Cottingham, H. | Search bioRxiv

bioRxiv2022

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

BackgroundOxford 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. MethodsWe 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 flowcells were basecalled using three alternative models (Fast, High-accuracy (HAC) and Super-accuracy (SUP), available within ONTs 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. ResultsThe 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; however, the definition of outbreak clusters from ONT-only assemblies was problematic. HAC basecalling + Medaka polishing performed similarly to SUP basecalling without polishing, and polishing introduced errors into HAC- or Fast-basecalled assemblies. Therefore, we recommend investing compute resources into basecalling (SUP model) over polishing, where compute resources and/or time are limiting. ConclusionsOverall, our results show that MLST, K type and AMR determinants can be reliably identified with ONT-only data. However, cluster detection remains challenging with this technology.

genomics↗

bioRxiv2021

Linear plasmids in Klebsiella and other Enterobacteriaceae

Linear plasmids are extrachromosomal DNA that have been found in a small number of bacterial species. To date, the only linear plasmids described in the Enterobacteriaceae family belong to Salmonella, first found in Salmonella Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. We used this collection to search public sequence databases and discovered an additional 74 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. Significance as a BioResource to the communityThis study provides the first report of linear plasmids identified within the Klebsiella pneumoniae species complex and the first report in Enterobacteriaceae besides Salmonella.We present the first comparative analysis of linear plasmid sequences in Enterobacteriaceae, however whilst this family is highly clinically significant, the functional and/or evolutionary importance of these plasmids is not yet clear. To facilitate future studies to address these questions, we have publicly deposited (i) the collection of linear plasmid sequence data; (ii) isolates representative of each of the distinct linear plasmid phylogroups. Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary information O_LIWhole genome sequence reads from Klebsiella pneumoniae isolates sequenced in this study have been deposited in NCBI SRA under the accession numbers listed in Table S1. C_LIO_LIRepresentative annotated sequences of one linear plasmid per phylogroup have been deposited in FigShare, doi 10.26180/16729126. C_LIO_LIA copy of all linear plasmid sequences that we assembled from publicly available genome sequence reads are available in FigShare, doi 10.26180/16531365. Read accessions for these are given in Table S1. C_LIO_LIEleven representative K. pneumoniae isolates harbouring linear plasmids described in this study have been deposited with the National Collection of Type Cultures (NCTC) and are available for purchase under the NCTC accession numbers listed in Table S1.K. pneumoniae 1194/11 (representative of phylogroup B) has been deposited in the Microorganisms Collection Center, Adolfo Lutz Institute, Sao Paulo, Brazil. To request strain 1194/11 (IAL 3063, SISGEN ABBF09B), contact: Microorganisms Collection Center Culture Collection Laboratory Instituto Adolfo Lutz, Sao Paulo State Department of Health Address: Av Dr Arnaldo, 351, 10 floor, room 1020 Phone number: +55 11 3068-2884 Zip code 01246-000, Sao Paulo, Brazil E-mail: colecaoial@ial.sp.gov.br C_LIO_LIAlignments of terminal inverted repeat sequences for each phylogroup can be found in Data S1, available on FigShare, doi 10.26180/16531371. C_LI

microbiology↗