Biochemistry and Pharmacology - Research Publications

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    Population genomics of hypervirulent Klebsiella pneumoniae clonal-group 23 reveals early emergence and rapid global dissemination
    Lam, MMC ; Wyres, KL ; Duchene, S ; Wick, RR ; Judd, LM ; Gan, Y-H ; Hoh, C-H ; Archuleta, S ; Molton, JS ; Kalimuddin, S ; Koh, TH ; Passet, V ; Brisse, S ; Holt, KE (NATURE PUBLISHING GROUP, 2018-07-13)
    Severe liver abscess infections caused by hypervirulent clonal-group CG23 Klebsiella pneumoniae have been increasingly reported since the mid-1980s. Strains typically possess several virulence factors including an integrative, conjugative element ICEKp encoding the siderophore yersiniabactin and genotoxin colibactin. Here we investigate CG23's evolutionary history, showing several deep-branching sublineages associated with distinct ICEKp acquisitions. Over 80% of liver abscess isolates belong to sublineage CG23-I, which emerged in ~1928 following acquisition of ICEKp10 (encoding yersiniabactin and colibactin), and then disseminated globally within the human population. CG23-I's distinguishing feature is the colibactin synthesis locus, which reportedly promotes gut colonisation and metastatic infection in murine models. These data show circulation of CG23 K. pneumoniae decades before the liver abscess epidemic was first recognised, and provide a framework for future epidemiological and experimental studies of hypervirulent K. pneumoniae. To support such studies we present an open access, completely sequenced CG23-I human liver abscess isolate, SGH10.
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    Morphological, genomic and transcriptomic responses of Klebsiella pneumoniae to the lastline antibiotic colistin
    Cain, AK ; Boinett, CJ ; Barquist, L ; Dordel, J ; Fookes, M ; Mayho, M ; Ellington, MJ ; Goulding, D ; Pickard, D ; Wick, RR ; Holt, KE ; Parkhill, J ; Thomson, NR (NATURE PUBLISHING GROUP, 2018-06-29)
    Colistin remains one of the few antibiotics effective against multi-drug resistant (MDR) hospital pathogens, such as Klebsiella pneumoniae. Yet resistance to this last-line drug is rapidly increasing. Characterized mechanisms of colR in K. pneumoniae are largely due to chromosomal mutations in two-component regulators, although a plasmid-mediated colR mechanism has recently been uncovered. However, the effects of intrinsic colistin resistance are yet to be characterized on a whole-genome level. Here, we used a genomics-based approach to understand the mechanisms of adaptive colR acquisition in K. pneumoniae. In controlled directed-evolution experiments we observed two distinct paths to colistin resistance acquisition. Whole genome sequencing identified mutations in two colistin resistance genes: in the known colR regulator phoQ which became fixed in the population and resulted in a single amino acid change, and unstable minority variants in the recently described two-component sensor crrB. Through RNAseq and microscopy, we reveal the broad range of effects that colistin exposure has on the cell. This study is the first to use genomics to identify a population of minority variants with mutations in a colR gene in K. pneumoniae.
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    Antimicrobial-Resistant Klebsiella pneumoniae Carriage and Infection in Specialized Geriatric Care Wards Linked to Acquisition in the Referring Hospital
    Gorrie, CL ; Mirceta, M ; Wick, RR ; Judd, LM ; Wyres, KL ; Thomson, NR ; Strugnell, RA ; Pratt, NF ; Garlick, JS ; Watson, KM ; Hunter, PC ; McGloughlin, SA ; Spelman, DW ; Jenney, AWJ ; Holt, KE (OXFORD UNIV PRESS INC, 2018-07-15)
    Background: Klebsiella pneumoniae is a leading cause of extended-spectrum β-lactamase (ESBL)-producing hospital-associated infections, for which elderly patients are at increased risk. Methods: We conducted a 1-year prospective cohort study, in which a third of patients admitted to 2 geriatric wards in a specialized hospital were recruited and screened for carriage of K. pneumoniae by microbiological culture. Clinical isolates were monitored via the hospital laboratory. Colonizing and clinical isolates were subjected to whole-genome sequencing and antimicrobial susceptibility testing. Results: K. pneumoniae throat carriage prevalence was 4.1%, rectal carriage 10.8%, and ESBL carriage 1.7%, and the incidence of K. pneumoniae infection was 1.2%. The isolates were diverse, and most patients were colonized or infected with a unique phylogenetic lineage, with no evidence of transmission in the wards. ESBL strains carried blaCTX-M-15 and belonged to clones associated with hospital-acquired ESBL infections in other countries (sequence type [ST] 29, ST323, and ST340). One also carried the carbapenemase blaIMP-26. Genomic and epidemiological data provided evidence that ESBL strains were acquired in the referring hospital. Nanopore sequencing also identified strain-to-strain transmission of a blaCTX-M-15 FIBK/FIIK plasmid in the referring hospital. Conclusions: The data suggest the major source of K. pneumoniae was the patient's own gut microbiome, but ESBL strains were acquired in the referring hospital. This highlights the importance of the wider hospital network to understanding K. pneumoniae risk and infection prevention. Rectal screening for ESBL organisms on admission to geriatric wards could help inform patient management and infection control in such facilities.
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    Gastrointestinal Carriage Is a Major Reservoir of Klebsiella pneumoniae Infection in Intensive Care Patients
    Gorrie, CL ; Mirceta, M ; Wick, RR ; Edwards, DJ ; Thomson, NR ; Strugnell, RA ; Pratt, NF ; Garlick, JS ; Watson, KM ; Pilcher, DV ; McGloughlin, SA ; Spelman, DW ; Jenney, AWJ ; Holt, KE (OXFORD UNIV PRESS INC, 2017-07-15)
    BACKGROUND: Klebsiella pneumoniae is an opportunistic pathogen and leading cause of hospital-associated infections. Intensive care unit (ICU) patients are particularly at risk. Klebsiella pneumoniae is part of the healthy human microbiome, providing a potential reservoir for infection. However, the frequency of gut colonization and its contribution to infections are not well characterized. METHODS: We conducted a 1-year prospective cohort study in which 498 ICU patients were screened for rectal and throat carriage of K. pneumoniae shortly after admission. Klebsiella pneumoniae isolated from screening swabs and clinical diagnostic samples were characterized using whole genome sequencing and combined with epidemiological data to identify likely transmission events. RESULTS: Klebsiella pneumoniae carriage frequencies were estimated at 6% (95% confidence interval [CI], 3%-8%) among ICU patients admitted direct from the community, and 19% (95% CI, 14%-51%) among those with recent healthcare contact. Gut colonization on admission was significantly associated with subsequent infection (infection risk 16% vs 3%, odds ratio [OR] = 6.9, P < .001), and genome data indicated matching carriage and infection isolates in 80% of isolate pairs. Five likely transmission chains were identified, responsible for 12% of K. pneumoniae infections in ICU. In sum, 49% of K. pneumoniae infections were caused by the patients' own unique strain, and 48% of screened patients with infections were positive for prior colonization. CONCLUSIONS: These data confirm K. pneumoniae colonization is a significant risk factor for infection in ICU, and indicate ~50% of K. pneumoniae infections result from patients' own microbiota. Screening for colonization on admission could limit risk of infection in the colonized patient and others.
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    Kaptive Web: User-Friendly Capsule and Lipopolysaccharide Serotype Prediction for Klebsiella Genomes
    Wick, RR ; Heinz, E ; Holt, KE ; Wyres, KL ; Diekema, DJ (AMER SOC MICROBIOLOGY, 2018-06-01)
    As whole-genome sequencing becomes an established component of the microbiologist's toolbox, it is imperative that researchers, clinical microbiologists, and public health professionals have access to genomic analysis tools for the rapid extraction of epidemiologically and clinically relevant information. For the Gram-negative hospital pathogens such as Klebsiella pneumoniae, initial efforts have focused on the detection and surveillance of antimicrobial resistance genes and clones. However, with the resurgence of interest in alternative infection control strategies targeting Klebsiella surface polysaccharides, the ability to extract information about these antigens is increasingly important. Here we present Kaptive Web, an online tool for the rapid typing of Klebsiella K and O loci, which encode the polysaccharide capsule and lipopolysaccharide O antigen, respectively. Kaptive Web enables users to upload and analyze genome assemblies in a web browser. The results can be downloaded in tabular format or explored in detail via the graphical interface, making it accessible for users at all levels of computational expertise. We demonstrate Kaptive Web's utility by analyzing >500 K. pneumoniae genomes. We identify extensive K and O locus diversity among 201 genomes belonging to the carbapenemase-associated clonal group 258 (25 K and 6 O loci). The characterization of a further 309 genomes indicated that such diversity is common among the multidrug-resistant clones and that these loci represent useful epidemiological markers for strain subtyping. These findings reinforce the need for rapid, reliable, and accessible typing methods such as Kaptive Web. Kaptive Web is available for use at http://kaptive.holtlab.net/, and the source code is available at https://github.com/kelwyres/Kaptive-Web.
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    Emergence and rapid global dissemination of CTX-M-15-associated Klebsiella pneumoniae strain ST307
    Wyres, KL ; Hawkey, J ; Hetland, MAK ; Fostervold, A ; Wick, RR ; Judd, LM ; Hamidian, M ; Howden, BP ; Lohr, IH ; Holt, KE (OXFORD UNIV PRESS, 2019-03-01)
    OBJECTIVES: Recent reports indicate the emergence of a new carbapenemase-producing Klebsiella pneumoniae clone, ST307. We sought to better understand the global epidemiology and evolution of this clone and evaluate its association with antimicrobial resistance (AMR) genes. METHODS: We collated information from the literature and public databases and performed a comparative analysis of 95 ST307 genomes (including 37 that were newly sequenced). RESULTS: We show that ST307 emerged in the mid-1990s (nearly 20 years prior to its first report), is already globally distributed and is intimately associated with a conserved plasmid harbouring the blaCTX-M-15 ESBL gene and several other AMR determinants. CONCLUSIONS: Our findings support the need for enhanced surveillance of this widespread ESBL clone in which carbapenem resistance has occasionally emerged.
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    Deepbinner: Demultiplexing barcoded Oxford Nanopore reads with deep convolutional neural networks
    Wick, RR ; Judd, LM ; Holt, KE ; Pertea, M (PUBLIC LIBRARY SCIENCE, 2018-11-01)
    Multiplexing, the simultaneous sequencing of multiple barcoded DNA samples on a single flow cell, has made Oxford Nanopore sequencing cost-effective for small genomes. However, it depends on the ability to sort the resulting sequencing reads by barcode, and current demultiplexing tools fail to classify many reads. Here we present Deepbinner, a tool for Oxford Nanopore demultiplexing that uses a deep neural network to classify reads based on the raw electrical read signal. This 'signal-space' approach allows for greater accuracy than existing 'base-space' tools (Albacore and Porechop) for which signals must first be converted to DNA base calls, itself a complex problem that can introduce noise into the barcode sequence. To assess Deepbinner and existing tools, we performed multiplex sequencing on 12 amplicons chosen for their distinguishability. This allowed us to establish a ground truth classification for each read based on internal sequence alone. Deepbinner had the lowest rate of unclassified reads (7.8%) and the highest demultiplexing precision (98.5% of classified reads were correctly assigned). It can be used alone (to maximise the number of classified reads) or in conjunction with other demultiplexers (to maximise precision and minimise false positive classifications). We also found cross-sample chimeric reads (0.3%) and evidence of barcode switching (0.3%) in our dataset, which likely arise during library preparation and may be detrimental for quantitative studies that use multiplexing. Deepbinner is open source (GPLv3) and available at https://github.com/rrwick/Deepbinner.
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    Tracking key virulence loci encoding aerobactin and salmochelin siderophore synthesis in Klebsiella pneumoniae
    Lam, MMC ; Wyres, K ; Judd, L ; Wick, R ; Jenney, A ; Brisse, S ; Holt, K (BMC, 2018-07-25)
    Background: Klebsiella pneumoniae is a recognised agent of multidrug-resistant (MDR) healthcare-associated infections, however individual strains vary in their virulence potential due to the presence of mobile accessory genes. In particular, gene clusters encoding the biosynthesis of siderophores aerobactin (iuc) and salmochelin (iro) are associated with invasive disease and are common amongst hypervirulent K. pneumoniae clones that cause severe community-associated infections such as liver abscess and pneumonia. Concerningly iuc has also been reported in MDR strains in the hospital setting, where it was associated with increased mortality, highlighting the need to understand, detect and track the mobility of these virulence loci in the K. pneumoniae population. Methods: Here we examined the genetic diversity, distribution and mobilisation of iuc and iro loci among 2503 K. pneumoniae genomes using comparative genomics approaches, and developed tools for tracking them via genomic surveillance. Results: Iro and iuc were detected at low prevalence (<10%). Considerable genetic diversity was observed, resolving into five iro and six iuc lineages that show distinct patterns of mobilisation and dissemination in the K. pneumoniae population. The major burden of iuc and iro amongst the genomes analysed was due to two linked lineages (iuc1/iro1, 74% and iuc2/iro2, 14%), each carried by a distinct non-self-transmissible IncFIBK virulence plasmid type that we designate KpVP-1 and KpVP-2. These dominant types also carry hypermucoidy (rmpA) determinants and include all previously described virulence plasmids of K. pneumoniae. The other iuc and iro lineages were associated with diverse plasmids, including some carrying FII conjugative transfer regions and some imported from E. coli; the exceptions were iro3 (mobilised by ICEKp1), and iuc4 (fixed in the chromosome of K. pneumoniae subspecies rhinoscleromatis). Iro/iuc MGEs appear to be stably maintained at high frequency within known hypervirulent strains (ST23, ST86, etc), but were also detected at low prevalence in others such as MDR strain ST258. Conclusions: Iuc and iro are mobilised in K. pneumoniae via a limited number of MGEs. This study provides a framework for identifying and tracking these important virulence loci, which will be important for genomic surveillance efforts including monitoring for the emergence of hypervirulent MDR K. pneumoniae strains.
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    Performance of neural network basecalling tools for Oxford Nanopore sequencing
    Wick, RR ; Judd, LM ; Holt, KE (BMC, 2019-06-24)
    BACKGROUND: Basecalling, the computational process of translating raw electrical signal to nucleotide sequence, is of critical importance to the sequencing platforms produced by Oxford Nanopore Technologies (ONT). Here, we examine the performance of different basecalling tools, looking at accuracy at the level of bases within individual reads and at majority-rule consensus basecalls in an assembly. We also investigate some additional aspects of basecalling: training using a taxon-specific dataset, using a larger neural network model and improving consensus basecalls in an assembly by additional signal-level analysis with Nanopolish. RESULTS: Training basecallers on taxon-specific data results in a significant boost in consensus accuracy, mostly due to the reduction of errors in methylation motifs. A larger neural network is able to improve both read and consensus accuracy, but at a cost to speed. Improving consensus sequences ('polishing') with Nanopolish somewhat negates the accuracy differences in basecallers, but pre-polish accuracy does have an effect on post-polish accuracy. CONCLUSIONS: Basecalling accuracy has seen significant improvements over the last 2 years. The current version of ONT's Guppy basecaller performs well overall, with good accuracy and fast performance. If higher accuracy is required, users should consider producing a custom model using a larger neural network and/or training data from the same species.
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    Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae
    Wyres, K ; Wick, R ; Judd, L ; Froumine, R ; Tokolyi, A ; Gorrie, C ; Lam, M ; Duchene, S ; Jenney, A ; Holt, K ; Hughes, D (Public Library of Science (PLoS), 2018-09-12)
    Klebsiella pneumoniae (Kp) has emerged as an important cause of two distinct public health threats: multi-drug resistant (MDR) healthcare-associated infections and community-acquired invasive infections, particularly pyogenic liver abscess. The majority of MDR hospital outbreaks are caused by a subset of Kp clones with a high prevalence of acquired antimicrobial resistance (AMR) genes, while the majority of community-acquired invasive infections are caused by hypervirulent clones that rarely harbour acquired AMR genes but have high prevalence of key virulence loci. Worryingly, the last few years have seen increasing reports of convergence of MDR and the key virulence genes within individual Kp strains, but it is not yet clear whether these represent a transient phenomenon or a significant ongoing threat. Here we perform comparative genomic analyses for 28 distinct Kp clones, including 6 hypervirulent and 8 MDR, to better understand their evolutionary histories and the risks of convergence. We show that MDR clones are highly diverse with frequent chromosomal recombination and gene content variability that far exceeds that of the hypervirulent clones. Consequently, we predict a much greater risk of virulence gene acquisition by MDR Kp clones than of resistance gene acquisition by hypervirulent clones.