Hybracter: enabling scalable, automated, complete and accurate bacterial genome assemblies.

Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- and long-reads) assembly approaches. Complete genomes allow a deeper understanding of bacterial evolution and genomic variation beyond single nucleotide variants. They are also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance genes. However, small plasmids are often missed or misassembled by long-read assembly algorithms. Here, we present Hybracter which allows for the fast, automatic and scalable recovery of near-perfect complete bacterial genomes using a long-read first assembly approach. Hybracter can be run either as a hybrid assembler or as a long-read only assembler. We compared Hybracter to existing automated hybrid and long-read only assembly tools using a diverse panel of samples of varying levels of long-read accuracy with manually curated ground truth reference genomes. We demonstrate that Hybracter as a hybrid assembler is more accurate and faster than the existing gold standard automated hybrid assembler Unicycler. We also show that Hybracter with long-reads only is the most accurate long-read only assembler and is comparable to hybrid methods in accurately recovering small plasmids.

Hybracter: Enabling Scalable, Automated, Complete and Accurate Bacterial Genome Assemblies.

Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- and long-reads) assembly approaches. Complete genomes allow a deeper understanding of bacterial evolution and genomic variation beyond single nucleotide variants (SNVs). They are also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance (AMR) genes. However, small plasmids are often missed or misassembled by long-read assembly algorithms. Here, we present Hybracter which allows for the fast, automatic, and scalable recovery of near-perfect complete bacterial genomes using a long-read first assembly approach. Hybracter can be run either as a hybrid assembler or as a long-read only assembler. We compared Hybracter to existing automated hybrid and long-read only assembly tools using a diverse panel of samples of varying levels of long-read accuracy with manually curated ground truth reference genomes. We demonstrate that Hybracter as a hybrid assembler is more accurate and faster than the existing gold standard automated hybrid assembler Unicycler. We also show that Hybracter with long-reads only is the most accurate long-read only assembler and is comparable to hybrid methods in accurately recovering small plasmids.

Discordance between different bioinformatic methods for identifying resistance genes from short-read genomic data, with a focus on Escherichia coli.

Several bioinformatics genotyping algorithms are now commonly used to characterize antimicrobial resistance (AMR) gene profiles in whole-genome sequencing (WGS) data, with a view to understanding AMR epidemiology and developing resistance prediction workflows using WGS in clinical settings. Accurately evaluating AMR in Enterobacterales, particularly Escherichia coli, is of major importance, because this is a common pathogen. However, robust comparisons of different genotyping approaches on relevant simulated and large real-life WGS datasets are lacking. Here, we used both simulated datasets and a large set of real E. coli WGS data (n=1818 isolates) to systematically investigate genotyping methods in greater detail. Simulated constructs and real sequences were processed using four different bioinformatic programs (ABRicate, ARIBA, KmerResistance and SRST2, run with the ResFinder database) and their outputs compared. For simulation tests where 3079 AMR gene variants were inserted into random sequence constructs, KmerResistance was correct for 3076 (99.9 %) simulations, ABRicate for 3054 (99.2 %), ARIBA for 2783 (90.4 %) and SRST2 for 2108 (68.5 %). For simulation tests where two closely related gene variants were inserted into random sequence constructs, KmerResistance identified the correct alleles in 35 338/46 318 (76.3 %) simulations, ABRicate identified them in 11 842/46 318 (25.6 %) simulations, ARIBA identified them in 1679/46 318 (3.6 %) simulations and SRST2 identified them in 2000/46 318 (4.3 %) simulations. In real data, across all methods, 1392/1818 (76 %) isolates had discrepant allele calls for at least 1 gene. In addition to highlighting areas for improvement in challenging scenarios, (e.g. identification of AMR genes at <10× coverage, identifying multiple closely related AMR genes present in the same sample), our evaluations identified some more systematic errors that could be readily soluble, such as repeated misclassification (i.e. naming) of genes as shorter variants of the same gene present within the reference resistance gene database. Such naming errors accounted for at least 2530/4321 (59 %) of the discrepancies seen in real data. Moreover, many of the remaining discrepancies were likely 'artefactual', with reporting of cut-off differences accounting for at least 1430/4321 (33 %) discrepants. Whilst we found that comparing outputs generated by running multiple algorithms on the same dataset could identify and resolve these algorithmic artefacts, the results of our evaluations emphasize the need for developing new and more robust genotyping algorithms to further improve accuracy and performance.

Plassembler: an automated bacterial plasmid assembly tool.

SUMMARY: With recent advances in sequencing technologies, it is now possible to obtain near-perfect complete bacterial chromosome assemblies cheaply and efficiently by combining a long-read-first assembly approach with short-read polishing. However, existing methods for assembling bacterial plasmids from long-read-first assemblies often misassemble or even miss bacterial plasmids entirely and accordingly require manual curation. Plassembler was developed to provide a tool that automatically assembles and outputs bacterial plasmids using a hybrid assembly approach. It achieves increased accuracy and computational efficiency compared to the existing gold standard tool Unicycler by removing chromosomal reads from the input read sets using a mapping approach. AVAILABILITY AND IMPLEMENTATION: Plassembler is implemented in Python and is installable as a bioconda package using 'conda install -c bioconda plassembler'. The source code is available on GitHub at https://github.com/gbouras13/plassembler. The full benchmarking pipeline can be found at https://github.com/gbouras13/plassembler_simulation_benchmarking, while the benchmarking input FASTQ and output files can be found at https://doi.org/10.5281/zenodo.7996690.

Integrated chromosomal and plasmid sequence analyses reveal diverse modes of carbapenemase gene spread among Klebsiella pneumoniae.

Molecular and genomic surveillance systems for bacterial pathogens currently rely on tracking clonally evolving lineages. By contrast, plasmids are usually excluded or analyzed with low-resolution techniques, despite being the primary vectors of antibiotic resistance genes across many key pathogens. Here, we used a combination of long- and short-read sequence data of Klebsiella pneumoniae isolates (n = 1,717) from a European survey to perform an integrated, continent-wide study of chromosomal and plasmid diversity. This revealed three contrasting modes of dissemination used by carbapenemase genes, which confer resistance to last-line carbapenems. First, blaOXA-48-like genes have spread primarily via the single epidemic pOXA-48-like plasmid, which emerged recently in clinical settings and spread rapidly to numerous lineages. Second, blaVIM and blaNDM genes have spread via transient associations of many diverse plasmids with numerous lineages. Third, blaKPC genes have transmitted predominantly by stable association with one successful clonal lineage (ST258/512) yet have been mobilized among diverse plasmids within this lineage. We show that these plasmids, which include pKpQIL-like and IncX3 plasmids, have a long association (and are coevolving) with the lineage, although frequent recombination and rearrangement events between them have led to a complex array of mosaic plasmids carrying blaKPC Taken altogether, these results reveal the diverse trajectories of antibiotic resistance genes in clinical settings, summarized as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage. Our study provides a framework for the much needed incorporation of plasmid data into genomic surveillance systems, an essential step toward a more comprehensive understanding of resistance spread.

Reconciling the Potentially Irreconcilable? Genotypic and Phenotypic Amoxicillin-Clavulanate Resistance in Escherichia coli.

Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, and yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 Escherichia coli bloodstream infection isolates from Oxfordshire, United Kingdom, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). A total of 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity, 23% [78/339]) but improved when genetic features associated with penicillinase hyperproduction (e.g., promoter mutations and copy number estimates) were considered (sensitivity, 82% [277/339]; P < 0.0001). Most discrepancies occurred in isolates with MICs within ±1 doubling dilution of the breakpoint. We investigated two potential causes: the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement (±1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes.

Illumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long-read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods affect hybrid assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the family Enterobacteriaceae, as these frequently have highly plastic, repetitive genetic structures, and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies, as well as comparing to long-read-only assembly with Flye followed by short-read polishing with Pilon. Hybrid assembly with either PacBio or ONT reads facilitated high-quality genome reconstruction, and was superior to the long-read assembly and polishing approach evaluated with respect to accuracy and completeness. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.

The Role of fosA in Challenges with Fosfomycin Susceptibility Testing of Multispecies Klebsiella pneumoniae Carbapenemase-Producing Clinical Isolates.

With multidrug-resistant (MDR) Enterobacterales on the rise, a nontoxic antimicrobial agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-Escherichia coli isolates in a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with 96 KPC-producing clinical isolates of multiple strains and species collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole-genome sequencing (WGS) data using NCBI's AMRFinder and custom HMM search. Susceptibility testing was performed using a glucose-6-phosphate-supplemented fosfomycin Etest and Kirby-Bauer disk diffusion (DD) assays, and the results were compared to those obtained by agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints for E. coli were applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by Etest, 70% (67/96) by DD, and 88% (84/96) by agar dilution. fosA was detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, and fosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibit fosA and showed significant increases in the zone diameter of DD testing for isolates that were fosA positive compared to those that were fosA negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate that fosA influences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory compared to agar dilution. Further research is needed to determine the impact of fosA on clinical outcomes.

Don't overlook the little guy: An evaluation of the frequency of small plasmids co-conjugating with larger carbapenemase gene containing plasmids.

As the spread of antimicrobial resistance (AMR) genes becomes an increasing global threat, improved understanding of mobile genetic elements which contribute to the spread of antimicrobial resistance genes, becomes more critical. We created transconjugants from the mating of three chromosomally isogenic Klebsiella pneumoniae carbapenemase (blaKPC) positive Citrobacter freundii isolates with a laboratory strain of Escherichia coli and evaluated the movement of small cryptic plasmids (SCPs), p3223 and p1916, when larger blaKPC-plasmids were transferred. In all of the 143 transconjugants, multiple plasmids, both large and small, transferred with each mating. When two blaKPC-plasmids were present in the host, frequently (87%; 98/113) both would be transferred during mating. p3223 is found in a wide range of bacterial hosts that harbor AMR genes; p1916 has been identified in only a limited number of publicly available sequences to date. From our evaluation, there is still much to learn about SCPs, and the high rate of co-transfer of multiple plasmids from real-world carbapenemase-producing Enterobacteriales.

Klebsiella quasipneumoniae Provides a Window into Carbapenemase Gene Transfer, Plasmid Rearrangements, and Patient Interactions with the Hospital Environment.

Several emerging pathogens have arisen as a result of selection pressures exerted by modern health care. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intraspecies transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10- and 5-year periods, respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from 8 patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subsp. quasipneumoniae (n = 23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then possible transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases.

TETyper: a bioinformatic pipeline for classifying variation and genetic contexts of transposable elements from short-read whole-genome sequencing data.

Much of the worldwide dissemination of antibiotic resistance has been driven by resistance gene associations with mobile genetic elements (MGEs), such as plasmids and transposons. Although increasing, our understanding of resistance spread remains relatively limited, as methods for tracking mobile resistance genes through multiple species, strains and plasmids are lacking. We have developed a bioinformatic pipeline for tracking variation within, and mobility of, specific transposable elements (TEs), such as transposons carrying antibiotic-resistance genes. TETyper takes short-read whole-genome sequencing data as input and identifies single-nucleotide mutations and deletions within the TE of interest, to enable tracking of specific sequence variants, as well as the surrounding genetic context(s), to enable identification of transposition events. A major advantage of TETyper over previous methods is that it does not require a genome reference. To investigate global dissemination of Klebsiella pneumoniae carbapenemase (KPC) and its associated transposon Tn4401, we applied TETyper to a collection of over 3000 publicly available Illumina datasets containing blaKPC. This revealed surprising diversity, with over 200 distinct flanking genetic contexts for Tn4401, indicating high levels of transposition. Integration of sample metadata revealed insights into associations between geographic locations, host species, Tn4401 sequence variants and flanking genetic contexts. To demonstrate the ability of TETyper to cope with high-copy-number TEs and to track specific short-term evolutionary changes, we also applied it to the insertion sequence IS26 within a defined K. pneumoniae outbreak. TETyper is implemented in python and is freely available at https://github.com/aesheppard/TETyper.

A Candida auris Outbreak and Its Control in an Intensive Care Setting.

BACKGROUND: Candida auris is an emerging and multidrug-resistant pathogen. Here we report the epidemiology of a hospital outbreak of C. auris colonization and infection. METHODS: After identification of a cluster of C. auris infections in the neurosciences intensive care unit (ICU) of the Oxford University Hospitals, United Kingdom, we instituted an intensive patient and environmental screening program and package of interventions. Multivariable logistic regression was used to identify predictors of C. auris colonization and infection. Isolates from patients and from the environment were analyzed by whole-genome sequencing. RESULTS: A total of 70 patients were identified as being colonized or infected with C. auris between February 2, 2015, and August 31, 2017; of these patients, 66 (94%) had been admitted to the neurosciences ICU before diagnosis. Invasive C. auris infections developed in 7 patients. When length of stay in the neurosciences ICU and patient vital signs and laboratory results were controlled for, the predictors of C. auris colonization or infection included the use of reusable skin-surface axillary temperature probes (multivariable odds ratio, 6.80; 95% confidence interval [CI], 2.96 to 15.63; P<0.001) and systemic fluconazole exposure (multivariable odds ratio, 10.34; 95% CI, 1.64 to 65.18; P=0.01). C. auris was rarely detected in the general environment. However, it was detected in isolates from reusable equipment, including multiple axillary skin-surface temperature probes. Despite a bundle of infection-control interventions, the incidence of new cases was reduced only after removal of the temperature probes. All outbreak sequences formed a single genetic cluster within the C. auris South African clade. The sequenced isolates from reusable equipment were genetically related to isolates from the patients. CONCLUSIONS: The transmission of C. auris in this hospital outbreak was found to be linked to reusable axillary temperature probes, indicating that this emerging pathogen can persist in the environment and be transmitted in health care settings. (Funded by the National Institute for Health Research Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Oxford University and others.).

High Rates of Human Fecal Carriage of mcr-1-Positive Multidrug-Resistant Enterobacteriaceae Emerge in China in Association With Successful Plasmid Families.

Background: mcr-1-mediated colistin resistance in Enterobacteriaceae is concerning, as colistin is used in treating multidrug-resistant Enterobacteriaceae infections. We identified trends in human fecal mcr-1-positivity rates and colonization with mcr-1-positive, third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae in Guangzhou, China, and investigated the genetic contexts of mcr-1 in mcr-1-positive 3GC-R strains. Methods: Fecal samples were collected from in-/out-patients submitting specimens to 3 hospitals (2011-2016). mcr-1 carriage trends were assessed using iterative sequential regression. A subset of mcr-1-positive isolates was sequenced (whole-genome sequencing [WGS], Illumina), and genetic contexts (flanking regions, plasmids) of mcr-1 were characterized. Results: Of 8022 fecal samples collected, 497 (6.2%) were mcr-1 positive, and 182 (2.3%) harbored mcr-1-positive 3GC-R Enterobacteriaceae. We observed marked increases in mcr-1 (0% [April 2011] to 31% [March 2016]) and more recent (since January 2014; 0% [April 2011] to 15% [March 2016]) increases in human colonization with mcr-1-positive 3GC-R Enterobacteriaceae (P < .001). mcr-1-positive 3GC-R isolates were commonly multidrug resistant. WGS of mcr-1-positive 3GC-R isolates (70 Escherichia coli, 3 Klebsiella pneumoniae) demonstrated bacterial strain diversity; mcr-1 in association with common plasmid backbones (IncI, IncHI2/HI2A, IncX4) and sometimes in multiple plasmids; frequent mcr-1 chromosomal integration; and high mobility of the mcr-1-associated insertion sequence ISApl1. Sequence data were consistent with plasmid spread among animal/human reservoirs. Conclusions: The high prevalence of mcr-1 in multidrug-resistant E. coli colonizing humans is a clinical threat; diverse genetic mechanisms (strains/plasmids/insertion sequences) have contributed to the dissemination of mcr-1, and will facilitate its persistence.

Severe infections emerge from commensal bacteria by adaptive evolution.

Bacteria responsible for the greatest global mortality colonize the human microbiota far more frequently than they cause severe infections. Whether mutation and selection among commensal bacteria are associated with infection is unknown. We investigated de novo mutation in 1163 Staphylococcus aureus genomes from 105 infected patients with nose colonization. We report that 72% of infections emerged from the nose, with infecting and nose-colonizing bacteria showing parallel adaptive differences. We found 2.8-to-3.6-fold adaptive enrichments of protein-altering variants in genes responding to rsp, which regulates surface antigens and toxin production; agr, which regulates quorum-sensing, toxin production and abscess formation; and host-derived antimicrobial peptides. Adaptive mutations in pathogenesis-associated genes were 3.1-fold enriched in infecting but not nose-colonizing bacteria. None of these signatures were observed in healthy carriers nor at the species-level, suggesting infection-associated, short-term, within-host selection pressures. Our results show that signatures of spontaneous adaptive evolution are specifically associated with infection, raising new possibilities for diagnosis and treatment.

Resolving plasmid structures in Enterobacteriaceae using the MinION nanopore sequencer: assessment of MinION and MinION/Illumina hybrid data assembly approaches.

This study aimed to assess the feasibility of using the Oxford Nanopore Technologies (ONT) MinION long-read sequencer in reconstructing fully closed plasmid sequences from eight Enterobacteriaceae isolates of six different species with plasmid populations of varying complexity. Species represented were Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia marcescens and Klebsiella oxytoca, with plasmid populations ranging from 1-11 plasmids with sizes of 2-330 kb. Isolates were sequenced using Illumina (short-read) and ONT's MinION (long-read) platforms, and compared with fully resolved PacBio (long-read) sequence assemblies for the same isolates. We compared the performance of different assembly approaches including SPAdes, plasmidSPAdes, hybridSPAdes, Canu, Canu+Pilon (canuPilon) and npScarf in recovering the plasmid structures of these isolates by comparing with the gold-standard PacBio reference sequences. Overall, canuPilon provided consistently good quality assemblies both in terms of assembly statistics (N50, number of contigs) and assembly accuracy [presence of single nucleotide polymorphisms (SNPs)/indels with respect to the reference sequence]. For plasmid reconstruction, Canu recovered 70 % of the plasmids in complete contigs, and combining three assembly approaches (Canu or canuPilon, hybridSPAdes and plasmidSPAdes) resulted in a total 78 % recovery rate for all the plasmids. The analysis demonstrated the potential of using MinION sequencing technology to resolve important plasmid structures in Enterobacteriaceae species independent of and in conjunction with Illumina sequencing data. A consensus assembly derived from several assembly approaches could present significant benefit in accurately resolving the greatest number of plasmid structures.

Complete genome sequence of the nematicidal Bacillus thuringiensis MYBT18247.

The Gram-positive spore forming bacterium Bacillus thuringiensis MYBT18247 encodes three cry toxin genes, (cry6Ba2, cry6Ba3 and cry21-like) which are active against nematodes. For a better understanding of the evolution of virulence and cry toxins, we present here the complete genome sequence of Bacillus thuringiensis MYBT18247. Various additional virulence factors such as bacteriocins, proteases and hemolysins were identified. In addition, the methylome and the metabolic potential of the strain were analyzed and the strain phylogenetically classified.

Complete Genome sequence of the nematicidal Bacillus thuringiensis MYBT18246.

10.1601/nm.5000 is a rod-shaped facultative anaerobic spore forming bacterium of the genus 10.1601/nm.4857. The defining feature of the species is the ability to produce parasporal crystal inclusion bodies, consisting of δ-endotoxins, encoded by cry-genes. Here we present the complete annotated genome sequence of the nematicidal 10.1601/nm.5000 strain MYBT18246. The genome comprises one 5,867,749 bp chromosome and 11 plasmids which vary in size from 6330 bp to 150,790 bp. The chromosome contains 6092 protein-coding and 150 RNA genes, including 36 rRNA genes. The plasmids encode 997 proteins and 4 t-RNA's. Analysis of the genome revealed a large number of mobile elements involved in genome plasticity including 11 plasmids and 16 chromosomal prophages. Three different nematicidal toxin genes were identified and classified according to the Cry toxin naming committee as cry13Aa2, cry13Ba1, and cry13Ab1. Strikingly, these genes are located on the chromosome in close proximity to three separate prophages. Moreover, four putative toxin genes of different toxin classes were identified on the plasmids p120510 (Vip-like toxin), p120416 (Cry-like toxin) and p109822 (two Bin-like toxins). A comparative genome analysis of 10.1601/nm.5000 MYBT18246 with three closely related 10.1601/nm.5000 strains enabled determination of the pan-genome of 10.1601/nm.5000 MYBT18246, revealing a large number of singletons, mostly represented by phage genes, morons and cryptic genes.

A curated dataset of complete Enterobacteriaceae plasmids compiled from the NCBI nucleotide database.

Thousands of plasmid sequences are now publicly available in the NCBI nucleotide database, but they are not reliably annotated to distinguish complete plasmids from plasmid fragments, such as gene or contig sequences; therefore, retrieving complete plasmids for downstream analyses is challenging. Here we present a curated dataset of complete bacterial plasmids from the clinically relevant Enterobacteriaceae family. The dataset was compiled from the NCBI nucleotide database using curation steps designed to exclude incomplete plasmid sequences, and chromosomal sequences misannotated as plasmids. Over 2000 complete plasmid sequences are included in the curated plasmid dataset. Protein sequences produced from translating each complete plasmid nucleotide sequence in all 6 frames are also provided. Further analysis and discussion of the dataset is presented in an accompanying research article: "Ordering the mob: insights into replicon and MOB typing…" (Orlek et al., 2017) [1]. The curated plasmid sequences are publicly available in the Figshare repository.

Enhanced Klebsiella pneumoniae Carbapenemase Expression from a Novel Tn4401 Deletion.

The Klebsiella pneumoniae carbapenemase gene (blaKPC) is typically located within mobile transposon Tn4401 Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of blaKPC Illumina sequences from blaKPC-positive clinical isolates from a single institution were mapped to a Tn4401b reference sequence, which carries no deletions. The novel isoform Tn4401h (188-bp deletion [between istB and blaKPC]) was present in 14% (39/281) of clinical isolates. MICs showed that Escherichia coli strains containing plasmids with Tn4401a and Tn4401h were more resistant to meropenem (≥16 and ≥16, respectively), ertapenem (≥8 and 4, respectively), and cefepime (≥64 and 4, respectively) than E. coli strains with Tn4401b (0.5, ≤0.5, and ≤1, respectively). Quantitative real-time PCR (qRT-PCR) demonstrated that Tn4401a had a 16-fold increase and Tn4401h a 4-fold increase in blaKPC mRNA levels compared to the reference Tn4401b. A lacZ reporter plasmid was used to test the activity of the promoter regions from the different variants, and the results showed that the Tn4401a and Tn4401h promoter sequences generated higher ß-galactosidase activity than the corresponding Tn4401b sequence. Further dissection of the promoter region demonstrated that putative promoter P1 was not functional. The activity of the isolated P2 promoter was greatly enhanced by inclusion of the P1-P2 intervening sequence. These studies indicated that gene expression could be an important consideration in understanding resistance phenotypes predicted by genetic signatures in the context of sequencing-based rapid diagnostics.