Drug resistance and vaccine target surveillance of Plasmodium falciparum using nanopore sequencing in Ghana.

Malaria results in over 600,000 deaths annually, with the highest burden of deaths in young children living in sub-Saharan Africa. Molecular surveillance can provide important information for malaria control policies, including detection of antimalarial drug resistance. However, genome sequencing capacity in malaria-endemic countries is limited. We designed and implemented an end-to-end workflow to detect Plasmodium falciparum antimalarial resistance markers and diversity in the vaccine target circumsporozoite protein (csp) using nanopore sequencing in Ghana. We analysed 196 clinical samples and showed that our method is rapid, robust, accurate and straightforward to implement. Importantly, our method could be applied to dried blood spot samples, which are readily collected in endemic settings. We report that P. falciparum parasites in Ghana are mostly susceptible to chloroquine, with persistent sulfadoxine-pyrimethamine resistance and no evidence of artemisinin resistance. Multiple single nucleotide polymorphisms were identified in csp, but their significance is uncertain. Our study demonstrates the feasibility of nanopore sequencing for malaria genomic surveillance in endemic countries.

A manually curated annotation characterises genomic features of P. falciparum lncRNAs.

BACKGROUND: Important regulation occurs at the level of transcription in Plasmodium falciparum and growing evidence suggests that these apicomplexan parasites have complex regulatory networks. Recent studies implicate long noncoding RNAs (lncRNAs) as transcriptional regulators in P. falciparum. However, due to limited research and the lack of necessary experimental tools, our understanding of their role in the malaria-causing parasite remains largely unelucidated. In this work, we address one of these limitations, the lack of an updated and improved lncRNA annotation in P. falciparum. RESULTS: We generated long-read RNA sequencing data and integrated information extracted and curated from multiple sources to manually annotate lncRNAs. We identified 1119 novel lncRNAs and validated and refined 1250 existing annotations. Utilising the collated datasets, we generated evidence-based ranking scores for each annotation and characterised the distinct genomic contexts and features of P. falciparum lncRNAs. Certain features indicated subsets with potential biological significance such as 25 lncRNAs containing multiple introns, 335 lncRNAs lacking mutations in piggyBac mutagenic studies and lncRNAs associated with specific biologic processes including two new types of lncRNAs found proximal to var genes. CONCLUSIONS: The insights and the annotation presented in this study will serve as valuable tools for researchers seeking to understand the role of lncRNAs in parasite biology through both bioinformatics and experimental approaches.

Nanopore Sequencing Indicates That Tandem Amplification of Chromosome 20q11.21 in Human Pluripotent Stem Cells Is Driven by Break-Induced Replication.

Copy number variants (CNVs) are genomic rearrangements implicated in numerous congenital and acquired diseases, including cancer. The appearance of culture-acquired CNVs in human pluripotent stem cells (PSCs) has prompted concerns for their use in regenerative medicine. A particular problem in PSC is the frequent occurrence of CNVs in the q11.21 region of chromosome 20. However, the exact mechanism of origin of this amplicon remains elusive due to the difficulty in delineating its sequence and breakpoints. Here, we have addressed this problem using long-read Nanopore sequencing of two examples of this CNV, present as duplication and as triplication. In both cases, the CNVs were arranged in a head-to-tail orientation, with microhomology sequences flanking or overlapping the proximal and distal breakpoints. These breakpoint signatures point to a mechanism of microhomology-mediated break-induced replication in CNV formation, with surrounding Alu sequences likely contributing to the instability of this genomic region.

Mechanisms generating cancer genome complexity from a single cell division error.

The chromosome breakage-fusion-bridge (BFB) cycle is a mutational process that produces gene amplification and genome instability. Signatures of BFB cycles can be observed in cancer genomes alongside chromothripsis, another catastrophic mutational phenomenon. We explain this association by elucidating a mutational cascade that is triggered by a single cell division error-chromosome bridge formation-that rapidly increases genomic complexity. We show that actomyosin forces are required for initial bridge breakage. Chromothripsis accumulates, beginning with aberrant interphase replication of bridge DNA. A subsequent burst of DNA replication in the next mitosis generates extensive DNA damage. During this second cell division, broken bridge chromosomes frequently missegregate and form micronuclei, promoting additional chromothripsis. We propose that iterations of this mutational cascade generate the continuing evolution and subclonal heterogeneity characteristic of many human cancers.

Complete Assembly of Escherichia coli Sequence Type 131 Genomes Using Long Reads Demonstrates Antibiotic Resistance Gene Variation within Diverse Plasmid and Chromosomal Contexts.

The incidence of infections caused by extraintestinal Escherichia coli (ExPEC) is rising globally, which is a major public health concern. ExPEC strains that are resistant to antimicrobials have been associated with excess mortality, prolonged hospital stays, and higher health care costs. E. coli sequence type 131 (ST131) is a major ExPEC clonal group worldwide, with variable plasmid composition, and has an array of genes enabling antimicrobial resistance (AMR). ST131 isolates frequently encode the AMR genes blaCTX-M-14, blaCTX-M-15, and blaCTX-M-27, which are often rearranged, amplified, and translocated by mobile genetic elements (MGEs). Short DNA reads do not fully resolve the architecture of repetitive elements on plasmids to allow MGE structures encoding blaCTX-M genes to be fully determined. Here, we performed long-read sequencing to decipher the genome structures of six E. coli ST131 isolates from six patients. Most long-read assemblies generated entire chromosomes and plasmids as single contigs, in contrast to more fragmented assemblies created with short reads alone. The long-read assemblies highlighted diverse accessory genomes with blaCTX-M-15, blaCTX-M-14, and blaCTX-M-27 genes identified in three, one, and one isolates, respectively. One sample had no blaCTX-M gene. Two samples had chromosomal blaCTX-M-14 and blaCTX-M-15 genes, and the latter was at three distinct locations, likely transposed by the adjacent MGEs: ISEcp1, IS903B, and Tn2 This study showed that AMR genes exist in multiple different chromosomal and plasmid contexts, even between closely related isolates within a clonal group such as E. coli ST131.IMPORTANCE Drug-resistant bacteria are a major cause of illness worldwide, and a specific subtype called Escherichia coli ST131 causes a significant number of these infections. ST131 bacteria become resistant to treatments by modifying their DNA and by transferring genes among one another via large packages of genes called plasmids, like a game of pass-the-parcel. Tackling infections more effectively requires a better understanding of what plasmids are being exchanged and their exact contents. To achieve this, we applied new high-resolution DNA sequencing technology to six ST131 samples from infected patients and compared the output to that of an existing approach. A combination of methods shows that drug resistance genes on plasmids are highly mobile because they can jump into ST131's chromosomes. We found that the plasmids are very elastic and undergo extensive rearrangements even in closely related samples. This application of DNA sequencing technologies illustrates at a new level the highly dynamic nature of ST131 genomes.

PlasmidTron: assembling the cause of phenotypes and genotypes from NGS data.

Increasingly rich metadata are now being linked to samples that have been whole-genome sequenced. However, much of this information is ignored. This is because linking this metadata to genes, or regions of the genome, usually relies on knowing the gene sequence(s) responsible for the particular trait being measured and looking for its presence or absence in that genome. Examples of this would be the spread of antimicrobial resistance genes carried on mobile genetic elements (MGEs). However, although it is possible to routinely identify the resistance gene, identifying the unknown MGE upon which it is carried can be much more difficult if the starting point is short-read whole-genome sequence data. The reason for this is that MGEs are often full of repeats and so assemble poorly, leading to fragmented consensus sequences. Since mobile DNA, which can carry many clinically and ecologically important genes, has a different evolutionary history from the host, its distribution across the host population will, by definition, be independent of the host phylogeny. It is possible to use this phenomenon in a genome-wide association study to identify both the genes associated with the specific trait and also the DNA linked to that gene, for example the flanking sequence of the plasmid vector on which it is encoded, which follows the same patterns of distribution as the marker gene/sequence itself. We present PlasmidTron, which utilizes the phenotypic data normally available in bacterial population studies, such as antibiograms, virulence factors, or geographical information, to identify traits that are likely to be present on DNA that can randomly reassort across defined bacterial populations. It is also possible to use this methodology to associate unknown genes/sequences (e.g. plasmid backbones) with a specific molecular signature or marker (e.g. resistance gene presence or absence) using PlasmidTron. PlasmidTron uses a k-mer-based approach to identify reads associated with a phylogenetically unlinked phenotype. These reads are then assembled de novo to produce contigs in a fast and scalable-to-large manner. PlasmidTron is written in Python 3 and is available under the open source licence GNU GPL3 from https://github.com/sanger-pathogens/plasmidtron.

Genomic survey of Clostridium difficile reservoirs in the East of England implicates environmental contamination of wastewater treatment plants by clinical lineages.

There is growing evidence that patients with Clostridiumdifficile-associated diarrhoea often acquire their infecting strain before hospital admission. Wastewater is known to be a potential source of surface water that is contaminated with C. difficile spores. Here, we describe a study that used genome sequencing to compare C. difficile isolated from multiple wastewater treatment plants across the East of England and from patients with clinical disease at a major hospital in the same region. We confirmed that C. difficile from 65 patients were highly diverse and that most cases were not linked to other active cases in the hospital. In total, 186 C. difficile isolates were isolated from effluent water obtained from 18 municipal treatment plants at the point of release into the environment. Whole genome comparisons of clinical and environmental isolates demonstrated highly related populations, and confirmed extensive release of toxigenic C. difficile into surface waters. An analysis based on multilocus sequence types (STs) identified 19 distinct STs in the clinical collection and 38 STs in the wastewater collection, with 13 of 44 STs common to both clinical and wastewater collections. Furthermore, we identified five pairs of highly similar isolates (≤2 SNPs different in the core genome) in clinical and wastewater collections. Strategies to control community acquisition should consider the need for bacterial control of treated wastewater.

Emergence of an Extensively Drug-Resistant Salmonella enterica Serovar Typhi Clone Harboring a Promiscuous Plasmid Encoding Resistance to Fluoroquinolones and Third-Generation Cephalosporins.

Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel S Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR S Typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the blaCTX-M-15 extended-spectrum ß-lactamase, and carrying the qnrS fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of S Typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally.IMPORTANCE Typhoid fever is a severe disease caused by the Gram-negative bacterium Salmonella enterica serovar Typhi. Antibiotic-resistant S Typhi strains have become increasingly common. Here, we report the first large-scale emergence and spread of a novel extensively drug-resistant (XDR) S Typhi clone in Sindh, Pakistan. The XDR S Typhi is resistant to the majority of drugs available for the treatment of typhoid fever. This study highlights the evolving threat of antibiotic resistance in S Typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases. We genetically characterized the XDR S Typhi to investigate the phylogenetic relationship between these isolates and a global collection of S Typhi isolates and to identify multiple genes linked to antibiotic resistance. This S Typhi clone harbored a promiscuous antibiotic resistance plasmid previously identified in other enteric bacteria. The increasing antibiotic resistance in S Typhi observed here adds urgency to the need for typhoid prevention measures.

Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MinION sequencing.

Dissemination of carbapenem resistance among pathogenic Gram-negative bacteria is a looming medical emergency. Efficient spread of resistance within and between bacterial species is facilitated by mobile genetic elements. We hypothesized that wastewater contributes to the dissemination of carbapenemase-producing Enterobacteriaceae (CPE), and studied this through a cross-sectional observational study of wastewater in the East of England. We isolated clinically relevant species of CPE in untreated and treated wastewater, confirming that waste treatment does not prevent release of CPE into the environment. We observed that CPE-positive plants were restricted to those in direct receipt of hospital waste, suggesting that hospital effluent may play a role in disseminating carbapenem resistance. We postulated that plasmids carrying carbapenemase genes were exchanged between bacterial hosts in sewage, and used short-read (Illumina) and long-read (MinION) technologies to characterize plasmids encoding resistance to antimicrobials and heavy metals. We demonstrated that different CPE species (Enterobacter kobei and Raoultella ornithinolytica) isolated from wastewater from the same treatment plant shared two plasmids of 63 and 280 kb. The former plasmid conferred resistance to carbapenems (blaOXA-48), and the latter to numerous drug classes and heavy metals. We also report the complete genome sequence for Enterobacter kobei. Small, portable sequencing instruments such as the MinION have the potential to improve the quality of information gathered on antimicrobial resistance in the environment.

Community outbreaks of group A Streptococcus revealed by genome sequencing.

The frequent occurrence of disease outbreaks in humans caused by group A Streptococcus (GAS) is an on-going public health threat. Conventional bacterial typing methods lack the discriminatory power to confidently confirm or refute outbreaks in hospital and community settings. Microbial whole genome sequencing (WGS) provides a potential solution to this, but, there has been limited population-based surveillance with accompanying sequence data. We performed retrospective genomic surveillance of 93 clinical GAS isolates from individuals in a defined geographic region. Detailed clinical information was obtained for closely related clusters of isolates. Genomic sequence data was contextualised through comparison with international data. We identified 18 different emm genotypes within our bacterial population, and revealed both highly diverse and closely related isolates. This high level of diversity was maintained even in the context of international sequence data. We also identified two emm1 clusters, and one emm3 cluster, of closely-related isolates that differed only by 1 to 4 single nucleotide polymorphisms. Analysis of clinical information identified no healthcare associated contact between patients, indicating cryptic community transmission. Our findings suggest that genomic surveillance of GAS would increase detection of transmission and highlight opportunities for intervention.

Comparison of bacterial genome assembly software for MinION data and their applicability to medical microbiology.

Translating the Oxford Nanopore MinION sequencing technology into medical microbiology requires on-going analysis that keeps pace with technological improvements to the instrument and release of associated analysis software. Here, we use a multidrug-resistant Enterobacter kobei isolate as a model organism to compare open source software for the assembly of genome data, and relate this to the time taken to generate actionable information. Three software tools (PBcR, Canu and miniasm) were used to assemble MinION data and a fourth (SPAdes) was used to combine MinION and Illumina data to produce a hybrid assembly. All four had a similar number of contigs and were more contiguous than the assembly using Illumina data alone, with SPAdes producing a single chromosomal contig. Evaluation of the four assemblies to represent the genome structure revealed a single large inversion in the SPAdes assembly, which also incorrectly integrated a plasmid into the chromosomal contig. Almost 50 %, 80 % and 90 % of MinION pass reads were generated in the first 6, 9 and 12 h, respectively. Using data from the first 6 h alone led to a less accurate, fragmented assembly, but data from the first 9 or 12 h generated similar assemblies to that from 48 h sequencing. Assemblies were generated in 2 h using Canu, indicating that going from isolate to assembled data is possible in less than 48 h. MinION data identified that genes responsible for resistance were carried by two plasmids encoding resistance to carbapenem and to sulphonamides, rifampicin and aminoglycosides, respectively.

Early insights into the potential of the Oxford Nanopore MinION for the detection of antimicrobial resistance genes.

OBJECTIVES: Genome sequencing will be increasingly used in the clinical setting to tailor antimicrobial prescribing and inform infection control outbreaks. A recent technological innovation that could reduce the delay between pathogen sampling and data generation is single molecule sequencing. An example of this technology, which is undergoing evaluation through an early access programme, is the Oxford Nanopore MinION. METHODS: We undertook a feasibility study on six clinically significant pathogens, comparing the MinION to the Illumina MiSeq and PacBio RSII platforms. Genomic DNA was prepared and sequenced using the MinION as instructed by the manufacturer, and Illumina MiSeq and PacBio sequencing was performed using established methods. RESULTS: An evaluation of the accuracy of the MinION based on sequencing of an MRSA isolate showed that error rates were higher in the MinION reads, but provided an even coverage across the entire genome length. The MinION detected all of the expected carbapenemases and ESBL genes in five Gram-negative isolates and the mecA gene in an MRSA isolate. CONCLUSIONS: The MinION can detect the presence of acquired resistance genes, but improvements in accuracy are needed so that antimicrobial resistance associated with mutations in chromosomal genes can be identified.