Therese Woodring - In Cold Blood: whole genome sequencing in a fatal septic transfusion reaction

Lightning talk: Therese Woodring, of the University of Illinois College of Medicine, Peoria, described the use of Oxford Nanopore’s MinION device in investigating the organism behind a fatal septic transfusion reaction (STR). She outlined the case of a woman who received a blood transfusion: after five minutes, the nurse could see that the patient’s breathing had changed and heart rate had increased, so stopped the transfusion. A transfusion reaction was suspected ~2 hours later, and Gram-negative rods were identified in the patient’s blood ~3 hours after this. After another ~5 hours, the patient was transferred to intensive care, and an hour and a half later was pronounced dead. Therese noted that fatalities from STRs are remarkably rare – in the single figures, despite the ~5 million transfusions performed each year, a credit to the work of blood banks, and also due to the inhospitable environment of blood packs. She described how, for example, the packed red blood cells used in transfusions are stored at 4C and accumulate waste products and reactive oxygen species – “not exactly a gentle culture media”. To investigate the kind of organism that could exploit this extreme enrivonment, Therese and her team performed whole-genome amplification of isolate DNA samples. Libraries were prepared using the Rapid Sequencing Kit (SQK-RAD004) and sequenced on the MinION device. Analysis was carried out on Therese’s laptop: assembly via Canu produced a 7.34 Mbn chromosome; 16S identification via three database searches indicated Pseudomonas, with two specifying P. poae, a ubiquitous soil bacterium often found in cold environments, including the Himalayas – not a human pathogen. Multilocus 16S analysis also placed the isolate in the P. fluorescens subgroup. Several cold tolerance genes were identified in the sequenced isolate; cold tolerance testing revealed growth at 4C and 25C, whilst the bacterium was killed at body temperature. This indicated that its pathogenicity in this case was an ecological problem, unlikely to occur outside of this environment. The team also identified four siderophore receptor genes, important in the acquisition of iron, which produced fluorescence under UV light. Therese concluded that whole genome sequencing of the isolate on the MinION helped to clarify the identification of the bacterium and characterise its behavioural repertoire – “a non-pathogen rendered lethal in cold blood” - whilst also improving understanding of the ecological context of STRs. The team hope their work will help inform future STR isolate investigations and encourage a “DIY” approach to the use of sequencing in medical education.