cgMLST-based Multiplex-PCR integrating MinION Sequencing of Legionella Pneumophila

Outbreaks of legionellosis caused by Legionella pneumophila (Lp) present a frequent threat with a 15-30% case fatality rate around the world. Vulnerable individuals are particularly susceptible to transmissions of this disease linking with various sources, including air conditioning units, water distribution systems, and water cooling towers. Epidemiological typing is critical for the investigation of pathogens during the outbreak assessment, which can provide an assessment of how closely cases are related to each other, thus providing the evidence for tracking down the source. Core-gene multilocus sequence typing (cgMLST) scheme with approximately 50 genes have been proved to be a sufficient typing method regarding the trade-off between the discriminatory power and epidemiological concordance. Due to the culturing difficulty of some pathogenic bacteria, the PCR amplification prior to sequencing with DNA extracted directly from samples is a crucial step to ensure the sensitivity and robustness of the overall source tracking experiment, particularly for environmental samples with low bacterial loads such as tap water, surface swabs or aerosol samples. Here we have developed a rapid 51 cgMLST scheme that couples with the improved primal-multiplex-PCR(pmPCR)-based MinION sequencing. The results show that with sensitively and specifically detecting Lp from samples containing as few as 3 genome copies using 51cgMLST-based pmPCR scheme, the subsequent MinION sequencing was able to recover all 51 core genes with 82% pseduo-genome coverage for precise ST investigation with at least 20K reads within one day. The deployment of our portable technique in the real hospital setting (Shenzhen, China) performed the rapid amplicon sequencing and offline bioinformatic analysis, successfully inferring the potential transmission of Lp in six different environment mediums across 8 departments at 2 seasons. It is proved that ST36 was the predominant typing in the hospital environment, and the Lp source was more likely from the water mediums with the potential transmission route via water-air-surface. This approach will be of use for rapid risk assessment of contaminated environments and outbreak investigations.