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 PORTFOLIO, 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 PORTFOLIO, 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)
    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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    Evolution of a clade of Acinetobacter baumannii global clone 1, lineage 1 via acquisition of carbapenem- and aminoglycoside-resistance genes and dispersion of ISAba1
    Hamidian, M ; Hawkey, J ; Wick, R ; Holt, KE ; Hall, RM (MICROBIOLOGY SOC, 2019-01)
    Resistance to carbapenem and aminoglycoside antibiotics is a critical problem in Acinetobacter baumannii, particularly when genes conferring resistance are acquired by multiply or extensively resistant members of successful globally distributed clonal complexes, such as global clone 1 (GC1) . Here, we investigate the evolution of an expanding clade of lineage 1 of the GC1 complex via repeated acquisition of carbapenem- and aminoglycoside-resistance genes. Lineage 1 arose in the late 1970s and the Tn6168/OCL3 clade arose in the late 1990s from an ancestor that had already acquired resistance to third-generation cephalosporins and fluoroquinolones. Between 2000 and 2002, two distinct subclades have emerged, and they are distinguishable via the presence of an integrated phage genome in subclade 1 and AbaR4 (carrying the oxa23 carbapenem-resistance gene in Tn2006) at a specific chromosomal location in subclade 2. Part or all of the original resistance gene cluster in the chromosomally located AbaR3 has been lost from some isolates, but plasmids carrying alternate resistance genes have been gained. In one group in subclade 2, the chromosomally located AbGRI3, carrying the armA aminoglycoside-resistance gene, has been acquired from a GC2 isolate and incorporated via homologous recombination. ISAba1 entered the common ancestor of this clade as part of the cephalosporin-resistance transposon Tn6168 and has dispersed differently in each subclade. Members of subclade 1 share an ISAba1 in one specific position in the chromosome and in subclade 2 two different ISAba1 locations are shared. Further shared ISAba1 locations distinguish further divisions, potentially providing simple markers for epidemiological studies.
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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)
    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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    GeneMates: an R package for detecting horizontal gene co-transfer between bacteria using gene-gene associations controlled for population structure
    Wan, Y ; Wick, RR ; Zobel, J ; Ingle, DJ ; Inouye, M ; Holt, KE (BMC, 2020-09-24)
    BACKGROUND: Horizontal gene transfer contributes to bacterial evolution through mobilising genes across various taxonomical boundaries. It is frequently mediated by mobile genetic elements (MGEs), which may capture, maintain, and rearrange mobile genes and co-mobilise them between bacteria, causing horizontal gene co-transfer (HGcoT). This physical linkage between mobile genes poses a great threat to public health as it facilitates dissemination and co-selection of clinically important genes amongst bacteria. Although rapid accumulation of bacterial whole-genome sequencing data since the 2000s enables study of HGcoT at the population level, results based on genetic co-occurrence counts and simple association tests are usually confounded by bacterial population structure when sampled bacteria belong to the same species, leading to spurious conclusions. RESULTS: We have developed a network approach to explore WGS data for evidence of intraspecies HGcoT and have implemented it in R package GeneMates ( github.com/wanyuac/GeneMates ). The package takes as input an allelic presence-absence matrix of interested genes and a matrix of core-genome single-nucleotide polymorphisms, performs association tests with linear mixed models controlled for population structure, produces a network of significantly associated alleles, and identifies clusters within the network as plausible co-transferred alleles. GeneMates users may choose to score consistency of allelic physical distances measured in genome assemblies using a novel approach we have developed and overlay scores to the network for further evidence of HGcoT. Validation studies of GeneMates on known acquired antimicrobial resistance genes in Escherichia coli and Salmonella Typhimurium show advantages of our network approach over simple association analysis: (1) distinguishing between allelic co-occurrence driven by HGcoT and that driven by clonal reproduction, (2) evaluating effects of population structure on allelic co-occurrence, and (3) direct links between allele clusters in the network and MGEs when physical distances are incorporated. CONCLUSION: GeneMates offers an effective approach to detection of intraspecies HGcoT using WGS data.
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    Complete Genome Sequence of WM99c, an Antibiotic-Resistant Acinetobacter baumannii Global Clone 2 (GC2) Strain Representing an Australian GC2 Lineage
    Nigro, SJ ; Wick, R ; Holt, KE ; Hall, RM ; Newton, ILG (AMER SOC MICROBIOLOGY, 2018-12)
    The extensively antibiotic-resistant Acinetobacter baumannii isolate WM99c recovered in Sydney, Australia, in 1999 is an early representative of a distinct lineage of global clone 2 (GC2) seen on the east coast of Australia. We present the complete 4.121-Mbp genome sequence (chromosome plus 2 plasmids), generated via long-read sequencing (PacBio).
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    Convergence of virulence and MDR in a single plasmid vector in MDR Klebsiella pneumoniae ST15
    Lam, MMC ; Wyres, KL ; Wick, RR ; Judd, LM ; Fostervold, A ; Holt, KE ; Lohr, IH (OXFORD UNIV PRESS, 2019-05)
    BACKGROUND: MDR and hypervirulence (hv) are typically observed in separate Klebsiella pneumoniae populations. However, convergent strains with both properties have been documented and potentially pose a high risk to public health in the form of invasive infections with limited treatment options. OBJECTIVES: Our aim was to characterize the genetic determinants of virulence and antimicrobial resistance (AMR) in two ESBL-producing K. pneumoniae isolates belonging to the international MDR clone ST15. METHODS: The complete genome sequences of both isolates, including their plasmids, were resolved using Illumina and Oxford Nanopore sequencing. RESULTS: Both isolates carried large mosaic plasmids in which AMR and virulence loci have converged within the same vector. These closely related mosaic hv-MDR plasmids include sequences typical of the K. pneumoniae virulence plasmid 1 (KpVP-1; including aerobactin synthesis locus iuc) fused with sequences typical of IncFIIK conjugative AMR plasmids. One hv-MDR plasmid carried three MDR elements encoding the ESBL gene blaCTX-M-15 and seven other AMR genes (blaTEM, aac3'-IIa, dfrA1, satA2, blaSHV, sul1 and aadA1). The other carried remnants of these elements encoding blaTEM and aac3'-IIa, and blaCTX-M-15 was located in a second plasmid in this isolate. The two isolates originated from patients hospitalized in Norway but have epidemiological and genomic links to Romania. CONCLUSIONS: The presence of both virulence and AMR determinants on a single vector enables simultaneous transfer in a single event and potentially rapid emergence of hv-MDR K. pneumoniae clones. This highlights the importance of monitoring for such convergence events with stringent genomic surveillance.