Complete Genome Sequences of Two Geographically Distinct Legionella micdadei Clinical Isolates

ABSTRACT Legionella is a highly diverse genus of intracellular bacterial pathogens that cause Legionnaire’s disease (LD), an often severe form of pneumonia. Two L. micdadei sp. clinical isolates, obtained from patients hospitalized with LD from geographically distinct areas, were sequenced using PacBio SMRT cell technology, identifying incomplete phage regions, which may impact virulence.

L egionella is a highly diverse genus of intracellular bacterial pathogens that can infect human lung macrophages, causing an often severe form of pneumonia known as Legionnaire's disease (LD). While L. pneumophila and L. longbeachae spp. are responsible for the majority of LD, improved diagnostic testing (PCR-based assays, which detect all species) has found cases caused by other species, such as L. micdadei, which is thought to be responsible for about 60% of LD cases not related to L. pneumophila or L. longbeachae (1). These have been predominantly isolated from immunocompromised patients (2,3). Recently, a complete prophage sequence was identified in the L. micdadei ATCC 33218 T genome, yet the prophage is absent from a separate Australian L. micdadei clinical isolate (4). Despite this, sequencing of L. micdadei strains has so far shown high genomic synteny, suggesting that L. micdadei genomes are highly conserved, except for their mobilome, which may vary for geographically distinct strains (4). Considering this, investigating the diversity within L. micdadei may contribute toward identifying mobilome features that may have strong implications for revealing the origin of a strain. Thus, to further elucidate the genomic diversity of Legionella spp. associated with LD, the full genomes of two geographically distinct clinical isolates of L. micdadei were sequenced.
Two L. micdadei isolates were obtained from patient sputum: LM2015 from Christchurch (South Island, New Zealand) and LM2016 from Waikato (North Island, New Zealand). Strains were sequenced with single-molecule real-time (SMRT) technology on PacBio RSII. The Canu version 1.3 assembler was used to generate single contigs and trim reads (5,6). Complete genomes were annotated with Prokka version 1.11 (7)(8)(9)(10). Genomic structural rearrangements between isolates were carried out using wholegenome BLAST analysis, and results were visualized using both the Artemis Comparison Tool (ACT) (11) and the MAUVE genome comparison tool version 2.4.0 (12). Sequences were analyzed for quality by using Qualimap version 2.2.1 (13). Multiple sequence alignments between LM2015 and LM2016 were generated using Mauve (12).
The 2015 and 2016 clinical isolates sequenced in this study were compared to the ATCC type strain genome for L. micdadei. Aligning these genomes revealed highly conserved genome content with some rearrangements of genomic regions. These rearrangements may be due to the insertion of mobile elements that are characteristic of the Legionella genus. This study expands our understanding of the diversity between strains within the species.
Accession number(s). These genomes have been deposited in GenBank under the accession numbers CP020614 and CP020615.

ACKNOWLEDGMENTS
We thank Trevor Anderson (Microbiology) at Canterbury Health Laboratories (CHL), and we acknowledge CHL for providing access to clinical isolates. P.P.G. is supported by a Rutherford Discovery Fellowship administered by the Royal Society of New Zealand. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.