Molecular characterization and descriptive analysis of carbapenemase-producing Gram-negative rod infections in Bogota, Colombia.

In this study, the genetic differences and clinical impact of the carbapenemase-encoding genes among the community and healthcare-acquired infections were assessed. This retrospective, multicenter cohort study was conducted in Colombia and included patients infected with carbapenem-resistant Gram-negative rods between 2017 and 2021. Carbapenem resistance was identified by Vitek, and carbapenemase-encoding genes were identified by whole-genome sequencing (WGS) to classify the alleles and sequence types (STs). Descriptive statistics were used to determine the association of any pathogen or gene with clinical outcomes. A total of 248 patients were included, of which only 0.8% (2/248) had community-acquired infections. Regarding the identified bacteria, the most prevalent pathogens were Pseudomonas aeruginosa and Klebsiella pneumoniae. In the WGS analysis, 228 isolates passed all the quality criteria and were analyzed. The principal carbapenemase-encoding gene was blaKPC, specifically blaKPC-2 [38.6% (88/228)] and blaKPC-3 [36.4% (83/228)]. These were frequently detected in co-concurrence with blaVIM-2 and blaNDM-1 in healthcare-acquired infections. Notably, the only identified allele among community-acquired infections was blaKPC-3 [50.0% (1/2)]. In reference to the STs, 78 were identified, of which Pseudomonas aeruginosa ST111 was mainly related to blaKPC-3. Klebsiella pneumoniae ST512, ST258, ST14, and ST1082 were exclusively associated with blaKPC-3. Finally, no particular carbapenemase-encoding gene was associated with worse clinical outcomes. The most identified genes in carbapenemase-producing Gram-negative rods were blaKPC-2 and blaKPC-3, both related to gene co-occurrence and diverse STs in the healthcare environment. Patients had several systemic complications and poor clinical outcomes that were not associated with a particular gene.IMPORTANCEAntimicrobial resistance is a pandemic and a worldwide public health problem, especially carbapenem resistance in low- and middle-income countries. Limited data regarding the molecular characteristics and clinical outcomes of patients infected with these bacteria are available. Thus, our study described the carbapenemase-encoding genes among community- and healthcare-acquired infections. Notably, the co-occurrence of carbapenemase-encoding genes was frequently identified. We also found 78 distinct sequence types, of which two were novel Pseudomonas aeruginosa, which could represent challenges in treating these infections. Our study shows that in low and middle-income countries, such as Colombia, the burden of carbapenem resistance in Gram-negative rods is a concern for public health, and regardless of the allele, these infections are associated with poor clinical outcomes. Thus, studies assessing local epidemiology, prevention strategies (including trials), and underpinning genetic mechanisms are urgently needed, especially in low and middle-income countries.

Tissue-associated and vertically transmitted bacterial symbiont in the coral Pocillopora acuta.

Coral microhabitats are colonized by a myriad of microorganisms, including diverse bacteria which are essential for host functioning and survival. However, the location, transmission, and functions of individual bacterial species living inside the coral tissues remain poorly studied. Here, we show that a previously undescribed bacterial symbiont of the coral Pocillopora acuta forms cell-associated microbial aggregates (CAMAs) within the mesenterial filaments. CAMAs were found in both adults and larval offspring, suggesting vertical transmission. In situ laser capture microdissection of CAMAs followed by 16S rRNA gene amplicon sequencing and shotgun metagenomics produced a near complete metagenome-assembled genome. We subsequently cultured the CAMA bacteria from Pocillopora acuta colonies, and sequenced and assembled their genomes. Phylogenetic analyses showed that the CAMA bacteria belong to an undescribed Endozoicomonadaceae genus and species, which we propose to name Candidatus Sororendozoicomonas aggregata gen. nov sp. nov. Metabolic pathway reconstruction from its genome sequence suggests this species can synthesize most amino acids, several B vitamins, and antioxidants, and participate in carbon cycling and prey digestion, which may be beneficial to its coral hosts. This study provides detailed insights into a new member of the widespread Endozoicomonadaceae family, thereby improving our understanding of coral holobiont functioning. Vertically transmitted, tissue-associated bacteria, such as Sororendozoicomonas aggregata may be key candidates for the development of microbiome manipulation approaches with long-term positive effects on the coral host.

Mosquitoes provide a transmission route between possums and humans for Buruli ulcer in southeastern Australia.

Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans, is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus. Spatial scanning statistics revealed overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.

High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks.

IMPORTANCE: This proof-of-concept study introduces a hybrid capture oligo panel for whole-genome sequencing of all six human pathogenic hepatitis A virus (HAV) subgenotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ability of hybrid capture to enrich multiple targets allows for a single, streamlined workflow, thus facilitating the potential harmonization of molecular surveillance of HAV with other enteric viruses. Even challenging sample matrices can be accommodated, making them suitable for broad implementation in clinical and public health laboratories. This innovative approach has significant implications for enhancing multijurisdictional outbreak investigations as well as our understanding of the global diversity and transmission dynamics of HAV.

High-level nitrofurantoin resistance in a clinical isolate of Klebsiella pneumoniae: a comparative genomics and metabolomics analysis.

Nitrofurantoin is a commonly used chemotherapeutic agent in the treatment of uncomplicated urinary tract infections caused by the problematic multidrug resistant Gram-negative pathogen Klebsiella pneumoniae. The present study aims to elucidate the mechanism of nitrofurantoin action and high-level resistance in K. pneumoniae using whole-genome sequencing (WGS), qPCR analysis, mutation structural modeling and untargeted metabolomic analysis. WGS profiling of evolved highly resistant mutants (nitrofurantoin minimum inhibitory concentrations > 256 mg/L) revealed modified expression of several genes related to membrane transport (porin ompK36 and efflux pump regulator oqxR) and nitroreductase activity (ribC and nfsB, involved in nitrofurantoin reduction). Untargeted metabolomics analysis of total metabolites extracted at 1 and 4 h post-nitrofurantoin treatment revealed that exposure to the drug caused a delayed effect on the metabolome which was most pronounced after 4 h. Pathway enrichment analysis illustrated that several complex interrelated metabolic pathways related to nitrofurantoin bacterial killing (aminoacyl-tRNA biosynthesis, purine metabolism, central carbohydrate metabolism, and pantothenate and CoA biosynthesis) and the development of nitrofurantoin resistance (riboflavin metabolism) were significantly perturbed. This study highlights for the first time the key role of efflux pump regulator oqxR in nitrofurantoin resistance and reveals global metabolome perturbations in response to nitrofurantoin, in K. pneumoniae.IMPORTANCEA quest for novel antibiotics and revitalizing older ones (such as nitrofurantoin) for treatment of difficult-to-treat Gram-negative bacterial infections has become increasingly popular. The precise antibacterial activity of nitrofurantoin is still not fully understood. Furthermore, although the prevalence of nitrofurantoin resistance remains low currently, the drug's fast-growing consumption worldwide highlights the need to comprehend the emerging resistance mechanisms. Here, we used multidisciplinary techniques to discern the exact mechanism of nitrofurantoin action and high-level resistance in Klebsiella pneumoniae, a common cause of urinary tract infections for which nitrofurantoin is the recommended treatment. We found that the expression of multiple genes related to membrane transport (including active efflux and passive diffusion of drug molecules) and nitroreductase activity was modified in nitrofurantoin-resistant strains, including oqxR, the transcriptional regulator of the oqxAB efflux pump. Furthermore, complex interconnected metabolic pathways that potentially govern the nitrofurantoin-killing mechanisms (e.g., aminoacyl-tRNA biosynthesis) and nitrofurantoin resistance (riboflavin metabolism) were significantly inhibited following nitrofurantoin treatment. Our study could help inform the improvement of nitrofuran derivatives, the development of new pharmacophores, or drug combinations to support the resurgence of nitrofurantoin in the management of multidrug resistant K. pneumouniae infection.

Functional potential and evolutionary response to long-term heat selection of bacterial associates of coral photosymbionts.

IMPORTANCE: Symbiotic microorganisms are crucial for the survival of corals and their resistance to coral bleaching in the face of climate change. However, the impact of microbe-microbe interactions on coral functioning is mostly unknown but could be essential factors for coral adaption to future climates. Here, we investigated interactions between cultured dinoflagellates of the Symbiodiniaceae family, essential photosymbionts of corals, and associated bacteria. By assessing the genomic potential of 49 bacteria, we found that they are likely beneficial for Symbiodiniaceae, through the production of B vitamins and antioxidants. Additionally, bacterial genes involved in host-symbiont interactions, such as secretion systems, accumulated mutations following long-term exposure to heat, suggesting symbiotic interactions may change under climate change. This highlights the importance of microbe-microbe interactions in coral functioning.

Bactabolize is a tool for high-throughput generation of bacterial strain-specific metabolic models.

Metabolic capacity can vary substantially within a bacterial species, leading to ecological niche separation, as well as differences in virulence and antimicrobial susceptibility. Genome-scale metabolic models are useful tools for studying the metabolic potential of individuals, and with the rapid expansion of genomic sequencing there is a wealth of data that can be leveraged for comparative analysis. However, there exist few tools to construct strain-specific metabolic models at scale. Here, we describe Bactabolize, a reference-based tool which rapidly produces strain-specific metabolic models and growth phenotype predictions. We describe a pan reference model for the priority antimicrobial-resistant pathogen, Klebsiella pneumoniae, and a quality control framework for using draft genome assemblies as input for Bactabolize. The Bactabolize-derived model for K. pneumoniae reference strain KPPR1 performed comparatively or better than currently available automated approaches CarveMe and gapseq across 507 substrate and 2317 knockout mutant growth predictions. Novel draft genomes passing our systematically defined quality control criteria resulted in models with a high degree of completeness (≥99% genes and reactions captured compared to models derived from matched complete genomes) and high accuracy (mean 0.97, n=10). We anticipate the tools and framework described herein will facilitate large-scale metabolic modelling analyses that broaden our understanding of diversity within bacterial species and inform novel control strategies for priority pathogens.

Direct inference and control of genetic population structure from RNA sequencing data.

RNAseq data can be used to infer genetic variants, yet its use for estimating genetic population structure remains underexplored. Here, we construct a freely available computational tool (RGStraP) to estimate RNAseq-based genetic principal components (RG-PCs) and assess whether RG-PCs can be used to control for population structure in gene expression analyses. Using whole blood samples from understudied Nepalese populations and the Geuvadis study, we show that RG-PCs had comparable results to paired array-based genotypes, with high genotype concordance and high correlations of genetic principal components, capturing subpopulations within the dataset. In differential gene expression analysis, we found that inclusion of RG-PCs as covariates reduced test statistic inflation. Our paper demonstrates that genetic population structure can be directly inferred and controlled for using RNAseq data, thus facilitating improved retrospective and future analyses of transcriptomic data.

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing.

A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism's genome-each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers, and polishers have brought perfect assemblies within reach. Here, we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools and manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).

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.

Improved Genome Sequence of Australian Methicillin-Resistant Staphylococcus aureus Strain JKD6159.

Staphylococcus aureus strain JKD6159 represents a prominent community-acquired methicillin-resistant S. aureus (MRSA) clone in Australia. Here, we report an improved assembly of the original S. aureus JKD6159 genome sequence. By using deep sequencing with multiple technologies combined with carefully curated assembly and polishing, we believe the assembly to contain zero errors.

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing

A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism's genome ­ each replicon sequence is complete and contains no errors of any scale. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers and polishers have brought perfect assemblies within reach. Here we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools with manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).

ESBL plasmids in Klebsiella pneumoniae: diversity, transmission and contribution to infection burden in the hospital setting.

BACKGROUND: Resistance to third-generation cephalosporins, often mediated by extended-spectrum beta-lactamases (ESBLs), is a considerable issue in hospital-associated infections as few drugs remain for treatment. ESBL genes are often located on large plasmids that transfer horizontally between strains and species of Enterobacteriaceae and frequently confer resistance to additional drug classes. Whilst plasmid transmission is recognised to occur in the hospital setting, the frequency and impact of plasmid transmission on infection burden, compared to ESBL + strain transmission, is not well understood. METHODS: We sequenced the genomes of clinical and carriage isolates of Klebsiella pneumoniae species complex from a year-long hospital surveillance study to investigate ESBL burden and plasmid transmission in an Australian hospital. Long-term persistence of a key transmitted ESBL + plasmid was investigated via sequencing of ceftriaxone-resistant isolates during 4 years of follow-up, beginning 3 years after the initial study. RESULTS: We found 25 distinct ESBL plasmids. We identified one plasmid, which we called Plasmid A, that carried blaCTX-M-15 in an IncF backbone similar to pKPN-307. Plasmid A was transmitted at least four times into different Klebsiella species/lineages and was responsible for half of all ESBL episodes during the initial 1-year study period. Three of the Plasmid A-positive strains persisted locally 3-6 years later, and Plasmid A was detected in two additional strain backgrounds. Overall Plasmid A accounted for 21% of ESBL + infections in the follow-up period. CONCLUSIONS: Here, we systematically surveyed ESBL strain and plasmid transmission over 1 year in a single hospital network. Whilst ESBL plasmid transmission events were rare in this setting, they had a significant and sustained impact on the burden of ceftriaxone-resistant and multidrug-resistant infections. If onward transmission of Plasmid A-carrying strains could have been prevented, this may have reduced the number of opportunities for Plasmid A to transmit and create novel ESBL + strains, as well as reducing overall ESBL infection burden.

Epidemiology and genomic analysis of Klebsiella oxytoca from a single hospital network in Australia.

BACKGROUND: Infections caused by Klebsiella oxytoca are the second most common cause of Klebsiella infections in humans. Most studies have focused on K. oxytoca outbreaks and few have examined the broader clinical context of K. oxytoca. METHODS: Here, we collected all clinical isolates identified as K. oxytoca in a hospital microbiological diagnostic lab across a 15-month period (n = 239). Whole genome sequencing was performed on a subset of 92 isolates (all invasive, third-generation cephalosporin resistant (3GCR) and non-urinary isolates collected > 48 h after admission), including long-read sequencing on a further six isolates with extended-spectrum beta-lactamase or carbapenemase genes. RESULTS: The majority of isolates were sensitive to antimicrobials, however 22 isolates were 3GCR, of which five were also carbapenem resistant. Genomic analyses showed those identified as K. oxytoca by the clinical laboratory actually encompassed four distinct species (K. oxytoca, Klebsiella michiganensis, Klebsiella grimontii and Klebsiella pasteurii), referred to as the K. oxytoca species complex (KoSC). There was significant diversity within the population, with only 10/67 multi-locus sequence types (STs) represented by more than one isolate. Strain transmission was rare, with only one likely event identified. Six isolates had extended spectrum beta-lactamase (blaSHV-12 and/or blaCTX-M-9) or carbapenemase (blaIMP-4) genes. One pair of K. michiganensis and K. pasteurii genomes carried identical blaIMP-4 IncL/M plasmids, indicative of plasmid transmission. CONCLUSION: Whilst antimicrobial resistance was rare, the resistance plasmids were similar to those found in other Enterobacterales, demonstrating that KoSC has access to the same plasmid reservoir and thus there is potential for multi-drug resistance. Further genomic studies are required to improve our understanding of the KoSC population and facilitate investigation into the attributes of successful nosocomial isolates.

IL-33 promotes gastric tumour growth in concert with activation and recruitment of inflammatory myeloid cells.

Interleukin-33 (IL-33) is an IL-1 family cytokine known to promote T-helper (Th) type 2 immune responses that are often deregulated in gastric cancer (GC). IL-33 is overexpressed in human gastric tumours suggesting a role in driving GC progression although a causal link has not been proven. Here, we investigated the impact of IL-33 genetic deficiency in the well-characterized gp130 F/F mouse model of GC. Expression of IL-33 (and it's cognate receptor, ST2) was increased in human and mouse GC progression. IL-33 deficient gp130 F/F /Il33 -/- mice had reduced gastric tumour growth and reduced recruitment of pro-tumorigenic myeloid cells including key mast cell subsets and type-2 (M2) macrophages. Cell sorting of gastric tumours revealed that IL-33 chiefly localized to gastric (tumour) epithelial cells and was absent from tumour-infiltrating immune cells (except modest IL-33 enrichment within CD11b+ CX3CR1+CD64+MHCII+ macrophages). By contrast, ST2 was absent from gastric epithelial cells and localized exclusively within the (non-macrophage) immune cell fraction together with mast cell markers, Mcpt1 and Mcpt2. Collectively, we show that IL-33 is required for gastric tumour growth and provide evidence of a likely mechanism by which gastric epithelial-derived IL-33 drives mobilization of tumour-promoting inflammatory myeloid cells.

Transmission of Klebsiella strains and plasmids within and between grey-headed flying fox colonies.

The grey-headed flying fox (Pteropus poliocephalus) is an endemic Australian fruit bat, known to carry zoonotic pathogens. We recently showed they harbour bacterial pathogen Klebsiella pneumoniae and closely related species in the K. pneumoniae species complex (KpSC); however, the dynamics of KpSC transmission and gene flow within flying fox colonies are poorly understood. High-resolution genome comparisons of 39 KpSC isolates from grey-headed flying foxes identified five putative strain transmission clusters (four intra- and one inter-colony). The instance of inter-colony strain transmission of K. africana was found between two flying fox populations within flying distance, indicating either direct or indirect transmission through a common food/water source. All 11 plasmids identified within the KpSC isolates showed 73% coverage (mean) and ≥95% identity to human-associated KpSC plasmids, indicating gene flow between human clinical and grey-headed flying fox isolates. Along with strain transmission, inter-species horizontal plasmid transmission between K. pneumoniae and Klebsiella africana was also identified within a flying fox colony. Finally, genome-scale metabolic models were generated to predict and compare substrate usage to previously published KpSC models, from human and environmental sources. These models indicated no distinction on the basis of metabolic capabilities. Instead, metabolic capabilities were consistent with population structure and ST/lineage.

Genomic dissection of Klebsiella pneumoniae infections in hospital patients reveals insights into an opportunistic pathogen.

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.

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.