isolates (representing 6.9% of an 855-isolate collection) underwent PCR-based phylotyping and whole-genome sequencing (WGS). Isolates from commensal-associated phylogenetic groups A (14/59, 24%) and B1 (18/59, 31%) were dominant, with ST224 (10/59, 17%), and ST744 (8/59, 14%) predominating. Less prevalent were phylogenetic groups D (12/59, 20%), with ST38 (8/59, 14%) predominating, and virulence-associated phylogenetic group B2 (7/59, 12%), with ST131 predominating (6/7, 86%) and no ST1193 isolates identified. In a WGS-based comparison of 20 cat and dog-source ST131 isolates with 188 reference human and animal ST131 isolates, the cat and dog-source isolates were phylogenetically diverse. Although cat and dog-source ST131 isolates exhibited some minor sub-clustering, most were closely related to human-source ST131 strains. Furthermore, the prevalence of ST131 as a cause of FQ infections in Australian companion animals was relatively constant between this study and the 5-year-earlier study of Platell et al. (2010) (9/125 isolates, 7.2%). Thus, although the high degree of clonal commonality among FQ clinical isolates from humans vs. companion animals suggests the possibility of bi-directional between-species transmission, the much higher reported prevalence of ST131 and ST1193 among FQ clinical isolates from humans as compared to companion animals suggests that companion animals are spillover hosts rather than being a primary reservoir for these lineages.
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http://dx.doi.org/10.3389/fmicb.2020.01968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492567PMC
September 2020

An alloy of zinc and innate immunity: Galvanising host defence against infection.

Cell Microbiol 2021 Jan 9;23(1):e13268. Epub 2020 Oct 9.

Institute for Molecular Bioscience (IMB), The University of Queensland, St. Lucia, Queensland, Australia.

Innate immune cells such as macrophages and neutrophils initiate protective inflammatory responses and engage antimicrobial responses to provide frontline defence against invading pathogens. These cells can both restrict the availability of certain transition metals that are essential for microbial growth and direct toxic concentrations of metals towards pathogens as antimicrobial responses. Zinc is important for the structure and function of many proteins, however excess zinc can be cytotoxic. In recent years, several studies have revealed that innate immune cells can deliver toxic concentrations of zinc to intracellular pathogens. In this review, we discuss the importance of zinc status during infectious disease and the evidence for zinc intoxication as an innate immune antimicrobial response. Evidence for pathogen subversion of this response is also examined. The likely mechanisms, including the involvement of specific zinc transporters that facilitate delivery of zinc by innate immune cells for metal ion poisoning of pathogens are also considered. Precise mechanisms by which excess levels of zinc can be toxic to microorganisms are then discussed, particularly in the context of synergy with other antimicrobial responses. Finally, we highlight key unanswered questions in this emerging field, which may offer new opportunities for exploiting innate immune responses for anti-infective development.
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http://dx.doi.org/10.1111/cmi.13268DOI Listing
January 2021

Restriction of chronic Escherichia coli urinary tract infection depends upon T cell-derived interleukin-17, a deficiency of which predisposes to flagella-driven bacterial persistence.

FASEB J 2020 11 9;34(11):14572-14587. Epub 2020 Sep 9.

School of Medical Sciences, And Menzies Health Institute Queensland, Griffith University, Parklands, QLD, Australia.

Urinary tract infections (UTI) frequently progress to chronicity in infected individuals but the mechanisms of pathogenesis underlying chronic UTI are not well understood. We examined the role of interleukin (IL)-17A in UTI because this cytokine promotes innate defense against uropathogenic Escherichia coli (UPEC). Analysis of UPEC persistence and pyelonephritis in mice deficient in IL-17A revealed that UPEC CFT073 caused infection at a rate higher than the multidrug resistant strain EC958. Il17a mice exhibited pyelonephritis with kidney bacterial burdens higher than those of wild-type (WT) mice. Synthesis of IL-17A in the bladder reflected a combination of γδ-T and T 17 cell responses. Analysis of circulating inflammatory mediators at 24h postinoculation identified predictors of progression to chronicity, including IL-6 and monocyte chemoattractant protein-1 (MCP-1). Histological analysis identified infiltrating populations of neutrophils, NK cells, and γδ T cells in the bladder, whereas neutrophils predominated in the kidney. Analysis of the contribution of flagella to chronicity using hyper-flagellated and fliC-deficient UPEC in WT and Il17a mice revealed that, in a host that is deficient for the production of IL-17A, flagella contribute to bacterial persistence. These findings show a role for IL-17A in defense against chronic UTI and a contribution of flagella to the pathogenesis of infection.
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http://dx.doi.org/10.1096/fj.202000760RDOI Listing
November 2020

Comprehensive analysis of IncC plasmid conjugation identifies a crucial role for the transcriptional regulator AcaB.

Nat Microbiol 2020 11 17;5(11):1340-1348. Epub 2020 Aug 17.

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia.

The IncC family of broad-host-range plasmids enables the spread of antibiotic resistance genes among human enteric pathogens. Although aspects of IncC plasmid conjugation have been well studied, many roles of conjugation genes have been assigned based solely on sequence similarity. We applied hypersaturated transposon mutagenesis and transposon-directed insertion-site sequencing to determine the set of genes required for IncC conjugation. We identified 27 conjugation genes, comprising 19 that were previously identified (including two regulatory genes, acaDC) and eight not previously associated with conjugation. We show that one previously unknown gene, acaB, encodes a transcriptional regulator that has a crucial role in the regulation of IncC conjugation. AcaB binds upstream of the acaDC promoter to increase acaDC transcription; in turn, AcaDC activates the transcription of IncC conjugation genes. We solved the crystal structure of AcaB at 2.9-Å resolution and used this to guide functional analyses that reveal how AcaB binds to DNA. This improved understanding of IncC conjugation provides a basis for the development of new approaches to reduce the spread of these multi-drug-resistance plasmids.
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http://dx.doi.org/10.1038/s41564-020-0775-0DOI Listing
November 2020

Oral cephalosporin and β-lactamase inhibitor combinations for ESBL-producing Enterobacteriaceae urinary tract infections.

J Antimicrob Chemother 2020 09;75(9):2384-2393

Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Royal Brisbane and Women's Hospital Campus, Brisbane, Australia.

ESBL-producing Enterobacteriaceae as uropathogens have given rise to a sizeable amount of global morbidity. Community and hospital surveillance studies continue to report increasing proportions of these organisms as causes of urinary tract infection (UTI). Due to limited treatment options and the presence of cross-resistance amongst oral antibiotics of different classes, patients often require IV therapy, thereby increasing healthcare costs and reducing the effectiveness of delivering healthcare. Oral cephalosporin antibiotics are well known for their ability to achieve high urinary concentrations, in addition to achieving clinical success for treatment of uncomplicated UTI with a drug-susceptible pathogen. Novel cephalosporin/β-lactamase inhibitor combinations have been developed and demonstrate good in vitro activity against ESBL-producing isolates. A pooled analysis of in vitro activity of existing oral cephalosporin/clavulanate combinations in ESBL-producing Enterobacteriaceae has shown MIC50s of 0.5-1, 0.125-1 and 0.25 mg/L for cefpodoxime, ceftibuten and cefixime, respectively. A novel cyclic boronic acid β-lactamase inhibitor, QPX7728, was able to produce MIC50 values of 0.5 and ≤0.06 mg/L when paired with cefpodoxime and ceftibuten, respectively. Other novel combinations, cefpodoxime/ETX0282 and ceftibuten/VNRX7145, have also demonstrated excellent activity against ESBL producers. Clinical trials are now awaited.
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http://dx.doi.org/10.1093/jac/dkaa183DOI Listing
September 2020

Frontline Science: LPS-inducible SLC30A1 drives human macrophage-mediated zinc toxicity against intracellular Escherichia coli.

J Leukoc Biol 2021 02 22;109(2):287-297. Epub 2020 May 22.

Institute for Molecular Bioscience (IMB), The University of Queensland, St. Lucia, Queensland, Australia.

TLR-inducible zinc toxicity is an antimicrobial mechanism utilized by macrophages, however knowledge of molecular mechanisms mediating this response is limited. Here, we show that E. coli exposed to zinc stress within primary human macrophages reside in membrane-bound vesicular compartments. Since SLC30A zinc exporters can deliver zinc into the lumen of vesicles, we examined LPS-regulated mRNA expression of Slc30a/SLC30A family members in primary mouse and human macrophages. A number of these transporters were dynamically regulated in both cell populations. In human monocyte-derived macrophages, LPS strongly up-regulated SLC30A1 mRNA and protein expression. In contrast, SLC30A1 was not LPS-inducible in macrophage-like PMA-differentiated THP-1 cells. We therefore ectopically expressed SLC30A1 in these cells, finding that this was sufficient to promote zinc-containing vesicle formation. The response was similar to that observed following LPS stimulation. Ectopically expressed SLC30A1 localized to both the plasma membrane and intracellular zinc-containing vesicles within LPS-stimulated THP-1 cells. Inducible overexpression of SLC30A1 in THP-1 cells infected with the Escherichia coli K-12 strain MG1655 augmented the zinc stress response of intracellular bacteria and promoted clearance. Furthermore, in THP-1 cells infected with an MG1655 zinc stress reporter strain, all bacteria contained within SLC30A1-positive compartments were subjected to zinc stress. Thus, SLC30A1 marks zinc-containing compartments associated with TLR-inducible zinc toxicity in human macrophages, and its ectopic over-expression is sufficient to initiate this antimicrobial pathway in these cells. Finally, SLC30A1 silencing did not compromise E. coli clearance by primary human macrophages, suggesting that other zinc exporters may also contribute to the zinc toxicity response.
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http://dx.doi.org/10.1002/JLB.2HI0420-160RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891337PMC
February 2021

Antimicrobial Resistance in ESKAPE Pathogens.

Clin Microbiol Rev 2020 06 13;33(3). Epub 2020 May 13.

School of Chemistry and Molecular Biosciences, The University of Queensland, QLD, Australia

Antimicrobial-resistant ESKAPE ( , , , , , and species) pathogens represent a global threat to human health. The acquisition of antimicrobial resistance genes by ESKAPE pathogens has reduced the treatment options for serious infections, increased the burden of disease, and increased death rates due to treatment failure and requires a coordinated global response for antimicrobial resistance surveillance. This looming health threat has restimulated interest in the development of new antimicrobial therapies, has demanded the need for better patient care, and has facilitated heightened governance over stewardship practices.
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http://dx.doi.org/10.1128/CMR.00181-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227449PMC
June 2020

Uncovering novel susceptibility targets to enhance the efficacy of third-generation cephalosporins against ESBL-producing uropathogenic Escherichia coli.

J Antimicrob Chemother 2020 06;75(6):1415-1423

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia.

Background: Uropathogenic Escherichia coli (UPEC) are a major cause of urinary tract infection (UTI), one of the most common infectious diseases in humans. UPEC are increasingly associated with resistance to multiple antibiotics. This includes resistance to third-generation cephalosporins, a common class of antibiotics frequently used to treat UTI.

Methods: We employed a high-throughput genome-wide screen using saturated transposon mutagenesis and transposon directed insertion-site sequencing (TraDIS) together with phenotypic resistance assessment to identify key genes required for survival of the MDR UPEC ST131 strain EC958 in the presence of the third-generation cephalosporin cefotaxime.

Results: We showed that blaCMY-23 is the major ESBL gene in EC958 responsible for mediating resistance to cefotaxime. Our screen also revealed that mutation of genes involved in cell division and the twin-arginine translocation pathway sensitized EC958 to cefotaxime. The role of these cell-division and protein-secretion genes in cefotaxime resistance was confirmed through the construction of mutants and phenotypic testing. Mutation of these genes also sensitized EC958 to other cephalosporins.

Conclusions: This work provides an exemplar for the application of TraDIS to define molecular mechanisms of resistance to antibiotics. The identification of mutants that sensitize UPEC to cefotaxime, despite the presence of a cephalosporinase, provides a framework for the development of new approaches to treat infections caused by MDR pathogens.
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http://dx.doi.org/10.1093/jac/dkaa023DOI Listing
June 2020

Integrating multiple genomic technologies to investigate an outbreak of carbapenemase-producing Enterobacter hormaechei.

Nat Commun 2020 01 24;11(1):466. Epub 2020 Jan 24.

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia.

Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of bla carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the bla gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak.
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http://dx.doi.org/10.1038/s41467-019-14139-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981164PMC
January 2020

Genomic analysis of carbapenemase-producing in Queensland reveals widespread transmission of on an IncHI2 plasmid.

Microb Genom 2020 01 19;6(1). Epub 2019 Dec 19.

Central Microbiology, Pathology Queensland, QLD, Australia.

Carbapenemase-producing (CPE) are an increasingly common cause of healthcare-associated infections and may occasionally be identified in patients without extensive healthcare exposure. is the most frequently detected carbapenemase gene in within Australia, but little is known about the mechanisms behind its persistence. Here we used whole genome sequencing (WGS) to investigate the molecular epidemiology of in Queensland, Australia. In total, 107 CPE were collected between 2014 and 2017 and sent for WGS on an Illumina NextSeq500. Resistance genes and plasmid types were detected using a combination of read mapping and nucleotide comparison of assemblies. Six isolates were additionally sequenced using Oxford Nanopore MinION to generate long-reads and fully characterize the context of the gene. Of 107 CPE, 93 carried the gene; 74/107 also carried an IncHI2 plasmid, suggesting carriage of the gene on an IncHI2 plasmid. Comparison of these isolates to a previously characterized IncHI2 plasmid pMS7884A (isolated from an strain in Brisbane) suggested that all isolates carried a similar plasmid. Five of six representative isolates sequenced using Nanopore long-read technology carried IncHI2 plasmids harbouring the gene. While the vast majority of isolates represented , several other species were also found to carry the IncHI2 plasmid, including species, and species. Several clonal groups of were also identified, suggesting that persistence of is driven by both presence on a common plasmid and clonal spread of certain lineages.
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http://dx.doi.org/10.1099/mgen.0.000321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067041PMC
January 2020

Rapid Bladder Interleukin-10 Synthesis in Response to Uropathogenic Escherichia coli Is Part of a Defense Strategy Triggered by the Major Bacterial Flagellar Filament FliC and Contingent on TLR5.

mSphere 2019 11 27;4(6). Epub 2019 Nov 27.

School of Medical Sciences and Menzies Health Institute Queensland, Griffith University, Parklands, Australia

Urinary tract infection (UTI) caused by uropathogenic (UPEC) engages interleukin-10 (IL-10) as an early innate immune response to regulate inflammation and promote the control of bladder infection. However, the mechanism of engagement of innate immunity by UPEC that leads to elicitation of IL-10 in the bladder is unknown. Here, we identify the major UPEC flagellar filament, FliC, as a key bacterial component sensed by the bladder innate immune system responsible for the induction of IL-10 synthesis. IL-10 responses of human as well as mouse bladder epithelial cell-monocyte cocultures were triggered by flagella of three major UPEC representative strains, CFT073, UTI89, and EC958. FliC purified to homogeneity induced IL-10 and as well as other functionally related cytokines, including IL-6. The genome-wide innate immunological context of FliC-induced IL-10 in the bladder was defined using RNA sequencing that revealed a network of transcriptional and antibacterial defenses comprising 1,400 genes that were induced by FliC. Of the FliC-responsive bladder transcriptome, altered expression of and 808 additional genes were dependent on Toll-like receptor 5 (TLR5), according to analysis of TLR5-deficient mice. Examination of the potential of FliC and associated innate immune signature in the bladder to boost host defense, based on prophylactic or therapeutic administration to mice, revealed significant benefits for the control of UPEC. We conclude that detection of FliC through TLR5 triggers rapid IL-10 synthesis in the bladder, and FliC represents a potential immune modulator that might offer benefit for the treatment or prevention of UPEC UTI. Interleukin-10 is part of the immune response to urinary tract infection (UTI) due to , and it is important in the early control of infection in the bladder. Defining the mechanism of engagement of the immune system by the bacteria that enables the protective IL-10 response is critical to exploring how we might exploit this mechanism for new infection control strategies. In this study, we reveal part of the bacterial flagellar apparatus (FliC) is an important component that is sensed by and responsible for induction of IL-10 in the response to UPEC. We show this response occurs in a TLR5-dependent manner. Using infection prevention and control trials in mice infected with , this study also provides evidence that purified FliC might be of value in novel approaches for the treatment of UTI or in preventing infection by exploiting the FliC-triggered bladder transcriptome.
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http://dx.doi.org/10.1128/mSphere.00545-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881718PMC
November 2019

Complex Multilevel Control of Hemolysin Production by Uropathogenic Escherichia coli.

mBio 2019 10 1;10(5). Epub 2019 Oct 1.

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia

Uropathogenic (UPEC) is the major cause of urinary tract infections. Nearly half of all UPEC strains secrete hemolysin, a cytotoxic pore-forming toxin. Here, we show that the prevalence of the hemolysin toxin gene () is highly variable among the most common 83 sequence types (STs) represented on the EnteroBase genome database. To explore this diversity in the context of a defined monophyletic lineage, we contextualized sequence variation of the operon within the genealogy of the globally disseminated multidrug-resistant ST131 clone. We show that sequence changes in and its newly defined 1.616-kb-long leader sequence correspond to phylogenetic designation, and that ST131 strains with the strongest hemolytic activity belong to the most extensive multidrug-resistant sublineage (clade C2). To define the set of genes involved in hemolysin production, the clade C2 strain S65EC was completely sequenced and subjected to a genome-wide screen by combining saturated transposon mutagenesis and transposon-directed insertion site sequencing with the capacity to lyse red blood cells. Using this approach, and subsequent targeted mutagenesis and complementation, 13 genes were confirmed to be specifically required for production of active hemolysin. New hemolysin-controlling elements included discrete sets of genes involved in lipopolysaccharide (LPS) inner core biosynthesis (, , , and ) and cytoplasmic chaperone activity ( and ), and we show these are required for hemolysin secretion. Overall, this work provides a unique description of hemolysin sequence diversity in a single clonal lineage and describes a complex multilevel system of regulatory control for this important toxin. Uropathogenic (UPEC) is the major cause of urinary tract infections and a frequent cause of sepsis. Nearly half of all UPEC strains produce the potent cytotoxin hemolysin, and its expression is associated with enhanced virulence. In this study, we explored hemolysin variation within the globally dominant UPEC ST131 clone, finding that strains from the ST131 sublineage with the greatest multidrug resistance also possess the strongest hemolytic activity. We also employed an innovative forward genetic screen to define the set of genes required for hemolysin production. Using this approach, and subsequent targeted mutagenesis and complementation, we identified new hemolysin-controlling elements involved in LPS inner core biosynthesis and cytoplasmic chaperone activity, and we show that mechanistically they are required for hemolysin secretion. These original discoveries substantially enhance our understanding of hemolysin regulation, secretion and function.
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http://dx.doi.org/10.1128/mBio.02248-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6775461PMC
October 2019

Bioinformatic and Molecular Analysis of Inverse Autotransporters from Escherichia coli.

mSphere 2019 08 28;4(4). Epub 2019 Aug 28.

School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia

Proteins secreted by the type V secretion system possess multiple functions, including the capacity to mediate adhesion, aggregation, and biolfilm formation. The type V secretion system can be divided into five subclasses, one of which is the type Ve system. Proteins of the type Ve secretion system are also referred to as inverse autotransporters (IATs). In this study, we performed an analysis of 126 completely sequenced genomes available in the NCBI database and identified several distinct IAT-encoding gene families whose distribution varied throughout the phylogeny. The genes included three characterized IATs (intimin, , and ) and four uncharacterized IATs (here named , , , and ). The four genes were cloned from the completely sequenced environmental strain SMS-3-5 and characterized. Three of these IAT proteins (IatB, IatC, and IatD) were expressed at the cell surface and possessed the capacity to mediate biofilm formation in a recombinant K-12 strain. Further analysis of the gene, which showed a unique association with extraintestinal strains, suggested that its regulation is controlled by the LeuO global regulator. Overall, this study provides new data describing the prevalence, sequence variation, domain structure, function, and regulation of IATs found in is one of the most prevalent facultative anaerobes of the human gut. normally exists as a harmless commensal but can also cause disease following the acquisition of genes that enhance its pathogenicity. Adhesion is an important first step in colonization of the host and is mediated by an array of cell surface components. In , these include a family of adhesins secreted by the type V secretion system. Here, we identified and characterized new proteins from an emerging subclass of the type V secretion system known as the inverse autotransporters (IATs). We found that IAT-encoding genes are present in a wide range of strains and showed that three novel IATs were localized on the cell surface and mediated biofilm formation. Overall, this study provides new insight into the prevalence, function, and regulation of IATs in .
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http://dx.doi.org/10.1128/mSphere.00572-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714894PMC
August 2019

Population dynamics of an Escherichia coli ST131 lineage during recurrent urinary tract infection.

Nat Commun 2019 08 13;10(1):3643. Epub 2019 Aug 13.

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 4072, Queensland, Australia.

Recurrent urinary tract infections (rUTIs) are extremely common, with ~ 25% of all women experiencing a recurrence within 1 year of their original infection. Escherichia coli ST131 is a globally dominant multidrug resistant clone associated with high rates of rUTI. Here, we show the dynamics of an ST131 population over a 5-year period from one elderly woman with rUTI since the 1970s. Using whole genome sequencing, we identify an indigenous clonal lineage (P1A) linked to rUTI and persistence in the fecal flora, providing compelling evidence of an intestinal reservoir of rUTI. We also show that the P1A lineage possesses substantial plasmid diversity, resulting in the coexistence of antibiotic resistant and sensitive intestinal isolates despite frequent treatment. Our longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure.
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http://dx.doi.org/10.1038/s41467-019-11571-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692316PMC
August 2019

A Retrospective Study Using the String of Pearls Tibial Plateau Levelling Osteotomy Locking Plate for the Treatment of Cranial Cruciate Ligament Disease.

Vet Comp Orthop Traumatol 2019 Nov 21;32(6):483-491. Epub 2019 Jun 21.

Northshore Veterinary Specialist Centre, Sydney, Australia.

Objective:  The aim of this study was to describe the use of String of Pearls (SOP) tibial plateau levelling osteotomy (TPLO) locking plates for the treatment of cranial cruciate ligament disease and retrospectively assess osteotomy healing, tibial plateau angle (TPA) change (c) and overall complication rates.

Methods:  Medical records and radiographic studies of 170 SOP TPLO surgical procedures were analysed. Radiographic measurement of TPAc and osteotomy union was determined 6 to 8 weeks postoperatively. Intra- and postoperative major and minor complications were identified. Factors influencing TPAc, osteotomy union and complications were assessed.

Results:  Mean (standard deviation) TPAc was 1.3° (1.61) and median osteotomy union was graded as 2 (26-50%) union. Improved osteotomy union was associated with retention of the antirotational pin (odds ratio [OR]: 2.3; 95% confidence interval [CI]: 1.3-4;  = 0.005) and reduced TPAc (OR: 0.8; 95%CI: 0.66-0.97;  = 0.02). Complications occurred in 42 (24.7%) procedures with 11 (6.5%) considered major and 31 (18.2%) minor.

Clinical Significance:  The use of the SOP TPLO plating system resulted in a median grade 2 (26-50%) radiographic osteotomy union score at 6 to 8 weeks' follow-up, less than previous TPLO locking plate studies. Osteotomy union was associated with retention of the antirotational pin and reduced TPAc. Major and minor complication rates were marginally greater than recent locking plate studies with fibular fracture and screw loosening common complications. The use of SOP TPLO plates is cautioned and further study is warranted.
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http://dx.doi.org/10.1055/s-0039-1692189DOI Listing
November 2019

Physical Extraction and Fast Protein Liquid Chromatography for Purifying Flagella Filament From Uropathogenic for Immune Assay.

Front Cell Infect Microbiol 2019 24;9:118. Epub 2019 Apr 24.

School of Medical Science, Griffith University, Southport, QLD, Australia.

Flagella are expressed on the surface of a wide range of bacteria, conferring motility and contributing to virulence and innate immune stimulation. Host-pathogen interaction studies of the roles of flagella in infection, including due to uropathogenic (UPEC), have used various methods to purify and examine the biology of the major flagella subunit protein, FliC. These studies have offered insight into the ways in which flagella proteins interact with host cells. However, previous methods used to extract and purify FliC, such as mechanical shearing, ultracentrifugation, heterologous expression in laboratory strains, and precipitation-inducing chemical treatments have various limitations; as a result, there are few observations based on highly purified, non-denatured FliC in the literature. This is especially relevant to host-pathogen interaction studies such as immune assays that are designed to parallel, as closely as possible, naturally-occurring interactions between host cells and flagella. In this study, we sought to establish a new, carefully optimized method to extract and purify non-denatured, native FliC from the reference UPEC strain CFT073 to be suitable for immune assays. To achieve purification of FliC to homogeneity, we used a mutant CFT073 strain containing deletions in four major chaperone-usher fimbriae operons (type 1, F1C and two P fimbrial gene clusters; CFT073Δ). A sequential flagella extraction method based on mechanical shearing, ultracentrifugation, size exclusion chromatography, protein concentration and endotoxin removal was applied to CFT073Δ. Protein purity and integrity was assessed using SDS-PAGE, Western blots with anti-flagellin antisera, and native-PAGE. We also generated a -deficient strain, CFT073ΔΔ, to enable the concurrent preparation of a suitable carrier control to be applied in downstream assays. Innate immune stimulation was examined by exposing J774A.1 macrophages to 0.05-1 μg of purified FliC for 5 h; the supernatants were analyzed for cytokines known to be induced by flagella, including TNF-α, IL-6, and IL-12; the results were assessed in the context of prior literature. Macrophage responses to purified FliC encompassed significant levels of several cytokines consistent with prior literature reports. The purification method described here establishes a new approach to examine highly purified FliC in the context of host-pathogen interaction model systems.
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http://dx.doi.org/10.3389/fcimb.2019.00118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6491459PMC
December 2019

Unique structural features of a bacterial autotransporter adhesin suggest mechanisms for interaction with host macromolecules.

Nat Commun 2019 04 29;10(1):1967. Epub 2019 Apr 29.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, 3086, VIC, Australia.

Autotransporters are the largest family of outer membrane and secreted proteins in Gram-negative bacteria. Most autotransporters are localised to the bacterial surface where they promote colonisation of host epithelial surfaces. Here we present the crystal structure of UpaB, an autotransporter that is known to contribute to uropathogenic E. coli (UPEC) colonisation of the urinary tract. We provide evidence that UpaB can interact with glycosaminoglycans and host fibronectin. Unique modifications to its core β-helical structure create a groove on one side of the protein for interaction with glycosaminoglycans, while the opposite face can bind fibronectin. Our findings reveal far greater diversity in the autotransporter β-helix than previously thought, and suggest that this domain can interact with host macromolecules. The relevance of these interactions during infection remains unclear.
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http://dx.doi.org/10.1038/s41467-019-09814-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488583PMC
April 2019

Variation in hemolysin A expression between uropathogenic isolates determines NLRP3-dependent -independent macrophage cell death and host colonization.

FASEB J 2019 06 14;33(6):7437-7450. Epub 2019 Mar 14.

Centre for Inflammation and Disease Research, Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland, Australia.

Uropathogenic (UPEC) is the major cause of urinary tract infections (UTIs). The multidrug-resistant sequence type 131 (ST131) clone is a serious threat to human health, yet its effects on immune responses are not well understood. Here we screened a panel of ST131 isolates, finding that only strains expressing the toxin hemolysin A (HlyA) killed primary human macrophages and triggered maturation of the inflammasome-dependent cytokine IL-1β. Using a representative strain, the requirement for the gene in these responses was confirmed. We also observed considerable heterogeneity in levels of cell death initiated by different HlyA ST131 isolates, and this correlated with secreted HlyA levels. Investigation into the biological significance of this variation revealed that an ST131 strain producing low levels of HlyA initiated cell death that was partly dependent on the nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, with this response being associated with a host-protective role in a mouse UTI model. When the same ST131 strain was engineered to overexpress high HlyA levels, macrophage cell death occurred even when NLRP3 function was abrogated, and bladder colonization was significantly increased. Thus, variation in HlyA expression in UPEC affects mechanisms by which macrophages die, as well as host susceptibility resistance to colonization.-Murthy, A. M. V., Sullivan, M. J., Nhu, N. T. K., Lo, A. W., Phan, M.-D., Peters, K. M., Boucher, D., Schroder, K., Beatson, S. A., Ulett, G. C., Schembri, M. A., Sweet, M. J. Variation in hemolysin A expression between uropathogenic isolates determines NLRP3-dependent -independent macrophage cell death and host colonization.
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http://dx.doi.org/10.1096/fj.201802100RDOI Listing
June 2019

Uropathogenic employs both evasion and resistance to subvert innate immune-mediated zinc toxicity for dissemination.

Proc Natl Acad Sci U S A 2019 03 7;116(13):6341-6350. Epub 2019 Mar 7.

Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD 4072, Australia;

Toll-like receptor (TLR)-inducible zinc toxicity is a recently described macrophage antimicrobial response used against bacterial pathogens. Here we investigated deployment of this pathway against uropathogenic (UPEC), the major cause of urinary tract infections. Primary human macrophages subjected EC958, a representative strain of the globally disseminated multidrug-resistant UPEC ST131 clone, to zinc stress. We therefore used transposon-directed insertion site sequencing to identify the complete set of UPEC genes conferring protection against zinc toxicity. Surprisingly, zinc-susceptible EC958 mutants were not compromised for intramacrophage survival, whereas corresponding mutants in the nonpathogenic K-12 strain MG1655 displayed significantly reduced intracellular bacterial loads within human macrophages. To investigate whether the intramacrophage zinc stress response of EC958 reflected the response of only a subpopulation of bacteria, we generated and validated reporter systems as highly specific sensors of zinc stress. Using these tools we show that, in contrast to MG1655, the majority of intramacrophage EC958 evades the zinc toxicity response, enabling survival within these cells. In addition, EC958 has a higher tolerance to zinc than MG1655, with this likely being important for survival of the minor subset of UPEC cells exposed to innate immune-mediated zinc stress. Indeed, analysis of zinc stress reporter strains and zinc-sensitive mutants in an intraperitoneal challenge model in mice revealed that EC958 employs both evasion and resistance against zinc toxicity, enabling its dissemination to the liver and spleen. We thus demonstrate that a pathogen of global significance uses multiple mechanisms to effectively subvert innate immune-mediated zinc poisoning for systemic spread.
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http://dx.doi.org/10.1073/pnas.1820870116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442554PMC
March 2019

Author Correction: A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance.

Nat Commun 2019 03 1;10(1):976. Epub 2019 Mar 1.

Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD, 4072, Australia.

This Article contains errors in Fig. 1, Table 1 and the Methods section. In panel c, the labels for PmScsC and EcDsbC in the upper two curves are interchanged. In Table 1 and the Methods section entitled 'Extended structure', the space group of the extended PmScsC structure is incorrectly referred to as H3 and should read H32. Correct versions of Fig. 1 and Table 1 are presented below; the errors have not been corrected in the Article.
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http://dx.doi.org/10.1038/s41467-019-08920-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397206PMC
March 2019

Chemical Synergy between Ionophore PBT2 and Zinc Reverses Antibiotic Resistance.

mBio 2018 12 11;9(6). Epub 2018 Dec 11.

School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer's and Huntington's disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A (GAS), methicillin-resistant (MRSA), and vancomycin-resistant (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens. The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant and vancomycin-resistant are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, , a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, "On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you."
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http://dx.doi.org/10.1128/mBio.02391-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299484PMC
December 2018

Clay nanoparticles co-deliver three antigens to promote potent immune responses against pathogenic Escherichia coli.

J Control Release 2018 12 7;292:196-209. Epub 2018 Nov 7.

Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD 4072, Australia. Electronic address:

Currently, there are few strategies for controlling pathogenic bacteria, especially the pathotypes of Escherichia coli which are an emerging threat to public health worldwide. Here, multivalent vaccine formulations are reported for control of pathogenic E. coli. The formulations utilised clay nanoparticles, either layered double hydroxides (LDH) or hectorite (HEC), to complex with a cocktail of three recombinant antigens, intimin β (IB), proprietary antigen 1 (PAg1) and proprietary antigen 2 (PAg2). Acting as nano-adjuvants, LDH and HEC were able to stimulate strong, durable and balanced immune responses in mice. Moreover, LDH-IB-PAg1-PAg2 and HEC-IB-PAg1-PAg2 immunised mice developed potent mucosal immune responses and efficiently prevented adherence of enterohemorrhagic E. coli serotype O26 to mammalian cells. Notably, the multi-faceted immune responses elicited by the clay nanoparticle formulations were significantly higher than those induced by a QuilA formulation, without antigenic competition observed for the first time. The results of this study suggest that LDH and HEC offer considerable promise as effective multivalent vaccine carriers against important pathogens such as enteropathogenic E. coli.
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http://dx.doi.org/10.1016/j.jconrel.2018.11.008DOI Listing
December 2018

Discovery of -Mediated Colistin Resistance in a Highly Virulent Escherichia coli Lineage.

mSphere 2018 10 10;3(5). Epub 2018 Oct 10.

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia

Resistance to last-line polymyxins mediated by the plasmid-borne mobile colistin resistance gene () represents a new threat to global human health. Here we present the complete genome sequence of an -positive multidrug-resistant strain (MS8345). We show that MS8345 belongs to serotype O2:K1:H4, has a large 241,164-bp IncHI2 plasmid that carries 15 other antibiotic resistance genes (including the extended-spectrum β-lactamase ) and 3 putative multidrug efflux systems, and contains 14 chromosomally encoded antibiotic resistance genes. MS8345 also carries a large ColV-like virulence plasmid that has been associated with bacteremia. Whole-genome phylogeny revealed that MS8345 clusters within a discrete clade in the sequence type 95 (ST95) lineage, and MS8345 is very closely related to the highly virulent O45:K1:H4 clone associated with neonatal meningitis. Overall, the acquisition of a plasmid carrying resistance to colistin and multiple other antibiotics in this virulent lineage is concerning and might herald an era where the empirical treatment of ST95 infections becomes increasingly more difficult. ST95 is a globally disseminated clone frequently associated with bloodstream infections and neonatal meningitis. However, the ST95 lineage is defined by low levels of drug resistance amongst clinical isolates, which normally provides for uncomplicated treatment options. Here, we provide the first detailed genomic analysis of an ST95 isolate that has both high virulence potential and resistance to multiple antibiotics. Using the genome, we predicted its virulence and antibiotic resistance mechanisms, which include resistance to last-line antibiotics mediated by the plasmid-borne gene. Finding an ST95 isolate resistant to nearly all antibiotics that also has a high virulence potential is of major clinical importance and underscores the need to monitor new and emerging trends in antibiotic resistance development in this important global lineage.
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http://dx.doi.org/10.1128/mSphere.00486-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180223PMC
October 2018

Discovery of New Genes Involved in Curli Production by a Uropathogenic Escherichia coli Strain from the Highly Virulent O45:K1:H7 Lineage.

mBio 2018 08 21;9(4). Epub 2018 Aug 21.

School of Chemistry and Molecular Biosciences, the University of Queensland, Brisbane, Queensland, Australia

Curli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and facilitate uropathogenic (UPEC) biofilm formation and protection against host innate defenses. Here we sequenced the genome of the curli-producing UPEC pyelonephritis strain MS7163 and showed it belongs to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. MS7163 produced curli at human physiological temperature, and this correlated with biofilm growth, resistance of sessile cells to the human cationic peptide cathelicidin, and enhanced colonization of the mouse bladder. We devised a forward genetic screen using CR staining as a proxy for curli production and identified 41 genes that were required for optimal CR binding, of which 19 genes were essential for curli synthesis. Ten of these genes were novel or poorly characterized with respect to curli synthesis and included genes involved in purine biosynthesis, a regulator that controls the Rcs phosphorelay system, and a novel repressor of curli production (referred to as ). The involvement of these genes in curli production was confirmed by the construction of defined mutants and their complementation. The mutants did not express the curli major subunit CsgA and failed to produce curli based on CR binding. Mutation of (the first gene in the purine biosynthesis pathway) and also led to attenuated colonization of the mouse bladder. Overall, this work has provided new insight into the regulation of curli and the role of these amyloid fibers in UPEC biofilm formation and pathogenesis. Uropathogenic (UPEC) strains are the most common cause of urinary tract infection, a disease increasingly associated with escalating antibiotic resistance. UPEC strains possess multiple surface-associated factors that enable their colonization of the urinary tract, including fimbriae, curli, and autotransporters. Curli are extracellular amyloid fibers that enhance UPEC virulence and promote biofilm formation. Here we examined the function and regulation of curli in a UPEC pyelonephritis strain belonging to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. Curli expression at human physiological temperature led to increased biofilm formation, resistance of sessile cells to the human cationic peptide LL-37, and enhanced bladder colonization. Using a comprehensive genetic screen, we identified multiple genes involved in curli production, including several that were novel or poorly characterized with respect to curli synthesis. In total, this study demonstrates an important role for curli as a UPEC virulence factor that promotes biofilm formation, resistance, and pathogenesis.
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http://dx.doi.org/10.1128/mBio.01462-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6106082PMC
August 2018