SRA

Currently available methods for the laboratory investigation of Legionella pneumophila outbreaks require organism culture. The ability to sequence L. pneumophila directly from clinical samples would significantly reduce delays. Here, we develop a method for targeted next generation sequencing (NGS) of selected L. pneumophila genes utilising a CRISPR/Cas9 based target enrichment system. We determine the utility of this method by typing cultured L. pneumophila isolates, and subsequently apply it directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods: (i) whole genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) NGS (FLASH-NGS) - sequencing 57 selected genes. The targeted NGS of 57 genes was more efficient than WGS, and phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole genome data. Furthermore, targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates. This provides proof of concept that targeted NGS can be used to sequence L. pneumophila directly from patient samples. Studies on a larger number of patient samples will help validate this method further. Nonetheless, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial outbreak investigations in the future, particularly in the context of difficult and slow growing organisms.