Genomic characterization and assessment of pathogenic potential of Legionella spp. isolates from environmental monitoring

Abstract

Several species in the genus Legionella are known to cause an acute pneumonia when the aerosols containing the bacteria from man-made water systems are inhaled. The disease is usually caused by Legionella pneumophila, but other species have been implicated in the infection. The disease is frequently manifested as an outbreak, which means several people are affected when exposed to the common source of Legionella contamination. Therefor environmental surveillance which includes isolation and identification of Legionella is performed routinely. However, usually no molecular or genome-based methods are employed in further characterization of the isolates during routine environmental monitoring. During several years of such monitoring, isolates from different geographical locations were collected and 39 of them were sequenced by hybrid de novo approach utilizing short and long sequencing reads. In addition, the isolates were typed by standard culture and MALDI-TOF method. The sequencing reads were assembled and annotated to produce high-quality genomes. By employing discriminatory genome typing, four potential new species in the Legionella genus were identified, which are yet to be biochemically and morphologically characterized. Moreover, functional annotations concerning virulence and antimicrobial resistance were performed on the sequenced genomes. The study contributes to the knowledge on little-known non-pneumophila species present in man-made water systems and establishes support for future genetic relatedness studies as well as understanding of their pathogenic potential.

Keywords: Legionella; environmental monitoring; novel species; virulence factors; whole-genome sequencing.

Figure 1

Core-genome dendrogram of Legionella type strains and the 39 newly sequenced Legionella isolates from this study. The core-genome was derived from 141 protein sequences encoded by single-copy genes. The tree was constructed, using Maximum Likelihood (ML) and the Approximate Likelihood-Ratio Test (aLRT).

Figure 2

Results of the virulence factor search against VFDB. Coverage represents percentage of the gene covered and identity percentage of exact nucleotide matches. (A) The number of putative virulence factors in the isolates displaying 60% coverage and 60% identity to virulence factors in the VFDB. (B) Virulence genes shared among all isolates sequenced in this study. (C) Biological role of virulence factors only found in Legionella pneumophila isolates compared to non-pneumophila isolates.

Figure 3

Antimicrobial resistance genes and sequence-based typing of Legionella isolates in this study grouped by pangenome tree constructed using the BPGA pipeline. Antimicrobial genes were predicted in silico searching the CARD database. Sequence-based typing was conducted in silico utilizing legsta tool which contains sequence base type profiles from the Public Health England database.

Figure 4

Comparative inter-species genome analysis of non-pneumophila genomes available from NCBI and genomes sequenced in this study. The pangenome trees were constructed with Bacterial Pan Genome Analysis Pipeline (BPGA). The number of core, accessory, unique, and exclusively absent genes was determined by the same pipeline.