Dataset Information

Rapid whole-genome sequencing for investigation of a suspected tuberculosis outbreak.

ABSTRACT: Two Southeast Asian students attending the same school in the United Kingdom presented with pulmonary tuberculosis. An epidemiological investigation failed to link the two cases, and drug resistance profiles of the Mycobacterium tuberculosis isolates were discrepant. Whole-genome sequencing of the isolates found them to be genetically identical, suggesting a missed transmission event.

SUBMITTER: Torok ME

PROVIDER: S-EPMC3553910 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Rapid whole-genome sequencing for investigation of a suspected tuberculosis outbreak.

Török M Estée ME Reuter Sandra S Bryant Josephine J Köser Claudio U CU Stinchcombe Sian V SV Nazareth Bernadette B Ellington Matthew J MJ Bentley Stephen D SD Smith Geoffrey P GP Parkhill Julian J Peacock Sharon J SJ

Journal of clinical microbiology 20121121 2

Two Southeast Asian students attending the same school in the United Kingdom presented with pulmonary tuberculosis. An epidemiological investigation failed to link the two cases, and drug resistance profiles of the Mycobacterium tuberculosis isolates were discrepant. Whole-genome sequencing of the isolates found them to be genetically identical, suggesting a missed transmission event. ...[more]

PMID: 23175259

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Project description:Whole genome sequencing (WGS) has become the new gold standard for bacterial outbreak investigation, due to the high resolution available for typing. While sequencing is currently predominantly performed on Illumina devices, the preceding library preparation can be performed using various protocols. Enzymatic fragmentation library preparation protocols are fast, have minimal hands-on time, and work with small quantities of DNA. The aim of our study was to compare three library preparation protocols for molecular typing: Nextera XT (Illumina); Nextera Flex (Illumina); and QIAseq FX (Qiagen). We selected 12 ATCC strains from human Gram-positive and Gram-negative pathogens with %G+C-content ranging from 27% (Fusobacterium nucleatum) to 73% (Micrococcus luteus), each having a high quality complete genome assembly available, to allow in-depth analysis of the resulting Illumina sequence data quality. Additionally, we selected isolates from previously analyzed cases of vancomycin-resistant Enterococcus faecium (VRE) (n = 7) and a local outbreak of Klebsiella aerogenes (n = 5). The number of protocol steps and time required were compared, in order to test the suitability for routine laboratory work. Data analyses were performed with standard tools commonly used in outbreak situations: Ridom SeqSphere+ for cgMLST; CLC genomics workbench for SNP analysis; and open source programs. Nextera Flex and QIAseq FX were found to be less sensitive than Nextera XT to variable %G+C-content, resulting in an almost uniform distribution of read-depth. Therefore, low coverage regions are reduced to a minimum resulting in a more complete representation of the genome. Thus, with these two protocols, more alleles were detected in the cgMLST analysis, producing a higher resolution of closely related isolates. Furthermore, they result in a more complete representation of accessory genes. In particular, the high data quality and relative simplicity of the workflow of Nextera Flex stood out in this comparison. This thorough comparison within an ISO/IEC 17025 accredited environment will be of interest to those aiming to optimize their clinical microbiological genome sequencing.

Project description:BACKGROUND: Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains. METHODS AND FINDINGS: During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed "Hamburg clone") started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance. CONCLUSIONS: Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context.

Dataset Information

Rapid whole-genome sequencing for investigation of a suspected tuberculosis outbreak.

Publications

Rapid whole-genome sequencing for investigation of a suspected tuberculosis outbreak.

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