By Monica Heger
This story was originally published Aug. 16.
The University of Münster team that was among the first to sequence the Escherichia coli strain responsible for the recent European outbreak has now used the Ion Torrent PGM to sequence a multidrug-resistant strain of Klebsiella pneumoniae that has infected more than 80 patients at the Maasstad Hospital in Rotterdam, the Netherlands.
The sequence information and analysis have since been used to develop a multiplex-PCR kit, which has been made available to all Dutch hospitals, representing a true "genomics for diagnostics application," microbiologist Dag Harmsen, who led the sequencing effort at the University of Münster, said in a statement.
Researchers at the University of Münster, Life Technologies, the Wellcome Trust Sanger Institute, and the Dutch National Institute for Public Health and the Environment collaborated for the sequencing and analysis of the outbreak strain and the subsequent development of PCR-based diagnostic assay.
The Münster team sequenced the outbreak strain on the PGM using the 316 chip and plans to make the data publicly available via the National Center Biotechnology Information's GenBank.
Bioinformaticians from Life Tech assembled a draft genome and identified 36 candidate signature sequences potentially unique to the K. pneumoniae Oxa-48 outbreak strain that could be used to develop diagnostic tests.
Further analysis was done at the Wellcome Trust Sanger Institute by Thomas Connor and Nicholas Thomson, who compared the 36 signature sequences to data from more than 200 publicly available Klebsiella genomes. This analysis revealed two regions that were entirely unique to the Dutch hospital outbreak.
Researchers at the Dutch National Institute for Public Health and the Environment then developed PCR diagnostic assays targeting those two unique sequences along with antibiotic-resistance genes, which they have field tested and made available to all Dutch hospitals.
The test has four targets, two of which are specific to the Dutch outbreak strain, including a 500-base region that encodes a subgroup of the CTX-M enzyme, which confers drug resistance to cefotaxime; and a homopolymer-rich 170-base pair fragment whose function is unknown. Additionally, the test targets a 600-base pair housekeeping gene present in all strains of Klebsiella, as well as a 400-base pair region that is specific for Oxa-48 strains.
Harmsen said that the time from culture to obtaining a whole-genome sequence took around 40 hours, but developing the PCR test, including choosing the primers and evaluating the test, took several weeks.
Additionally, the Klebsiella outbreak was different from the German E. coli outbreak because while E. coli is a well studied organism, "not much was known about the [Klebsiella] bug in advance," Harmsen wrote in an e-mail. Therefore, whole-genome sequence data "was absolutely essential in developing a rapid molecular screening test."
The tests are currently being used by Dutch hospitals to screen referred patients. Those patients who test positive are isolated to prevent further spread of the infection, Harmsen said.
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