While long-read sequencing allows for the complete assembly of bacterial genomes, long-read assemblies contain a variety of errors. Here, we present Trycycler, a tool which produces a consensus assembly from multiple input assemblies of the same genome. Benchmarking showed that Trycycler assemblies contained fewer errors than assemblies constructed with a single tool. Post-assembly polishing further reduced errors and Trycycler+polishing assemblies were the most accurate genomes in our study. As Trycycler requires manual intervention, its output is not deterministic. However, we demonstrated that multiple users converge on similar assemblies that are consistently more accurate than those produced by automated assembly tools.

Klebsiella pneumoniae is 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 of K. pneumoniae clinical isolates in hospital patients. Whole-genome sequence (WGS) data reveals a diverse pathogen population, including other species within the K. pneumoniae species complex (18%). Several infections were caused by K. variicola/K. pneumoniae hybrids, one of which shows evidence of nosocomial transmission. A wide range of antimicrobial resistance (AMR) phenotypes are observed, and diverse genetic mechanisms identified (mainly plasmid-borne genes). ESBLs are correlated with presence of other acquired AMR genes (median n = 10). Bacterial genomic features associated with nosocomial onset are ESBLs (OR 2.34, p = 0.015) and rhamnose-positive capsules (OR 3.12, p < 0.001). Virulence plasmid-encoded features (aerobactin, hypermucoidy) are observed at low-prevalence (<3%), mostly in community-onset cases. WGS-confirmed nosocomial transmission is implicated in just 10% of cases, but strongly associated with ESBLs (OR 21, p < 1 × 10−11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate that K. pneumoniae infections in hospitalised patients are due largely to opportunistic infections with diverse strains, with an additional burden from nosocomially-transmitted AMR strains and community-acquired hypervirulent strains.

Background: K. pneumoniae is a leading cause of blood stream infection (BSI). Strains producing extended spectrum beta--lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial resistant (AMR) K. pneumoniae, and also for the characteristically antimicrobial sensitive, community--acquired 'hypervirulent' strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide. Methods: We conducted a retrospective genomic epidemiology study of 365 BSI K. pneumoniae from seven major healthcare facilities across South and Southeast Asia, extracting clinically relevant information (AMR, virulence, K and O antigen loci) using Kleborate. Findings: K. pneumoniae BSI isolates were highly diverse, comprising 120 multi--locus sequence types (STs) and 63 K--loci. ESBL and carbapenemase gene frequencies were 47% and 17%, respectively. The aerobactin synthesis locus (iuc), associated with hypervirulence, was detected in 28% of isolates. Importantly, 7% of isolates harboured iuc plus ESBL and/or carbapenemase genes. The latter represent genotypic AMR--virulence convergence, which is generally considered a rare phenomenon but was particularly common amongst South Asian BSI (17%). Of greatest concern, we identified seven novel plasmids carrying both iuc and AMR genes, raising the prospect of co--transfer of these phenotypes amongst K. pneumoniae. Interpretation: South and Southeast Asia are high--risk regions for the emergence of AMR and convergent AMR--hypervirulent K. pneumoniae. Enhanced surveillance efforts, reporting STs, AMR and virulence information are urgently required to monitor this public health threat. BackgroundKlebsiella pneumoniae is now regarded globally by the World Health Organisation (WHO) and others, as a priority antimicrobial resistant (AMR) pathogen requiring new control strategies 1 . These include rapid identification and containment of high--risk AMR clones such as the carbapenemase--producing (CP) variants, augmented with vaccines, bacteriophages, or immunotherapies that target conserved surface antigens. However, K. pneumoniae is highly diverse, hindering the development of such strategies and our ability to study its molecular epidemiology in a short time frame. This diverse bacterial species is generally associated with a range of differing community and healthcare--associated infections, but can be particularly problematic when the organisms gain access to sterile sites such as the cerebrospinal fluid, internal body cavities, and the bloodstream. Such infections are often characterised by rapid onset and multi--drug resistance (MDR), including resistance to third generation cephalosporins and/or carbapenems. Antimicrobials are the primary treatment strategy but options are severely limited by AMR. Concomitant w...