Population Genomics of Legionella longbeachae and Hidden Complexities of Infection Source Attribution

Abstract

Legionella longbeachae is the primary cause of legionellosis in Australasia and Southeast Asia and an emerging pathogen in Europe and the United States; however, our understanding of the population diversity of L. longbeachae from patient and environmental sources is limited. We analyzed the genomes of 64 L. longbeachae isolates, of which 29 were from a cluster of legionellosis cases linked to commercial growing media in Scotland in 2013 and 35 were non-outbreak-associated isolates from Scotland and other countries. We identified extensive genetic diversity across the L. longbeachae species, associated with intraspecies and interspecies gene flow, and a wide geographic distribution of closely related genotypes. Of note, we observed a highly diverse pool of L. longbeachae genotypes within compost samples that precluded the genetic establishment of an infection source. These data represent a view of the genomic diversity of L. longbeachae that will inform strategies for investigating future outbreaks.

Keywords: Legionella longbeachae; bacteria; environmental genotypes; epidemiology; genomic diversity; legionella; legionellosis; outbreaks; pathogenic genotypes; phylogeny; plasmids; recombination; source attribution.

Figure 1

16S rRNA gene–based phylogenetic tree of the sequenced genomes and all the cultured and type Legionella spp. strains available in the ribosomal database project (http://rdp.cme.msu.edu/), as accessed in May 2015. Scale bar indicates the mean number of nucleotide substitutions per site.

Figure 2

Core genome–based maximum-likelihood phylogeny of Legionella longbeachae serogroup 1 isolates corrected for recombination; source, country, year of isolation, relatedness and plasmid carriage are indicated. Related isolates are shown in the same color; those from the 2013 outbreak are indicated by gray. Isolates from the same patient are clustered together but do not co-segregate with cognate compost samples. Scale bar indicates the mean number of nucleotide substitutions per site.

Figure 3

Legionella longbeachae plasmid analysis: contigs networks reconstructions for 6 representative L. longbeachae types of plasmid content. The networks of the contigs representing the main chromosome and plasmids comprising the genome obtained by using PLACNET (38), a program enabling reconstruction of plasmids from whole-genome sequence datasets. The sizes of the contig nodes (in gray) are proportional to their lengths; continuous lines correspond to scaffold links. Dashed lines represent BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) hits to the L. longbeachae (blue) or L. pneumophila (red) strains; intensity of the line is proportional to the hit (white indicates low, black indicates high). Green lines correspond to plasmid contigs. Background colors indicate species relatedness for the main chromosome and plasmids (blue for L. longbeachae, red for L. pneumophila, pink for a combination of both, and yellow for previously unidentified genomic content).

Figure 4

Variation in gene content between environmental and patient Legionella longbeachae samples. A) Increase in pangenome size with every addition of a L. longbeachae genome. Environmental isolates pangenomes (green) are larger and continue increasing after the addition of 5 genomes, consistent with an open pangenome, but the within-patient pangenome plateaus quickly, consistent with a more closed pangenome. B) Average gene content of environmental isolates is significantly higher than that of clinical isolates (p = 0.01474).