1. Introduction to Legionellosis Diagnosis and Control in the Genomic Era

Rachel E. Gibbs and Jacob Moran-Gilad

Legionella bacteria are a leading cause of infectious disease and mortality worldwide. Moreover, from the perspectives of bacteriology, public health, epidemiology and clinical medicine, Legionella is an exceptional and unique micro-organism. Historically, the study of Legionella is inherently multidisciplinary as leading up to human infection, the bacteria interacts with freshwater and other various ecosystems, protozoa, complex molecular secretion systems, man-made water systems and finally, human colonization and antibiotic treatment. These multifaceted interactions often render the studies of Legionella as an applicable model for the prevention, assessment and control of other infectious diseases causing outbreaks. Since the discovery of the organism in 1977, the techniques which specialists use for research on Legionella have rapidly transformed. With chapters written by a diverse array of specialists, this book exemplifies the dynamic nature of Legionella while new methods such as genomics revolutionize this domain.

2. Freshwater Ecology of Legionella pneumophila

Rafael A. Garduño

Legionella pneumophila is a Gram-negative freshwater bacterium that emerged in the mid-1970s as an opportunistic human pathogen and the causal agent of Legionnaires' disease. Legionnaires' disease is an atypical pneumonia which is always acquired from the environment. L. pneumophila is principally not transmitted from person-to-person which indicates that L. pneumophila is not adapted to the human host. In addition, L. pneumophila is an intracellular pathogen of amoebae, and infection of humans is merely accidental. This chapter discusses L. pneumophila as a highly adapted intracellular parasite whose existence depends on its ability to replicate intracellularly in free-living freshwater amoebae. The complex ecology of L. pneumophila is explored through its interactions with both the abiotic and the biotic components of the freshwater environment. Since L. pneumophila is a pleomorphic organism with many developmental, morphological and(or) physiological forms, a comparative analysis of the unique ecologies of relevant forms will be presented. Control measures used to mitigate the transmission of Legionnaires' disease and the risk management of water systems will be discussed in further chapters, though the information provided in this chapter contributes to the improvement of control initiatives.

3. A 'Secreted Army' for the Invasion and Survival of Legionella pneumophila Within Host Cells

Elisabeth Kay, Virginie Lelogeais, Sophie Jarraud and Christophe Gilbert and Patricia Doublet

Intracellular multiplication within protozoans is an essential step in the emergence of pathogenic Legionella pneumophila strains. Key features of intracellular survival and multiplication are secretion systems propelling virulence factors and other important substances into their surroundings. These systems are highly diverse and likely contribute to the versatility of Legionella species which replicate in a wide spectrum of hosts. This chapter discusses secretion systems with emphasis on the highly conserved Dot/Icm T4BSS and its regulators. First, the unique features of each type of secretion system employed by Legionella is described. Then, a review of the current knowledge about the Dot/ICM T4BSS highlighting the cohort of secreted proteins which determine the intracellular fate of Legionella is presented. In addition, regulators coordinating responses to environmental cues and determining virulence will be discussed. This shows how Legionella is an excellent model for the study of intracellular pathogens, as they survive inside a wide variety of hosts and even manipulate the intracellular environment to increase virulence.

4. Epidemiology of Legionellosis and a Historical Perspective on Legionella pneumophila Strains for the Genomic Era

Natalia A. Kozak-Muiznieks, Jeffrey W. Mercante and Brian H. Raphael

As a relatively newly discovered pathogen, Legionella, the cause of Legionnaires' disease and Pontiac fever, emerged at a time of rapidly evolving diagnostic techniques. The sequencing of the first L. pneumophila strain in 2004 brought extensive new findings on this unique pathogen regarding virulence, metabolism and diversity of this predominant species. This chapter describes the major L. pneumophila reference strains, OLDA, Pontiac-1, Philadelphia, Paris, Lens, Lorraine, Detroit-1 and the strains representing L. pneumophila subspecies pascillei in the historical contexts in which they were discovered. Each strain contributed significant findings to our current understanding of Legionella in the environment and pathologically. The distinct outbreaks leading to the discovery of each strain show the multifarious nature of this organism along with the major public health significance of Legionella research. Facing the era of genomics, much remains to be uncovered including pathogenicity profiles and resolution of the evident diversity within strains.

5. Clinical Symptoms and Treatment of Legionellosis

Giancarlo Ceccarelli, Mario Venditti, Maria Scaturro and Maria Luisa Ricci

Legionellosis causes a unique clinical course which can pose challenges for prompt diagnosis and treatment. This chapter discusses the clinical symptoms of the two forms of legionellosis, Legionnaires' disease and Pontiac fever, along with the current antibiotic treatment regimens for Legionnaires' disease. Legionella pneumonia does not have clear distinguishing features apart from other cases of bacterial pneumonia. Clinical scores and predicting tools have been developed to predict the likelihood of Legionella infection and determine the severity of infection. Antibiotics effective against Legionella must be active and concentrated in the intracellular space as well as distributed to infected tissues. Currently, fluoroquinolones and macrolides are the first line treatment with preference for fluoroquinolones and particularly, levofloxacin. However, immunocompromised patients may have unique clinical features requiring a more intensive antibiotic regimen. Lastly, the nonspecific symptoms of legionellosis may lead to misdiagnosis and treatment with antibiotics such as, beta-lactams which may cause a brief clinical improvement followed by a severe deterioration. Due to this dangerous phenomenon and the rise of antibiotic resistance, diagnosis and treatment regimens must be carefully considered.

6. Laboratory Diagnosis of Legionellosis

Giancarlo Ceccarelli, Mario Venditti, Maria Scaturro and Maria Luisa Ricci

Despite efforts to find improved clinical and laboratory predictors for diagnosing cases of legionellosis, the clinical picture of Legionnaires disease, in particular, remains nonspecific. Due to the severity of Legionella pneumonia and the distinct antibiotic treatment regimen, prompt and accurate diagnosis is important to improve patient prognosis. For this reason, laboratory diagnostic methods are most essential tools for diagnosing legionellosis. This chapter discusses the diagnostic criteria along with an explanation of each laboratory method for the diagnosis of legionellosis including culture, urinary antigen, antibody detection and direct immunofluorescence. As there are benefits and disadvantages to each technique, this chapter describes how certain methods can be used in conjunction with one another in order to gain the most sensitive and specific diagnostic strategy.

7. Clinical Significance of (non-Legionella pneumophila) Legionella Species

Diane S.J. Lindsay

Despite most human cases of clinical legionellosis being caused by Legionella pneumophila, there are many non-L. pneumophila species of the family, Legionellaceae, which have clinical, environmental and public health relevance. This chapter explores the history, notable features and geographical distribution of the most common non-L. pneumophila species of the Legionella genus with emphasis on L. longbeachae, L. bozemanae, L. micdadei, L. dumoffii and L. anisa. The Legionella species and L. pneumophila can be indistinguishable in terms of the clinical features of the disease that they cause and the respective treatments they require. However, infections caused by Legionella species predominate in the immunosuppressed and these infections can feature distinct clinical and radiologic findings such as an association with extra pulmonary sites of infection. Current diagnostic tools such as the urinary antigen test are biased towards detection of L. pneumophila serogroup 1 and increases the likelihood of cases caused by Legionella species being under or mis-diagnosed. Therefore, the true incidence of legionellosis cases caused by non-L. pneumophila species is probably under reported.

8. Regulatory and Risk Management Strategies for Control of Legionella

Susanne Surman-Lee and James T. Walker

Despite four decades passing since the discovery of Legionella, Legionnaires' disease (LD) is the deadliest waterborne disease in the United States, and according to the WHO, Legionella still has the highest burden of all water borne pathogens. This is despite years of improved understanding of the microorganism, engineering practices and control measures. A number of large outbreaks in The Netherlands, UK, and USA resulted in changes in legislation to enable the publication of regulatory documents for the control of Legionella in water systems. Countries where regulatory documents have been published often lack resources to monitor the implementation across wide ranges of water systems that could pose health risks. The WHO advised that Water Safety Groups (WSGs) be initiated, and this has been adopted into guidance in the UK for large healthcare establishments. The advantage of this approach is that it enables teams with the required expertise to undertake or engage with other professionals to carry out risk assessments to identify the hazards to vulnerable patients.

9. European Surveillance of Legionnaires' Disease   Free download

Birgitta de Jong and Lara Payne Hallström

Legionnaires' disease and Pontaic fever are both diseases with important public health implications and require prompt and thorough responses to outbreaks for future prevention. Effective methods for defining, diagnosing, reporting and responding to legionellosis outbreaks ideally should be standardized across countries. Therefore, the European Union surveillance methods for countering legionellosis is a useful model for legionellosis control especially for travel-associated Legionnaires' disease (TALD) cases which are on the rise. Multi-country surveillance in the EU and European Economic Area has evolved since the first organizational efforts in the 1980's to the currently responsible, European Legionnaires' Disease Surveillance Network (ELDSNet). This chapter outlines the practices of the EU surveillance of legionellosis including their schemata, definitions, responsibilities of participating members, methods and the results of the data collected since the program's inception. Lastly, improvements must still be made as the incidence of Legionnaires' disease in the EU is likely underestimated as most cases are reported from only four countries, France, Germany, Italy and Spain, which represent 50% of the EU population due to underreporting and/or underdiagnosis.

10. Epidemiological Genotyping of Legionella pneumophila: from Plasmids to Sequence-Based Typing

Norman K. Fry and Sophie Jarraud

Genotyping is used for the identification and differentiation of Legionella types within a species and most often for the species, L. pneumophila, which causes most cases of human infection. Due to the complex nature of Legionella outbreaks and sporadic infections which are often associated with travel, effective genotyping methods must be transportable and accessible with enough resolution to identify closely related strains. This chapter describes each genotyping method developed for the discernment of L. pneumophila including the discovery of diversity within plasmid genomes, restriction endonuclease analysis, pulsed-field gel electrophoresis, restriction fragment length polymorphism analysis and ribotyping, polymerase chain reaction (PCR) typing, amplified fragment length polymorphism analysis and finally sequence-based typing (SBT) which is the recent gold-standard. The shortfalls of each typing method will be discussed and how these flaws lead us into the era of next generation sequencing (NGS) which has already begun to revolutionize typing methodology.

11. Typing of Legionella Isolates in the Genomic Era

Daniel Wüthrich, Helena M.B. Seth-Smith and Adrian Egli

Precise typing of Legionella clinical and environmental isolates is vastly important, as L. pneumophila is a highly conserved species with clonal groups such as the sequence type (ST) 1. This chapter discusses the typing of Legionella isolates and with emphasis on the new and advantageous genome-based methods. Previously relied on methods such as serotyping, pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) differentiate between L. pneumophila isolates. However, these techniques are flawed when evaluating closely related L. pneumophila types. This suggests that a new gold-standard is needed for effective surveillance of Legionella and prevention of future outbreaks. With the emergence of next-generation sequencing (NGS) and genomics, boundless conclusions can be drawn from the heightened resolution. The four current techniques constantly being applied to novel situations are core genome MLST (cgMLST), whole genome MLST (wgMLST), core genome SNP-tree, and the whole genome SNP-tree. The broad application of WGS is exemplified with mention of prior outbreak investigations utilizing WGS for analysis in conjunction with epidemiological data in varying scenarios.