Department of Microbiology and Infectious Diseases, Liverpool Hospitalgrid.415994.4, Liverpool, New South Wales, Australia.
NSW Health Pathology, Microbiology, Liverpool Hospitalgrid.415994.4, Liverpool, New South Wales, Australia.
Microbiol Spectr. 2022 Aug 31;10(4):e0035922. doi: 10.1128/spectrum.00359-22. Epub 2022 Jul 11.
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 utilizing a CRISPR/Cas9-based target enrichment system. We determine the method's utility by typing cultured L. pneumophila isolates and subsequently apply the method directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods, (i) whole-genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) inding ow-bundance equences by ybridization (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 further validate this method. 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. 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 investigation of Legionella outbreaks directly from patient samples, without the need for prior microbiological culture, which causes delays. Our study aims to address this problem. We have utilized a CRISPR/Cas9-based targeted next-generation sequencing (NGS) method that can be applied directly on human specimens. Furthermore, we show that analysis of the sequences of a small number of targeted genes offers the same classification of L. pneumophila as that based on data derived from the whole genome. Given the rising interest globally in sequencing pathogens directly from human samples, 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.
目前用于军团菌暴发的实验室调查的方法需要进行生物体培养。直接从临床样本中对嗜肺军团菌进行测序的能力将显著减少延迟。在这里，我们开发了一种利用基于 CRISPR/Cas9 的靶向富集系统对选定的嗜肺军团菌基因进行靶向下一代测序 (NGS) 的方法。我们通过对培养的嗜肺军团菌分离物进行分型来确定该方法的实用性，然后直接将该方法应用于患者样本。我们使用两种方法对 10 株嗜肺军团菌分离物进行了测序，（i）全基因组测序 (WGS) 和（ii）基于 CRISPR/Cas9 的靶向杂交低丰度序列（FLASH）-NGS，对 57 个选定基因进行测序。靶向 57 个基因的 NGS 比 WGS 更有效，并且对 57 个基因的系统发育分析与基于全基因组数据的分析产生了相同的嗜肺军团菌分离物分类。此外，直接对患者呼吸道样本进行嗜肺军团菌靶向 NGS 可以根据相应的培养分离物正确地对患者进行分类。这证明了靶向 NGS 可直接从患者样本中对嗜肺军团菌进行测序。对更多患者样本的研究将进一步验证该方法。尽管如此，基于 CRISPR/Cas9 的靶向 NGS 方法具有在未来广泛应用于微生物暴发调查的潜力，特别是在难以培养和生长缓慢的生物体的情况下。
嗜肺军团菌是一种引起严重且危及生命的肺炎（称为军团病）的细菌。需要新的分子方法，以便能够直接从患者样本中调查军团菌暴发，而无需事先进行微生物培养，因为这会导致延迟。我们的研究旨在解决这个问题。我们利用了一种基于 CRISPR/Cas9 的靶向下一代测序（NGS）方法，可以直接应用于人类标本。此外，我们还表明，对少数靶向基因的序列分析提供了与基于全基因组数据的分析相同的嗜肺军团菌分类。鉴于全球对直接从人类样本中测序病原体的兴趣日益浓厚，基于 CRISPR/Cas9 的靶向 NGS 方法在未来具有广泛应用于微生物暴发调查的潜力，特别是在难以培养和生长缓慢的生物体的情况下。
