Evaluation of Whole-Genome Sequencing for Identification and Typing of Vibrio cholerae

ABSTRACT Epidemiological and microbiological data on Vibrio cholerae strains isolated between April 2004 and March 2018 (n = 836) and held at the Public Health England culture archive were reviewed. The traditional biochemical species identification and serological typing results were compared with the genome-derived species identification and serotype for a subset of isolates (n = 152). Of the 836 isolates, 750 (89.7%) were from a fecal specimen, 206 (24.6%) belonged to serogroup O1, and 7 (0.8%) were serogroup O139; 792 (94.7%) isolates were from patients reporting recent travel abroad, most commonly to India (n = 209) and Pakistan (n = 104). Of the 152 V. cholerae isolates identified by use of kmer, 149 (98.1%) were concordant with those identified using the traditional biochemical approach. Traditional serotyping results were 100% concordant with those of the whole-genome sequencing (WGS) analysis for the identification of serogroups O1 and O139 and classical and El Tor biotypes. ctxA was detected in all isolates of V. cholerae O1 El Tor and O139 belonging to sequence type 69 (ST69) and in V. cholerae O1 classical variants belonging to ST73. A phylogeny of isolates belonging to ST69 from U.K. travelers clustered geographically, with isolates from India and Pakistan located on separate branches. Moving forward, WGS data from U.K. travelers will contribute to global surveillance programs and the monitoring of emerging threats to public health and the global dissemination of pathogenic lineages. At the national level, these WGS data will inform the timely reinforcement of direct public health messaging to travelers and mitigate the impact of imported infections and the associated risks to public health.

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Evaluation of whole genome sequencing for the identification and typing of Vibrio cholerae

10.1101/327973 ◽

2018 ◽

Author(s):

David R. Greig ◽

Ulf Schafer ◽

Sophie Octavia ◽

Ebony Hunter ◽

Marie A. Chattaway ◽

...

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Vibrio Cholerae ◽

Species Identification ◽

Genome Sequencing ◽

National Level ◽

Whole Genome ◽

Surveillance Programs ◽

El Tor ◽

The Impact

AbstractEpidemiological and microbiological data on Vibrio cholerae isolated between 2004 and 2017 (n=836) and held in the Public Health England culture archive were reviewed. The traditional biochemical species identification and serological typing results were compared with the genome derived species identification and serotype for a sub-set of isolates (n=152). Of the 836 isolates, 750 (89.7%) were from faecal specimens, 206 (24.6%) belonged to serogroup O1 and seven (0.8%) were serogroup O139, and 792 (94.7%) isolates from patients reporting recent travel abroad, most commonly to India (n=209) and Pakistan (n=104). Of the 152 isolates of V. cholerae speciated by kmer identification, 149 (98.1%) were concordant with the traditional biochemical approach. Traditional serotyping results were 100% concordant with the whole genome sequencing (WGS) analysis for identification of serogroups O1 and O139 and Classical and El Tor biotypes. ctxA was detected in all isolates of V. cholerae O1 El Tor and O139 belonging to sequence type (ST) 69, and in V. cholerae O1 Classical variants belonging to ST73. A phylogeny of isolates belonging to ST69 from UK travellers clustered geographically, with isolates from India and Pakistan located on separate branches. Moving forward, WGS data from UK travellers will contribute to global surveillance programs, and the monitoring of emerging threats to public health and the global dissemination of pathogenic lineages. At the national level, these WGS data will inform the timely reinforcement of direct public health messaging to travellers and mitigate the impact of imported infections and the associated risks to public health.

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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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Whole-Genome Sequencing of Vibrio cholerae O1 El Tor Strains Isolated in Ukraine (2011) and Russia (2014)

Genome Announcements ◽

10.1128/genomea.01640-16 ◽

2017 ◽

Vol 5 (8) ◽

Cited By ~ 3

Author(s):

Nina I. Smirnova ◽

Yaroslav M. Krasnov ◽

Elena Y. Agafonova ◽

Elena Y. Shchelkanova ◽

Zhanna V. Alkhova ◽

...

Keyword(s):

Vibrio Cholerae ◽

Genome Sequencing ◽

Genetic Similarity ◽

Whole Genome Sequence ◽

Whole Genome ◽

Content Type ◽

Vibrio Cholerae O1 ◽

El Tor ◽

Highly Virulent ◽

High Genetic Similarity

ABSTRACT Here, we present the draft whole-genome sequence of Vibrio cholerae O1 El Tor strains 76 and M3265/80, isolated in Mariupol, Ukraine, and Moscow, Russia. The presence of various mutations detected in virulence-associated mobile elements indicates high genetic similarity of the strains reported here with new highly virulent variants of the cholera agent V. cholerae.

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Genomic Analysis of a Pan-Resistant Isolate ofKlebsiella pneumoniae, United States 2016

mBio ◽

10.1128/mbio.00440-18 ◽

2018 ◽

Vol 9 (2) ◽

Cited By ~ 16

Author(s):

Tom J. B. de Man ◽

Joseph D. Lutgring ◽

David R. Lonsway ◽

Karen F. Anderson ◽

Julia A. Kiehlbauch ◽

...

Keyword(s):

Public Health ◽

United States ◽

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

The United States ◽

Whole Genome ◽

Beta Lactams ◽

Content Type

ABSTRACTAntimicrobial resistance is a threat to public health globally and leads to an estimated 23,000 deaths annually in the United States alone. Here, we report the genomic characterization of an unusualKlebsiella pneumoniae, nonsusceptible to all 26 antibiotics tested, that was isolated from a U.S. patient. The isolate harbored four known beta-lactamase genes, including plasmid-mediatedblaNDM-1andblaCMY-6, as well as chromosomalblaCTX-M-15andblaSHV-28, which accounted for resistance to all beta-lactams tested. In addition, sequence analysis identified mechanisms that could explain all other reported nonsusceptibility results, including nonsusceptibility to colistin, tigecycline, and chloramphenicol. Two plasmids, IncA/C2 and IncFIB, were closely related to mobile elements described previously and isolated from Gram-negative bacteria from China, Nepal, India, the United States, and Kenya, suggesting possible origins of the isolate and plasmids. This is one of the firstK. pneumoniaeisolates in the United States to have been reported to the Centers for Disease Control and Prevention (CDC) as nonsusceptible to all drugs tested, including all beta-lactams, colistin, and tigecycline.IMPORTANCEAntimicrobial resistance is a major public health threat worldwide. Bacteria that are nonsusceptible or resistant to all antimicrobials available are of major concern to patients and the public because of lack of treatment options and potential for spread. AKlebsiella pneumoniaestrain that was nonsusceptible to all tested antibiotics was isolated from a U.S. patient. Mechanisms that could explain all observed phenotypic antimicrobial resistance phenotypes, including resistance to colistin and beta-lactams, were identified through whole-genome sequencing. The large variety of resistance determinants identified demonstrates the usefulness of whole-genome sequencing for detecting these genes in an outbreak response. Sequencing of isolates with rare and unusual phenotypes can provide information on how these extremely resistant isolates develop, including whether resistance is acquired on mobile elements or accumulated through chromosomal mutations. Moreover, this provides further insight into not only detecting these highly resistant organisms but also preventing their spread.

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Draft Genome Sequence of Erysipelothrix rhusiopathiae, Isolated from a Canine Case of Diskospondylitis

Microbiology Resource Announcements ◽

10.1128/mra.00592-20 ◽

2020 ◽

Vol 9 (26) ◽

Author(s):

Sara V. Little ◽

Andrew E. Hillhouse ◽

Sara D. Lawhon

Keyword(s):

Whole Genome Sequencing ◽

Species Identification ◽

Genome Sequencing ◽

Ribosomal Dna ◽

Draft Genome ◽

Dna Amplification ◽

Whole Genome ◽

Erysipelothrix Rhusiopathiae ◽

Content Type ◽

16S Ribosomal Dna

ABSTRACT This is the draft genome of an Erysipelothrix rhusiopathiae strain isolated from the blood of a canine. Initial 16S ribosomal DNA amplification identified the isolate as belonging to the Erysipelothrix genus but could not elucidate the species due to previous misidentification of E. rhusiopathiae and E. tonsillarum. The species identification was confirmed by whole-genome sequencing.

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The Future of Whole-Genome Sequencing for Public Health and the Clinic

Journal of Clinical Microbiology ◽

10.1128/jcm.01082-16 ◽

2016 ◽

Vol 54 (8) ◽

pp. 1946-1948 ◽

Cited By ~ 24

Author(s):

Marc W. Allard

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Microbial Forensics ◽

Microbial Taxonomy ◽

Support Mechanisms ◽

American Society ◽

American Society For Microbiology ◽

The Impact

An American Society for Microbiology (ASM) conference titled the Conference on Rapid Next-Generation Sequencing and Bioinformatic Pipelines for Enhanced Molecular Epidemiological Investigation of Pathogens provided a venue for discussing how technologies surrounding whole-genome sequencing (WGS) are advancing microbiology. Several applications in microbial taxonomy, microbial forensics, and genomics for public health pathogen surveillance were presented at the meeting and are reviewed. All of these studies document that WGS is revolutionizing applications in microbiology and that the impact of these technologies will be profound. ASM is providing support mechanisms to promote discussions of WGS techniques to foster applications and interpretations.

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Validation and Implementation of Clinical Laboratory Improvements Act-Compliant Whole-Genome Sequencing in the Public Health Microbiology Laboratory

Journal of Clinical Microbiology ◽

10.1128/jcm.00361-17 ◽

2017 ◽

Vol 55 (8) ◽

pp. 2502-2520 ◽

Cited By ~ 46

Author(s):

Varvara K. Kozyreva ◽

Chau-Linda Truong ◽

Alexander L. Greninger ◽

John Crandall ◽

Rituparna Mukhopadhyay ◽

...

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Laboratory ◽

Limit Of Detection ◽

Control Measures ◽

Whole Genome ◽

Content Type ◽

Specificity And Sensitivity ◽

Performance Specifications

ABSTRACT Public health microbiology laboratories (PHLs) are on the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole-genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards. Here, we describe the validation of WGS on the Illumina platform for routine use in PHLs according to Clinical Laboratory Improvements Act (CLIA) guidelines for laboratory-developed tests (LDTs). We developed a validation panel comprising 10 Enterobacteriaceae isolates, 5 Gram-positive cocci, 5 Gram-negative nonfermenting species, 9 Mycobacterium tuberculosis isolates, and 5 miscellaneous bacteria. The genome coverage range was 15.71× to 216.4× (average, 79.72×; median, 71.55×); the limit of detection (LOD) for single nucleotide polymorphisms (SNPs) was 60×. The accuracy, reproducibility, and repeatability of base calling were >99.9%. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from multilocus sequence typing (MLST) and genome-wide SNP-based phylogenetic assays were 100%. The following objectives were accomplished: (i) the establishment of the performance specifications for WGS applications in PHLs according to CLIA guidelines, (ii) the development of quality assurance and quality control measures, (iii) the development of a reporting format for end users with or without WGS expertise, (iv) the availability of a validation set of microorganisms, and (v) the creation of a modular template for the validation of WGS processes in PHLs. The validation panel, sequencing analytics, and raw sequences could facilitate multilaboratory comparisons of WGS data. Additionally, the WGS performance specifications and modular template are adaptable for the validation of other platforms and reagent kits.

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Mycobacterium tuberculosis Next-Generation Whole Genome Sequencing: Opportunities and Challenges

Tuberculosis Research and Treatment ◽

10.1155/2018/1298542 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Thato Iketleng ◽

Richard Lessells ◽

Mlungisi Thabiso Dlamini ◽

Tuelo Mogashoa ◽

Lucy Mupfumi ◽

...

Keyword(s):

Public Health ◽

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Patient Management ◽

Drug Susceptibility ◽

Whole Genome ◽

Next Generation ◽

The Impact

Mycobacterium tuberculosis drug resistance is a threat to global tuberculosis (TB) control. Comprehensive and timely drug susceptibility determination is critical to inform appropriate treatment of drug-resistant tuberculosis (DR-TB). Phenotypic drug susceptibility testing (DST) is the gold standard for M. tuberculosis drug resistance determination. M. tuberculosis whole genome sequencing (WGS) has the potential to be a one-stop method for both comprehensive DST and epidemiological investigations. We discuss in this review the tremendous opportunities that next-generation WGS presents in terms of understanding the molecular epidemiology of tuberculosis and mechanisms of drug resistance. The potential clinical value and public health impact in the areas of DST for patient management and tracing of transmission chains for timely public health intervention are also discussed. We present the current challenges for the implementation of WGS in low and middle-income settings. WGS analysis has already been adapted routinely in laboratories to inform patient management and public health interventions in low burden high-income settings such as the United Kingdom. We predict that the technology will be adapted similarly in high burden settings where the impact on the epidemic will be greatest.

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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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Whole genome sequencing reveals great diversity of Vibrio spp in prawns at retail

Microbial Genomics ◽

10.1099/mgen.0.000647 ◽

2021 ◽

Vol 7 (9) ◽

Author(s):

Nicol Janecko ◽

Samuel J. Bloomfield ◽

Raphaëlle Palau ◽

Alison E. Mather

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Type Species ◽

Whole Genome ◽

Content Type ◽

Link Type ◽

Critical Control Points ◽

Antimicrobial Resistance Genes

Consumption of prawns as a protein source has been on the rise worldwide with seafood identified as the predominant attributable source of human vibriosis. However, surveillance of non-cholera Vibrio is limited both in public health and in food. Using a population- and market share-weighted study design, 211 prawn samples were collected and cultured for Vibrio spp. Contamination was detected in 46 % of samples, and multiple diverse Vibrio isolates were obtained from 34 % of positive samples. Whole genome sequencing (WGS) and phylogenetic analysis illustrated a comprehensive view of Vibrio species diversity in prawns available at retail, with no known pathogenicity markers identified in Vibrio parahaemolyticus and V. cholerae . Antimicrobial resistance genes were found in 77 % of isolates, and 12 % carried genes conferring resistance to three or more drug classes. Resistance genes were found predominantly in V. parahaemolyticus , though multiple resistance genes were also identified in V. cholerae and V. vulnificus . This study highlights the large diversity in Vibrio derived from prawns at retail, even within a single sample. Although there was little evidence in this study that prawns are a major source of vibriosis in the UK, surveillance of non-cholera Vibrio is very limited. This study illustrates the value of expanding WGS surveillance efforts of non-cholera Vibrios in the food chain to identify critical control points for food safety through the production system and to determine the full extent of the public health impact.

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