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Oxford Nanopore Technologies (ONT) sequencing has rich potential for genomic epidemiology and public health investigations of bacterial pathogens, particularly in low-resource settings and at the point of care, due to its portability and affordability. However, low base-call accuracy has limited the reliability of ONT data for critical tasks such as antimicrobial resistance (AMR) and virulence gene detection and typing, serotype prediction, and cluster identification. Thus, Illumina sequencing remains the standard for genomic surveillance despite higher capital and running costs. We tested the accuracy of ONT-only assemblies for common applied bacterial genomics tasks (genotyping and cluster detection, implemented via Kleborate, Kaptive and Pathogenwatch), using data from 54 unique Klebsiella pneumoniae isolates. ONT reads generated via MinION with R9.4.1 flowcells were basecalled using three alternative models [Fast, High-accuracy (HAC) and Super-accuracy (SUP), available within ONT’s Guppy software], assembled with Flye and polished using Medaka. Accuracy of typing using ONT-only assemblies was compared with that of Illumina-only and hybrid ONT+Illumina assemblies, constructed from the same isolates as reference standards. The most resource-intensive ONT-assembly approach (SUP basecalling, with or without Medaka polishing) performed best, yielding reliable capsule (K) type calls for all strains (100 % exact or best matching locus), reliable multi-locus sequence type (MLST) assignment (98.3 % exact match or single-locus variants), and good detection of acquired AMR genes and mutations (88–100 % correct identification across the various drug classes). Distance-based trees generated from SUP+Medaka assemblies accurately reflected overall genetic relationships between isolates. The definition of outbreak clusters from ONT-only assemblies was problematic due to inflation of SNP counts by high base-call errors. However, ONT data could be reliably used to ‘rule out’ isolates of distinct lineages from suspected transmission clusters. HAC basecalling + Medaka polishing performed similarly to SUP basecalling without polishing. Therefore, we recommend investing compute resources into basecalling (SUP model), wherever compute resources and time allow, and note that polishing is also worthwhile for improved performance. Overall, our results show that MLST, K type and AMR determinants can be reliably identified with ONT-only R9.4.1 flowcell data. However, cluster detection remains challenging with this technology.

Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family Enterobacteriaceae belong to Salmonella , first found in Salmonella enterica Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. Screening of assembly graphs assembled from public read sets identified linear plasmid structures in a further 13 K . pneumoniae species complex genomes. We used these 25 linear plasmid sequences to query all bacterial genome assemblies in the National Center for Biotechnology Information database, and discovered an additional 61 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function; however, each phylogroup carried its own unique toxin–antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug-resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.

Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family

The outer polysaccharide capsule and lipopolysaccharide (LPS) antigens are key targets for novel control strategies targeting

Bacterial capsules and lipopolysaccharides are diverse surface polysaccharides (SPs) that serve as the frontline for interactions with the outside world. While SPs can evolve rapidly, their diversity and evolutionary dynamics across different taxonomic scales has not been investigated in detail. Here, we focused on the bacterial order Enterobacteriales (including the medically relevant Enterobacteriaceae), to carry out comparative genomics of two SP locus synthesis regions, cps and kps, using 27,334 genomes from 45 genera. We identified high-quality cps loci in 22 genera and kps in 11 genera, around 4% of which were detected in multiple species. We found SP loci to be highly dynamic genetic entities: their evolution was driven by high rates of horizontal gene transfer (HGT), both of whole loci and component genes, and relaxed purifying selection, yielding large repertoires of SP diversity. In spite of that, we found the presence of (near-)identical locus structures in distant taxonomic backgrounds that could not be explained by recent exchange, pointing to long-term selective preservation of locus structures in some populations. Our results reveal differences in evolutionary dynamics driving SP diversity within different bacterial species, with lineages of Escherichia coli, Enterobacter hormaechei and Klebsiella aerogenes most likely to share SP loci via recent exchange; and lineages of Salmonella enterica, Cronobacter sakazakii and Serratia marcescens most likely to share SP loci via other mechanisms such as long-term preservation. Overall, the evolution of SP loci in Enterobacteriales is driven by a range of evolutionary forces and their dynamics and relative importance varies between different species.

Klebsiella pneumoniaeis a major cause of opportunistic healthcare-associated infections, which are increasingly complicated by the presence of extended-spectrum beta-lactamases (ESBLs) and carbapenem resistance. We conducted a year-long prospective surveillance study ofK. pneumoniaeclinical isolates identified in a hospital microbiological diagnostic laboratory. Disease burden was two-thirds urinary tract infections (UTI; associated with female sex and age), followed by pneumonia (15%), wound (10%) and disseminated infections/sepsis (10%). Whole-genome sequencing (WGS) revealed a diverse pathogen population, including other species within theK. pneumoniaecomplex (18%). Several infections were caused byK. variicola/K. pneumoniaespecies hybrids, one of which showed evidence of nosocomial transmission, indicating fitness to transmit and cause disease despite a lack of acquired antimicrobial resistance (AMR). A wide range of AMR phenotypes were observed and, in most cases, corresponding mechanisms were identified in the genomes, mainly in the form of plasmid-borne genes. ESBLs were correlated with presence of other acquired AMR genes (median 10). Bacterial genomic features associated with nosocomial onset of disease were ESBL genes (OR 2.34, p=0.015) and rhamnose-positive capsules (OR 3.12, p-11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate the underlying burden ofK. pneumoniaedisease in hospitalised patients is due largely to opportunistic infections with diverse strains. However, we also identified several successful lineages that were overrepresented but not due to nosocomial transmission. These lineages were associated with ESBL, yersiniabactin, mannose+ K loci and rhamnose- K loci; most are also common in public clinical genome collections, suggesting enhanced propensity for colonisation and spread in the human population.

The outer polysaccharide capsule and lipopolysaccharide antigens are key targets for novel control strategies targeting Klebsiella pneumoniae and related taxa from the K. pneumoniae species complex (KpSC), including vaccines, phage and monoclonal antibody therapies. Given the importance and growing interest in these highly diverse surface antigens, we had previously developed Kaptive, a tool for rapidly identifying and typing capsule (K) and outer lipopolysaccharide (O) loci from whole genome sequence data. Here, we report two significant updates, now freely available in Kaptive 2.0 (github.com/katholt/kaptive); i) the addition of 16 novel K locus sequences to the K locus reference database following an extensive search of >17,000 KpSC genomes; and ii) enhanced O locus typing to enable prediction of the clinically relevant O2 antigen (sub)types, for which the genetic determinants have been recently described. We applied Kaptive 2.0 to a curated dataset of >12,000 public KpSC genomes to explore for the first time the distribution of predicted O (sub)types across species, sampling niches and clones, which highlighted key differences in the distributions that warrant further investigation. As the uptake of genomic surveillance approaches continues to expand globally, the application of Kaptive 2.0 will generate novel insights essential for the design of effective KpSC control strategies.Significance as a BioResource to the communityKlebsiella pneumoniae is a major cause of bacterial healthcare associated infections globally, with increasing rates of antimicrobial resistance, including strains with resistance to the drugs of last resort. The latter have therefore been flagged as priority pathogens for the development of novel control strategies.K. pneumoniae produce two key surface antigen sugars (capsular polysaccharide and lipopolysaccharide (LPS)) that are immunogenic and targets for novel controls such as a vaccines and phage therapy. However, there is substantial antigenic diversity in the population and relatively little is understood about the distribution of antigen types geographically and among strains causing different types of infections. Whereas laboratory-based antigen typing is difficult and rarely performed, information about the relevant synthesis loci can be readily extracted from whole genome sequence data. We have previously developed Kaptive, a freely available tool for rapid typing of Klebsiella capsule and LPS loci from genome sequences.Kaptive is now used widely in the global research community and has facilitated new insights into Klebsiella capsule and LPS diversity. Here we present an update to Kaptive facilitating i) the identification of 16 additional novel capsule loci, and ii) the prediction of immunologically relevant LPS O2 antigen subtypes. These updates will enable enhanced sero-epidemiological surveillance for K. pneumoniae, to inform the design of vaccines and other novel Klebsiella control strategies.Data summaryThe updated code and reference databases for Kaptive are available at https://github.com/katholt/KaptiveGenome accessions from which reference sequences of novel K loci were defined are listed in Supplementary Table 1, and genomes from which these loci were detected (along with the corresponding Kaptive output) are listed in Supplementary Table 2.Accessions for the genomes screened for O types/subtypes (along with the corresponding Kaptive output) are listed in Supplementary Table 3.The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.Repositories1.1RepositoriesGenome sequence from which the novel K locus KL182 was defined has been deposited under the accession JAJHNT000000000.

Long-read-only bacterial genome assemblies usually contain residual errors, most commonly homopolymer-length errors. Short-read polishing tools can use short reads to fix these errors, but most rely on short-read alignment which is unreliable in repeat regions. Errors in such regions are therefore challenging to fix and often remain after short-read polishing. Here we introduce Polypolish, a new short-read polisher which uses all-per-read alignments to repair errors in repeat sequences that other polishers cannot. Polypolish performed well in benchmarking tests using both simulated and real reads, and it almost never introduced errors during polishing. The best results were achieved by using Polypolish in combination with other short-read polishers.Author summaryRecent improvements in Oxford Nanopore Technologies sequencing platforms and assembly algorithms have made it easier than ever to generate complete bacterial genome sequences. However, Oxford Nanopore genome sequences suffer from errors that limit their utility in downstream analyses. To fix these errors, one can ‘polish’ the genome with Illumina sequencing, exploiting the fact that Oxford Nanopore and Illumina sequencing have different error profiles. There are several polishing tools which can fix most errors in an Oxford Nanopore genome, but they struggle with errors in repetitive regions of the genome. With this in mind, we have developed a polisher, Polypolish, which uses a novel approach that allows it to fix more errors in genomic repeats. Our results show that Polypolish is both effective at repairing sequence errors and very unlikely to introduce new errors. Polypolish can often fix errors that other polishers cannot and vice versa, so the best results come from using a combination of tools. Polypolish therefore has an important role in bacterial genome assembly methods that aim for the highest possible sequence accuracy.

Background Third-generation cephalosporin-resistant Gram-negatives (3GCR-GN) and vancomycin-resistant enterococci (VRE) are common causes of multi-drug resistant healthcare-associated infections, for which gut colonisation is considered a prerequisite. However, there remains a key knowledge gap about colonisation and infection dynamics in high-risk settings such as the intensive care unit (ICU), thus hampering infection prevention efforts. Methods We performed a three-month prospective genomic survey of infecting and gut-colonising 3GCR-GN and VRE among patients admitted to an Australian ICU. Bacteria were isolated from rectal swabs (n = 287 and n = 103 patients ≤2 and > 2 days from admission, respectively) and diagnostic clinical specimens between Dec 2013 and March 2014. Isolates were subjected to Illumina whole-genome sequencing (n = 127 3GCR-GN, n = 41 VRE). Multi-locus sequence types (STs) and antimicrobial resistance determinants were identified from de novo assemblies. Twenty-three isolates were selected for sequencing on the Oxford Nanopore MinION device to generate completed reference genomes (one for each ST isolated from ≥2 patients). Single nucleotide variants (SNVs) were identified by read mapping and variant calling against these references. Results Among 287 patients screened on admission, 17.4 and 8.4% were colonised by 3GCR-GN and VRE, respectively. Escherichia coli was the most common species (n = 36 episodes, 58.1%) and the most common cause of 3GCR-GN infection. Only two VRE infections were identified. The rate of infection among patients colonised with E. coli was low, but higher than those who were not colonised on admission (n = 2/33, 6% vs n = 4/254, 2%, respectively, p = 0.3). While few patients were colonised with 3GCR- Klebsiella pneumoniae or Pseudomonas aeruginosa on admission (n = 4), all such patients developed infections with the colonising strain. Genomic analyses revealed 10 putative nosocomial transmission clusters (≤20 SNVs for 3GCR-GN, ≤3 SNVs for VRE): four VRE, six 3GCR-GN, with epidemiologically linked clusters accounting for 21 and 6% of episodes, respectively (OR 4.3, p = 0.02). Conclusions 3GCR-E. coli and VRE were the most common gut colonisers. E. coli was the most common cause of 3GCR-GN infection, but other 3GCR-GN species showed greater risk for infection in colonised patients. Larger studies are warranted to elucidate the relative risks of different colonisers and guide the use of screening in ICU infection control.

Assembly of bacterial genomes from long-read data (generated by Oxford Nanopore or Pacific Biosciences platforms) can often be complete: a single contig for each chromosome or plasmid in the genome. However, even complete bacterial genome assemblies constructed solely from long reads still contain a variety of errors, and different assemblies of the same genome often contain different errors. Here, we present Trycycler, a tool which produces a consensus assembly from multiple input assemblies of the same genome. Benchmarking using both simulated and real sequencing reads showed that Trycycler consensus assemblies contained fewer errors than any of those constructed with a single long-read assembler. Post-assembly polishing with Medaka and Pilon further reduced errors and yielded the most accurate genome assemblies in our study. As Trycycler can require human judgement and manual intervention, its output is not deterministic, and different users can produce different Trycycler assemblies from the same input data. However, we demonstrated that multiple users with minimal training converge on similar assemblies that are consistently more accurate than those produced by automated assembly tools. We therefore recommend Trycycler+Medaka+Pilon as an ideal approach for generating high-quality bacterial reference genomes.Data availabilitySupplementary figures, tables and code can be found at: github.com/rrwick/Trycycler-paperReads, assemblies and reference sequences can be found at: bridges.monash.edu/articles/dataset/Trycycler_paper_dataset/14890734

Klebsiella pneumoniae is a leading cause of antimicrobial-resistant (AMR) healthcare-associated infections, neonatal sepsis and community-acquired liver abscess, and is associated with chronic intestinal diseases. Its diversity and complex population structure pose challenges for analysis and interpretation of K. pneumoniae genome data. Here we introduce Kleborate, a tool for analysing genomes of K. pneumoniae and its associated species complex, which consolidates interrogation of key features of proven clinical importance. Kleborate provides a framework to support genomic surveillance and epidemiology in research, clinical and public health settings. To demonstrate its utility we apply Kleborate to analyse publicly available Klebsiella genomes, including clinical isolates from a pan-European study of carbapenemase-producing Klebsiella, highlighting global trends in AMR and virulence as examples of what could be achieved by applying this genomic framework within more systematic genomic surveillance efforts. We also demonstrate the application of Kleborate to detect and type K. pneumoniae from gut metagenomes., Klebsiella pneumoniae is a pathogen of increasing public health concern and antimicrobial resistance is becoming more prevalent. Here, the authors describe a K. pneumoniae genotyping tool, Kleborate, that can be used to identify lineages and detect antimicrobial resistance and virulence loci.

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. Electronic supplementary material The online version of this article (10.1186/s13059-019-1727-y) contains supplementary material, which is available to authorized users.

Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (Impact statementResearchers who use Oxford Nanopore Technologies (ONT) platforms to sequence bacterial genomes can currently choose from two library preparation methods. The first is a ligation-based approach, which uses ligase to attach sequencing adapters to the ends of DNA molecules. The second is a rapid approach, which uses a transposase enzyme to cleave DNA and attach adapters in a single step. There are advantages to each preparation, for example ligation can produce better yields but rapid is a simpler procedure. Our study reveals another advantage of rapid preparations: they are more effective at sequencing small plasmids. We show that sequencing of ligation-based libraries yields fewer reads derived from small plasmids, making such plasmids harder to detect in bacterial genomes. Since small plasmids can contain clinically relevant genes, including antimicrobial resistance (AMR) or virulence determinants, their exclusion could lead to unreliable conclusions that have serious consequences for AMR surveillance and prediction. We therefore recommend that researchers performing ONT-only sequencing of bacterial genomes should consider using rapid preparations whenever small plasmid recovery is important.Data summarySupplementary figures, tables, data and code can be found at: github.com/rrwick/Small-plasmid-Nanopore

Background. Hypervirulent Klebsiella pneumoniae causes severe disseminated infections, typically with hepatic and central nervous system involvement including endophthalmitis. Case Presentation. We report a fatal case of an undocumented Chinese migrant in her 60s who presented to the emergency department with abdominal pain, lethargy, and headache over the preceding two weeks. She had a new diagnosis of diabetes mellitus on admission. Computed tomography scan of the thorax, abdomen, and pelvis showed bilateral pneumonia with liver abscesses. The patient was treated with empirical broad-spectrum antibiotics before K. pneumoniae was isolated from cerebrospinal fluid and blood cultures. The isolate was further characterised as a ST23 (ST: sequence type), serotype K1 hypervirulent strain using Nanopore sequencing. Despite admission to the intensive care unit, the patient died within 48 hrs of admission. Conclusions. This case demonstrates the need for increased awareness of hypervirulent K. pneumoniae, even in countries where it occurs infrequently. Novel, rapid, sequencing technologies can support diagnosis in unusual presentations.

K. pneumoniae is a leading cause of antimicrobial-resistant (AMR) healthcare-associated infections, neonatal sepsis and community-acquired liver abscess, and is associated with chronic intestinal diseases. Its diversity and complex population structure pose challenges for analysis and interpretation of K. pneumoniae genome data. Here we introduce Kleborate, a tool for analysing genomes of K. pneumoniae and its associated species complex, which consolidates interrogation of key features of proven clinical importance. Kleborate provides a framework to support genomic surveillance and epidemiology in research, clinical and public health settings. To demonstrate its utility we apply Kleborate to analyse publicly available Klebsiella genomes, including clinical isolates from a pan-European study of carbapenemase-producing Klebsiella, highlighting global trends in AMR and virulence as examples of what could be achieved by applying this genomic framework within more systematic genomic surveillance efforts. We also demonstrate the application of Kleborate to detect and type K. pneumoniae from gut metagenomes.

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.

Geothermal systems emit substantial amounts of aqueous, gaseous, and methylated mercury, but little is known about microbial influences on mercury speciation. Here, we report results from genome-resolved metagenomics and mercury speciation analysis of acidic warm springs in the Ngawha Geothermal Field (

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400 S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei. We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations (gyrA-S83L, parC-S80I, and gyrA-D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance., Shigella sonnei is one of the main species causing shigellosis worldwide. Here the authors analyse nearly 400 S. sonnei genome sequences and carry out experimental evolution experiments to shed light into the evolutionary processes underlying the recent emergence of fluoroquinolone resistance in this pathogen.

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., Since the 1980s, hypervirulent clonal-group CG23 serotype K1 Klebsiella pneumoniae has been recognised as a prominent cause of community-acquired liver abscess and other severe infections. Here, the authors investigate the genomic evolutionary history of CG23 and suggest a new reference strain for CG23.

Objectives mcr-9.1 is a newly described mobile colistin resistance gene. We have noted its presence in multiple species of carbapenem-resistant Enterobacterales (CRE) from our institution. We aimed to determine the clinical features, genomic context and phenotypic impact of mcr-9.1 carriage in a series of patients between 2010 and 2019. Methods We identified 32 patients with mcr-9.1-carrying CRE isolates (mCRE) and collected demographic, antimicrobial exposure and infection data. Whole-genome sequencing (including short and long reads) was performed on 32 isolates. We assessed sequence similarity of mcr-9.1-harbouring plasmids, then compared our findings with plasmids for which sequence data were publicly available. Results There was no colistin exposure in patients prior to isolation of mCRE. mcr-9.1 was identified on IncHI2 plasmids across four different bacterial species and was co-located with blaIMP-4 in 23/30 plasmids studied. mCRE isolates did not demonstrate phenotypic colistin resistance, either at baseline or following sublethal colistin exposure, thus showing that mcr-9.1 alone is not sufficient for resistance. Publicly available sequence data indicated the presence of carbapenemase genes in 236/619 mcr-9.1-carrying genomes (38%). IncHI2 plasmids carrying mcr-9.1 and carbapenemase genes were detected in genomes from North America, Europe, North Africa, Asia and Oceania. Conclusions Spread of mcr-9.1 in CRE from our institution was driven by IncHI2 ‘superplasmids’, so termed because of their large size and their prolific carriage of resistance determinants. These were also detected in global CRE genomes. Phenotypic colistin resistance was not detected in our isolates but remains to be determined from global mCRE.

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.

Acinetobacter baumannii isolate A388, recovered in Greece in 2002, represents a distinct antibiotic-resistant lineage of global clone 1 (GC1) producing the OXA-58 carbapenemase. We present the complete 4.332-Mbp genome sequence (chromosome plus 1 plasmid), generated by combining long (MinION) and short (Illumina HiSeq) read sequencing data.

2.AbstractThe Acinetobacter baumannii global clone 1 (GC1) isolate AB307-0294, recovered in the USA in 1994, and the global clone 2 (GC2) isolate ACICU, isolated in 2005 in Italy, were among the first A. baumannii isolates to be completely sequenced. AB307-0294 is susceptible to most antibiotics and has been used in many genetic studies and ACICU belongs to a rare GC2 lineage. The complete genome sequences, originally determined using 454 pyrosequencing technology which is known to generate sequencing errors, were re-determined using Illumina MiSeq and MinION (ONT) technologies and a hybrid assembly generated using Unicycler. Comparison of the resulting new high-quality genomes to the earlier 454-sequenced version identified a large number of nucleotide differences affecting protein coding features, and allowed the sequence of the long and highly-repetitive bap and blp1 genes to be properly resolved for the first time in ACICU. Comparisons of the annotations of the original and revised genomes revealed a large number of differences in the protein coding features (CDSs), underlining the impact of sequence errors on protein sequence predictions and core gene determination. On average, 400 predicted CDSs were longer or shorter in the revised genomes and about 200 CDS features were no longer present.3.Impact statementThe genomes of the first 10 A. baumannii strains to be completely sequenced underpin a large amount of published genetic and genomic analysis. However, most of their genome sequences contain substantial numbers of errors as they were sequenced using 454 pyrosequencing, which is known to generate errors particularly in homopolymer regions; and employed manual PCR and capillary sequencing steps to bridge contig gaps and repetitive regions in order to finish the genomes. Assembly of the very large and internally repetitive gene for the biofilm-associated proteins Bap and BLP1 was a recurring problem. As these strains continue to be used for genetic studies and their genomes continue to be used as references in phylogenomics studies including core gene determination, there is value in improving the quality of their genome sequences. To this end, we re-sequenced two such strains that belong to the two major globally distributed clones of A. baumannii, using a combination of highly-accurate short-read and gap-spanning long-read technologies. Annotation of the revised genome sequences eliminated hundreds of incorrect CDS feature annotations and corrected hundreds more. Given that these revisions affected hundreds of non-existent or incorrect CDS features currently cluttering GenBank protein databases, it can be envisaged that similar revision of other early bacterial genomes that were sequenced using error-prone technologies will affect thousands of CDS currently listed in GenBank and other databases. These corrections will impact the quality of predicted protein sequence data stored in public databases. The revised genomes will also improve the accuracy of future genetic and comparative genomic analyses incorporating these clinically important strains.4.Data summaryThe corrected complete genome sequence of A. baumannii AB307-0294 has been deposited in GenBank; GenBank accession number CP001172.2 (chromosome url - https://www.ncbi.nlm.nih.gov/nuccore/CP001172.2).The corrected complete genome sequence of ACICU has been deposited in GenBank under the GenBank accession numbers CP031380 (chromosome; url - https://www.ncbi.nlm.nih.gov/nuccore/CP031380), CP031381 (pACICU1; url - https://www.ncbi.nlm.nih.gov/nuccore/CP031381) and CP031382 (pACICU2; url - https://www.ncbi.nlm.nih.gov/nuccore/CP031382).The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Assessing the taxonomic composition of metagenomic samples is an important first step in understanding the biology and ecology of microbial communities in complex environments. Despite a wealth of algorithms and tools for metagenomic classification, relatively little effort has been put into the critical task of improving the quality of reference indices to which metagenomic reads are assigned. Here, we inferred the taxonomic composition of 404 publicly available metagenomes from human, marine and soil environments, using custom index databases modified according to two factors: the number of reference genomes used to build the databases, and the monophyletic strictness of species definitions. Index databases built following the NCBI taxonomic system were also compared to others using Genome Taxonomy Database (GTDB) taxonomic redefinitions. We observed a considerable increase in the rate of read classification using modified reference index databases as compared to a default NCBI RefSeq database, with up to a 4.4-, 6.4- and 2.2-fold increase in classified reads per sample for human, marine and soil metagenomes, respectively. Importantly, targeted correction for 70 common human pathogens and bacterial genera in the index database increased their specific detection levels in human metagenomes. We also show the choice of index database can influence downstream diversity and distance estimates for microbiome data. Overall, the study shows a large amount of accessible information in metagenomes remains unexploited using current methods, and that the same data analysed using different index databases could potentially lead to different conclusions. These results have implications for the power and design of individual microbiome studies, and for comparison and meta-analysis of microbiome datasets.

Klebsiella pneumoniae (Kp) is an infamous cause of multi-drug resistant (MDR) healthcare-associated infections and several MDR clones are globally distributed. A small number of drug-susceptible clones have also become globally distributed, causing severe community-acquired infections. These ‘hypervirulent’ clones are distinguished by expression of highly serum-resistant K1/K2 capsules, plus high prevalence of acquired virulence determinants. While hypervirulence and drug resistance are usually mutually exclusive, there are now increasing reports of convergent strains that are both highly virulent and MDR – a potentially disastrous combination. To better understand the risks of MDR-virulence convergence, we leveraged a collection of >2200 Kp genomes to identify 28 common clones (n≥10 genomes each), and performed a genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified by acquired resistance and virulence gene prevalence. Chromosomal recombination, capsule locus diversity, pan-genome, plasmid and phage dynamics were compared. MDR clones were highly diverse, with frequent chromosomal recombination generating extensive capsule locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which were each associated with only a single capsule locus and showed significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. These data suggest that hypervirulent clones are subject to some sort of constraint for horizontal gene-transfer. Hence we predict MDR clones pose the greatest risk for MDR-virulence convergence because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.

A retrospective genomic study led to the identification of two carbapenem-resistant K. pneumoniae isolates (KPN535 and KPC45) carrying bla KPC-2 genes on non-conjugative plasmids.….

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. publishedVersion

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).

OBJECTIVES: The objective of this study was to assess the presence of mcr-1 in Shigella sonnei isolated in Vietnam. METHODS: WGS data were analysed for the presence of the mcr-1 gene sequence. The association of mcr-1 with a plasmid was assessed by PCR and by conjugation. RESULTS: Through genome sequencing we identified a plasmid-associated inactive form of mcr-1 in a 2008 Vietnamese isolate of Shigella sonnei. The plasmid was conjugated into Escherichia coli and mcr-1 was activated upon exposure to colistin, resulting in highly colistin-resistant transconjugants. CONCLUSIONS: This is the first description of the mcr-1 gene in Shigella, which is atypical given that colistin is not ordinarily used to treat diarrhoea. Our data suggest the mcr-1 gene has been circulating in human-restricted pathogens for some time but likely carries a selective fitness cost.

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.

Klebsiella pneumoniae has emerged as an important cause of two distinct public health threats: multi-drug resistant (MDR) healthcare-associated infections and drug susceptible community-acquired invasive infections. These pathotypes are generally associated with two distinct subsets of K. pneumoniae lineages or ‘clones’ that are distinguished by the presence of acquired resistance genes and several key virulence loci. Genomic evolutionary analyses of the most notorious MDR and invasive community-associated (‘hypervirulent’) clones indicate differences in terms of chromosomal recombination dynamics and capsule polysaccharide diversity, but it remains unclear if these differences represent generalised trends. Here we leverage a collection of >2200 K. pneumoniae genomes to identify 28 common clones (n ≥ 10 genomes each), and perform the first genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified on the basis of acquired resistance and virulence gene prevalence. Chromosomal recombination, surface polysaccharide locus diversity, pan-genome, plasmid and phage dynamics were characterised and compared. The data showed that MDR clones were highly diverse, with frequent chromosomal recombination generating extensive surface polysaccharide locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which also showed a significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. Hence the data indicate that hypervirulent clones may be subject to some sort of constraint for horizontal gene transfer that does not apply to the MDR clones. Our findings are relevant for understanding the risk of emergence of individual K. pneumoniae strains carrying both virulence and acquired resistance genes, which have been increasingly reported and cause highly virulent infections that are extremely difficult to treat. Specifically, our data indicate that MDR clones pose the greatest risk, because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes., Author summary Klebsiellla pneumoniae is classified by the World Health Organization as a priority drug-resistant organism because it causes a significant burden of hospital infections that are extremely difficult to treat. However, outside of the hospital setting this bacterium is also an important cause of severe drug-susceptible infections. Until recently these two infection types were associated with distinct subsets of the K. pneumoniae population harbouring high prevalence of drug-resistance and virulence genes, respectively. However, there are now increasing reports of highly-virulent and difficult-to-treat K. pneumoniae strains that carry both resistance and virulence genes. In this study, we used genomic analyses to characterise and compare the evolutionary histories of drug-resistant and virulent K. pneumoniae. We show that the former are highly diverse, frequently acquiring novel genes through the processes of chromosomal recombination, plasmid and bacteriophage acquisition. In comparison, the latter show considerably lower gene content diversity, suggesting that they may be subject to some sort of limitation for gene acquisition. Consequently, we predict that drug-resistant K. pneumoniae are more likely to acquire virulence genes than virulent K. pneumoniae are to acquire drug-resistance genes.

SYNOPSISBackgroundMultidrug resistance (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.ObjectivesTo characterize the genetic determinants of virulence and antimicrobial resistance (AMR) in two ESBL-producing K. pneumoniae isolates belonging to the international MDR clone ST15.MethodsThe complete genome sequences of both isolates, including their plasmids, were resolved using Illumina and Oxford Nanopore sequencing.ResultsBoth 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 eight other AMR genes (blaTEM, aac3’-IIa, aph3’-Ia, dfrA1, satA2, blaSHV, sul1, 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.ConclusionsThe 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.