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Genome sequencing of pathogens is now ubiquitous in microbiology, and the sequence archives are effectively no longer searchable for arbitrary sequences. Furthermore, the exponential increase of these archives is likely to be further spurred by automated diagnostics. To unlock their use for scientific research and real-time surveillance we have combined knowledge about bacterial genetic variation with ideas used in web-search, to build a DNA search engine for microbial data that can growdoi:10.1101/234955 fatcat:p6wjflaikzhczlsjs2nro4sh2i
more »... ntally. We indexed the complete global corpus of bacterial and viral whole genome sequence data (447,833 genomes), using four orders of magnitude less storage than previous methods. The method allows future scaling to millions of genomes. This renders the global archive accessible to sequence search, which we demonstrate with three applications: ultra-fast search for resistance genes MCR1-3, analysis of host-range for 2827 plasmids, and quantification of the rise of antibiotic resistance prevalence in the sequence archives.
Colistin represents one of the very few available drugs for treating infections caused by carbapenem resistant Enterobacteriaceae (CRE). As such, the recent plasmid-mediated spread of the mobilized colistin resistance gene mcr-1 poses a significant public health threat requiring global monitoring and surveillance. In this work, we characterize the global distribution of mcr-1 using a dataset of 457 mcr-1 positive sequenced isolates consisting of currently publicly available mcr-1 carryingdoi:10.1101/220921 fatcat:46h42nosqzavvpoeq6cejymdtu
more »... ces combined with an additional 110 newly sequenced mcr-1 positive isolates from China. We find mcr-1 in a diversity of plasmid backgrounds but identify an immediate background common to all mcr-1 sequences. Our analyses establish that all mcr-1 elements in circulation descend from the same initial mobilization of mcr-1 by an ISApl1 transposon in the mid 2000s (2002-2008; 95% higher posterior density), followed by a dramatic demographic expansion, which led to its current global distribution. Our results provide the first systematic phylogenetic analysis of the origin and spread of mcr-1, and emphasize the importance of understanding the movement of mobile elements carrying antibiotic resistance genes across multiple levels of genomic organization.
Colistin represents one of the few available drugs for treating infections caused by carbapenem-resistant Enterobacteriaceae. As such, the recent plasmid-mediated spread of the colistin resistance gene mcr-1 poses a significant public health threat, requiring global monitoring and surveillance. Here, we characterize the global distribution of mcr-1 using a data set of 457 mcr-1-positive sequenced isolates. We find mcr-1 in various plasmid types but identify an immediate background common to alldoi:10.1038/s41467-018-03205-z pmid:29563494 pmcid:PMC5862964 fatcat:rcebbr4uhjflrkgjgss5uokymq
more »... mcr-1 sequences. Our analyses establish that all mcr-1 elements in circulation descend from the same initial mobilization of mcr-1 by an ISApl1 transposon in the mid 2000s (2002)(2003)(2004)(2005)(2006)(2007)(2008); 95% highest posterior density), followed by a marked demographic expansion, which led to its current global distribution. Our results provide the first systematic phylogenetic analysis of the origin and spread of mcr-1, and emphasize the importance of understanding the movement of antibiotic resistance genes across multiple levels of genomic organization.
CC-BY 4.0 International license It is made available under a Walker TM, Kohl TA, Omar SV, Hedge J, del Ojo Elias C, Bradley P, Iqbal Z, 671 Feuerriegel S, Niehaus KE, Wilson DJ, Clifton DA, Kapatai ...doi:10.1101/094789 fatcat:cgpheqkk3varpjevgcbaxuv77u
The clinical phenotype of zoonotic tuberculosis and its contribution to the global burden of disease are poorly understood and probably underestimated. This shortcoming is partly because of the inability of currently available laboratory and in silico tools to accurately identify all subspecies of the Mycobacterium tuberculosis complex (MTBC). We present SNPs to Identify TB (SNP-IT), a single-nucleotide polymorphism-based tool to identify all members of MTBC, including animal clades. Bydoi:10.3201/eid2503.180894 pmid:30789126 pmcid:PMC6390766 fatcat:77yn3bkfljaildufb5yyt6gyzi
more »... SNP-IT to a collection of clinical genomes from a UK reference laboratory, we detected an unexpectedly high number of M. orygis isolates. M. orygis is seen at a similar rate to M. bovis, yet M. orygis cases have not been previously described in the United Kingdom. From an international perspective, it is possible that M. orygis is an underestimated zoonosis. Accurate identification will enable study of the clinical phenotype, host range, and transmission mechanisms of all subspecies of MTBC in greater detail.
Zamin Iqbal was funded by a Wellcome Trust/Royal Society Sir Henry Dale Fellowship (grant 102541/Z/13/Z) and Phelim Bradley was funded by a Wellcome Trust PhD studentship. ...doi:10.1099/jmm.0.000664 pmid:29458686 pmcid:PMC5882078 fatcat:llabmwccfbbnpgd3epyk2xp5y4
Bradley and Z. Iqbal (9) . ... Bradley et al. (9) found good concordance between Mykrobe and SeqSphere (23) , an allele-based method that detects the presence/absence of a limited number of resistance and virulence markers. ...doi:10.1128/jcm.01815-17 pmid:29925638 fatcat:7y23fdkk2zhybem7elerxb6xoi
Routine full characterization of Mycobacterium tuberculosis 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 the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeqdoi:10.1128/jcm.02483-16 pmid:28275074 pmcid:PMC5405248 fatcat:wrecdzv6cjgf7k6w2v5cj7b3xm
more »... equencer (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 were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of 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 DNA in direct samples.
Rapid and accurate detection of antibiotic resistance in pathogens is an urgent need, affecting both patient care and population-scale control. Microbial genome sequencing promises much, but many barriers exist to its routine deployment. Here, we address these challenges, using a de Bruijn graph comparison of clinical isolate and curated knowledge-base to identify species and predict resistance profile, including minor populations. This is implemented in a package, Mykrobe predictor, for S.doi:10.1101/018564 fatcat:5bfwvv6tknhqdnggbf7peqwwsm
more »... us and M. tuberculosis, running in under three minutes on a laptop from raw data. For S. aureus, we train and validate in 495/471 samples respectively, finding error rates comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.3%/99.5% across 12 drugs. For M. tuberculosis, we identify species and predict resistance with specificity of 98.5% (training/validating on 1920/1609 samples). Sensitivity of 82.6% is limited by current understanding of genetic mechanisms. We also show that analysis of minor populations increases power to detect phenotypic resistance in second-line drugs without appreciable loss of specificity. Finally, we demonstrate feasibility of an emerging single-molecule sequencing technique.
The clinical phenotype of zoonotic tuberculosis, its contribution to the global burden of disease and prevalence is poorly understood and probably underestimated. This is partly because currently available laboratory and in silico tools have not been calibrated to accurately distinguish between all subspecies of the Mycobacterium tuberculosis complex (Mtbc). We here present the first such tool, SNPs to Identify TB (SNP-IT). Applying SNP-IT to a collection of clinical genomes from a UK referencedoi:10.1101/213850 fatcat:p6ftauxponhofev3qe4jnemy2e
more »... laboratory, we demonstrate an unexpectedly high number of M. orygis isolates. These are seen at a similar rate to M. bovis which attracts much health protection resource and yet M. orygis cases have not been previously described in the UK. From an international perspective it is likely that M. orygis is an underestimated zoonosis. As whole genome sequencing is increasingly integrated into the clinical setting, accurate subspecies identification with SNP-IT will allow the clinical phenotype, host range and transmission mechanisms of subspecies of the Mtbc to be studied in greater detail.
The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package ('Mykrobe predictor') that takes raw sequence data as input, and generates adoi:10.1038/ncomms10063 pmid:26686880 pmcid:PMC4703848 fatcat:khm73m3lzbfhdfsiosveldvha4
more »... linician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n ¼ 470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n ¼ 1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes.
The method was previously described in Bradley et al. 1 . Lineage identification with MTBC. Mykrobe currently uses the lineage-informative SNPs from 47 to assign lineage within the MTBC. ... 13 14 15 16 17 18 19 20 21 22 23 24 25 02 Dec 2019, :191 ( First published: 4 ) https:: Conceptualization, Methodology, Resources, Software, Writing -Review & Editing; Bradley ...doi:10.12688/wellcomeopenres.15603.1 pmid:32055708 pmcid:PMC7004237 fatcat:xtefgqwfs5de3j7pydtllhmuie
Use of whole-genome sequencing (WGS) for routine mycobacterial species identification and drug susceptibility testing (DST) is becoming a reality. We compared the performances of WGS and standard laboratory workflows prospectively, by parallel processing at a major mycobacterial reference service over the course of 1 year, for species identification, first-line Mycobacterium tuberculosis resistance prediction, and turnaround time. Among 2,039 isolates with line probe assay results for speciesdoi:10.1128/jcm.01480-17 pmid:29167290 pmcid:PMC5786738 fatcat:rqxi4k6tenhgdaqfnuimo6ig5y
more »... entification, 74 (3.6%) failed sequencing or WGS species identification. Excluding these isolates, clinically important species were identified for 1,902 isolates, of which 1,825 (96.0%) were identified as the same species by WGS and the line probe assay. A total of 2,157 line probe test results for detection of resistance to the first-line drugs isoniazid and rifampin were available for 728 M. tuberculosis complex isolates. Excluding 216 (10.0%) cases where there were insufficient sequencing data for WGS to make a prediction, overall concordance was 99.3% (95% confidence interval [CI], 98.9 to 99.6%), sensitivity was 97.6% (91.7 to 99.7%), and specificity was 99.5% (99.0 to 99.7%). A total of 2,982 phenotypic DST results were available for 777 M. tuberculosis complex isolates. Of these, 356 (11.9%) had no WGS comparator due to insufficient sequencing data, and in 154 (5.2%) cases the WGS prediction was indeterminate due to discovery of novel, previously uncharacterized mutations. Excluding these data, overall concordance was 99.2% (98.7 to 99.5%), sensitivity was 94.2% (88.4 to 97.6%), and specificity was 99.4% (99.0 to 99.7%). Median processing times for the routine laboratory tests versus WGS were similar overall, i.e., 20 days (interquartile range [IQR], 15 to 31 days) and 21 days (15 to 29 days), respectively ( P = 0.41). In conclusion, WGS predicts species and drug susceptibility with great accuracy, but work is needed to increase the proportion of predictions made.
Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drugsusceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistancedetermining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all fi rst-line and second-line drugs for tuberculosis. Methods Between Sept 1, 2010, and Dec 1, 2013, we sequenced a trainingdoi:10.1016/s1473-3099(15)00062-6 pmid:26116186 pmcid:PMC4579482 fatcat:2ihlhjn2mjfexmro2przyrmcwq
more »... of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identifi ed from the drug-resistance scientifi c literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. Findings We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7-93·7) and 98·4% specifi city (98·1-98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identifi ed among mutations under selection pressure in non-candidate genes. Interpretation A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workfl ows, phasing out phenotypic drugsusceptibility testing while reporting drug resistance early.
why hast thou, Phelim, no slumbering bed? ... Bradley takes a pair of ro'lers about fifteen inches in diameter, which have been pre- viously drilied and turned with four grooves, requisite for manufacturing the sort of skelps required, and fixes them ...
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