Bacteria of the genus Legionella cause water-based infections resulting in severe pneumonia. Here we analyze and compare the bacterial microbiome of sputum samples from pneumonia patients in relation to the presence and abundance of the genus Legionella. The prevalence of Legionella species was determined by culture, PCR, and Next Generation Sequencing (NGS). Nine sputum samples out of the 133 analyzed were PCR-positive using Legionella genus-specific primers. Only one sample was positive by culture. Illumina MiSeq 16S rRNA gene sequencing analyses of Legionella-positive and Legionella-negative sputum samples, confirmed that indeed, Legionella was present in the PCR-positive sputum samples. This approach allowed the identification of the sputum microbiome at the genus level, and for Legionella genus at the species and sub-species level. 42% of the sputum samples were dominated by Streptococcus. Legionella was never the dominating genus and was always accompanied by other respiratory pathogens. Interestingly, sputum samples that were Legionella positive were inhabited by aquatic bacteria that have been observed in an association with amoeba, indicating that amoeba might have transferred Legionella from the drinking water together with its microbiome. This is the first study that demonstrates the sputum major bacterial commensals and pathogens profiles with regard to Legionella presence.

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