Real-time rapid response

With 35 deaths and thousands in hospital, time was of the essence – what was behind the recent E.coli outbreak in Germany?

Genetics has entered a new era and the recent real-time response to a public health crisis in Germany is another example of how fast that pace of change is.  Here was an Escherichia coli outbreak that was baffling experts. Where had it come from and why was it so aggressive?

In an information vacuum, Life Technologies set out to discover the facts. Clearly, the shiga toxin–producing E. coli had serious health implications, causing the deaths of at least 35 people in northern Germany at the end of June 2011. Thousands more people had been hospitalised. This particular isolate can cause haemolytic-uremic syndrome (HUS), a type of kidney failure that inevitably increases the mortality rate in affected populations.

Multiple teams across the globe have been analysing this outbreak with the ultimate goals of rapidly generating a complete genomic sequence, annotating the sequence, explaining  the reasons for high mortality, and, most pressingly, developing a specific assay for detecting the strain in food samples.

With a broad portfolio of assays and a plurality of sequencing solutions at its disposal, Life Technologies was well positioned to assist with the development of a complete workflow solution – from initial sequence elucidation to an isolate-specific assay. In collaboration with multiple clinicians and public health centres, scientists deployed complementary technologies and associated bioinformatics to speed the development of an assay.

The National Consulting Laboratory on Hemolytic Uremic Syndrome in Münster, Germany, initially characterised the infectious agent using classical serotyping and Sanger-based capillary electrophoresis (CE) sequencing and was one of the centres within Germany Life Technologies that was using an Applied Biosystems 3130 Genetic Analyser. Using this initial information, the strain was characterised as E. coli O104:H4, stx2-positive, eae-negative, iha-positive, ESBL-positive, but gentamicin- and fluoroquinolone-susceptible.

In order to assist in dealing with the outbreak, Life Technologies was able to make available a previously designed TaqMan based Stx2 assay – the TaqMan VT2 (stx2) Detection Assay. This assay enables the rapid screening of food sources for the potential presence of the Stx2 gene, which is present in the E. coli O104:H4 strain responsible for the outbreak.

Even though the initial characterisation of the strain was complete, the organism’s full genomic sequence was required to develop a strain-specific assay, as well as to understand, on a scientific level, the pathophysiology of the strain. Such an assay would provide rapid answers regarding contaminated food and determine if E. coli O104 is actually present. A strain-specific assay would also assist in identifying the source of infection, important for both public health strategies and to minimise potential diplomatic complications.

With the advent of next-generation sequencing, large amounts of sequence data can be generated very rapidly. In fact, the Ion Personal Genome Machine sequencer allows researchers to generate high-quality sequence data from a genome of this size in about two hours and each individual run on an Ion 316 chip generates at least 100 Mb of high-quality sequence, which will deconvolute one E-coli genome in one run. Combined with simple upstream sample preparation, this technology can deliver a complete research workflow – from DNA sample to sequence data – in a single day.

Following sequence generation, the sequences were assembled to yield a 5.4 Mb draft genome with an N50 of 182 kb and 18x average coverage. This draft assembly was submitted to the National Centre for Biotechnology Information (NCBI) (accession number AFOB00000000.1). A second sequencing project was carried out using the Ion PGM sequencer by the Beijing Genome Institute (BGI) and is available at NCBI with accession number SRX067313.

The true power of this process is demonstrated by the fact that this initial sequence generation was achieved in days, both in Europe and in China. Access to this sequence enabled the teams to rapidly pinpoint key features of the virulent strain. As noted by Dr. Alexander Mellmann, a scientist at the German National Consulting Laboratory for Haemolytic Uremic Syndrome, “The rapid whole-genome sequencing results enabled us to discover within days a unique combination of virulence traits, and makes this German outbreak clone a unique hybrid of different E. coli pathovars”.

Armed with the newly acquired reference sequence, scientists executed a fast assay design process, which allowed an assay for specifically detecting the deadly E. coli O104:H4 strain in food to be designed, tested and made available within a week.

The TaqMan E. coli O104:H4 Detection Assay is ideal for food contamination detection, as it has two inherent levels of specificity – from binding of the PCR primers, and binding and subsequent hydrolysis of the TaqMan probe. This allows for highly accurate identification of a virulent strain in foods without the confounding false positives that can arise from closely related strains. The TaqMan assay approach is also exquisitely sensitive with a wide linear range.

So by utilising a broad portfolio of assay and sequencing technologies, it has been possible to proceed from an isolate of a pathogenic bacterium to a specific assay within two weeks. Throughout this serious health threat, Life Technologies was able to demonstrate its commitment to clinical research and work collaboratively with public health centres, facilitating rapid identification and full genetic characterisation of the organism and development of a reliable, strain-specific screening assay, helping to save lives.