Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples

ABSTRACT 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 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 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.

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

10.1101/094789 ◽

2016 ◽

Cited By ~ 5

Author(s):

Antonina A. Votintseva ◽

Phelim Bradley ◽

Louise Pankhurst ◽

Carlos del Ojo Elias ◽

Matthew Loose ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antibiotic Susceptibility ◽

Point Of Care ◽

Low Cost ◽

Illumina Miseq ◽

Turnaround Time ◽

Reference Laboratory ◽

Whole Genome ◽

Specific Pcr

AbstractRoutine 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.We 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 £96/£198 per sample.We 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.

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Whole-Genome Sequencing of Mycobacterium tilburgii Strain MEPHI

Microbiology Resource Announcements ◽

10.1128/mra.00933-19 ◽

2019 ◽

Vol 8 (40) ◽

Author(s):

Jamal Saad ◽

Michel Drancourt ◽

Margaret M. Hannan ◽

Patrick J. Stapleton ◽

Simon Grandjean Lapierre

Keyword(s):

Whole Genome Sequencing ◽

Clinical Isolate ◽

Genome Sequencing ◽

Gc Content ◽

Whole Genome ◽

Content Type ◽

Phylogenetic Placement ◽

Mycobacterium Simiae

Mycobacterium tilburgii is a fastidious mycobacterium which has previously been reported to cause severe disseminated infections. Genome sequencing of the M. tilburgii MEPHI clinical isolate yielded 3.14 Mb, with 66.3% GC content, and confirmed phylogenetic placement within the Mycobacterium simiae complex.

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Whole-Genome Sequencing of Salmonella enterica Serovar Infantis and Kentucky Isolates Obtained from Layer Poultry Farms in Ecuador

Microbiology Resource Announcements ◽

10.1128/mra.00091-20 ◽

2020 ◽

Vol 9 (13) ◽

Author(s):

William Calero-Cáceres ◽

Joyce Villacís ◽

Maria Ishida ◽

Elton Burnett ◽

Christian Vinueza-Burgos

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Salmonella Enterica ◽

Egg Production ◽

Illumina Miseq ◽

Whole Genome ◽

Genome Sequences ◽

Content Type ◽

Poultry Farms

Five strains of Salmonella enterica subsp. enterica serovar Infantis and two strains of S. enterica subsp. enterica serovar Kentucky isolated in 2017 from Ecuadorian layer poultry farms were sequenced using Illumina MiSeq technology. These isolates were collected on layer farms in central Ecuador, one of the most important areas of egg production in the country. The genome sequences of these isolates show valuable information for surveillance purposes.

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Streptococcus gallolyticus subsp. gallolyticus from Human and Animal Origins: Genetic Diversity, Antimicrobial Susceptibility, and Characterization of a Vancomycin-Resistant Calf Isolate Carrying avanA-Tn1546-Like Element

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04083-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2006-2015 ◽

Cited By ~ 12

Author(s):

Beatriz Romero-Hernández ◽

Ana P. Tedim ◽

José Francisco Sánchez-Herrero ◽

Pablo Librado ◽

Julio Rozas ◽

...

Keyword(s):

Genetic Diversity ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antibiotic Susceptibility ◽

Resistant Isolate ◽

Comparative Genomic ◽

Gene Gain ◽

Whole Genome ◽

Content Type ◽

Streptococcus Gallolyticus

ABSTRACTThe aim of this work was to characterize the antibiotic susceptibility and genetic diversity of 41Streptococcus gallolyticussubsp.gallolyticusisolates: 18 isolates obtained from animals and 23 human clinical isolates. Antibiotic susceptibility was determined by the semiautomatic Wider system and genetic diversity by pulsed-field gel electrophoresis (PFGE) with SmaI. Animal isolates grouped separately in the PFGE analysis, but no statistical differences in antimicrobial resistance were found between the two groups. The LMG 17956 sequence type 28 (ST28) strain recovered from the feces of a calf exhibited high levels of resistance to vancomycin and teicoplanin (MIC, ≥256 mg/liter). Its glycopeptide resistance mechanism was characterized by Southern blot hybridization and a primer-walking strategy, and finally its genome, determined by whole-genome sequencing, was compared with four closely relatedS. gallolyticussubsp.gallolyticusgenomes. Hybridization experiments demonstrated that a Tn1546-like element was integrated into the bacterial chromosome. In agreement with this finding, whole-genome sequencing confirmed a partial deletion of thevanY-vanZregion and partial duplication of thevanHgene. The comparative genomic analyses revealed that the LMG 17956 ST28 strain had acquired an unusually high number of transposable elements and had experienced extensive chromosomal rearrangements, as well as gene gain and loss events. In conclusion,S. gallolyticussubsp.gallolyticusisolates from animals seem to belong to lineages separate from those infecting humans. In addition, we report a glycopeptide-resistant isolate from a calf carrying a Tn1546-like element integrated into its chromosome.

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Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures

Journal of Clinical Microbiology ◽

10.1128/jcm.03073-14 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1137-1143 ◽

Cited By ~ 47

Author(s):

Antonina A. Votintseva ◽

Louise J. Pankhurst ◽

Luke W. Anson ◽

Marcus R. Morgan ◽

Deborah Gascoyne-Binzi ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Dna Extraction ◽

Genome Sequencing ◽

Solid Phase ◽

Clinical Samples ◽

Reference Laboratory ◽

Whole Genome ◽

Mycobacterial Species ◽

Accurate Identification ◽

Content Type

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) asMycobacterium tuberculosiswere successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

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Draft Genome Sequence of Weissella viridescens UCO-SMC3, Isolated from the Slime of Helix aspersa Müller Snails

Microbiology Resource Announcements ◽

10.1128/mra.01654-18 ◽

2019 ◽

Vol 8 (11) ◽

Author(s):

Apolinaria Garcia-Cancino ◽

Leonardo Albarracin ◽

Marcela Espinoza-Monje ◽

Jorge Campos-Martin ◽

Valeria Garcia-Castillo ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Sequence ◽

Draft Genome ◽

Illumina Miseq ◽

Helix Aspersa ◽

Draft Genome Sequence ◽

Whole Genome ◽

Content Type

This report describes the draft genome sequence of Weissella viridescens UCO-SMC3, isolated from Helix aspersa Müller slime. The reads were generated by a whole-genome sequencing (WGS) strategy on an Illumina MiSeq sequencer and were assembled into contigs with a total estimated size of 1,612,814 bp.

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DNA Thermo-Protection Facilitates Whole-Genome Sequencing of Mycobacteria Direct from Clinical Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.00670-20 ◽

2020 ◽

Vol 58 (10) ◽

Author(s):

Sophie George ◽

Yifei Xu ◽

Gillian Rodger ◽

Marcus Morgan ◽

Nicholas D. Sanderson ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Low Cost ◽

Clinical Samples ◽

Heat Inactivation ◽

Whole Genome ◽

Genome Coverage ◽

Content Type ◽

C 30 ◽

Validation Experiments

ABSTRACT Mycobacterium tuberculosis is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from M. tuberculosis-positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 105 Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved M. tuberculosis detection at 101 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 104 BCG cells/ml; >91% coverage (1× depth) occurred at 103 BCG cells/ml. Final validation employed M. tuberculosis-positive clinical samples (n = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer.

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Validation of Whole-Genome Sequencing for Identification and Characterization of Shiga Toxin-ProducingEscherichia coliTo Produce Standardized Data To Enable Data Sharing

Journal of Clinical Microbiology ◽

10.1128/jcm.01388-17 ◽

2017 ◽

Vol 56 (3) ◽

Cited By ~ 5

Author(s):

Anne Holmes ◽

Timothy J. Dallman ◽

Sharif Shabaan ◽

Mary Hanson ◽

Lesley Allison

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Shiga Toxin ◽

Reference Laboratory ◽

Whole Genome ◽

Public Health Action ◽

Data Set ◽

Content Type ◽

E Coli

ABSTRACTWhole-genome sequencing (WGS) is rapidly becoming the method of choice for outbreak investigations and public health surveillance of microbial pathogens. The combination of improved cluster resolution and prediction of resistance and virulence phenotypes provided by a single tool is extremely advantageous. However, the data produced are complex, and standard bioinformatics pipelines are required to translate the output into easily interpreted epidemiologically relevant information for public health action. The main aim of this study was to validate the implementation of WGS at the ScottishEscherichia coliO157/STEC Reference Laboratory (SERL) using the Public Health England (PHE) bioinformatics pipeline to produce standardized data to enable interlaboratory comparison of results generated at two national reference laboratories. In addition, we evaluated the BioNumerics whole-genome multilocus sequence typing (wgMLST) andE. coligenotyping plug-in tools using the same data set. A panel of 150 well-characterized isolates of Shiga toxin-producingE. coli(STEC) that had been sequenced and analyzed at PHE using the PHE pipeline and database (SnapperDB) was assembled to provide identification and typing data, including serotype (O:H type), sequence type (ST), virulence genes (eaeand Shiga toxin [stx] subtype), and a single-nucleotide polymorphism (SNP) address. To validate the implementation of sequencing at the SERL, DNA was reextracted from the isolates and sequenced and analyzed using the PHE pipeline, which had been installed at the SERL; the output was then compared with the PHE data. The results showed a very high correlation between the data, ranging from 93% to 100%, suggesting that the standardization of WGS between our reference laboratories is possible. We also found excellent correlation between the results obtained using the PHE pipeline and BioNumerics, except for the detection ofstx2aandstx2cwhen these subtypes are both carried by strains.

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Revolutionising Public Health Reference Microbiology using Whole Genome Sequencing: Salmonella as an exemplar

10.1101/033225 ◽

2015 ◽

Cited By ~ 14

Author(s):

Philip Ashton ◽

Satheesh Nair ◽

Tansy Peters ◽

Rediat Tewolde ◽

Martin Day ◽

...

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

High Throughput Sequencing ◽

Low Cost ◽

Bacterial Pathogens ◽

Reference Laboratory ◽

Whole Genome ◽

The United Kingdom ◽

Primary Test

Advances in whole genome sequencing (WGS) platforms and DNA library preparation have led to the development of methods for high throughput sequencing of bacterial genomes at a relatively low cost (Loman et al. 2012; Medini et al. 2008). WGS offers unprecedented resolution for determining degrees of relatedness between strains of bacterial pathogens and has proven a powerful tool for microbial population studies and epidemiological investigations (Harris et al. 2010; Lienau et al. 2011; Holt et al. 2009; Ashton, Peters, et al. 2015). The potential utility of WGS to public health microbiology has been highlighted previously (Koser et al. 2012; Kwong et al. 2013; Reuter et al. 2013; Joensen et al. 2014; Nair et al. 2014; Bakker et al. 2014; D Auria et al. 2014). Here we report, for the first time, the routine use of WGS as the primary test for identification, surveillance and outbreak investigation by a national reference laboratory. We present data on how this has revolutionised public health microbiology for one of the most common bacterial pathogens in the United Kingdom, the Salmonellae.

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Expanding U.S. Laboratory Capacity for Neisseria gonorrhoeae Antimicrobial Susceptibility Testing and Whole-Genome Sequencing through the CDC's Antibiotic Resistance Laboratory Network

Journal of Clinical Microbiology ◽

10.1128/jcm.01461-19 ◽

2020 ◽

Vol 58 (4) ◽

Cited By ~ 2

Author(s):

Ellen N. Kersh ◽

Cau D. Pham ◽

John R. Papp ◽

Robert Myers ◽

Richard Steece ◽

...

Keyword(s):

Public Health ◽

Antibiotic Resistance ◽

Neisseria Gonorrhoeae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Whole Genome ◽

Content Type ◽

Laboratory Capacity

ABSTRACT U.S. gonorrhea rates are rising, and antibiotic-resistant Neisseria gonorrhoeae (AR-Ng) is an urgent public health threat. Since implementation of nucleic acid amplification tests for N. gonorrhoeae identification, the capacity for culturing N. gonorrhoeae in the United States has declined, along with the ability to perform culture-based antimicrobial susceptibility testing (AST). Yet AST is critical for detecting and monitoring AR-Ng. In 2016, the CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to shore up the national capacity for detecting several resistance threats including N. gonorrhoeae. AR-Ng testing, a subactivity of the CDC’s AR Lab Network, is performed in a tiered network of approximately 35 local laboratories, four regional laboratories (state public health laboratories in Maryland, Tennessee, Texas, and Washington), and the CDC’s national reference laboratory. Local laboratories receive specimens from approximately 60 clinics associated with the Gonococcal Isolate Surveillance Project (GISP), enhanced GISP (eGISP), and the program Strengthening the U.S. Response to Resistant Gonorrhea (SURRG). They isolate and ship up to 20,000 isolates to regional laboratories for culture-based agar dilution AST with seven antibiotics and for whole-genome sequencing of up to 5,000 isolates. The CDC further examines concerning isolates and monitors genetic AR markers. During 2017 and 2018, the network tested 8,214 and 8,628 N. gonorrhoeae isolates, respectively, and the CDC received 531 and 646 concerning isolates and 605 and 3,159 sequences, respectively. In summary, the AR Lab Network supported the laboratory capacity for N. gonorrhoeae AST and associated genetic marker detection, expanding preexisting notification and analysis systems for resistance detection. Continued, robust AST and genomic capacity can help inform national public health monitoring and intervention.

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