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April 09, 2013

Diagnostic metagenomics in bacteriology: our proof–of–principle paper

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A Culture-Independent Sequence-Based Metagenomics Approach to the Investigation of an Outbreak of Shiga-Toxigenic Escherichia coli O104:H4

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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

JAMA. 2013;309(14):1502-1510

Ever since Hans Christian Gram and Robert Koch published their seminal papers in the early 1880s (1, 2), clinical diagnostic bacteriology has relied primarily on microscopy and culture in the laboratory to detect, identify and characterize human pathogens. Yet, there are many drawbacks to the use of these nineteenth-century approaches in the twenty-first century. Some important pathogenic bacteria can be isolated only after prolonged incubation (e.g. Mycobacterium tuberculosis) or under exacting culture conditions (e.g. anaerobic or microaerophilic organisms like Clostridium difficile or Campylobacter jejuni); in fact, several important pathogenic bacteria cannot be grown in vitro at all, including those that cause syphilis or leprosy. Variation in cultural requirements creates a need for many different culture media and many different procedures when processing clinical specimens and isolates—this results in complex and onerous workflows and a requirement for skilled staff (3).

So, what’s the solution? Well, 20 years ago, the polymerase chain reaction (PCR) brought the promise of a new model of diagnosis that might take us beyond the need for microscopy and culture.(4, 5) Gene-specific tests can be used to detect specific genetic determinants of importance in virulence and antimicrobial resistance. However, this approach suffers from the problem of finding only what you are looking for, so is not ideal for finding unknown virulence or resistance factors or even new combinations of known genetic determinants. Also, like culture, this approach requires a plethora of protocols each optimized for a particular pathogen or gene.

Today, we have published a paper in the Journal of the American Medical Association (JAMA) that presents proof of principle for a new approach that might augment or even replace the Koch-Gram paradigm that has dominated diagnostic bacteriology for so long. Metagenomics—the recovery of sequences directly from mixed populations of organisms without culture—has already proven a powerful approach to the analysis of complex microbial communities, particularly with the advent of high-throughput sequencing, which has delivered ultra-deep shotgun sequencing with unprecedented ease and cost-effectiveness (6, 7). This approach has seen widespread application in environmental microbiology and most recently in the cataloguing and investigation of the microbial communities associated with humans and other animals (8-10).

Diagnostic metagenomics—high-throughput sequencing of DNA extracted from clinical samples—provides an attractive alternative to the Gram-Koch paradigm in medical and public health bacteriology, avoiding the artifacts and inconvenience of culture and PCR, while potentially providing a simple but highly informative, one-size-fits-all approach to sample preparation and analysis. We have explored the potential of this approach by sequencing DNA extracted from a variety of human fecal samples, collected during and after the German STEC outbreak of 2011, in the hope of detecting and characterizing the outbreak strain and other bacterial pathogens without the need for microscopy, culture or PCR.

So how well did we do? Well, you can find out for yourself by reading the paper and associated commentary and blog post (coming soon from Bridget Kuehn) on the JAMA web site! Do not be put off by David Relman’s downbeat list of caveats: he is merely letting clinicians know that this is not a technique that is going to work out of the box in a routine diagnostic lab in the next week or two. But I would like to hope that there are parallels with the Wright brothers at Kitty Hawk: one day diagnostic metagenomics will be as routine as heavier-than-air human flight!


1. Koch R. Zur Untersuchung von pathogenen Organismen. Mitth. a. d. Kaiserl. Gesundheitsampte 1881; 1: 1-48. English translation Methods for the study of pathogenic organisms In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p101.

2. Gram HC. Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten. Fortschritte der Medizin 1884; 2: 185–189. English translation in Methods for the study of pathogenic organisms In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998. 1884.

3. Didelot X, Bowden R, Wilson DJ, Peto TE, Crook DW. Transforming clinical microbiology with bacterial genome sequencing. Nat Rev Genet. 2012;13:601-612.

4. Pallen MJ, Butcher PD. New strategies in microbiological diagnosis. J Hosp Infect. 1991;18 Suppl A:147-158.

5. Fredericks DN, Relman DA. Sequence-based identification of microbial pathogens: a reconsideration of Koch's postulates. Clin Microbiol Rev. 1996;9:18-33.

6. Gill SR, Pop M, Deboy RT et al. Metagenomic analysis of the human distal gut microbiome. Science. 2006;312:1355-1359.

7. Snyder LA, Loman N, Pallen MJ, Penn CW. Next-generation sequencing--the promise and perils of charting the great microbial unknown. Microb Ecol. 2009;57:1-3.

8. Caporaso JG, Lauber CL, Walters WA et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 2012;6:1621-1624.

9. Qin J, Li R, Raes J et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature. 2010;464:59-65.

10. A framework for human microbiome research. Nature. 2012;486:215-221.

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  • I have just seen this paper on the ~7–thousand–year auroochs genome:… by Mark Pallen on this entry
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