Experts at RIVM teamed up with scientists of the Medical Faculty of the University Münster (Germany), and bioinformaticans from Life Technologies Corporation (United States of America) and the Wellcome Trust Sanger Institute (United Kingdom) to develop a highly specific, and rapid molecular screening test to detect and prevent further spread of the superbug.
Hajo Grundmann, epidemiologist at the RIVM, coordinated this joint task force. “It was essential to quickly bring together the right people and resources, so that we were able to respond to the potential spread of this multi-resistant bacterium among patients in Dutch hospitals. We are especially pleased about the role of rapid whole genome sequencing of the outbreak strain,” he explained.
Microbiologist Dag Harmsen from the Department of Periodontology at the University Münster led the team responsible for sequencing on an Ion Torrent PGM™. "When we first used the PGM™ during the German EHEC 2011 outbreak, this was just a proof of principle (PLoS ONE. 2011 6(7): e22751; PubMed). However, this time the rapid bacterial whole genome sequencing will make a difference for the protection of patients’ health. The sequence data laid the ground for the development of a specific molecular test. That is a true first real-time ‘genomics for diagnostics’ application,” he said.
Bioinformaticans from Life Technologies used the data from a single Ion 316™ chip to assemble a draft genome of the K. pneumoniae Oxa-48 outbreak strain (isolate 1191100241), which is available to the public via NCBI. “Using our TaqMan® assay development pipeline, we were able to rapidly compare this genome to other publicly available Klebsiella genomes and identify 36 candidate signature sequences that could be used to develop an outbreak strain-specific multiplex PCR test,” said Craig Cummings from Life Technologies.
By comparing these candidate signature sequences against the publically available data for over two-hundred additional Klebsiella genomes from an ongoing worldwide population study, Thomas Connor and Nicholas Thomson from the Wellcome Trust Sanger Institute in Cambridge identified two regions that remain entirely specific for the Dutch hospital outbreak strain. PCR assays targeting these two unique sequences together with the antibiotic resistance genes were then evaluated and field-tested by Grundmann’s team at the RIVM to develop a multiplex molecular screening test. Today, the protocol for this test and test kits have been made available to all Dutch hospitals.
“Overall, this is a good example of how rapid next generation sequencing can help controlling outbreaks and how joint forces from different countries and continents can achieve quick results to prevent further infections for the sake of the Dutch hospital patients,” commented Hajo Grundmann from the RIVM