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ID Deadly Pathogens Without Growing Bacteria

Metagenomics has allowed researchers to reconstruct the genome sequence of a deadly Shiga-toxigenic E. coli outbreak without having to grow bacteria in the lab.

“The outbreak of Shiga-toxigenic Escherichia coli illustrated the effects of a bacterial epidemic on a wealthy, modern, industrialized society, with more than 3,000 cases and more than 50 deaths reported in Germany between May and June of 2011,” says Mark Pallen, professor of microbial genomics at Warwick Medical School.

“During an outbreak such as this, rapid and accurate pathogen identification and characterization is essential for the management of individual cases and the outbreak as a whole.

“Traditionally, clinical bacteriology has relied primarily on laboratory isolation of bacteria in pure culture to identify and characterize an outbreak strain.”

The team of researchers was able to reconstruct the genome sequence through the direct sequencing of DNA extracted from microbiologically complex samples.

The study, published in a genomics-themed issue of JAMA, highlights the potential of this approach to identify and characterize bacterial pathogens directly from clinical specimens.

Metagenomics has been used previously in a clinical diagnostic setting to identify the cause of outbreaks of viral infection, but this is its first reported use in an outbreak of bacterial infection.

Often, the process of laboratory culture proves slow and the recognition of an outbreak strain can be difficult if it belongs to an unknown variety or species for which specific laboratory tests and diagnostic criteria don’t already exist.

“There are numerous drawbacks to the use of nineteenth-century approaches such as microscopy and culture when it comes to classification,” says Pallen.

“Our results illustrate the potential of metagenomics as an open-ended, culture-independent approach for the identification and characterization of bacterial pathogens during an outbreak. There are challenges, of course, including speeding up and simplifying workflows, reducing costs, and improving diagnostic sensitivity.

“However, given the dizzying pace of progress in high-throughput sequencing, these are not likely to remain problems for very long.”
Researchers from the University of Birmingham, the University of Glasgow, the University Medical Centre Hamburg-Eppendorf in Germany and the sequencing company Illumina contributed to the findings.

Source: University of Warwick

The JAMA article may be reviewed at Below is the abstract:

A Culture-Independent Sequence-Based Metagenomics Approach to the Investigation of an Outbreak of Shiga-Toxigenic Escherichia coli O104:H4. Nicholas J. Loman, MBBS, PhD; Chrystala Constantinidou, PhD; Martin Christner, MD; Holger Rohde, MD; Jacqueline Z.-M. Chan, PhD; Joshua Quick, BSc; Jacqueline C. Weir, MSci; Christopher Quince, PhD; Geoffrey P. Smith, PhD; Jason R. Betley, PhD; Martin Aepfelbacher, MD; Mark J. Pallen, MA, MD, PhD

Importance  Identification of the bacterium responsible for an outbreak can aid in disease management. However, traditional culture-based diagnosis can be difficult, particularly if no specific diagnostic test is available for an outbreak strain.

Objective  To explore the potential of metagenomics, which is the direct sequencing of DNA extracted from microbiologically complex samples, as an open-ended clinical discovery platform capable of identifying and characterizing bacterial strains from an outbreak without laboratory culture.

Design, Setting, and Patients  In a retrospective investigation, 45 samples were selected from fecal specimens obtained from patients with diarrhea during the 2011 outbreak of Shiga-toxigenic Escherichia coli (STEC) O104:H4 in Germany. Samples were subjected to high-throughput sequencing (August-September 2012), followed by a 3-phase analysis (November 2012-February 2013). In phase 1, a de novo assembly approach was developed to obtain a draft genome of the outbreak strain. In phase 2, the depth of coverage of the outbreak strain genome was determined in each sample. In phase 3, sequences from each sample were compared with sequences from known bacteria to identify pathogens other than the outbreak strain.

Main Outcomes and Measures  The recovery of genome sequence data for the purposes of identification and characterization of the outbreak strain and other pathogens from fecal samples.

Results  During phase 1, a draft genome of the STEC outbreak strain was obtained. During phase 2, the outbreak strain genome was recovered from 10 samples at greater than 10-fold coverage and from 26 samples at greater than 1-fold coverage. Sequences from the Shiga-toxin genes were detected in 27 of 40 STEC-positive samples (67%). In phase 3, sequences from Clostridium difficile, Campylobacter jejuni, Campylobacter concisus, and Salmonella enterica were recovered.

Conclusions and Relevance  These results suggest the potential of metagenomics as a culture-independent approach for the identification of bacterial pathogens during an outbreak of diarrheal disease. Challenges include improving diagnostic sensitivity, speeding up and simplifying workflows, and reducing costs.

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