Biology subjects

Crook, D. W.

Publications and source records attributed to Crook, D. W..

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bioRxiv2019

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 impact on 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 from SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the Enterobacteriaceae family, 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. Both strategies facilitate high-quality genome reconstruction. 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. IMPACT STATEMENTIllumina short-read sequencing is frequently used for tasks in bacterial genomics, such as assessing which species are present within samples, checking if specific genes of interest are present within individual isolates, and reconstructing the evolutionary relationships between strains. However, while short-read sequencing can reveal significant detail about the genomic content of bacterial isolates, it is often insufficient for assessing genomic structure: how different genes are arranged within genomes, and particularly which genes are on plasmids - potentially highly mobile components of the genome frequently carrying antimicrobial resistance elements. This is because Illumina short reads are typically too short to span repetitive structures in the genome, making it impossible to accurately reconstruct these repetitive regions. One solution is to complement Illumina short reads with long reads generated with SMRT Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) sequencing platforms. Using this approach, called hybrid assembly, we show that we can automatically fully reconstruct complex bacterial genomes of Enterobacteriaceae isolates in the majority of cases (best-performing method: 17/20 isolates). In particular, by comparing different methods we find that using the assembler Unicycler with Illumina and ONT reads represents a low-cost, high-quality approach for reconstructing bacterial genomes using publicly available software. DATA SUMMARYRaw sequencing data and assemblies have been deposited in NCBI under BioProject Accession PRJNA422511 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA422511). We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

bioinformatics↗

bioRxiv2019

Prediction of pyrazinamide resistance in Mycobacterium tuberculosis using structure-based machine learning approaches.

SynopsisO_ST_ABSBackgroundC_ST_ABSPyrazinamide is one of four first-line antibiotics used to treat tuberculosis, however antibiotic susceptibility testing for pyrazinamide is challenging. Resistance to pyrazinamide is primarily driven by genetic variation in pncA, an enzyme that converts pyrazinamide into its active form. MethodsWe curated a dataset of 664 non-redundant, missense amino acid mutations in pncA with associated high-confidence phenotypes from published studies and then trained three different machine learning models to predict pyrazinamide resistance. All models had access to a range of protein structural-, chemical- and sequence-based features. ResultsThe best model, a gradient-boosted decision tree, achieved a sensitivity of 80.2% and a specificity of 76.9% on the hold-out Test dataset. The clinical performance of the models was then estimated by predicting the binary pyrazinamide resistance phenotype of 4,027 samples harboring 367 unique missense mutations in pncA derived from 24,231 clinical isolates. ConclusionsThis work demonstrates how machine learning can enhance the sensitivity/specificity of pyrazinamide resistance prediction in genetics-based clinical microbiology workflows, highlights novel mutations for future biochemical investigation, and is a proof of concept for using this approach in other drugs.

microbiology↗

bioRxiv2019

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, 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 E. coli bloodstream infection isolates from Oxfordshire, UK, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). 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 hyper-production (e.g. promoter mutations, copy number estimates) were considered (sensitivity 82% (277/339); p<0.0001). Most discrepancies occurred in isolates with peri-breakpoint MICs. 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.

microbiology↗

bioRxiv2018

Nanopore metagenomic sequencing of full length human metapneumovirus (HMPV) within a unique sub-lineage

Human metapneumovirus (HMPV) has been recognized as an important pathogen which can cause a spectrum of respiratory tract disease. Here, we report Nanopore metagenomic sequencing of the first full length HMPV genome directly from a throat swab from a UK patient with complex lung disease and immunocompromise. We found a predominance (26.4%) of HMPV reads in the metagenomic sequencing data and consequently assembled the full genome at a high depth of coverage (mean 4,786). Through phylogenetic analyses, we identified this HMPV strain to originate from a unique genetic group in A2b, showing the presence of this group in the UK. Our study demonstrated the effectiveness of Nanopore metagenomic sequencing for diagnosing infectious diseases and recovering complete sequences for genomic characterization, highlighting the applicability of Nanopore sequencing in clinical settings.\n\nImportanceNanopore metagenomic sequencing has the potential to evolve as a point-of-care test for a range of infectious diseases. Here, we report the first full length human metapneumovirus (HMPV) genome in the UK sequenced by Nanopore from a non-invasive sample from an immunocompromised patient. We demonstrate the presence of HMPV from a unique genetic group not previously reported from the UK. Our study demonstrates the effectiveness of Nanopore sequencing for diagnosing an infection that was not detected by routine first-line tests in the clinical microbiology laboratory. We report sufficient genomic data to provide insight into the epidemiology of infection and with the potential to inform treatment decisions.

microbiology↗

bioRxiv2018

MinION Nanopore Sequencing of Multiple Displacement Amplified Mycobacteria DNA Direct from Sputum

Sequencing of pathogen DNA directly from clinical samples offers the possibilities of rapid diagnosis, faster antimicrobial resistance prediction and enhanced outbreak investigation. The approach is especially advantageous for infections caused by species which grow very slowly in culture, such as Mycobacteria tuberculosis. Since the pathogen of interest may represent as little as 0.01% of the total DNA, enrichment of the input material for target sequences by specific amplification and, or depletion of non-target DNA (human, other bacteria) is essential for success. Here, we investigated the potential of isothermal multiple displacement amplification by Phi29 polymerase. We directed the amplification reaction towards Mycobacteria DNA in sputum samples by exploiting in our oligonucleotide primer design, their high GC content (approximately 65%) relative to human DNA. Amplified DNA was then sequenced using the Oxford Nanopore Technology MinION. In addition, a model system comprising standardised mock clinical samples was designed. Pooled infection negative human sputum samples were spiked with enumerated Mycobacterium bovis (BCG) Pasteur strain at concentrations spanning the typical range at which Mycobacterium tuberculosis is found in human sputum samples (106 - 101 BCG cells/ml). To assess the amount of BCG sequence enrichment achieved, sample DNA was sequenced both before, and after amplification. Reads from amplified samples, which mapped to a BCG reference genome, comprised short repeated sequences - apparently transcribed multiple times from the same fragment of BCG DNA. Therefore post-amplification, the samples were enriched for BCG sequences relative to unamplified sequences (8,101 BCG reference mapped reads, increasing to 28,617 at 106 BCG cells/ml sample), but BCG genome coverage declined markedly (for example 89.4% to 4.1%). In summary, the use of standardised mock clinical samples allowed direct comparison of data from different Mycobacteria enrichment experiments and sequencing runs. However, optimal conditions for multiple displacement amplification of minority Mycobacteria DNAs, remain to be identified.

microbiology↗

bioRxiv2018

Klebsiella quasipneumonaiae provides a window into carbapenemase gene transfer, plasmid rearrangements and nosocomial acquisition from the hospital environment

Several emerging pathogens have arisen as a result of selection pressures exerted by modern healthcare. 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 intra-species 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 five-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 eight patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subspecies 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 probable 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.

microbiology↗

bioRxiv2018

Identifying mixed Mycobacterium tuberculosis infection and laboratory cross-contamination during Mycobacterial sequencing programs

IntroductionDetecting laboratory cross-contamination and mixed tuberculosis infection are important goals of clinical Mycobacteriology laboratories.\n\nObjectivesTo develop a method detecting mixtures of different M. tuberculosis lineages in laboratories performing Mycobacterial next generation sequencing (NGS).\n\nSettingPublic Health England National Mycobacteriology Laboratory Birmingham, which performs Illumina sequencing on DNA extracted from positive Mycobacterial Growth Indicator tubes.\n\nMethodsWe analysed 4,156 samples yielding M. tuberculosis from 663 MiSeq runs, obtained during development and production use of a diagnostic process using NGS. Counts of the most common (major) variant, and all other variants (non-major variants) were determined from reads mapping to positions defining M. tuberculosis lineages. Expected variation was estimated during process development.\n\nResultsFor each sample we determined the non-major variant proportions at 55 sets of lineage defining positions. The non-major variant proportion in the two most mixed lineage defining sets (F2 metric) was compared with that in the 47 least mixed lineage defining sets (F47 metric). Three patterns were observed: (i) not mixed by either metric, (ii) high F47 metric suggesting mixtures of multiple lineages, and (iii) samples compatible with mixtures of two lineages, detected by differential F2 metric elevation relative to F47. Pattern (ii) was observed in batches, with similar patterns in the H37Rv control present in each run, and is likely to reflect cross-contamination. During production, the proportions of samples in each pattern were 97%, 2.8%, and 0.001%, respectively.\n\nConclusionThe F2 and F47 metrics described could be used for laboratory process control in laboratories sequencing M. tuberculosis.

microbiology↗

bioRxiv2018

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. To investigate global dissemination of Klebsiella pneumoniae carbapenemase (KPC) and its associated transposon Tn4401, we applied TETyper to a collection of >3000 publicly available Illumina datasets containing blaKPC. This revealed surprising diversity, with >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.

bioinformatics↗

bioRxiv2018

A role for tetracycline selection in the evolution of Clostridium difficile PCR-ribotype 078

Farm animals have been identified as reservoirs of Clostridium difficile PCR-ribotype 078 (RT078). Since 2005, the incidence of human clinical cases (frequently severe), with this genotype has increased. We aimed to understand this change, by studying the recent evolutionary history of RT078. Phylogenetic analysis of international genomes (isolates from 2006-2014) revealed several recent clonal expansions. A common ancestor of each expansion had independently acquired different alleles of the tetracycline resistance gene tetM. Consequently, an unusually high proportion of RT078 genomes were tetM positive (76.5%). Additional tetracycline resistance determinants were also identified, some for the first time in C. difficile (efflux pump tet40). Each tetM-clonal expansion lacked geographic structure, indicating rapid international spread. Resistance determinants for C. difficile-infection-triggering antimicrobials including fluoroquinolones and clindamycin were comparatively rare in RT078. Tetracyclines are used intensively in agriculture; this selective pressure, plus rapid spread via the food-chain may explain the increased RT078 prevalence in humans.

microbiology↗

bioRxiv2018

A quantitative evaluation of MIRU-VNTR typing against whole-genome sequencing for identifying Mycobacterium tuberculosis transmission: A prospective observational cohort study

SummaryO_ST_ABSBackgroundC_ST_ABSMycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing is widely used in high-income countries for Mycobacterium tuberculosis typing. Whole-genome sequencing (WGS) is known to deliver greater specificity, but no quantitative prospective comparison has yet been undertaken.\n\nMethodsWe studied isolates from the English Midlands, sampled consecutively between 1 January 2012 and 31 December 2015. In addition to routinely performed MIRU-VNTR typing, DNA was extracted from liquid cultures and sequenced using Illumina technology. Demographic and epidemiological data were extracted from the Enhanced Tuberculosis Surveillance system maintained by Public Health England. Closely related samples, defined using a threshold of five single nucleotide variants (SNVs), were compared to samples with identical MIRU-VNTR profiles, with shared epidemiological risk factors, and to those with both characteristics.\n\nFindings1,999 patients were identified for whom at least one M. tuberculosis isolate had been MIRU-VNTR typed and sequenced. Comparing epidemiological risk factors with close genetic relatedness, only coresidence had a positive predictive value of over 5%. Excluding co-resident individuals, 18.6% of patients with identical MIRU-VNTR profiles were within 5 SNVs. Where patients also shared social risk factors and ethnic group, this rose to 48%. Only 8% of MIRU-VNTR linked pairs in lineage 1 were within 5 SNV, compared to 31% in lineage 4.\n\nInterpretationIn the setting studied, MIRU-VNTR typing and epidemiological risk factors are poorly predictive of close genomic relatedness, assessed by SNV. MIRU-VNTR performance varies markedly by lineage.\n\nFundingPublic Health England, National Institute of Health Research Oxford Biomedical Research Centre.

microbiology↗

bioRxiv2018

Control of artefactual variation in reported inter-sample relatedness during clinical use of a Mycobacterium tuberculosis sequencing pipeline

Contact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artefactual variation between M. tuberculosis isolates during routine next generation sequencing of Mycobacterium spp, we investigated its basis in 2,018 consecutive M. tuberculosis isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted, sequenced, reads mapped, and consensus sequences determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 M. tuberculosis genomic regions, we quantified the relationship between the minor variant depth and the amount of non-Mycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of non-Mycobacterial bacterial DNA, we found significant increases in minor variant frequencies of more than 1.5 fold in 242 regions covering 5.1% of the M. tuberculosis genome. Included within these were four high variation regions strongly influenced by the amount of non-Mycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we have demonstrated an approach identifying critical genomic regions contributing to clinically relevant artefactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multi-step laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

microbiology↗

bioRxiv2018

Validation of a 14-drug microtitre plate that includes delamanid and bedaquiline for susceptibility testing of M. tuberculosis

UKMYC5 is a 96-well microtitre plate designed by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to enable the measurement of minimum inhibitory concentrations (MICs) of 14 different anti-TB compounds for >30,000 clinical tuberculosis isolates. Unlike the MYCOTB plate, on which UKMYC5 is based, the plate included two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents using a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers using three methods (mirrored-box, microscope and Vizion Digital viewing system) after 7, 10, 14 and 21 days incubation. In addition, all EQA strains were whole-genome sequenced and phenotypically characterized by 7H10/7H11 agar proportion method (APM) and MGIT960. We conclude that the UKMYC5 plate is optimally read using the Vizion system after 14 days incubation, achieving an inter-reader agreement of 97.9% and intra- and inter-laboratory reproducibilities of 95.6% and 93.1%, respectively. The mirrored-box had similar reproducibilities. Strains classified as resistant by APM, MGIT960 or the presence of mutations known to confer resistance consistently record elevated MICs compared with those strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain which the APM measured MICs close the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds due to specific genetic variation.

microbiology↗

bioRxiv2017

Automated detection of Mycobacterial growth on 96-well plates for rapid and accurate Tuberculosis drug susceptibility testing

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the minimum inhibitory concentrations (MICs) to be elucidated. The two participating laboratories each inoculated ten 96-well plates with the standard H37Rv reference strain and, after two weeks incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (> 30,000) of samples of M. tuberculosis from patients over the next few years.

microbiology↗

bioRxiv2017

Molecular diagnosis of orthopaedic device infection direct from sonication fluid by metagenomic sequencing

Culture of multiple periprosthetic tissue samples is the current gold-standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through sonication fluid culture of explants. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of complete bacterial DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI.\n\nWe compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopaedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 samples (derivation set), we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and validated our findings in 47 independent samples.\n\nCompared to sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69(88%,95%CI 77-94%) (derivation samples 35/38[92%,79-98%]; validation 26/31[84%,66-95%]), and genus-level sensitivity was 64/69(93%,84-98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97(88%,79-93%) (derivation 43/50[86%,73-94%], validation 42/47[89%,77-96%]). High levels of human DNA contamination were seen despite use of laboratory methods to remove it. Rigorous laboratory good practice was required to prevent bacterial DNA contamination.\n\nWe demonstrate metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings combined with increasing availability of portable, random-access sequencing technology offers the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.

microbiology↗

bioRxiv2017

Severe infections emerge from the microbiome by adaptive evolution

Bacteria responsible for the greatest global mortality colonize the human microbiome far more frequently than they cause severe infections. Whether mutation and selection within the microbiome accompany 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 microbiome, with infecting and nose-colonizing bacteria showing parallel adaptive differences. We found 2.8-to-3.6-fold enrichments of protein-altering variants in genes responding to rsp, which regulates surface antigens and toxicity; agr, which regulates quorum-sensing, toxicity 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 disease-associated, short-term, within-host selection pressures. Our results show that infection, like a cancer of the microbiome, emerges through spontaneous adaptive evolution, raising new possibilities for diagnosis and treatment.\n\nOne Sentence SummaryLife-threatening S. aureus infections emerge from nose microbiome bacteria in association with repeatable adaptive evolution.

genomics↗

bioRxiv2017

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common human infections. blaKPC, encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10kb). Here we characterize the genetic features of the emergence of blaKPC in global E. coli, 2008-2013, using both long-and short-read whole genome sequencing.\n\nAmongst 43/45 successfully sequenced blaKPC-E. coli strains, we identified high strain (n=21 sequence types, 18% of annotated genes in the core genome); plasmid ([≥]9 replicon types); and blaKPC-associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of interspecies, regional and international plasmid spread. In several cases blaKPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures.\n\nE. coli is a common human pathogen, but also a commensal in a multiple environmental and animal reservoirs, and easily transmissible. The association of blaKPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. blaTEM, blaCTX-M) suggests that it is likely to become similarly prevalent.

microbiology↗

bioRxiv2017

High rates of human faecal carriage of mcr-1-positive multi-drug resistant isolates emerge in China in association with successful plasmid families

SynopsisO_ST_ABSBackgroundC_ST_ABSmcr-1-mediated transmissible colistin resistance in Enterobacteriaceae is concerning, given colistin is frequently used as a treatment of last resort in multidrug-resistant Enterobacteriaceae infections. Reported rates of human mcr-1 gastrointestinal carriage have historically been low.\n\nObjectivesTo identify trends in human gastrointestinal carriage of mcr-1 positive and mcr-1-positive/cefotaxime-resistant Enterobacteriaceae in Guangzhou, China, 2011-2016, and investigate the genetic contexts of mcr-1 in a subset of mcr-1-positive/cefotaxime-resistant strains using whole genome sequencing (WGS).\n\nMethodsOf 8,022 faecal samples collected, 497 (6.2%) were mcr-1- positive, and 182 (2.3%) mcr-1-positive/cefotaxime-resistant. Trends in carriage were assessed using iterative sequential regression. A subset of mcr-1-positive isolates was sequenced (Illumina), and genetic contexts of mcr-1 were characterised.\n\nResultsWe observed marked increases in mcr-1 (now ~30% prevalence) and more recent (since January 2014) increases in mcr-1-positive/third-generation cephalosporin-resistant Enterobacteriaceae human colonisation (p<0.001). Sub-cultured mcr-1-positive/third-generation cephalosporin-resistant isolates were commonly multi-drug resistant.\n\nWGS of 50 mcr-1/third-generation cephalosporin-resistant isolates (49 Escherichia coli; 1 Klebsiella pneumoniae) demonstrated bacterial strain diversity (39 E. coli sequence types); mcr-1 in association with common plasmid backbones (IncI, IncHI2/HI2A, IncX4) and sometimes in multiple plasmids; frequent mcr-1 chromosomal integration; and loss of the mcr-1-associated insertion sequence ISApl1 in some plasmids. Significant sequence similarity with published mcr-1 plasmid sequences was consistent with spread amongst pig, chicken and human reservoirs.\n\nConclusionsThe high positivity rate (~10%) of mcr-1 in multidrug-resistant E. coli colonising humans is a clinical threat; the diverse genetic mechanisms (strains/plasmids/insertion sequences) associated with mcr-1 have likely contributed to its dissemination, and will facilitate its persistence.

microbiology↗

bioRxiv2016

Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples.

Routine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care.\n\nWe demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction was obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. Using an Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a cost of {pound}96/{pound}198 per sample.\n\nWe then employed a non-specific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis BCG strain (BCG), and to combined culture-negative sputum DNA and BCG DNA. For the latest flowcell, the estimated turnaround time from patient to identification of BCG was 6 hours, with full susceptibility and surveillance results 2 hours later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of the MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis in direct samples.

microbiology↗