The bacterium Legionella pneumophila is responsible for outbreaks of serious and life-threatening pneumonia called Legionnaires’ disease. There is a need for new molecular methods that allow for investigation of L. pneumophila outbreaks directly from patient samples, without prior microbiological culture which causes delays.
The aim of this research project was to develop a method for targeted next generation sequencing (NGS) of 57 L. pneumophila genes, utilising a CRISPR/Cas9 based target enrichment system. The main goal being to improve patient outcomes and care through the development of a molecular method that can be applied directly to patient samples rather than cultured isolates. This would effectively enable faster investigation of L. pneumophila outbreaks and identification of the source.
After extensive research and an incredible amount of work led by Ana and the collaborating parties*, the results showed that targeted NGS of 57 genes is more efficient than whole genome sequencing. Phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole-genome data. Targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates.
* Collaborator acknowledgements: This project was performed in collaboration with Department of Infectious Diseases and Microbiology, NSW Health Pathology, Liverpool and the Antibiotic Resistance and Mobile Elements Group, Ingham Institute of Applied Medical Research, Sydney, Australia. Professor Slade Jensen and Dr Michael Radzietta from the Ingham Institute provided advice and access to bench space and equipment that was necessary to perform some of the experimental genomic work.