Whole-Genome Mapping as a Novel High-Resolution Typing Tool for Legionella pneumophila

Abstract

bLegionella is the causative agent for Legionnaires' disease (LD) and is responsible for several large outbreaks in the world. More than 90% of LD cases are caused by Legionella pneumophila, and studies on the origin and transmission routes of this pathogen rely on adequate molecular characterization of isolates. Current typing of L. pneumophila mainly depends on sequence-based typing (SBT). However, studies have shown that in some outbreak situations, SBT does not have sufficient discriminatory power to distinguish between related and nonrelated L. pneumophila isolates. In this study, we used a novel high-resolution typing technique, called whole-genome mapping (WGM), to differentiate between epidemiologically related and nonrelated L. pneumophila isolates. Assessment of the method by various validation experiments showed highly reproducible results, and WGM was able to confirm two well-documented Dutch L. pneumophila outbreaks. Comparison of whole-genome maps of the two outbreaks together with WGMs of epidemiologically nonrelated L. pneumophila isolates showed major differences between the maps, and WGM yielded a higher discriminatory power than SBT. In conclusion, WGM can be a valuable alternative to perform outbreak investigations of L. pneumophila in real time since the turnaround time from culture to comparison of the L. pneumophila maps is less than 24 h. Legionella is a rod-shaped, Gram-negative bacterial pathogen that is ubiquitous in aquatic reservoirs. It is the causative agent for Legionnaires' disease (LD), an acute pneumonia, characterized by clinical symptoms such as cough, fever, and radiological signs of infiltration that do not differ from pneumonia caused by other pathogens. LD is thought to account for 2% to 15% of all community-acquired pneumonias (1-3) and proves fatal in about 6% of cases (4).Several large outbreaks of LD have been reported worldwide. Examples include Murcia, Spain (449 confirmed cases); Barrowin-Furness, United Kingdom (179 confirmed cases); and Quebec City, Canada (182 confirmed cases). These outbreaks often involved contaminated cooling towers that can infect hundreds of people within a short time period, until the source of infection is detected and appropriate control measures are taken (5-7). In the source investigation of such outbreaks, epidemiological analyses together with genotypic comparisons between clinical and environmental isolates are essential (8, 9).More than 90% of LD cases are caused by Legionella pneumophila, and as this species is commonly found in the environment, adequate typing methods are needed to differentiate between isolates in order to confirm or reject a potential source of infection (10). Sequence-based typing (SBT), a variant of the classic multilocus sequence typing schemes (11), is an internationally recognized procedure for genotyping L. pneumophila isolates. It is a rapid, discriminatory, and reproducible seven-gene molecular typing method that is recommended as the method of choice by the European Society of Clinical Microbiology and...

Comparison of in situ sequence type analysis of Legionella pneumophila in respiratory tract secretions and environmental samples of a hospital in East Jerusalem

“…On the other hand, the occurrence of same L. pneumophila STs in the environmental samples poses a risk, and sustainable monitoring of hospital water systems is recommended [33]. Nowadays, WGS has been successfully applied for L. pneumophila providing the highest conceivable discriminatory power to distinguish outbreak from non-outbreak isolates [34, 35]. To this end, further genomic and metagenomics studies are needed using DNA from the clinical and environmental samples used in this study [36].…”

Population structure and minimum core genome typing of Legionella pneumophila

“…Recently, whole genome sequencing (WGS) has been applied to subtype various pathogens including L. pneumophila . For L. pneumophila subtyping, WGS-based methods provided better discriminatory power to distinguish outbreak isolates from non-outbreak isolates 17 19 20 21 .…”

Molecular typing of Legionella pneumophila isolates from environmental water samples and clinical samples using a five-gene sequence typing and standard Sequence-Based Typing

“…Although there have been many studies probing new typing methods for L . pneumophila , such as SNP-based approach [ 56 ], whole-genome mapping (WGM) [ 17 ], cgMLST [ 13 , 50 ] and rMLST [ 59 ], these schemes required to sequence a great many of gene loci, and the cost and bioinformatics infrastructure might be issues in some laboratories. In this study, we reported a five-gene MLST scheme for genotyping of L .…”

“…For example, subtyping the isolates belonging to a same prevalent ST required combinatory approach, including PFGE, AFLP, monoclonal antibody-based (MAb) subgrouping and some other genome sequence-based typing schemes [ 14 – 16 ]. A large proportion of LD cases is caused by just a small number of common STs (e.g., ST1); the SBT can lack discriminatory power [ 13 , 17 , 18 ]. Therefore, research and improvement of molecular typing methods for L .…”