Biology subjects

Judd, L. M.

Publications and source records attributed to Judd, L. M..

11 recordsLinked to original sources

bioRxiv2018

Yersiniabactin, Colibactin and Wider Resistome Contribute to Enhanced Virulence and Persistence of KPC-2-Producing Klebsiella pneumoniae CG258 in South America

The emergence and dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a worrisome public health issue compromising the treatment and outcome of infections caused by this pathogen. We performed a detailed virulome and resistome analysis of representative KPC- and/or CTX-M-producing K. pneumoniae belonging to clonal group (CG) 258 (sequence types ST11, ST258, ST340, ST437), circulating in Argentina, Brazil, Chile, Colombia and Peru; with further evaluation of the virulence behavior using the Galleria mellonella infection model. Genomic analysis of K. pneumoniae strains recovered from the human-animal-environment interface revealed a wide resistome characterized by the presence of genes and mutations conferring resistance to human and veterinary antibiotics, quaternary ammonium compounds (QACs) and heavy metals. Plasmid Inc typing revealed the presence of a wide diversity of replicon types with IncF, IncN, IncR and Col-like being frequently detected. Moreover, KPC-2-producing K. pneumoniae belonging to ST11 (KL-64 andKL-105) and ST340 (KL-15) carried multiple variants of distinct yersiniabactin siderophore (ybt) and/or genotoxic colibactin (clb) genes. In this regard, ICEKp3, ICEKp4 and ICEKp12 were identified in strains belonging to ST11 and ST340, recovered from Argentina, Brazil, Chile and Colombia; whereas ybt 17 and a novel ybt sequence type (YbST346) were identified together with clb in ICEKp10 structures from ST11 and ST258, from Brazil and Colombia, respectively. K. pneumoniae ST11 (ICEKp10/YbST346 and ICEKp4/ybt 10) strains killed 100% of wax moth larvae, in a similar way to hypervirulent K1/ST23 strain (ybt- and clb-negative) carrying the pLVPK-like plasmid, indicating enhanced virulence. In summary, our results indicate that yersiniabactin, colibactin and an expanded resistome have contributed to enhanced virulence and persistence of KPC-2-producing K. pneumoniae CG258 in South America. Therefore, active surveillance of hospital-associated lineages of K. pneumoniae should not only focus on clonal origin and antimicrobial resistance, but also on the virulence factors ybt and clb.

microbiology↗

bioRxiv2018

Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae

Klebsiella pneumoniae (Kp) has emerged as an important cause of two distinct public health threats: multidrug resistant (MDR) healthcare-associated infections1 and community-acquired invasive infections, particularly pyogenic liver abscess2. 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 loci3-5. Worryingly, the last few years have seen increasing reports of convergence of MDR and the key virulence genes within individual Kp strains6, 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.

microbiology↗

bioRxiv2018

Tracking key virulence loci encoding aerobactin and salmochelin siderophore synthesis in Klebsiella pneumoniae

BackgroundKlebsiella 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.\n\nMethodsHere 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.\n\nResultsIro 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.\n\nConclusionsIuc 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.

genomics↗

bioRxiv2018

Deepbinner: Demultiplexing barcoded Oxford Nanopore reads with deep convolutional neural networks

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.

bioinformatics↗

bioRxiv2018

Emergence and rapid global dissemination of CTX-M-15-associated Klebsiella pneumoniae strain ST307

Recent reports indicate the emergence of a new carbapenemase producing Klebsiella pneumoniae clone, ST307. Here 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 extended-spectrum beta-lactamase (ESBL) gene plus other antimicrobial resistance determinants. Our findings support the need for enhanced surveillance of this widespread ESBL clone in which carbapenem resistance is now emerging.

microbiology↗

bioRxiv2017

Population genomics of hypervirulent Klebsiella pneumoniae clonal group 23 reveals early emergence and rapid global dissemination

Since the mid-1980s there have been increasing reports of severe community-acquired pyogenic liver abscess, meningitis and bloodstream infections caused by hypervirulent Klebsiella pneumoniae, predominantly encompassing clonal group (CG) 23 serotype K1 strains. Common features of CG23 include a virulence plasmid associated with iron scavenging and hypermucoidy, and a chromosomal integrative and conjugative element (ICE) encoding the siderophore yersiniabactin and the genotoxin colibactin. Here we investigate the evolutionary history and genomic diversity of CG23 based on comparative analysis of 98 genomes. Contrary to previous reports with more limited samples, we show that CG23 comprises several deep branching sublineages dating back to the 1870s, many of which are associated with distinct chromosomal insertions of ICEs encoding yersiniabactin. We find that most liver abscess isolates (>80%) belong to a dominant sublineage, CG23-I, which emerged in the 1920s following acquisition of ICEKp10 (encoding colibactin in addition to yersiniabactin) and has undergone clonal expansion and global dissemination within the human population. The unique genomic feature of CG23-I is the production of colibactin, which has been reported previously as a promoter of gut colonisation and dissemination to the liver and brain in a mouse model of CG23 K. pneumoniae infection, and has been linked to colorectal cancer. We also identify an antibiotic-resistant subclade of CG23-I associated with sexually-transmitted infections in horses dating back to the 1980s. These data show that hypervirulent CG23 K. pneumoniae was circulating in humans for decades before the liver abscess epidemic was first recognised, and has the capacity to acquire and maintain AMR plasmids. These data provide a framework for future epidemiological and experimental studies of hypervirulent K. pneumoniae. To further support such studies we present an open access and completely sequenced human liver abscess isolate, SGH10, which is typical of the globally disseminated CG23-I sublineage.

genomics↗

bioRxiv2017

Dynamics of the upper airway microbiome in the pathogenesis of asthma-associated persistent wheeze in preschool children

Repeated cycles of infection-associated lower airway inflammation drives the pathogenesis of persistent wheezing disease in children. Tracking these events across a birth cohort during their first five years, we demonstrate that >80% of infectious events indeed involve viral pathogens, but are accompanied by a shift in the nasopharyngeal microbiome (NPM) towards dominance by a small range of pathogenic bacterial genera. Unexpectedly, this change in NPM frequently precedes the appearance of viral pathogens and acute symptoms. In non-sensitized children these events are associated only with \"transient wheeze\" that resolves after age three. In contrast, in children developing early allergic sensitization, they are associated with ensuing development of persistent wheeze, which is the hallmark of the asthma phenotype. This suggests underlying pathogenic interactions between allergic sensitization and antibacterial mechanisms.

genetics↗

bioRxiv2017

Antimicrobial resistant Klebsiella pneumoniae carriage and infection in specialized geriatric care wards linked to acquisition in the referring hospital

BackgroundKlebsiella pneumoniae is a leading cause of extended-spectrum beta-lactamase (ESBL) producing hospital-associated infections, for which elderly patients are at increased risk.\n\nMethodsWe conducted a 1-year prospective cohort study, in which a third of patients admitted to two 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.\n\nResultsK. pneumoniae throat carriage prevalence was 4.1%, rectal carriage 10.8% and ESBL carriage 1.7%. K. pneumoniae infection incidence 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 (ST29, ST323, ST340).\n\nOne 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.\n\nConclusionsThe data suggest the major source of K. pneumoniae was the patients 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 control. Rectal screening for ESBL organisms upon admission to geriatric wards could help inform patient management and infection control in such facilities.\n\nSummaryPatients own gut microbiota were the major source of K. pneumoniae, but extended-spectrum beta-lactamase strains were acquired in the referring hospital. This highlights the potential for rectal screening, and the importance of the wider hospital network, for local risk management.

microbiology↗

bioRxiv2017

Completing bacterial genome assemblies with multiplex MinION sequencing

Illumina sequencing platforms have enabled widespread bacterial whole genome sequencing. While Illumina data is appropriate for many analyses, its short read length limits its ability to resolve genomic structure. This has major implications for tracking the spread of mobile genetic elements, including those which carry antimicrobial resistance determinants. Fully resolving a bacterial genome requires long-read sequencing such as those generated by Oxford Nanopore Technologies (ONT) platforms. Here we describe our use of the ONT MinION to sequence 12 isolates of Klebsiella pneumoniae on a single flow cell. We assembled each genome using a combination of ONT reads and previously available Illumina reads, and little to no manual intervention was needed to achieve fully resolved assemblies using the Unicycler hybrid assembler. Assembling only ONT reads with Canu was less effective, resulting in fewer resolved genomes and higher error rates even following error correction with Nanopolish. We demonstrate that multiplexed ONT sequencing is a valuable tool for high-throughput bacterial genome finishing. Specifically, we advocate the use of Illumina sequencing as a first analysis step, followed by ONT reads as needed to resolve genomic structure.\n\nData summaryO_LISequence read files for all 12 isolates have been deposited in SRA, accessible through these NCBI BioSample accession numbers: SAMEA3357010, SAMEA3357043, SAMN07211279, SAMN07211280, SAMEA3357223, SAMEA3357193, SAMEA3357346, SAMEA3357374, SAMEA3357320, SAMN07211281, SAMN07211282, SAMEA3357405.\nC_LIO_LIA full list of SRA run accession numbers (both Illumina reads and ONT reads) for these samples are available in Table S1.\nC_LIO_LIAssemblies and sequencing reads corresponding to each stage of processing and analysis are provided in the following figshare project: https://figshare.com/projects/Completing_bacterial_genome_assemblies_with_multiplex_MinION_sequencing/23068\nC_LIO_LISource code is provided in the following public GitHub repositories: https://github.com/rrwick/Bacterial-genome-assemblies-with-multiplex-MinION-sequencing https://github.com/rrwick/Porechop https://github.com/rrwick/Fast5-to-Fastq\nC_LI\n\nImpact StatementLike many research and public health laboratories, we frequently perform large-scale bacterial comparative genomics studies using Illumina sequencing, which assays gene content and provides the high-confidence variant calls needed for phylogenomics and transmission studies. However, problems often arise with resolving genome assemblies, particularly around regions that matter most to our research, such as mobile genetic elements encoding antibiotic resistance or virulence genes. These complexities can often be resolved by long sequence reads generated with PacBio or Oxford Nanopore Technologies (ONT) platforms. While effective, this has proven difficult to scale, due to the relatively high costs of generating long reads and the manual intervention required for assembly. Here we demonstrate the use of barcoded ONT libraries sequenced in multiplex on a single ONT MinION flow cell, coupled with hybrid assembly using Unicycler, to resolve 12 large bacterial genomes. Minor manual intervention was required to fully resolve small plasmids in five isolates, which we found to be underrepresented in ONT data. Cost per sample for the ONT sequencing was equivalent to Illumina sequencing, and there is potential for significant savings by multiplexing more samples on the ONT run. This approach paves the way for high-throughput and cost-effective generation of completely resolved bacterial genomes to become widely accessible.

bioinformatics↗

bioRxiv2017

Frequent emergence of pathogenic lineages of Klebsiella pneumoniae viamobilisation of yersiniabactin and colibactin

Mobile genetic elements (MGEs) that frequently transfer within and between bacterial species play a critical role in bacterial evolution, and often carry key accessory genes that associate with a bacterias ability to cause disease. MGEs carrying antimicrobial resistance (AMR) and/or virulence determinants are common in opportunistic pathogen Klebsiella pneumoniae, which are a leading cause of highly drug-resistant infections in hospitals. Well-characterised virulence determinants in K. pneumoniae include the polyketide synthesis loci ybt and clb (also known as pks), encoding the iron-scavenging siderophore yersiniabactin and genotoxin colibactin respectively. These loci are located within an MGE called ICEKp, which is the most common virulence-associated MGE of K. pneumoniae, providing a mechanism for these virulence factors to spread within the population.\n\nHere we apply population genomics to investigate the prevalence, evolution and mobility of ybt and clb in K. pneumoniae populations through comparative analysis of 2,498 whole genome sequences. The ybt locus was detected in 40% of K. pneumoniae genomes, particularly amongst those associated with invasive infections. We identified 17 distinct ybt lineages and 3 clb lineages, each associated with one of 14 different structural variants of ICEKp. Comparison with the wider Enterobacteriaceae population showed occasional ICEKp acquisition by other members. The clb locus was present in 14% of all K. pneumoniae and 38.4% of ybt+ genomes. Hundreds of independent ICEKp integration events were detected affecting hundreds of phylogenetically distinct K. pneumoniae lineages, including [≥]19 in the globally-disseminated carbapenem-resistant clone CG258. A novel plasmid-encoded form of ybt was also identified, representing a new mechanism for ybt dispersal in K. pneumoniae populations. These data show that MGEs carrying ybt and clb circulate freely in the K. pneumoniae population, including among multidrug-resistant strains, and should be considered a target for genomic surveillance along with AMR determinants.\n\nAUTHOR SUMMARYKlebsiella pneumoniae infections are becoming increasingly difficult to treat with antibiotics. Some K. pneumoniae strains also carry extra genes that allow them to synthesise yersiniabactin, an iron-scavenging molecule, which enhances their ability to cause disease. These genes are located on a genetic element that can easily transfer between strains. Here, we screened 2498 K. pneumoniae genome sequences and found substantial diversity in the yersiniabactin genes and the associated genetic elements, including a novel mechanism of transfer, and detected hundreds of distinct yersiniabactin acquisition events between K. pneumoniae strains. We show that these yersiniabactin mobile genetic elements are specifically adapted to the K. pneumoniae population but also occasionally acquired by other bacterial members belonging to the Enterobacteriaceae family such as E. coli. These insights into the movement and genetics of yersiniabactin genes allow tracking of the evolution and spread of yersiniabactin in global K. pneumoniae populations and monitoring for acquisition of yersiniabactin in antibiotic-resistant strains.

microbiology↗

bioRxiv2016

Unicycler: resolving bacterial genome assemblies from short and long sequencing reads

1.The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce more complete genome assemblies, but the sequencing is more expensive and error prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate \"hybrid\" assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler utilises a novel semi-global aligner, which is used to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.

bioinformatics↗