Corrigendum: 'Nanopore-only assemblies for genomic surveillance of the global priority drug-resistant pathogen, Klebsiella pneumoniae'.

In the published version of the article, there were errors in the read accessions provided in Table S1, which are now fixed. The updated file (TableS1_v2_13March2023.csv) can be accessed on Figshare using the DOI: 10.6084/m9.figshare.19745608. Furthermore, we would like to correct the following sentence in the Data Summary section of the published version: ‘Illumina reads have been deposited in the NCBI SRA, under the BioProject ID: PRJEB6891, and ONT reads (SUP basecalled) have been deposited under BioProject PRJNA646837.’ The sentence should read: ‘Illumina and ONT reads have been deposited in the NCBI SRA, under the BioProject IDs: PRJEB6891, PRJNA646837 and PRJNA351909.’

Nanopore-only assemblies for genomic surveillance of the global priority drug-resistant pathogen, Klebsiella pneumoniae

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

Rehabilitation versus surgical reconstruction for non-acute anterior cruciate ligament injury (ACL SNNAP): a pragmatic randomised controlled trial

BackgroundAnterior cruciate ligament (ACL) rupture is a common debilitating injury that can cause instability of the knee. We aimed to investigate the best management strategy between reconstructive surgery and non-surgical treatment for patients with a non-acute ACL injury and persistent symptoms of instability.MethodsWe did a pragmatic, multicentre, superiority, randomised controlled trial in 29 secondary care National Health Service orthopaedic units in the UK. Patients with symptomatic knee problems (instability) consistent with an ACL injury were eligible. We excluded patients with meniscal pathology with characteristics that indicate immediate surgery. Patients were randomly assigned (1:1) by computer to either surgery (reconstruction) or rehabilitation (physiotherapy but with subsequent reconstruction permitted if instability persisted after treatment), stratified by site and baseline Knee Injury and Osteoarthritis Outcome Score—4 domain version (KOOS4). This management design represented normal practice. The primary outcome was KOOS4 at 18 months after randomisation. The principal analyses were intention-to-treat based, with KOOS4 results analysed using linear regression. This trial is registered with ISRCTN, ISRCTN10110685, and ClinicalTrials.gov, NCT02980367.FindingsBetween Feb 1, 2017, and April 12, 2020, we recruited 316 patients. 156 (49%) participants were randomly assigned to the surgical reconstruction group and 160 (51%) to the rehabilitation group. Mean KOOS4 at 18 months was 73·0 (SD 18·3) in the surgical group and 64·6 (21·6) in the rehabilitation group. The adjusted mean difference was 7·9 (95% CI 2·5–13·2; p=0·0053) in favour of surgical management. 65 (41%) of 160 patients allocated to rehabilitation underwent subsequent surgery according to protocol within 18 months. 43 (28%) of 156 patients allocated to surgery did not receive their allocated treatment. We found no differences between groups in the proportion of intervention-related complications.InterpretationSurgical reconstruction as a management strategy for patients with non-acute ACL injury with persistent symptoms of instability was clinically superior and more cost-effective in comparison with rehabilitation management

Linear plasmids in Klebsiella and other Enterobacteriaceae.

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