Culture of Clinical Specimens Reveals Extensive Diversity of Legionella pneumophila Strains in Arizona

Abstract

Between 2000 and 2017, a total of 236 Legionella species isolates from Arizona were submitted to the CDC for reference testing. Most of these isolates were recovered from bronchoalveolar lavage specimens. Although the incidence of legionellosis in Arizona is less than the overall U.S. incidence, Arizona submits the largest number of isolates to the CDC for testing compared to those from other states. In addition to a higher proportion of culture confirmation of legionellosis cases in Arizona than in other states, all Legionellapneumophila isolates are forwarded to the CDC for confirmatory testing. Compared to that from other states, a higher proportion of isolates from Arizona were identified as belonging to L. pneumophila serogroups 6 (28.2%) and 8 (8.9%). Genome sequencing was conducted on 113 L. pneumophila clinical isolates not known to be associated with outbreaks in order to understand the genomic diversity of strains causing legionellosis in Arizona. Whole-genome multilocus sequence typing (wgMLST) revealed 17 clusters of isolates sharing at least 99% identical allele content. Only two of these clusters contained isolates from more than one individual with exposure at the same facility. Additionally, wgMLST analysis revealed a group of 31 isolates predominantly belonging to serogroup 6 and containing isolates from three separate clusters. Single nucleotide polymorphism (SNP) and pangenome analysis were used to further resolve genome sequences belonging to a subset of isolates. This study demonstrates that culture of clinical specimens for Legionella spp. reveals a highly diverse population of strains causing legionellosis in Arizona which could be underappreciated using other diagnostic approaches. IMPORTANCE Culture of clinical specimens from patients with Legionnaires’ disease is rarely performed, restricting our understanding of the diversity and ecology of Legionella. Culture of Legionella from patient specimens in Arizona revealed a greater proportion of non-serogroup 1 Legionellapneumophila isolates than in other U.S. isolates examined. Disease caused by such isolates may go undetected using other diagnostic methods. Moreover, genome sequence analysis revealed that these isolates were genetically diverse, and understanding these populations may help in future environmental source attribution studies.

Genome analysis of Legionella pneumophila ST23 from various countries reveals highly similar strains

“…This was based on mapping sequence reads to reference genomes followed by the analysis of either single-nucleotide polymorphism (SNP) or a gene-by-gene approach that reproduces on a larger scale the multi-locus sequence typing (MLST). Whole-genome MLST (wgMLST) has been applied to study the genomic diversity of Lp clinical strains, unrelated to specific epidemic events, and interestingly previously unknown clusters were revealed (Raphael et al, 2019). WGS also showed that a single Lp1 clone was the cause of three outbreaks when SNP, core genome MLST and pangenome approach was used (Timms et al, 2018).…”

From Investigating a Case of Cellulitis to Exploring Nosocomial Infection Control of ST1 <em>Legionella pneumophila</em> Using Genomic Approaches

“…Raw data found on online repositories can be used by different key users with different approaches. We excluded several isolates from our analysis after bad quality results for de novo assemblies based on N50 < 100 000, which generally was associated to discordant genome length and /or high N bases numbers, nevertheless they were presented by studies considering these isolates of reliable quality after using another de novo alignment protocol (Supplementary Table S1) [15,28].…”

“…Quality scores of each method for each isolate are found in the Supplementary Table S1. N-50 was superior to 100,000, Q-score at least 30 (32)(33)(34)(35)(36)(37)(38) and average de novo covering was superior to 40 (46 -203) [15,28].…”

“…Nevertheless, issues remain for highly represented STs like ST1 [7,10,13,14]. Indeed, ST1 is known to be both genetically highly conserved and ubiquitous, but its population structure remains poorly described [18]. We carried out a mini review of L. pneumophila genomic studies conducted since 2015 in order to explore the methods and threshold for clustering used to investigate both outbreaks and long-term surveillances to identify which to apply to our specific question (Table 1).…”

From Investigating a Case of Cellulitis to Exploring Nosocomial Infection Control of ST1 Legionella pneumophila Using Genomic Approaches

“…Nevertheless, issues remain for highly represented STs like ST1 [8,12,15,16]. Indeed, ST1 is known to be both genetically highly conserved and ubiquitous, but its population structure remains poorly described [19]. We carried out a mini review of L. pneumophila genomic studies conducted since 2015 in order to explore the methods and clustering thresholds used to investigate both outbreaks and long-term surveillances of L. pneumophila to identify which to apply to our specific question (Table 1).…”