92 results on '"Forde BM"'
Search Results
2. Optimising Treatment Outcomes for Children and Adults Through Rapid Genome Sequencing of Sepsis Pathogens. A Study Protocol for a Prospective, Multi-Centre Trial (DIRECT)
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Irwin, AD, Coin, LJM, Harris, PNA, Cotta, MO, Bauer, MJ, Buckley, C, Balch, R, Kruger, P, Meyer, J, Shekar, K, Brady, K, Fourie, C, Sharp, N, Vlad, L, Whiley, D, Beatson, SA, Forde, BM, Paterson, D, Clark, J, Hajkowicz, K, Raman, S, Bialasiewicz, S, Lipman, J, Schlapbach, LJ, Roberts, JA, Irwin, AD, Coin, LJM, Harris, PNA, Cotta, MO, Bauer, MJ, Buckley, C, Balch, R, Kruger, P, Meyer, J, Shekar, K, Brady, K, Fourie, C, Sharp, N, Vlad, L, Whiley, D, Beatson, SA, Forde, BM, Paterson, D, Clark, J, Hajkowicz, K, Raman, S, Bialasiewicz, S, Lipman, J, Schlapbach, LJ, and Roberts, JA
- Abstract
BACKGROUND: Sepsis contributes significantly to morbidity and mortality globally. In Australia, 20,000 develop sepsis every year, resulting in 5,000 deaths, and more than AUD$846 million in expenditure. Prompt, appropriate antibiotic therapy is effective in improving outcomes in sepsis. Conventional culture-based methods to identify appropriate therapy have limited yield and take days to complete. Recently, nanopore technology has enabled rapid sequencing with real-time analysis of pathogen DNA. We set out to demonstrate the feasibility and diagnostic accuracy of pathogen sequencing direct from clinical samples, and estimate the impact of this approach on time to effective therapy when integrated with personalised software-guided antimicrobial dosing in children and adults on ICU with sepsis. METHODS: The DIRECT study is a pilot prospective, non-randomized multicentre trial of an integrated diagnostic and therapeutic algorithm combining rapid direct pathogen sequencing and software-guided, personalised antibiotic dosing in children and adults with sepsis on ICU. PARTICIPANTS AND INTERVENTIONS: DIRECT will collect microbiological and pharmacokinetic samples from approximately 200 children and adults with sepsis admitted to one of four ICUs in Brisbane. In Phase 1, we will evaluate Oxford Nanopore Technologies MinION sequencing direct from blood in 50 blood culture-proven sepsis patients recruited from consecutive patients with suspected sepsis. In Phase 2, a further 50 consecutive patients with suspected sepsis will be recruited in whom MinION sequencing will be combined with Bayesian software-guided (ID-ODS) personalised antimicrobial dosing. OUTCOME MEASURES: The primary outcome is time to effective antimicrobial therapy, defined as trough drug concentrations above the MIC of the pathogen. Secondary outcomes are diagnostic accuracy of MinION sequencing from whole blood, time to pathogen identification and susceptibility testing using sequencing direct from whole bl
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- 2021
3. Predicting nitroimidazole antibiotic resistance mutations in Mycobacterium tuberculosis with protein engineering
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Boshoff, HI, Lee, BM, Harold, LK, Almeida, DV, Afriat-Jurnou, L, Aung, HL, Forde, BM, Hards, K, Pidot, SJ, Ahmed, FH, Mohamed, AE, Taylor, MC, West, NP, Stinear, TP, Greening, C, Beatson, SA, Nuermberger, EL, Cook, GM, Jackson, CJ, Boshoff, HI, Lee, BM, Harold, LK, Almeida, DV, Afriat-Jurnou, L, Aung, HL, Forde, BM, Hards, K, Pidot, SJ, Ahmed, FH, Mohamed, AE, Taylor, MC, West, NP, Stinear, TP, Greening, C, Beatson, SA, Nuermberger, EL, Cook, GM, and Jackson, CJ
- Abstract
Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.
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- 2020
4. Successful treatment of a chronic oroantral fistula infected with extensively drug resistant bacteria using long‐term oesophageal tube feeding and several non‐conventional treatments in a horse.
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Fletcher, JR, Yuen, KY, Stewart, AJ, Young, AC, Gibson, JS, James, OA, Medina‐Torres, CE, Forde, BM, and Sole‐Guitart, A
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TUBE feeding ,AMIKACIN ,TREATMENT effectiveness ,FEEDING tubes ,FEED contamination ,CIDER vinegar ,ENTERAL feeding - Abstract
Background: Chronic oroantral fistulae (OAF) with secondary sinusitis can occur following repulsion of cheek teeth in horses. Case Report: An 8‐year‐old Andalusian cross gelding presented with an iatrogenic clinical crown fracture of tooth 209, which underwent repulsion of its apical portion (day 0). The horse was treated with intramuscular penicillin and intravenous gentamicin (5 days), followed by oral trimethoprim‐sulphonamide (10 days) and then oral doxycycline (14 days). The acute iatrogenic OAF created during the initial repulsion persisted; a chronic OAF was identified on day 24. On day 48, septic sinusitis with multidrug‐resistant (MDR) Escherichia coli was confirmed. Although susceptible to enrofloxacin in vitro, 30 days of therapy was unsuccessful. Subsequent serial cultures grew multiple MDR and extensively drug‐resistant (XDR) gram‐negative microorganisms. Whole‐genome sequencing (WGS) revealed multiple sequence types of E. coli, with a range of resistance and virulence genes. The orientation of the OAF, regional osteomyelitis and septic sinusitis were confirmed with computed tomography on day 70. On day 74, enteral nutrition was provided through a cervical oesophagostomy tube for 3 months for prevention of oral feed contamination. The OAF was treated with various alternative therapeutics, including apple cider vinegar, propolis and amikacin impregnated products, until resolution on day 116. Conclusion: These non‐conventional therapeutics, antimicrobials and long‐term oesophagostomy contributed to the successful treatment of a complicated OAF. In the future, WGS may be useful to inform antimicrobial selection when MDR or XDR organisms are identified. [ABSTRACT FROM AUTHOR]
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- 2022
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5. Complete Genome Sequence of Serotype III Streptococcus agalactiae Sequence Type 17 Strain 874391
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Sullivan, MJ, Forde, BM, Prince, DW, Ipe, DS, Ben Zakour, NL, Davies, MR, Dougan, G, Beatson, SA, Ulett, GC, Sullivan, MJ, Forde, BM, Prince, DW, Ipe, DS, Ben Zakour, NL, Davies, MR, Dougan, G, Beatson, SA, and Ulett, GC
- Abstract
Here we report the complete genome sequence of Streptococcus agalactiae strain 874391. This serotype III isolate is a member of the hypervirulent sequence type 17 (ST-17) lineage that causes a disproportionate number of cases of invasive disease in humans and mammals. A brief historical context of the strain is discussed.
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- 2017
6. Mechanisms involved in acquisition of blaNDM genes by IncA/C2 and IncFIIY plasmids
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Wailan, AM, Sidjabat, HE, Yam, WK, Alikhan, NF, Petty, NK, Sartor, AL, Williamson, DA, Forde, BM, Schembri, MA, Beatson, SA, Paterson, DL, Walsh, TR, and Partridge, SR
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Klebsiella pneumoniae ,Acinetobacter ,Carbapenems ,RNA, Ribosomal, 16S ,Drug Resistance, Multiple, Bacterial ,Enterobacter cloacae ,Escherichia coli ,DNA Transposable Elements ,Microbial Sensitivity Tests ,Microbiology ,beta-Lactamases ,Anti-Bacterial Agents ,Plasmids - Abstract
Copyright © 2016, American Society for Microbiology. All Rights Reserved. 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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- 2016
7. Lineage-Specific Methyltransferases Define the Methylome of the Globally Disseminated Escherichia coli ST131 Clone
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Sperandio, V, Forde, BM, Minh-Duy, P, Gawthorne, JA, Ashcroft, MM, Stanton-Cook, M, Sarkar, S, Peters, KM, Chan, K-G, Chong, TM, Yin, W-F, Upton, M, Schembri, MA, Beatson, SA, Sperandio, V, Forde, BM, Minh-Duy, P, Gawthorne, JA, Ashcroft, MM, Stanton-Cook, M, Sarkar, S, Peters, KM, Chan, K-G, Chong, TM, Yin, W-F, Upton, M, Schembri, MA, and Beatson, SA
- Abstract
UNLABELLED: Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located. IMPORTANCE: DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-spe
- Published
- 2015
8. Transfer of scarlet fever-associated elements into the group A Streptococcus M1T1 clone
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Ben Zakour, NL, Davies, MR, You, Y, Chen, JHK, Forde, BM, Stanton-Cook, M, Yang, R, Cui, Y, Barnett, TC, Venturini, C, Ong, C-IY, Tse, H, Dougan, G, Zhang, J, Yuen, K-Y, Beatson, SA, Walker, MJ, Ben Zakour, NL, Davies, MR, You, Y, Chen, JHK, Forde, BM, Stanton-Cook, M, Yang, R, Cui, Y, Barnett, TC, Venturini, C, Ong, C-IY, Tse, H, Dougan, G, Zhang, J, Yuen, K-Y, Beatson, SA, and Walker, MJ
- Abstract
The group A Streptococcus (GAS) M1T1 clone emerged in the 1980s as a leading cause of epidemic invasive infections worldwide, including necrotizing fasciitis and toxic shock syndrome. Horizontal transfer of mobile genetic elements has played a central role in the evolution of the M1T1 clone, with bacteriophage-encoded determinants DNase Sda1 and superantigen SpeA2 contributing to enhanced virulence and colonization respectively. Outbreaks of scarlet fever in Hong Kong and China in 2011, caused primarily by emm12 GAS, led to our investigation of the next most common cause of scarlet fever, emm1 GAS. Genomic analysis of 18 emm1 isolates from Hong Kong and 16 emm1 isolates from mainland China revealed the presence of mobile genetic elements associated with the expansion of emm12 scarlet fever clones in the M1T1 genomic background. These mobile genetic elements confer expression of superantigens SSA and SpeC, and resistance to tetracycline, erythromycin and clindamycin. Horizontal transfer of mobile DNA conferring multi-drug resistance and expression of a new superantigen repertoire in the M1T1 clone should trigger heightened public health awareness for the global dissemination of these genetic elements.
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- 2015
9. Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition
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Beatson, SA, Ben Zakour, NL, Totsika, M, Forde, BM, Watts, RE, Mabbett, AN, Szubert, JM, Sarkar, S, Phan, MD, Peters, KM, Petty, NK, Alikhan, NF, Sullivan, MJ, Gawthorne, JA, Stanton-Cook, M, Nhu, NTK, Chong, TM, Yin, WF, Chan, KG, Hancock, V, Ussery, DW, Ulett, GC, Schembri, MA, Beatson, SA, Ben Zakour, NL, Totsika, M, Forde, BM, Watts, RE, Mabbett, AN, Szubert, JM, Sarkar, S, Phan, MD, Peters, KM, Petty, NK, Alikhan, NF, Sullivan, MJ, Gawthorne, JA, Stanton-Cook, M, Nhu, NTK, Chong, TM, Yin, WF, Chan, KG, Hancock, V, Ussery, DW, Ulett, GC, and Schembri, MA
- Abstract
© 2015, American Society for Microbiology. Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo.We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.
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- 2015
10. Global dissemination of a multidrug resistant Escherichia coli clone
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Mark A. Schembri, Gordon Dougan, Minh-Duy Phan, Jesús Rodríguez-Baño, David L. Paterson, Danilo Gomes Moriel, Elizabeth Skippington, Álvaro Pascual, Nicola K. Petty, Brian M. Forde, Scott A. Beatson, Johann D. D. Pitout, Benjamin A. Rogers, Mark R. Davies, Kate M. Peters, Timothy R. Walsh, Mathew Upton, Mitchell Stanton-Cook, Nouri L. Ben Zakour, Makrina Totsika, [Petty,NK, Ben Zakour,NL, Stanton-Cook,M, Skippington,E, Totsika,M, Forde,BM, Phan,BM, Gomes Moriel,D, Peters,KM, Davies,M, Paterson,DL, Schembri,MA, Beatson,SA] Australian Infectious Diseases Research Centre, Brisbane, Australia. [Petty,NK, Beatson,SA] School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia. [Petty,NK] The ithree institute, University of Technology Sydney, Sydney, Australia. [Davies,M, Dougan,G] Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom. [Rogers,BA, Paterson,DL] University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital Campus, The University of Queensland, Brisbane, Australia. [Rodriguez-Baño,J, Pascual,A] Unidad Clínica de Enfermedades Infecciosas y Microbiología, Hospital Universitario Virgen Macarena, Seville, Spain, Departmentos de Medicina y Microbiología, Universidad de Sevilla, Seville, Spain. [Pitout,JDD] Division of Microbiology, Calgary Laboratory Services, Calgary, Canada, Department of Pathology and Laboratory Medicine, and Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Canada. [Upton,M] School of Biomedical and Healthcare Sciences, Plymouth University, Plymouth, United Kingdom. [Walsh,TR] School of Medicine, Cardiff University, Cardiff, United Kingdom., Universidad de Sevilla. Departamento de Medicina, and Universidad de Sevilla. Departamento de Microbiología
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medicine.disease_cause ,Genome ,Chemicals and Drugs::Heterocyclic Compounds::Heterocyclic Compounds, 2-Ring::Quinolines::Quinolones::Fluoroquinolones [Medical Subject Headings] ,Bacterial evolution ,Drug Resistance, Multiple, Bacterial ,Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Epidemiologic Methods::Statistics as Topic::Models, Statistical::Likelihood Functions [Medical Subject Headings] ,Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome, Microbial::Genome, Bacterial [Medical Subject Headings] ,Phylogeny ,Genetics ,Disciplines and Occupations::Natural Science Disciplines::Biological Science Disciplines::Biology::Genetics::Genetics, Population::Phylogeography [Medical Subject Headings] ,Likelihood Functions ,Multidisciplinary ,Chemicals and Drugs::Enzymes and Coenzymes::Enzymes::Hydrolases::Amidohydrolases::beta-Lactamases [Medical Subject Headings] ,Genomics ,Biological Sciences ,Organisms::Bacteria::Gram-Negative Bacteria::Gram-Negative Facultatively Anaerobic Rods::Enterobacteriaceae::Escherichia::Escherichia coli [Medical Subject Headings] ,Phenomena and Processes::Microbiological Phenomena::Bacterial Physiological Phenomena::Drug Resistance, Bacterial::Drug Resistance, Multiple, Bacterial [Medical Subject Headings] ,Phylogeography ,Farmacorresistencia bacteriana múltiple ,Disciplines and Occupations::Natural Science Disciplines::Biological Science Disciplines::Biology::Computational Biology [Medical Subject Headings] ,Fluoroquinolonas ,Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Sequence Analysis::Sequence Analysis, DNA [Medical Subject Headings] ,Fluoroquinolones ,Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Models, Theoretical::Models, Biological::Models, Genetic [Medical Subject Headings] ,Sequence analysis ,Molecular Sequence Data ,Virulence ,Biology ,Polymorphism, Single Nucleotide ,beta-Lactamases ,Antibiotic resistance ,Species Specificity ,medicine ,Phenomena and Processes::Biological Phenomena::Species Specificity [Medical Subject Headings] ,Escherichia coli ,Secuencia de bases ,Phenomena and Processes::Genetic Phenomena::Genetic Variation::Polymorphism, Genetic::Polymorphism, Single Nucleotide [Medical Subject Headings] ,Molecular epidemiology ,Base Sequence ,Models, Genetic ,Computational Biology ,Sequence Analysis, DNA ,Genomic epidemiology ,Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Sequence Alignment [Medical Subject Headings] ,Multiple drug resistance ,Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Molecular Structure::Base Sequence [Medical Subject Headings] ,Information Science::Information Science::Information Services::Documentation::Molecular Sequence Data [Medical Subject Headings] ,Phenomena and Processes::Genetic Phenomena::Phylogeny [Medical Subject Headings] ,Mobile genetic elements ,Sequence Alignment ,Genome, Bacterial - Abstract
Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum β-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000-2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL-resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen. Australian National Health and Medical Research Council (NHMRC); the Australian Infectious Diseases Research Centre; ARC Discovery Early Career Researcher Award; Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, the Spanish Network for Research in Infectious Diseases; Fondo de Investigación Sanitaria and Junta de Andalucía. Yes
- Published
- 2014
11. Misregulation of mitochondrial 6mA promotes the propagation of mutant mtDNA and causes aging in C. elegans.
- Author
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Hahn A, Hung GCC, Ahier A, Dai CY, Kirmes I, Forde BM, Campbell D, Lee RSY, Sucic J, Onraet T, and Zuryn S
- Abstract
In virtually all eukaryotes, the mitochondrial DNA (mtDNA) encodes proteins necessary for oxidative phosphorylation (OXPHOS) and RNAs required for their synthesis. The mechanisms of regulation of mtDNA copy number and expression are not completely understood but crucially ensure the correct stoichiometric assembly of OXPHOS complexes from nuclear- and mtDNA-encoded subunits. Here, we detect adenosine N6-methylation (6mA) on the mtDNA of diverse animal and plant species. This modification is regulated in C. elegans by the DNA methyltransferase DAMT-1 and demethylase ALKB-1. Misregulation of mtDNA 6mA through targeted modulation of these activities inappropriately alters mtDNA copy number and transcript levels, impairing OXPHOS function, elevating oxidative stress, and shortening lifespan. Compounding these defects, mtDNA 6mA hypomethylation promotes the cross-generational propagation of a deleterious mtDNA. Together, these results reveal that mtDNA 6mA is highly conserved among eukaryotes and regulates lifespan by influencing mtDNA copy number, expression, and heritable mutation levels in vivo., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)
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- 2024
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12. The Genomic Epidemiology of Clinical Burkholderia pseudomallei Isolates in North Queensland, Australia.
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Gassiep I, Chatfield MD, Permana B, Burnard D, Bauer MJ, Cuddihy T, Forde BM, Mayer-Coverdale J, Norton RE, and Harris PNA
- Abstract
Background: Burkholderia pseudomallei , the causative agent of melioidosis, is highly genetically recombinant, resulting in significant genomic diversity. Multiple virulence factors have been associated with specific disease presentations. To date, there are limited data relating to genomic diversity and virulence factors associated with melioidosis cases in North Queensland, Australia. Aim: To describe the genetic diversity of B. pseudomallei and identify virulence factors associated with clinical risk factors and patient outcomes. Methods: Whole genome sequencing of clinical isolates was performed and analysed with clinical data obtained from a retrospective melioidosis cohort study. Results: Fifty-nine distinct sequence types (STs) were identified from the 128 clinical isolates. Six STs comprised 64/128 (50%) isolates. Novel STs accounted for 38/59 (64%) STs, with ST TSV-13 as the most prevalent (n = 7), and were less likely to possess an LPS A genotype or YLF gene cluster ( p < 0.001). These isolates were most likely to be found outside the inner city (aOR: 4.0, 95% CI: 1.7-9.0, p = 0.001). ST TSV-13 was associated with increased mortality (aOR: 6.1, 95% CI: 1.2-30.9, p = 0.03). Patients with a history of alcohol excess were less likely to be infected by fhaB 3 (aOR 0.2, 95% CI: 0.1-0.7, p = 0.01) or YLF (aOR: 0.4, 95% CI: 0.2-0.9, p = 0.04) positive isolates. Conclusions: There are a significant number of novel sequence types in Townsville, Australia. An emerging novel ST appears to have an association with geographic location and mortality. Ongoing investigation is required to further understand the impact of this ST on the Townsville region.
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- 2024
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13. Demographic, clinical and molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli bloodstream infections in Central Australia.
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Langham F, Tsai D, Forde BM, Camilleri S, Harris PNA, Roberts JA, and Chiong F
- Abstract
We describe the demographics, clinical and molecular epidemiology of extended-spectrum β-lactamase (ESBL) Escherichia coli bloodstream infections (BSI) in Central Australia. All ESBL-producing E. coli bloodstream isolates from January 2018 to December 2020 were retrospectively identified. Demographic and clinical information was extracted by chart review. Whole-genome sequencing was performed for multi-locus sequence typing, antibiotic-resistance genes, and phylogenetic relationships. We identified 41 non-duplicate episodes of ESBL E. coli BSI. Median age was 55 years (IQR 47-63), 78% were female, 93% were Aboriginal, and half came from a remote community. Infections were predominantly urinary (68%, 28/41). In the 12 months prior, 70% (26/37) of identified patients had been hospitalised and 81% (30/37) prescribed antibiotics. Meropenem and piperacillin-tazobactam susceptibility was maintained in 100% and 95% of isolates, respectively. Co-resistance to non-β-lactam antibiotics was 32% to gentamicin, 61% to trimethoprim/sulfamethoxazole, and 68% to ciprofloxacin. For sequenced isolates, 41% (16/35) were sequence type 131 (ST131). Mean acquired antibiotic-resistance genes for each isolate was 12.3 (SD 3.1). Four isolates carried an OXA-1 gene. Only non-ST131 isolates carried AmpC and acquired quinolone-resistance genes. There was some evidence of clustering of closely related strains, but no evidence of community or healthcare admission overlap. ESBL rates are rapidly rising in Central Australia, which is a conducive environment for antibiotic resistance development (e.g., overcrowding, socioeconomic disadvantages, high healthcare exposure and high antibiotic use). Future research is required to explore resistance-transmission dynamics in this unique setting., (Copyright © 2024 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)
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- 2024
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14. Correction: Achievement of therapeutic antibiotic exposures using Bayesian dosing software in critically unwell children and adults with sepsis.
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Chai MG, Tu Q, Cotta MO, Bauer MJ, Balch R, Okafor C, Comans T, Kruger P, Meyer J, Shekar K, Brady K, Fourie C, Sharp N, Vlad L, Whiley D, Ungerer JPJ, Mcwhinney BC, Farkas A, Paterson DL, Clark JE, Hajkowicz K, Raman S, Bialasiewicz S, Lipman J, Forde BM, Harris PNA, Schlapbach LJ, Coin L, Roberts JA, and Irwin AD
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- 2024
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15. High-risk Escherichia coli clones that cause neonatal meningitis and association with recrudescent infection.
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Nhu NTK, Phan MD, Hancock SJ, Peters KM, Alvarez-Fraga L, Forde BM, Andersen SB, Miliya T, Harris PNA, Beatson SA, Schlebusch S, Bergh H, Turner P, Brauner A, Westerlund-Wikström B, Irwin AD, and Schembri MA
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- Infant, Newborn, Humans, Escherichia coli genetics, Virulence genetics, Clone Cells, Escherichia coli Infections, Meningitis
- Abstract
Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974-2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection., Competing Interests: NN, MP, SH, KP, LA, BF, SA, TM, PH, SB, SS, HB, PT, AB, BW, AI, MS No competing interests declared, (© 2023, Nhu et al.)
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- 2024
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16. Achievement of therapeutic antibiotic exposures using Bayesian dosing software in critically unwell children and adults with sepsis.
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Chai MG, Tu Q, Cotta MO, Bauer MJ, Balch R, Okafor C, Comans T, Kruger P, Meyer J, Shekar K, Brady K, Fourie C, Sharp N, Vlad L, Whiley D, Ungerer JPJ, Mcwhinney BC, Farkas A, Paterson DL, Clark JE, Hajkowicz K, Raman S, Bialasiewicz S, Lipman J, Forde BM, Harris PNA, Schlapbach LJ, Coin L, Roberts JA, and Irwin AD
- Subjects
- Adult, Child, Humans, Bayes Theorem, Critical Illness therapy, Intensive Care Units, Pediatric, Prospective Studies, Software, Anti-Bacterial Agents therapeutic use, Sepsis drug therapy
- Abstract
Purpose: Early recognition and effective treatment of sepsis improves outcomes in critically ill patients. However, antibiotic exposures are frequently suboptimal in the intensive care unit (ICU) setting. We describe the feasibility of the Bayesian dosing software Individually Designed Optimum Dosing Strategies (ID-ODS™), to reduce time to effective antibiotic exposure in children and adults with sepsis in ICU., Methods: A multi-centre prospective, non-randomised interventional trial in three adult ICUs and one paediatric ICU. In a pre-intervention Phase 1, we measured the time to target antibiotic exposure in participants. In Phase 2, antibiotic dosing recommendations were made using ID-ODS™, and time to target antibiotic concentrations were compared to patients in Phase 1 (a pre-post-design)., Results: 175 antibiotic courses (Phase 1 = 123, Phase 2 = 52) were analysed from 156 participants. Across all patients, there was no difference in the time to achieve target exposures (8.7 h vs 14.3 h in Phase 1 and Phase 2, respectively, p = 0.45). Sixty-one courses in 54 participants failed to achieve target exposures within 24 h of antibiotic commencement (n = 36 in Phase 1, n = 18 in Phase 2). In these participants, ID-ODS™ was associated with a reduction in time to target antibiotic exposure (96 vs 36.4 h in Phase 1 and Phase 2, respectively, p < 0.01). These patients were less likely to exhibit subtherapeutic antibiotic exposures at 96 h (hazard ratio (HR) 0.02, 95% confidence interval (CI) 0.01-0.05, p < 0.01). There was no difference observed in in-hospital mortality., Conclusions: Dosing software may reduce the time to achieve target antibiotic exposures. It should be evaluated further in trials to establish its impact on clinical outcomes., (© 2024. The Author(s).)
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- 2024
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17. Emergence and clonal expansion of a qacA-harbouring sequence type 45 lineage of methicillin-resistant Staphylococcus aureus.
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Nong Y, Steinig E, Pollock GL, Taiaroa G, Carter GP, Monk IR, Pang S, Daley DA, Coombs GW, Forde BM, Harris PNA, Sherry NL, Howden BP, Pasricha S, Baines SL, and Williamson DA
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- Humans, Staphylococcus aureus genetics, Bayes Theorem, Phylogeny, Membrane Transport Proteins genetics, Bacterial Proteins genetics, Australia, Methicillin-Resistant Staphylococcus aureus genetics, Staphylococcal Infections epidemiology
- Abstract
The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus, we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA, predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn552 (carrying blaZ) and Tn4001 (carrying aac(6')-aph(2")) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA., (© 2024. The Author(s).)
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- 2024
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18. TcrXY is an acid-sensing two-component transcriptional regulator of Mycobacterium tuberculosis required for persistent infection.
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Stupar M, Tan L, Kerr ED, De Voss CJ, Forde BM, Schulz BL, and West NP
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- Animals, Humans, Mice, Antitubercular Agents chemistry, Isoniazid, Persistent Infection, Rifampin, Genes, Bacterial physiology, Mycobacterium tuberculosis genetics
- Abstract
The ability of Mycobacterium tuberculosis (Mtb) to persist in the host complicates and prolongs tuberculosis (TB) patient chemotherapy. Here we demonstrate that a neglected two-component system (TCS) of Mtb, TcrXY, is an autoregulated acid-sensing TCS that controls a functionally diverse 70-gene regulon required for bacterial persistence. Characterisation of two representatives of this regulon, Rv3706c and Rv3705A, implicate these genes as key determinants for the survival of Mtb in vivo by serving as important effectors to mitigate redox stress at acidic pH. We show that genetic silencing of the response regulator tcrX using CRISPR interference attenuates the persistence of Mtb during chronic mouse infection and improves treatment with the two front-line anti-TB drugs, rifampicin and isoniazid. We propose that targeting TcrXY signal transduction blocks the ability of Mtb to sense and respond to acid stress, resulting in a disordered program of persistence to render the organism vulnerable to existing TB chemotherapy., (© 2024. The Author(s).)
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- 2024
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19. Genomic epidemiology reveals geographical clustering of multidrug-resistant Escherichia coli ST131 associated with bacteraemia in Wales.
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White RT, Bull MJ, Barker CR, Arnott JM, Wootton M, Jones LS, Howe RA, Morgan M, Ashcroft MM, Forde BM, Connor TR, and Beatson SA
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- Humans, Escherichia coli, Wales epidemiology, Genotype, Genomics, beta-Lactamases genetics, Cluster Analysis, Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents therapeutic use, Drug Resistance, Multiple, Bacterial genetics, Escherichia coli Infections epidemiology, Escherichia coli Proteins genetics, Bacteremia epidemiology
- Abstract
Antibiotic resistance is a significant global public health concern. Uropathogenic Escherichia coli sequence type (ST)131, a widely prevalent multidrug-resistant clone, is frequently associated with bacteraemia. This study investigates third-generation cephalosporin resistance in bloodstream infections caused by E. coli ST131. From 2013-2014 blood culture surveillance in Wales, 142 E. coli ST131 genomes were studied alongside global data. All three major ST131 clades were represented across Wales, with clade C/H30 predominant (n = 102/142, 71.8%). Consistent with global findings, Welsh strains of clade C/H30 contain β-lactamase genes from the bla
CTX-M-1 group (n = 65/102, 63.7%), which confer resistance to third-generation cephalosporins. Most Welsh clade C/H30 genomes belonged to sub-clade C2/H30Rx (58.3%). A Wales-specific sub-lineage, named GB-WLS.C2, diverged around 1996-2000. An introduction to North Wales around 2002 led to a localised cluster by 2009, depicting limited genomic diversity within North Wales. This investigation emphasises the value of genomic epidemiology, allowing the detection of genetically similar strains in local areas, enabling targeted and timely public health interventions., (© 2024. The Author(s).)- Published
- 2024
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20. Rapid nanopore sequencing and predictive susceptibility testing of positive blood cultures from intensive care patients with sepsis.
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Harris PNA, Bauer MJ, Lüftinger L, Beisken S, Forde BM, Balch R, Cotta M, Schlapbach L, Raman S, Shekar K, Kruger P, Lipman J, Bialasiewicz S, Coin L, Roberts JA, Paterson DL, and Irwin AD
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- Humans, Blood Culture methods, Microbial Sensitivity Tests, Anti-Bacterial Agents, Critical Care, Nanopore Sequencing, Sepsis microbiology
- Abstract
We aimed to evaluate the performance of Oxford Nanopore Technologies (ONT) sequencing from positive blood culture (BC) broths for bacterial identification and antimicrobial susceptibility prediction. Patients with suspected sepsis in four intensive care units were prospectively enrolled. Human-depleted DNA was extracted from positive BC broths and sequenced using ONT (MinION). Species abundance was estimated using Kraken2, and a cloud-based system (AREScloud) provided in silico predictive antimicrobial susceptibility testing (AST) from assembled contigs. Results were compared to conventional identification and phenotypic AST. Species-level agreement between conventional methods and AST predicted from sequencing was 94.2% (49/52), increasing to 100% in monomicrobial infections. In 262 high-quality AREScloud AST predictions across 24 samples, categorical agreement (CA) was 89.3%, with major error (ME) and very major error (VME) rates of 10.5% and 12.1%, respectively. Over 90% CA was achieved for some taxa (e.g., Staphylococcus aureus ) but was suboptimal for Pseudomonas aeruginosa . In 470 AST predictions across 42 samples, with both high quality and exploratory-only predictions, overall CA, ME, and VME rates were 87.7%, 8.3%, and 28.4%. VME rates were inflated by false susceptibility calls in a small number of species/antibiotic combinations with few representative resistant isolates. Time to reporting from sequencing could be achieved within 8-16 h from BC positivity. Direct sequencing from positive BC broths is feasible and can provide accurate predictive AST for some species. ONT-based approaches may be faster but significant improvements in accuracy are required before it can be considered for clinical use.IMPORTANCESepsis and bloodstream infections carry a high risk of morbidity and mortality. Rapid identification and susceptibility prediction of causative pathogens, using Nanopore sequencing direct from blood cultures, may offer clinical benefit. We assessed this approach in comparison to conventional phenotypic methods and determined the accuracy of species identification and susceptibility prediction from genomic data. While this workflow holds promise, and performed well for some common bacterial species, improvements in sequencing accuracy and more robust predictive algorithms across a diverse range of organisms are required before this can be considered for clinical use. However, results could be achieved in timeframes that are faster than conventional phenotypic methods., Competing Interests: Lukas Lüftinger and Stephan Beisken are employees of Ares Genetics. Patrick Harris reports research grants from Gilead, has served on advisory boards for OpGen, Merck, and Sandoz, and has received honoraria from OpGen, Sandoz, Pfizer, and BioMerieux. David Paterson reports grants from Shionogi, Pfizer, Merck and bioMerieux, and consultancies with the AMR Action Fund, Entasis, QPex, Spero, VenatoRx, Pfizer, Merck, Gilead, bioMerieux, and Accelerate Diagnostics. Jason Roberts reports grants from Qpex, Gilead, Pfizer, Sandoz, MSD, Summit Pharma, and Cipla. Adam Irwin has received research grants and honoraria from Gilead, and honoraria from bioMerieux unrelated to this work.
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- 2024
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21. HAIviz: an interactive dashboard for visualising and integrating healthcare-associated genomic epidemiological data.
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Permana B, Harris PNA, Roberts LW, Cuddihy T, Paterson DL, Beatson SA, and Forde BM
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- Humans, Phylogeny, Disease Outbreaks, Pandemics, Genomics, Cross Infection epidemiology
- Abstract
Existing tools for phylogeographic and epidemiological visualisation primarily provide a macro-geographic view of epidemic and pandemic transmission events but offer little support for detailed investigation of outbreaks in healthcare settings. Here, we present HAIviz, an interactive web-based application designed for integrating and visualising genomic epidemiological information to improve the tracking of healthcare-associated infections (HAIs). HAIviz displays and links the outbreak timeline, building map, phylogenetic tree, patient bed movements, and transmission network on a single interactive dashboard. HAIviz has been developed for bacterial outbreak investigations but can be utilised for general epidemiological investigations focused on built environments for which visualisation to customised maps is required. This paper describes and demonstrates the application of HAIviz for HAI outbreak investigations.
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- 2024
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22. Melioidosis Queensland: An analysis of clinical outcomes and genomic factors.
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Gassiep I, Burnard D, Permana B, Bauer MJ, Cuddihy T, Forde BM, Chatfield MD, Ling W, Norton R, and Harris PNA
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- Humans, Aged, Queensland epidemiology, Australia epidemiology, Genomics, Melioidosis epidemiology, Burkholderia pseudomallei genetics
- Abstract
Background: The clinical and genomic epidemiology of melioidosis varies across regions., Aim: To describe the clinical and genetic diversity of B. pseudomallei across Queensland, Australia., Methods: Whole genome sequencing of clinical isolates stored at the melioidosis reference lab from 1996-2020 was performed and analysed in conjunction with available clinical data., Results: Isolates from 292 patients were analysed. Bacteraemia was present in 71% and pneumonia in 65%. The case-fatality rate was 25%. Novel sequence types (ST) accounted for 51% of all isolates. No association was identified between the variable virulence factors assessed and patient outcome. Over time, the proportion of First Nation's patients declined from 59% to 26%, and the proportion of patients aged >70 years rose from 13% to 38%., Conclusion: This study describes a genomically diverse and comparatively distinct collection of B. pseudomallei clinical isolates from across Queensland, Australia. An increasing incidence of melioidosis in elderly patients may be an important factor in the persistently high case-fatality in this region and warrants further investigation and directed intervention., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gassiep 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.)
- Published
- 2023
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23. Using Genomics To Investigate an Outbreak of Vancomycin-Resistant Enterococcus faecium ST78 at a Large Tertiary Hospital in Queensland.
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Permana B, Harris PNA, Runnegar N, Lindsay M, Henderson BC, Playford EG, Paterson DL, Beatson SA, and Forde BM
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- Humans, Vancomycin, Queensland epidemiology, Tertiary Care Centers, Phylogeny, Australia epidemiology, Genomics, Disease Outbreaks, Enterococcus faecium genetics, Vancomycin-Resistant Enterococci genetics, Cross Infection epidemiology, Gram-Positive Bacterial Infections epidemiology
- Abstract
To investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) sequence type 78 (ST78) in a large tertiary Australian hospital. A collection of 63 VREfm ST78 isolates, identified during a routine genomic surveillance program, were subjected to genomic epidemiological analysis based on whole-genome sequencing (WGS) data. The population structure was reconstructed using phylogenetic analysis, and a collection of publicly available VREfm ST78 genomes were used to provide global context. Core genome single nucleotide polymorphism (SNP) distances and available clinical metadata were used to characterize outbreak clusters and reconstruct transmission events. In silico genotyping confirmed that all study isolates were vanB -type VREfm carrying virulence characteristics of the hospital-associated E. faecium. Phylogenetic analysis identified two distinct phylogenetic clades, only one of which was responsible for a hospital outbreak. Four outbreak subtypes could be defined with examples of recent transmissions. Inference on transmission trees suggested complex transmission routes with unknown environmental reservoirs mediating the outbreak. WGS-based cluster analysis with publicly available genomes identified closely related Australian ST78 and ST203 isolates, highlighting the capacity for WGS to resolve complex clonal relationships between the VREfm lineages. Whole genome-based analysis has provided a high-resolution description of an outbreak of vanB -type VREfm ST78 in a Queensland hospital. Combined routine genomic surveillance and epidemiological analysis have facilitated better understanding of the local epidemiology of this endemic strain, providing valuable insight for better targeted control of VREfm. IMPORTANCE Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of health care-associated infections (HAIs) globally. In Australia, the spread of hospital-adapted VREfm is largely driven by a single clonal group (clonal complex [CC]), CC17, to which the lineage ST78 belongs. While implementing a genomic surveillance program in Queensland, we observed increased incidence of ST78 colonizations and infections among patients. Here, we demonstrate the use of real-time genomic surveillance as a tool to support and enhance infection control (IC) practices. Our results show that real-time whole-genome sequencing (WGS) can efficiently disrupt outbreaks by identifying transmission routes that in turn can be targeted using resource-limited interventions. Additionally, we demonstrate that by placing local outbreaks in a global context, high-risk clones can be identified and targeted prior to them becoming established within clinical environments. Finally, the persistence of these organism within the hospital highlights the need for routine genomic surveillance as a management tool to control VRE transmission., Competing Interests: The authors declare no conflict of interest.
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- 2023
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24. GraphSNP: an interactive distance viewer for investigating outbreaks and transmission networks using a graph approach.
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Permana B, Beatson SA, and Forde BM
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- Web Browser, Genome, Disease Outbreaks, Software, Genomics methods
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Background: Cluster and transmission analysis utilising pairwise SNP distance are increasingly used in genomic epidemiological studies. However, current methods are often challenging to install and use, and lack interactive functionalities for easy data exploration., Results: GraphSNP is an interactive visualisation tool running in a web browser that allows users to rapidly generate pairwise SNP distance networks, investigate SNP distance distributions, identify clusters of related organisms, and reconstruct transmission routes. The functionality of GraphSNP is demonstrated using examples from recent multi-drug resistant bacterial outbreaks in healthcare settings., Conclusions: GraphSNP is freely available at https://github.com/nalarbp/graphsnp . An online version of GraphSNP, including demonstration datasets, input templates, and quick start guide is available for use at https://graphsnp.fordelab.com ., (© 2023. The Author(s).)
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- 2023
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25. Treatment Failure Due to Adaptive Resistance Mechanisms in a Severe and Complicated Bloodstream Infection Due to Elizabethkingia meningoseptica .
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Stewart AG, Roberts JA, Forde BM, Bergh H, Kidd TJ, Wright H, and Harris PNA
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- Humans, Anti-Bacterial Agents pharmacology, Microbial Sensitivity Tests, Treatment Failure, Flavobacteriaceae Infections drug therapy, Flavobacteriaceae Infections microbiology, Chryseobacterium, Sepsis drug therapy, Sepsis microbiology
- Abstract
Elizabethkingia meningoseptica is an uncommonly encountered multidrug-resistant gram-negative bacterium that causes infections primarily among vulnerable hosts. A true opportunistic pathogen, its ability to cause severe sepsis and complicated infection in selected patients has been noted. Very limited preclinical and clinical data exist with regard to suitable therapeutic options. In this study, we present the case of prolonged bloodstream and central nervous system infection due to E. meningoseptica treated with dose-optimized combination antibiotic therapy, with evidence of microbiological (including development of adaptive resistance mechanisms) and clinical failure.
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- 2023
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26. Clinical Implementation of Routine Whole-genome Sequencing for Hospital Infection Control of Multi-drug Resistant Pathogens.
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Forde BM, Bergh H, Cuddihy T, Hajkowicz K, Hurst T, Playford EG, Henderson BC, Runnegar N, Clark J, Jennison AV, Moss S, Hume A, Leroux H, Beatson SA, Paterson DL, and Harris PNA
- Subjects
- Adult, Humans, Child, Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents therapeutic use, Multilocus Sequence Typing, Tertiary Care Centers, Cross Infection epidemiology, Methicillin-Resistant Staphylococcus aureus genetics
- Abstract
Background: Prospective whole-genome sequencing (WGS)-based surveillance may be the optimal approach to rapidly identify transmission of multi-drug resistant (MDR) bacteria in the healthcare setting., Methods: We prospectively collected methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), carbapenem-resistant Acinetobacter baumannii (CRAB), extended-spectrum beta-lactamase (ESBL-E), and carbapenemase-producing Enterobacterales (CPE) isolated from blood cultures, sterile sites, or screening specimens across three large tertiary referral hospitals (2 adult, 1 paediatric) in Brisbane, Australia. WGS was used to determine in silico multi-locus sequence typing (MLST) and resistance gene profiling via a bespoke genomic analysis pipeline. Putative transmission events were identified by comparison of core genome single nucleotide polymorphisms (SNPs). Relevant clinical meta-data were combined with genomic analyses via customised automation, collated into hospital-specific reports regularly distributed to infection control teams., Results: Over 4 years (April 2017 to July 2021) 2660 isolates were sequenced. This included MDR gram-negative bacilli (n = 293 CPE, n = 1309 ESBL), MRSA (n = 620), and VRE (n = 433). A total of 379 clinical reports were issued. Core genome SNP data identified that 33% of isolates formed 76 distinct clusters. Of the 76 clusters, 43 were contained to the 3 target hospitals, suggesting ongoing transmission within the clinical environment. The remaining 33 clusters represented possible inter-hospital transmission events or strains circulating in the community. In 1 hospital, proven negligible transmission of non-multi-resistant MRSA enabled changes to infection control policy., Conclusions: Implementation of routine WGS for MDR pathogens in clinical laboratories is feasible and can enable targeted infection prevention and control interventions., Competing Interests: Potential conflicts of interest. P. N. A. H. reports research grants from Merck, Sandoz, and Shionogi, outside the submitted work; has served on advisory boards for Sandoz and Merck, and has received speaker's fees from Pfizer, Sandoz, and Sumitomo. D. L. P reports research grants from Merck, Pfizer, and Shionogi outside the submitted work; has received honoraria for advisory board membership from Merck, Pfizer, Shionogi, GSK, QPex, Entasis, VenatoRx, BioMerieux, and Accelerate. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2023
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27. Case report: a fatal case of Aspergillus felis infection in an immunocompetent host.
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Parkes-Smith J, Bauer MJ, Bergh H, Eidan A, Forde BM, Hilton J, Kidd TJ, Schmidt C, Stewart AG, and Harris PNA
- Abstract
We report a fatal case of Aspergillus felis invasive rhinosinusitis with secondary cerebral abscesses in an immunocompetent host despite aggressive surgical debridement and combination antifungals. Whilst this organism is known to cause fatalities in cats, only a few cases in humans have been documented, all of which had significant immunosuppression. This is the first human death due to A. felis described in an immunocompetent host., Competing Interests: The authors declare that there are no conflicts of interest., (© 2022 The Authors.)
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- 2022
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28. Optimized Method for Bacterial Nucleic Acid Extraction from Positive Blood Culture Broth for Whole-Genome Sequencing, Resistance Phenotype Prediction, and Downstream Molecular Applications.
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Bauer MJ, Peri AM, Lüftinger L, Beisken S, Bergh H, Forde BM, Buckley C, Cuddihy T, Tan P, Paterson DL, Whiley DM, and Harris PNA
- Subjects
- Microbial Sensitivity Tests, Anti-Bacterial Agents pharmacology, Phenotype, Blood Culture methods, Nucleic Acids
- Abstract
The application of direct metagenomic sequencing from positive blood culture broth may solve the challenges of sequencing from low-bacterial-load blood samples in patients with sepsis. Forty prospectively collected blood culture broth samples growing Gram-negative bacteria were extracted using commercially available kits to achieve high-quality DNA. Species identification via metagenomic sequencing and susceptibility prediction via a machine-learning algorithm (AREScloud) were compared to conventional methods and other rapid diagnostic platforms (Accelerate Pheno and blood culture identification [BCID] panel). A two-kit method (using MolYsis Basic and Qiagen DNeasy UltraClean kits) resulted in optimal extractions. Taxonomic profiling by direct metagenomic sequencing matched conventional identification in 38/40 (95%) samples. In two polymicrobial samples, a second organism was missed by sequencing. Prediction models were able to accurately infer susceptibility profiles for 6 common pathogens against 17 antibiotics, with an overall categorical agreement (CA) of 95% (increasing to >95% for 5/6 of the most common pathogens, if Klebsiella oxytoca was excluded). The performance of whole-genome sequencing (WGS)-antimicrobial susceptibility testing (AST) was suboptimal for uncommon pathogens (e.g., Elizabethkingia ) and some β-lactamase inhibitor antibiotics (e.g., ticarcillin-clavulanate). The time to pathogen identification was the fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a susceptibility result in approximately 8 h. Illumina-based direct sequencing methods provided results in time frames similar to those of conventional culture-based methods. Direct metagenomic sequencing from blood cultures for pathogen detection and susceptibility prediction is feasible. Additional work is required to optimize algorithms for uncommon species and complex resistance genotypes as well as to streamline methods to provide more rapid results.
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- 2022
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29. Proof-of-concept, rapid, instrument-free molecular detection of Neisseria gonorrhoeae and ciprofloxacin susceptibility.
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Ayfan AKS, Macdonald J, Irwin AD, Zowawi HM, Forde BM, Paterson DL, Lahra MM, and Whiley DM
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- Humans, Ciprofloxacin pharmacology, Microbial Sensitivity Tests, Drug Resistance, Bacterial, Anti-Bacterial Agents pharmacology, Neisseria gonorrhoeae genetics, Gonorrhea diagnosis
- Abstract
Objectives: To develop instrument-free point-of-care methods using recombinase polymerase amplification (RPA) technology coupled with a simple lateral flow detection system to detect Neisseria gonorrhoeae and susceptibility to ciprofloxacin., Methods: For identification of gonococcal infection, an RPA-based method was developed targeting the gonococcal porA pseudogene (NG-porA-RPA). For ciprofloxacin susceptibility, predictive WT sequences at codons 91 and 95 of the gonococcal gyrA DNase gene were targeted. Given the known complexities of SNP detection using RPA (e.g. the ability to accommodate mismatches) we trialled several different assays incorporating various additional non-template mismatches in the oligonucleotide sequences to reduce affinity for the mutant (resistant) gyrA sequences. Assays were evaluated using a bank of N. gonorrhoeae (n = 10) and non-gonococcal (n = 5) isolates and a panel of N. gonorrhoeae nucleic acid amplification test (NAAT)-positive clinical sample extracts (n = 40)., Results: The NG-porA-RPA assay was specific to N. gonorrhoeae and provided a positive percentage agreement (PPA) of 87.5% (35/40) compared with a commercial N. gonorrhoeae NAAT when applied to the 40 clinical sample extracts. For gyrA, the non-template bases successfully reduced banding intensity for double-mutant strains (mutations at both 91 and 95), but not for rarer single-mutant (91 only) strains. The most promising gyrA assay, NG-gyrA-RPA08, correctly detected 83% (25/30) of infections from NAAT-positive clinical samples confirmed to have WT gyrA sequences based on Sanger sequencing., Conclusions: These proof-of-concept data show that RPA technology has considerable promise for detecting N. gonorrhoeae and associated antibiotic susceptibility and would offer a diagnostic-based stewardship strategy identified as urgently needed by the WHO., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
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- 2022
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30. Pharmacodynamic evaluation of piperacillin/tazobactam versus meropenem against extended-spectrum β-lactamase-producing and non-producing Escherichia coli clinical isolates in a hollow-fibre infection model.
- Author
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Islam K, Sime FB, Wallis SC, Bauer MJ, Forde BM, Harris P, Shirin T, Habib ZH, Flora MS, and Roberts JA
- Subjects
- Anti-Bacterial Agents therapeutic use, Carbapenems therapeutic use, Humans, Meropenem pharmacology, Meropenem therapeutic use, Microbial Sensitivity Tests, Penicillanic Acid pharmacology, Penicillanic Acid therapeutic use, Piperacillin, Piperacillin, Tazobactam Drug Combination therapeutic use, beta-Lactamases, Escherichia coli, Escherichia coli Infections drug therapy, Escherichia coli Infections microbiology
- Abstract
Background: Urosepsis caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is increasing worldwide. Carbapenems are commonly recommended for the treatment of ESBL infections; however, to minimize the emergence of carbapenem resistance, interest in alternative treatments has heightened., Objectives: This study compared pharmacodynamics of piperacillin/tazobactam versus meropenem against ESBL-producing and non-producing E. coli clinical isolates., Methods: E. coli isolates, obtained from national reference laboratory in Bangladesh, were characterized by phenotypic tests, WGS, susceptibility tests and mutant frequency analysis. Three ESBL-producing and two non-producing E. coli were exposed to piperacillin/tazobactam (4.5 g, every 6 h and every 8 h, 30 min infusion) and meropenem (1 g, every 8 h, 30 min infusion) in a hollow-fibre infection model over 7 days., Results: Piperacillin/tazobactam regimens attained ∼4-5 log10 cfu/mL bacterial killing within 24 h and prevented resistance emergence over the experiment against ESBL-producing and non-producing E. coli. However, compared with 8 hourly meropenem, the 6 hourly piperacillin/tazobactam attained ∼1 log10 lower bacterial kill against one of three ESBL-producing E. coli (CTAP#173) but comparable killing for the other two ESBL-producing (CTAP#168 and CTAP#169) and two non-producing E. coli (CTAP#179 and CTAP#180). The 6 hourly piperacillin/tazobactam regimen attained ∼1 log10 greater bacterial kill compared with the 8 hourly regimen against CTAP#168 and CTAP#179 at 24 h., Conclusions: Our study suggests piperacillin/tazobactam may be a potential alternative to carbapenems to treat urosepsis caused by ESBL-producing E. coli, although clinical trials with robust design are needed to confirm non-inferiority of outcome., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)
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- 2022
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31. Pharmacodynamic evaluation of piperacillin/tazobactam against extended-spectrum β-lactamase (ESBL)-producing versus non-ESBL-producing Escherichia coli in a hollow-fibre infection model.
- Author
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Islam K, Sime FB, Bauer MJ, Forde BM, Wallis SC, Harris P, Naicker S, Shirin T, Habib ZH, Flora MS, and Roberts JA
- Subjects
- Anti-Bacterial Agents pharmacology, Humans, Microbial Sensitivity Tests, Piperacillin, Tazobactam Drug Combination, beta-Lactamases genetics, Escherichia coli, Escherichia coli Infections drug therapy, Escherichia coli Infections microbiology
- Abstract
Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are a global public-health concern. We evaluated the pharmacodynamic activity of piperacillin/tazobactam (TZP) dosing regimens against ESBL-producing versus non-ESBL-producing E. coli. Five E. coli clinical isolates were obtained from Bangladesh. Broth microdilution and whole-genome sequencing (WGS) were performed on the five studied isolates. Three TZP-susceptible ESBL-producing and two non-ESBL-producing E. coli were exposed to TZP regimens of 4.5 g every 6 h (q6h) and every 8 h (q8h) as a 30-min infusion in a dynamic hollow-fibre infection model over 7 days. The extent of bacterial killing was ∼4-5 log
10 CFU/mL against ESBL-producing and non-ESBL-producing E. coli with TZP q6h and q8h regimens over the first 8 h. Bacterial killing was similar between two of three ESBL-producing (CTAP#168 and CTAP#169) and two non-ESBL-producing (CTAP#179 and CTAP#180) E. coli clinical isolates over the course of the experiment. ESBL-producing CTAP#173 E. coli was poorly killed (∼1 log) compared with two non-ESBL-producing E. coli over 168 h. WGS revealed that ESBL-producing E. coli isolates co-harboured multiple antimicrobial resistance genes such as blaCTX-M-15 , blaEC , blaOXA-1 , blaTEM-1 and aac(6')-Ib-cr5. Overall, TZP q6h and q8h dosing regimens attained >3 log bacterial kill against all ESBL-producing or non-ESBL-producing E. coli within 24 h and maintained and prevented the emergence of resistance through the end of the experiment. In conclusion, TZP standard regimens resulted in similar bacterial killing and prevented the emergence of resistance against CTX-M-15-type ESBL-producing and non-ESBL-producing E. coli clinical isolates., Competing Interests: Competing interests None declared., (Copyright © 2022 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)- Published
- 2022
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32. Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli.
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Hancock SJ, Lo AW, Ve T, Day CJ, Tan L, Mendez AA, Phan MD, Nhu NTK, Peters KM, Richards AC, Fleming BA, Chang C, Ngu DHY, Forde BM, Haselhorst T, Goh KGK, Beatson SA, Jennings MP, Mulvey MA, Kobe B, and Schembri MA
- Subjects
- Adhesins, Bacterial metabolism, Adhesins, Escherichia coli genetics, Escherichia coli genetics, Escherichia coli metabolism, Fimbriae, Bacterial genetics, Fimbriae, Bacterial metabolism, Humans, Intestinal Diseases, Polysaccharides metabolism, Escherichia coli Infections metabolism, Extraintestinal Pathogenic Escherichia coli genetics, Extraintestinal Pathogenic Escherichia coli metabolism
- Abstract
Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics., Competing Interests: The authors have declared that no competing interests exist.
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- 2022
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33. CATHAI: cluster analysis tool for healthcare-associated infections.
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Cuddihy T, Harris PNA, Permana B, Beatson SA, and Forde BM
- Abstract
Motivation: Whole genome sequencing (WGS) is revolutionizing disease surveillance where it facilitates high-resolution clustering of related organism and outbreak detection. However, visualizing and efficiently communicating genomic data back to clinical staff is crucial for the successful deployment of a targeted infection control response., Results: CATHAI (cluster analysis tool for healthcare-associated infections) is an interactive web-based visualization platform that couples WGS informed clustering with associated metadata, thereby converting sequencing data into informative and accessible clinical information for the management of healthcare-associated infections (HAI) and nosocomial outbreaks., Availability and Implementation: All code associated with this application are free available from https://github.com/FordeGenomics/cathai. A demonstration version of CATHAI is available online at https://cathai.fordelab.com., (© The Author(s) 2022. Published by Oxford University Press.)
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- 2022
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34. Strengths and caveats of identifying resistance genes from whole genome sequencing data.
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Forde BM, De Oliveira DMP, Falconer C, Graves B, and Harris PNA
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- Anti-Bacterial Agents pharmacology, Humans, Microbial Sensitivity Tests, Whole Genome Sequencing, Artificial Intelligence, Drug Resistance, Bacterial genetics
- Abstract
Introduction: Antimicrobial resistance (AMR) continues to present major challenges to modern healthcare. Recent advances in whole-genome sequencing (WGS) have made the rapid molecular characterization of AMR a realistic possibility for diagnostic laboratories; yet major barriers to clinical implementation exist., Areas Covered: We describe and compare short- and long-read sequencing platforms, typical components of bioinformatics pipelines, tools for AMR gene detection and the relative merits of read- or assembly-based approaches. The challenges of characterizing mobile genetic elements from genomic data are outlined, as well as the complexities inherent to the prediction of phenotypic resistance from WGS. Practical obstacles to implementation in diagnostic laboratories, the critical role of quality control and external quality assurance, as well as standardized reporting standards are also discussed. Future directions, such as the application of machine-learning and artificial intelligence algorithms, linked to clinically meaningful outcomes, may offer a new paradigm for the clinical application of AMR prediction., Expert Opinion: AMR prediction from WGS data presents an exciting opportunity to advance our capacity to comprehensively characterize infectious pathogens in a rapid manner, ultimately aiming to improve patient outcomes. Collaborative efforts between clinicians, scientists, regulatory bodies and healthcare administrators will be critical to achieve the full promise of this approach.
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- 2022
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35. A high-quality reference genome for the fish pathogen Streptococcus iniae .
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Alsheikh-Hussain AS, Ben Zakour NL, Forde BM, Rudenko O, Barnes AC, and Beatson SA
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- Animals, DNA Transposable Elements, Phylogeny, Sequence Analysis, DNA, Streptococcus genetics, Streptococcus iniae genetics
- Abstract
Fish mortality caused by Streptococcus iniae is a major economic problem in aquaculture in warm and temperate regions globally. There is also risk of zoonotic infection by S. iniae through handling of contaminated fish. In this study, we present the complete genome sequence of S. iniae strain QMA0248, isolated from farmed barramundi in South Australia. The 2.12 Mb genome of S. iniae QMA0248 carries a 32 kb prophage, a 12 kb genomic island and 92 discrete insertion sequence (IS) elements. These include nine novel IS types that belong mostly to the IS 3 family. Comparative and phylogenetic analysis between S. iniae QMA0248 and publicly available complete S. iniae genomes revealed discrepancies that are probably due to misassembly in the genomes of isolates ISET0901 and ISNO. Long-range PCR confirmed five rRNA loci in the PacBio assembly of QMA0248, and, unlike S. iniae 89353, no tandemly repeated rRNA loci in the consensus genome. However, we found sequence read evidence that the tandem rRNA repeat existed within a subpopulation of the original QMA0248 culture. Subsequent nanopore sequencing revealed that the tandem rRNA repeat was the most prevalent genotype, suggesting that there is selective pressure to maintain fewer rRNA copies under uncertain laboratory conditions. Our study not only highlights assembly problems in existing genomes, but provides a high-quality reference genome for S. iniae QMA0248, including manually curated mobile genetic elements, that will assist future S. iniae comparative genomic and evolutionary studies.
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- 2022
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36. Phase variation in the glycosyltransferase genes of Pasteurella multocida associated with outbreaks of fowl cholera on free-range layer farms.
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Omaleki L, Blackall PJ, Cuddihy T, White RT, Courtice JM, Turni C, Forde BM, and Beatson SA
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- Australia epidemiology, Disease Outbreaks veterinary, Farms, Glycosyltransferases genetics, Humans, Lipopolysaccharides genetics, Phase Variation, Cholera epidemiology, Pasteurella Infections epidemiology, Pasteurella Infections veterinary, Pasteurella multocida genetics
- Abstract
Fowl cholera caused by Pasteurella multocida has re-emerged in Australian poultry production since the increasing adoption of free-range production systems. Currently, autogenous killed whole-cell vaccines prepared from the isolates previously obtained from each farm are the main preventative measures used. In this study, we use whole-genome sequencing and phylogenomic analysis to investigate outbreak dynamics, as well as monitoring and comparing the variations in the lipopolysaccharide (LPS) outer core biosynthesis loci of the outbreak and vaccine strains. In total, 73 isolates from two different free-range layer farms were included. Our genomic analysis revealed that all investigated isolates within the two farms (layer A and layer B) carried LPS type L3, albeit with a high degree of genetic diversity between them. Additionally, the isolates belonged to five different sequence types (STs), with isolates belonging to ST9 and ST20 being the most prevalent. The isolates carried ST-specific mutations within their LPS type L3 outer core biosynthesis loci, including frameshift mutations in the outer core heptosyltransferase gene ( htpE ) (ST7 and ST274) or galactosyltransferase gene ( gatG ) (ST20). The ST9 isolates could be separated into three groups based on their LPS outer core biosynthesis loci sequences, with evidence for potential phase variation mechanisms identified. The potential phase variation mechanisms included a tandem repeat insertion in natC and a single base deletion in a homopolymer region of gatG . Importantly, our results demonstrated that two of the three ST9 groups shared identical rep-PCR (repetitive extragenic palindromic PCR) patterns, while carrying differences in their LPS outer core biosynthesis loci region. In addition, we found that ST9 isolates either with or without the natC tandem repeat insertion were both associated with a single outbreak, which would indicate the importance of screening more than one isolate within an outbreak. Our results strongly suggest the need for a metagenomics culture-independent approach, as well as a genetic typing scheme for LPS, to ensure an appropriate vaccine strain with a matching predicted LPS structure is used.
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- 2022
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37. Rescuing Tetracycline Class Antibiotics for the Treatment of Multidrug-Resistant Acinetobacter baumannii Pulmonary Infection.
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De Oliveira DMP, Forde BM, Phan MD, Steiner B, Zhang B, Zuegg J, El-Deeb IM, Li G, Keller N, Brouwer S, Harbison-Price N, Cork AJ, Bauer MJ, Alquethamy SF, Beatson SA, Roberts JA, Paterson DL, McEwan AG, Blaskovich MAT, Schembri MA, McDevitt CA, von Itzstein M, and Walker MJ
- Subjects
- Humans, Animals, Mice, Tigecycline pharmacology, Tetracycline pharmacology, Pandemics, Drug Resistance, Multiple, Bacterial, Anti-Bacterial Agents pharmacology, Carbapenems pharmacology, beta-Lactams pharmacology, Microbial Sensitivity Tests, Zinc pharmacology, Acinetobacter baumannii, Pneumonia, Ventilator-Associated drug therapy, Pneumonia, Ventilator-Associated microbiology, Acinetobacter Infections microbiology, COVID-19
- Abstract
Acinetobacter baumannii causes high mortality in ventilator-associated pneumonia patients, and antibiotic treatment is compromised by multidrug-resistant strains resistant to β-lactams, carbapenems, cephalosporins, polymyxins, and tetracyclines. Among COVID-19 patients receiving ventilator support, a multidrug-resistant A. baumannii secondary infection is associated with a 2-fold increase in mortality. Here, we investigated the use of the 8-hydroxyquinoline ionophore PBT2 to break the resistance of A. baumannii to tetracycline class antibiotics. In vitro , the combination of PBT2 and zinc with either tetracycline, doxycycline, or tigecycline was shown to be bactericidal against multidrug-resistant A. baumannii, and any resistance that did arise imposed a fitness cost. PBT2 and zinc disrupted metal ion homeostasis in A. baumannii, increasing cellular zinc and copper while decreasing magnesium accumulation. Using a murine model of pulmonary infection, treatment with PBT2 in combination with tetracycline or tigecycline proved efficacious against multidrug-resistant A. baumannii. These findings suggest that PBT2 may find utility as a resistance breaker to rescue the efficacy of tetracycline-class antibiotics commonly employed to treat multidrug-resistant A. baumannii infections. IMPORTANCE Within intensive care unit settings, multidrug-resistant (MDR) Acinetobacter baumannii is a major cause of ventilator-associated pneumonia, and hospital-associated outbreaks are becoming increasingly widespread. Antibiotic treatment of A. baumannii infection is often compromised by MDR strains resistant to last-resort β-lactam (e.g., carbapenems), polymyxin, and tetracycline class antibiotics. During the on-going COVID-19 pandemic, secondary bacterial infection by A. baumannii has been associated with a 2-fold increase in COVID-19-related mortality. With a rise in antibiotic resistance and a reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. Rescuing the efficacy of existing therapies for the treatment of MDR A. baumannii infection represents a financially viable pathway, reducing time, cost, and risk associated with drug innovation.
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- 2022
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38. Differential Afa/Dr Fimbriae Expression in the Multidrug-Resistant Escherichia coli ST131 Clone.
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Alvarez-Fraga L, Phan MD, Goh KGK, Nhu NTK, Hancock SJ, Allsopp LP, Peters KM, Forde BM, Roberts LW, Sullivan MJ, Totsika M, Beatson SA, Ulett GC, and Schembri MA
- Subjects
- Humans, Adhesins, Bacterial metabolism, Anti-Bacterial Agents metabolism, Clone Cells, DNA Transposable Elements, Virulence genetics, Drug Resistance, Multiple, Bacterial genetics, Escherichia coli Infections genetics, Urinary Tract Infections genetics, Uropathogenic Escherichia coli genetics, Uropathogenic Escherichia coli pathogenicity
- Abstract
Many antibiotic resistant uropathogenic Escherichia coli (UPEC) strains belong to clones defined by their multilocus sequence type (ST), with ST131 being the most dominant. Although we have a good understanding of resistance development to fluoroquinolones and third-generation cephalosporins by ST131, our understanding of the virulence repertoire that has contributed to its global dissemination is limited. Here we show that the genes encoding Afa/Dr fimbriae, a group of adhesins strongly associated with UPEC that cause gestational pyelonephritis and recurrent cystitis, are found in approximately one third of all ST131 strains. Sequence comparison of the AfaE adhesin protein revealed a unique allelic variant carried by 82.9% of afa -positive ST131 strains. We identify the afa regulatory region as a hotspot for the integration of insertion sequence (IS) elements, all but one of which alter afa transcription. Close investigation demonstrated that the integration of an IS 1 element in the afa regulatory region leads to increased expression of Afa/Dr fimbriae, promoting enhanced adhesion to kidney epithelial cells and suggesting a mechanism for altered virulence. Finally, we provide evidence for a more widespread impact of IS 1 on ST131 genome evolution, suggesting that IS dynamics contribute to strain level microevolution that impacts ST131 fitness. IMPORTANCE E. coli ST131 is the most common antibiotic resistant UPEC clone associated with human urinary tract and bloodstream infections. Understanding the features of ST131 that have driven its global dissemination remains a critical priority if we are to counter its increasing antibiotic resistance. Here, we utilized a large collection of ST131 isolates to investigate the prevalence, regulation, and function of Afa/Dr fimbriae, a well-characterized UPEC colonization and virulence factor. We show that the afa genes are found frequently in ST131 and demonstrate how the integration of IS elements in the afa regulatory region modulates Afa expression, presenting an example of altered virulence capacity. We also exploit a curated set of ST131 genomes to map the integration of the antibiotic resistance-associated IS 1 element in the ST131 pangenome, providing evidence for its widespread impact on ST131 genome evolution.
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- 2022
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39. Plasmid-Mediated Ciprofloxacin Resistance Imparts a Selective Advantage on Escherichia coli ST131.
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Phan MD, Peters KM, Alvarez Fraga L, Wallis SC, Hancock SJ, Nhu NTK, Forde BM, Bauer MJ, Paterson DL, Beatson SA, Lipman J, and Schembri MA
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- Anti-Bacterial Agents pharmacology, Ciprofloxacin pharmacology, Drug Resistance, Multiple, Bacterial genetics, Humans, Microbial Sensitivity Tests, Plasmids genetics, Escherichia coli genetics, Escherichia coli Infections drug therapy
- Abstract
Escherichia coli ST131 is a recently emerged antibiotic resistant clone responsible for high rates of urinary tract and bloodstream infections. Despite its global dominance, the precise mechanisms that have driven the rapid dissemination of ST131 remain unknown. Here, we show that the plasmid-associated resistance gene encoding the AAC(6')-Ib-cr enzyme that inactivates the fluoroquinolone (FQ) antibiotic ciprofloxacin is present in >70% of strains from the most rapidly expanding subgroup of multidrug resistant ST131. Using a series of genome-edited and plasmid-cured isogenic strains, we demonstrate that the aac(6')-Ib-cr gene confers a selective advantage on ST131 in the presence of ciprofloxacin, even in strains containing chromosomal GyrA and ParC FQ-resistance mutations. Further, we identify a pattern of emerging carbapenem resistance in other common E. coli clones carrying both aac(6')-Ib-cr and chromosomal FQ-resistance mutations, suggesting this dual resistance combination may also impart a selective advantage on these non-ST131 antibiotic resistant lineages.
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- 2022
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40. Fatal Respiratory Diphtheria Caused by ß-Lactam-Resistant Corynebacterium diphtheriae.
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Forde BM, Henderson A, Playford EG, Looke D, Henderson BC, Watson C, Steen JA, Sidjabat HE, Laurie G, Muttaiyah S, Nimmo GR, Lampe G, Smith H, Jennison AV, McCall B, Carroll H, Cooper MA, Paterson DL, and Beatson SA
- Subjects
- Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents therapeutic use, Humans, Lactams therapeutic use, Microbial Sensitivity Tests, Penicillins therapeutic use, Corynebacterium diphtheriae genetics, Diphtheria drug therapy
- Abstract
Background: Diphtheria is a potentially fatal respiratory disease caused by toxigenic Corynebacterium diphtheriae. Although resistance to erythromycin has been recognized, β-lactam resistance in toxigenic diphtheria has not been described. Here, we report a case of fatal respiratory diphtheria caused by toxigenic C. diphtheriae resistant to penicillin and all other β-lactam antibiotics, and describe a novel mechanism of inducible carbapenem resistance associated with the acquisition of a mobile resistance element., Methods: Long-read whole-genome sequencing was performed using Pacific Biosciences Single Molecule Real-Time sequencing to determine the genome sequence of C. diphtheriae BQ11 and the mechanism of β-lactam resistance. To investigate the phenotypic inducibility of meropenem resistance, short-read sequencing was performed using an Illumina NextSeq500 sequencer on the strain both with and without exposure to meropenem., Results: BQ11 demonstrated high-level resistance to penicillin (benzylpenicillin minimum inhibitory concentration [MIC] ≥ 256 μg/ml), β-lactam/β-lactamase inhibitors and cephalosporins (amoxicillin/clavulanic acid MIC ≥ 256 μg/mL; ceftriaxone MIC ≥ 8 μg/L). Genomic analysis of BQ11 identified acquisition of a novel transposon carrying the penicillin-binding protein (PBP) Pbp2c, responsible for resistance to penicillin and cephalosporins. When strain BQ11 was exposed to meropenem, selective pressure drove amplification of the transposon in a tandem array and led to a corresponding change from a low-level to a high-level meropenem-resistant phenotype., Conclusions: We have identified a novel mechanism of inducible antibiotic resistance whereby isolates that appear to be carbapenem susceptible on initial testing can develop in vivo resistance to carbapenems with repeated exposure. This phenomenon could have significant implications for the treatment of C. diphtheriae infection, and may lead to clinical failure., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)
- Published
- 2021
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41. Meropenem Versus Piperacillin-Tazobactam for Definitive Treatment of Bloodstream Infections Caused by AmpC β-Lactamase-Producing Enterobacter spp, Citrobacter freundii , Morganella morganii , Providencia spp, or Serratia marcescens : A Pilot Multicenter Randomized Controlled Trial (MERINO-2).
- Author
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Stewart AG, Paterson DL, Young B, Lye DC, Davis JS, Schneider K, Yilmaz M, Dinleyici R, Runnegar N, Henderson A, Archuleta S, Kalimuddin S, Forde BM, Chatfield MD, Bauer MJ, Lipman J, Harris-Brown T, and Harris PNA
- Abstract
Background: Carbapenems are recommended treatment for serious infections caused by AmpC-producing gram-negative bacteria but can select for carbapenem resistance. Piperacillin-tazobactam may be a suitable alternative., Methods: We enrolled adult patients with bloodstream infection due to chromosomal AmpC producers in a multicenter randomized controlled trial. Patients were assigned 1:1 to receive piperacillin-tazobactam 4.5 g every 6 hours or meropenem 1 g every 8 hours. The primary efficacy outcome was a composite of death, clinical failure, microbiological failure, and microbiological relapse at 30 days., Results: Seventy-two patients underwent randomization and were included in the primary analysis population. Eleven of 38 patients (29%) randomized to piperacillin-tazobactam met the primary outcome compared with 7 of 34 patients (21%) in the meropenem group (risk difference, 8% [95% confidence interval {CI}, -12% to 28%]). Effects were consistent in an analysis of the per-protocol population. Within the subcomponents of the primary outcome, 5 of 38 (13%) experienced microbiological failure in the piperacillin-tazobactam group compared to 0 of 34 patients (0%) in the meropenem group (risk difference, 13% [95% CI, 2% to 24%]). In contrast, 0% vs 9% of microbiological relapses were seen in the piperacillin-tazobactam and meropenem arms, respectively. Susceptibility to piperacillin-tazobactam and meropenem using broth microdilution was found in 96.5% and 100% of isolates, respectively. The most common AmpC β-lactamase genes identified were bla
CMY-2 , blaDHA-17 , blaCMH-3 , and blaACT-17 . No ESBL, OXA, or other carbapenemase genes were identified., Conclusions: Among patients with bloodstream infection due to AmpC producers, piperacillin-tazobactam may lead to more microbiological failures, although fewer microbiological relapses were seen., Clinical Trials Registration: NCT02437045., (© The Author(s) 2021. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)- Published
- 2021
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42. MicroPIPE: validating an end-to-end workflow for high-quality complete bacterial genome construction.
- Author
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Murigneux V, Roberts LW, Forde BM, Phan MD, Nhu NTK, Irwin AD, Harris PNA, Paterson DL, Schembri MA, Whiley DM, and Beatson SA
- Subjects
- Computational Biology, High-Throughput Nucleotide Sequencing, Sequence Analysis, DNA, Workflow, Escherichia coli, Genome, Bacterial
- Abstract
Background: Oxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers due to the accessibility and affordability of its devices. However, easy and automated construction of high-quality bacterial genomes using nanopore reads remains challenging. Here we aimed to create a reproducible end-to-end bacterial genome assembly pipeline using ONT in combination with Illumina sequencing., Results: We evaluated the performance of several popular tools used during genome reconstruction, including base-calling, filtering, assembly, and polishing. We also assessed overall genome accuracy using ONT both natively and with Illumina. All steps were validated using the high-quality complete reference genome for the Escherichia coli sequence type (ST)131 strain EC958. Software chosen at each stage were incorporated into our final pipeline, MicroPIPE. Further validation of MicroPIPE was carried out using 11 additional ST131 E. coli isolates, which demonstrated that complete circularised chromosomes and plasmids could be achieved without manual intervention. Twelve publicly available Gram-negative and Gram-positive bacterial genomes (with available raw ONT data and matched complete genomes) were also assembled using MicroPIPE. We found that revised basecalling and updated assembly of the majority of these genomes resulted in improved accuracy compared to the current publicly available complete genomes., Conclusions: MicroPIPE is built in modules using Singularity container images and the bioinformatics workflow manager Nextflow, allowing changes and adjustments to be made in response to future tool development. Overall, MicroPIPE provides an easy-access, end-to-end solution for attaining high-quality bacterial genomes. MicroPIPE is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe .
- Published
- 2021
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43. Optimising Treatment Outcomes for Children and Adults Through Rapid Genome Sequencing of Sepsis Pathogens. A Study Protocol for a Prospective, Multi-Centre Trial (DIRECT).
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Irwin AD, Coin LJM, Harris PNA, Cotta MO, Bauer MJ, Buckley C, Balch R, Kruger P, Meyer J, Shekar K, Brady K, Fourie C, Sharp N, Vlad L, Whiley D, Beatson SA, Forde BM, Paterson D, Clark J, Hajkowicz K, Raman S, Bialasiewicz S, Lipman J, Schlapbach LJ, and Roberts JA
- Subjects
- Adult, Anti-Bacterial Agents therapeutic use, Australia, Bayes Theorem, Child, Humans, Multicenter Studies as Topic, Pilot Projects, Prospective Studies, Treatment Outcome, Sepsis diagnosis, Sepsis drug therapy
- Abstract
Background: Sepsis contributes significantly to morbidity and mortality globally. In Australia, 20,000 develop sepsis every year, resulting in 5,000 deaths, and more than AUD$846 million in expenditure. Prompt, appropriate antibiotic therapy is effective in improving outcomes in sepsis. Conventional culture-based methods to identify appropriate therapy have limited yield and take days to complete. Recently, nanopore technology has enabled rapid sequencing with real-time analysis of pathogen DNA. We set out to demonstrate the feasibility and diagnostic accuracy of pathogen sequencing direct from clinical samples, and estimate the impact of this approach on time to effective therapy when integrated with personalised software-guided antimicrobial dosing in children and adults on ICU with sepsis., Methods: The DIRECT study is a pilot prospective, non-randomized multicentre trial of an integrated diagnostic and therapeutic algorithm combining rapid direct pathogen sequencing and software-guided, personalised antibiotic dosing in children and adults with sepsis on ICU., Participants and Interventions: DIRECT will collect microbiological and pharmacokinetic samples from approximately 200 children and adults with sepsis admitted to one of four ICUs in Brisbane. In Phase 1, we will evaluate Oxford Nanopore Technologies MinION sequencing direct from blood in 50 blood culture-proven sepsis patients recruited from consecutive patients with suspected sepsis. In Phase 2, a further 50 consecutive patients with suspected sepsis will be recruited in whom MinION sequencing will be combined with Bayesian software-guided (ID-ODS) personalised antimicrobial dosing., Outcome Measures: The primary outcome is time to effective antimicrobial therapy, defined as trough drug concentrations above the MIC of the pathogen. Secondary outcomes are diagnostic accuracy of MinION sequencing from whole blood, time to pathogen identification and susceptibility testing using sequencing direct from whole blood and from positive blood culture broth., Discussion: Rapid pathogen sequencing coupled with antimicrobial dosing software has great potential to overcome the limitations of conventional diagnostics which often result in prolonged inappropriate antimicrobial therapy. Reduced time to optimal antimicrobial therapy may reduce sepsis mortality and ICU length of stay. This pilot study will yield key feasibility data to inform further, urgently needed sepsis studies. Phase 2 of the trial protocol is registered with the ANZCTR (ACTRN12620001122943)., Trial Registration: Registered with the Australia New Zealand Clinical Trials Registry Number ACTRN12620001122943., Competing Interests: AI has received research funding and teaching honoraria from Gilead Sciences inc. unrelated to this work. DP has received research funding from Pfizer, Merck and Shionogi and funding for advisory boards or speaking engagements from Merck, Pfizer, BioMerieux, Sumitomo, Accelerate, QPex and Entasis, unrelated to this work. LC has received research funding from Oxford Nanopore Technologies unrelated to this work, and received travel reimbursement to travel to a conference. JR has consulted for or received grants from The Medicines Company, MSD, Biomerieux, QPEX, Pfizer and Discuva. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Irwin, Coin, Harris, Cotta, Bauer, Buckley, Balch, Kruger, Meyer, Shekar, Brady, Fourie, Sharp, Vlad, Whiley, Beatson, Forde, Paterson, Clark, Hajkowicz, Raman, Bialasiewicz, Lipman, Schlapbach and Roberts.)
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- 2021
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44. Completing the Picture-Capturing the Resistome in Antibiotic Clinical Trials.
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Stewart AG, Satlin MJ, Schlebusch S, Isler B, Forde BM, Paterson DL, and Harris PNA
- Subjects
- Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents therapeutic use, Clinical Trials as Topic, Drug Resistance, Microbial, Humans, Anti-Infective Agents pharmacology, Microbiota genetics
- Abstract
Despite the accepted dogma that antibiotic use is the largest contributor to antimicrobial resistance (AMR) and human microbiome disruption, our knowledge of specific antibiotic-microbiome effects remains basic. Detection of associations between new or old antimicrobials and specific AMR burden is patchy and heterogeneous. Various microbiome analysis tools are available to determine antibiotic effects on microbial communities in vivo. Microbiome analysis of treatment groups in antibiotic clinical trials, powered to measure clinically meaningful endpoints would greatly assist the antibiotic development pipeline and clinician antibiotic decision making., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)
- Published
- 2021
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45. Molecular Epidemiology of Clinical and Colonizing Methicillin-Resistant Staphylococcus Isolates in Companion Animals.
- Author
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Rynhoud H, Forde BM, Beatson SA, Abraham S, Meler E, Soares Magalhães RJ, and Gibson JS
- Abstract
In this study, we aimed to investigate the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) clinical and colonizing isolates of dogs and cats to profile contributing factors associated with their isolation. Nasal and rectal samples were collected from dogs and cats between 2015 and 2017 to identify colonizing isolates. Clinical isolates collected between 2003 and 2016 were retrieved from a Queensland university veterinary diagnostic laboratory. All isolates were identified using standard microbiological and molecular methods and were characterized by whole genome sequencing. Phylogenetic relationships and differences in epidemiological factors were investigated. Seventy-two MRSP isolates out of 1,460 colonizing samples and nine MRSP clinical isolates were identified. No MRSA was isolated. ST496 and ST749 were the most commonly isolated sequence types with different SCCmec types. ST496 clones spread both along the coast and more inland where ST749 was more centered in Brisbane. The resistance and virulence factors differed significantly between the two sequence types. ST496 colonizing and clinical isolates were similarly multidrug resistant. The virulence genes of ST749 colonizing and clinical isolates were similar as both contained the gene nanB for sialidase. There were no differences in the individual and clinical factors between predominant sequence types. High levels of antimicrobial resistance occurred in the majority of isolates, which is of potential concern to human and veterinary health. The phylogenetic clustering of isolates from this study and others previously identified in countries, particularly New Zealand, with which Australia has high volume of pet movements could suggest the importation of clones, which needs further investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rynhoud, Forde, Beatson, Abraham, Meler, Soares Magalhães and Gibson.)
- Published
- 2021
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46. Genomic surveillance, characterization and intervention of a polymicrobial multidrug-resistant outbreak in critical care.
- Author
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Roberts LW, Forde BM, Hurst T, Ling W, Nimmo GR, Bergh H, George N, Hajkowicz K, McNamara JF, Lipman J, Permana B, Schembri MA, Paterson D, Beatson SA, and Harris PNA
- Subjects
- Acinetobacter baumannii classification, Acinetobacter baumannii drug effects, Acinetobacter baumannii isolation & purification, Adult, Aged, Anti-Bacterial Agents pharmacology, Critical Care statistics & numerical data, Disease Outbreaks, Female, Genome, Bacterial, Genomics, Humans, Klebsiella pneumoniae classification, Klebsiella pneumoniae drug effects, Klebsiella pneumoniae isolation & purification, Male, Middle Aged, Phylogeny, Pseudomonas aeruginosa classification, Pseudomonas aeruginosa drug effects, Pseudomonas aeruginosa isolation & purification, Serratia marcescens classification, Serratia marcescens drug effects, Serratia marcescens isolation & purification, Whole Genome Sequencing, Acinetobacter baumannii genetics, Cross Infection microbiology, Drug Resistance, Multiple, Bacterial, Gram-Negative Bacterial Infections microbiology, Klebsiella pneumoniae genetics, Pseudomonas aeruginosa genetics, Serratia marcescens genetics
- Abstract
Background. Infections caused by carbapenem-resistant Acinetobacter baumannii (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. Methods . A. baumannii, Klebsiella pneumoniae, Serratia marcescens and Pseudomonas aeruginosa 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. Results. 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 K. pneumoniae 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. Conclusion. We implemented a comprehensive WGS and metagenomics investigation that resolved a persistent CR-Ab outbreak in a critical care setting.
- Published
- 2021
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47. Outbreak of multi-drug-resistant (MDR) Shigella flexneri in northern Australia due to an endemic regional clone acquiring an IncFII plasmid.
- Author
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Guglielmino CJD, Kakkanat A, Forde BM, Rubenach S, Merone L, Stafford R, Graham RMA, Beatson SA, and Jennison AV
- Subjects
- Adolescent, Australia epidemiology, Humans, Plasmids, Disease Outbreaks, Drug Resistance, Multiple, Bacterial genetics, Dysentery, Bacillary epidemiology, Dysentery, Bacillary microbiology, Endemic Diseases, Shigella flexneri genetics, Shigella flexneri isolation & purification
- Abstract
Epidemiological surveillance of Shigella spp. in Australia is conducted to inform public health response. Multi-drug resistance has recently emerged as a contributing factor to sustained local transmission of Shigella spp. All data were collected as part of routine public health surveillance, and strains were whole-genome sequenced for further molecular characterisation. 108 patients with an endemic regional Shigella flexneri strain were identified between 2016 and 2019. The S. flexneri phylogroup 3 strain endemic to northern Australia acquired a multi-drug resistance conferring bla
DHA plasmid, which has an IncFII plasmid backbone with virulence and resistance elements typically found in IncR plasmids. This is the first report of multi-drug resistance in Shigella sp. in Australia that is not associated with men who have sex with men. This strain caused an outbreak of multi-drug-resistant S. flexneri in northern Australia that disproportionality affects Aboriginal and Torres Strait Islander children. Community controlled public health action is recommended.- Published
- 2021
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48. Repurposing a neurodegenerative disease drug to treat Gram-negative antibiotic-resistant bacterial sepsis.
- Author
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De Oliveira DMP, Bohlmann L, Conroy T, Jen FE, Everest-Dass A, Hansford KA, Bolisetti R, El-Deeb IM, Forde BM, Phan MD, Lacey JA, Tan A, Rivera-Hernandez T, Brouwer S, Keller N, Kidd TJ, Cork AJ, Bauer MJ, Cook GM, Davies MR, Beatson SA, Paterson DL, McEwan AG, Li J, Schembri MA, Blaskovich MAT, Jennings MP, McDevitt CA, von Itzstein M, and Walker MJ
- Subjects
- Animals, Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents therapeutic use, Bacteria, Colistin pharmacology, Drug Repositioning, Drug Resistance, Bacterial, Drug Resistance, Multiple, Bacterial, Escherichia coli, Klebsiella pneumoniae, Mice, Microbial Sensitivity Tests, Escherichia coli Proteins pharmacology, Neurodegenerative Diseases, Pharmaceutical Preparations, Sepsis drug therapy
- Abstract
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 Klebsiella pneumoniae , Escherichia coli , Acinetobacter baumannii , and Pseudomonas aeruginosa 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 E. coli containing a plasmid-borne mcr-1 gene or K. pneumoniae carrying a chromosomal mgrB mutation. Using a highly invasive K. pneumoniae strain engineered for polymyxin resistance through mgrB 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., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2020
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49. Comprehensive analysis of IncC plasmid conjugation identifies a crucial role for the transcriptional regulator AcaB.
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Hancock SJ, Phan MD, Luo Z, Lo AW, Peters KM, Nhu NTK, Forde BM, Whitfield J, Yang J, Strugnell RA, Paterson DL, Walsh TR, Kobe B, Beatson SA, and Schembri MA
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Drug Resistance, Multiple, Bacterial, Escherichia coli genetics, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Mutagenesis, Mutation, Promoter Regions, Genetic, Protein Structure, Secondary, Trans-Activators chemistry, Trans-Activators genetics, Transcription Factors chemistry, Transcription Factors genetics, Transcription, Genetic, Conjugation, Genetic genetics, Escherichia coli Proteins metabolism, Plasmids genetics, Trans-Activators metabolism, Transcription Factors metabolism
- Abstract
The IncC family of broad-host-range plasmids enables the spread of antibiotic resistance genes among human enteric pathogens
1-3 . Although aspects of IncC plasmid conjugation have been well studied4-9 , 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.- Published
- 2020
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50. Antimicrobial Resistance in ESKAPE Pathogens.
- Author
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De Oliveira DMP, Forde BM, Kidd TJ, Harris PNA, Schembri MA, Beatson SA, Paterson DL, and Walker MJ
- Subjects
- Bacterial Infections microbiology, Humans, Anti-Bacterial Agents pharmacology, Bacteria drug effects, Bacteria genetics, Bacterial Infections drug therapy, Drug Discovery, Drug Resistance, Multiple, Bacterial
- Abstract
Antimicrobial-resistant ESKAPE ( E nterococcus faecium , S taphylococcus aureus , K lebsiella pneumoniae , A cinetobacter baumannii , P seudomonas aeruginosa , and E nterobacter 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., (Copyright © 2020 American Society for Microbiology.)
- Published
- 2020
- Full Text
- View/download PDF
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