Dataset Information

Validation of Whole-Genome Sequencing for Identification and Characterization of Shiga Toxin-Producing Escherichia coli To Produce Standardized Data To Enable Data Sharing.

ABSTRACT: Whole-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 Scottish Escherichia coli O157/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) and E. coli genotyping plug-in tools using the same data set. A panel of 150 well-characterized isolates of Shiga toxin-producing E. 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 (eae and 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 of stx2a and stx2c when these subtypes are both carried by strains.

SUBMITTER: Holmes A

PROVIDER: S-EPMC5824047 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Validation of Whole-Genome Sequencing for Identification and Characterization of Shiga Toxin-Producing Escherichia coli To Produce Standardized Data To Enable Data Sharing.

Holmes Anne A Dallman Timothy J TJ Shabaan Sharif S Hanson Mary M Allison Lesley L

Journal of clinical microbiology 20180222 3

Whole-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 hea ...[more]

PMID: 29263202

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Project description:Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection of stx1, stx2, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection of stx and serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having complete stxA and stxB subunits. However, truncated stx fragments occurred in twenty-two other isolates, some having multiple stx fragments in the genome. Isolates with complete stx by WGS had consistent stx1 and stx2 detection by PCR, although one also having a stx2 fragment had inconsistent stx2 PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates were Citrobacter spp. Results demonstrate that stx fragments associated with stx-carrying bacteriophages in the E. coli genome may contribute to inconsistent detection of stx1 and stx2 by PCR. Fourteen isolates had integrated stx bacteriophage but lacked complete or fragmentary stx possibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) where stx was located, likely increasing the stability of stx in the bacterial genome and its detection by PCR.

Dataset Information

Validation of Whole-Genome Sequencing for Identification and Characterization of Shiga Toxin-Producing Escherichia coli To Produce Standardized Data To Enable Data Sharing.

Publications

Validation of Whole-Genome Sequencing for Identification and Characterization of Shiga Toxin-Producing Escherichia coli To Produce Standardized Data To Enable Data Sharing.

Similar Datasets

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