Legionella Pneumophila: From Environment to Disease : from Environment to Disease [1 ed.] 9781617612770, 9781608769476

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Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Legionella Pneumophila: From Environment to Disease : from Environment to Disease, Nova Science Publishers, Incorporated,

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Legionella Pneumophila: From Environment to Disease : from Environment to Disease, Nova Science Publishers, Incorporated,

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LEGIONELLA PNEUMOPHILA: FROM ENVIRONMENT TO DISEASE

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Legionella Pneumophila: From Environment to Disease Atac Uzel and E. Esin Hames-Kocabas 2010. ISBN: 978-1-60876-947-6

Legionella Pneumophila: From Environment to Disease : from Environment to Disease, Nova Science Publishers,

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Legionella Pneumophila: From Environment to Disease : from Environment to Disease, Nova Science Publishers,

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LEGIONELLA PNEUMOPHILA: FROM ENVIRONMENT TO DISEASE

ATAC UZEL AND

E. ESIN HAMES-KOCABAS

Nova Biomedical Books New York

Legionella Pneumophila: From Environment to Disease : from Environment to Disease, Nova Science Publishers,

Copyright © 2010 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Legionella pneumophila : from environment to disease / authors, Atac Uzel, E. Esin Hames-Kocabas. p. ; cm. Includes bibliographical references and index. ISBN H%RRN 1. Legionella pneumophila. 2. Legionnaires' disease. I. Uzel, Atac. II. Hames-Kocabas, E. Esin. [DNLM: 1. Legionella pneumophila. 2. Legionnaires' Disease. QW 131 L5145 2009] QR201.L44L435 2009 616.2'41--dc22 2009048909

Published by Nova Science Publishers, Inc.  New York

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Dedicated to my family Ataç Uzel

To the memory of my father

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E. Esin Hameş-Kocabaş

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Contents

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Preface

xi

Chapter I

Introduction

1

Chapter II

Microbial Ecology

5

Chapter III

Environmental Analysis

13

Chapter IV

Identification

23

Chapter V

Environmental Control

31

Chapter VI

Epidemiology

37

Chapter VII

Pathogenity

43

Chapter VIII

Diagnose

49

Conclusion

55

References

57

Index

87

Legionella Pneumophila: From Environment to Disease : from Environment to Disease, Nova Science Publishers,

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Preface Since the discovery of Legionella pneumophila in 1976 its significance increased rapidly in terms of public health and became an important pathogen. With the discovery and description as a new kind of species, the life cycle of Legionella pneumophila has been investigated and interestingly it was found that humans are an accidental host of the life cycle of these bacteria. L. pneumophila are normally present in fresh water environments, but when entering in man-made systems it multiplies and causes Legionellosis. Nowadays, L. pneumophila constitute an important part of community originated cases of atypical pneumonia and travel associated diseases therefore, it must be under strict control in manmade habitats. Understanding the biology of L. pneumophila is critical for the development of more effective combat methods. During the preparation of this book, a large number of valuable works were examined and all aspects of L. pneumophila were basically introduced. The different features of this interesting microbe’s journey from the existence in the environment to the diseases caused in humans were trying to be discussed and the prevention methods also have been mentioned. We believe that this book is a good start for researchers who want to have a first overlook at this subject.

Abstract Legionella pneumophila was recognized as an important human pathogen after the first discovery during an investigation of a pneumonia outbreak among American Legion convention in 1976 in Philadelphia, USA. L. pneumophila is a gram-negative, mesophilic, facultative intracellular parasitic and nonspore-

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xii

Preface

forming rod-shaped bacterium belonging to the gamma-subgroup of proteobacteria. L. pneumophila inhabits natural freshwater environments at low concentration. Along with the transfer from natural aquatic habitats into manmade water systems such as cooling towers, evaporative condensers, water distribution systems, whirlpool spas and hot water tanks, L. pneumophila reaches high cell density and can cause Legionnaires’ disease (pneumonic legionellosis) or Pontiac fever (severe influenza-like illness). Infection occurs primarily via the inhalation of L. pneumophila-contaminated aerosols. In aquatic habitats, L. pneumophila cells are intracellular parasites of freshwater protozoa and use a similar mechanism to multiply within mammalian cells. L. pneumophila can also multiply extracellularly within biofilms and can persist within these microbial communities for years. Transmission to human primarily occurs via the inhalation of L. pneumophila containing aerosols. The bacterium enters to human phagocytic cells by coiling or conventional phagocytosis then inhibits phagosome-lysosome fusion and multiplies in the phagosome. A number of virulence factors have been described for L. pneumophila such as surface proteins, secreted factors and putative virulence factors. L. pneumophila can be identified by using cultural, serologic and various molecular techniques such as DNA sequencing and DNADNA hybridization. Diagnosis can be made by culture, direct fluorescent antibody staining, serological tests, urinary antigen detection or nucleic acid detection and various subtyping techniques. In order to eradicate L. pneumophila from contaminated water systems several methods are available; Thermal or chemical shock disinfection, UV irradiation, ozone treatment, silver-copper ionization, anodic oxidation and chlorine dioxide application.

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Chapter I

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Introduction A severe pneumonia outbreak among the participants of an American legion convention in Philadelphia in 1976 resulted in the identification of Legionnaries’ Disease (LD). Among 221 people who became sick, 34 subsequently died and it was obvious that this new type of pneumonia did not respond to treatment with beta-lactam antibiotics. The search for conventional infectious agents was unsuccessful and only the guinea pig experiments which were originally designed to detect the Rickettsiae yielded a gram negative bacterium with previously unknown characteristics. This bacterium showed complex growth requirements including cysteine and iron salts dependency in media (Brenner et al., 1979). The causative agent, what would come to be known as Legionella pneumophila, was isolated and given its own genus (Legionella referring to the legionnaires who were infected at the convention, pneumophila meaning “lung loving”). Subsequently, by isolating the causative bacterium, it was also possible to conduct seroepidemiological studies, which resulted in the recognition of earlier outbreaks of the same pneumonic infection (Mc Dade et al., 1977). Retrospective studies revealed the previous outbreaks of pneumonia and other respiratory illnesses dating back as far as 1957. The first known isolation of L. pneumophila occurred in 1947 from a guinea pig, which had been inoculated with blood from a patient with an unknown febrile illness (Bozeman et al., 1968). After the discovery of this new family Legionellaceae currently consisting four genera including Fluoribacter, Legionella, Sarcobium, Tatlockia, the species number in the genus Legionella have increased rapidly. Furthermore, several new serogroups of L. pneumophila and other Legionella species have been discovered. Currently, the genus comprises 52 species and 3 subspecies with more than 60

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serogroups of which 15 belong to L. pneumophila (IJSEM, 2009). While more than half of these species/serogroups have been associated with human disease (Parry et al., 1985, Doebbeling et al., 1989), L. pneumophila, the first Legionella bacterial species identified, accounts for approximately 90% of infections, with illnesses most frequently associated with serogroups 1, 4, and 6 (Reingold et al., 1984). L. pneumophila is a non-spore-forming, aerobic and mesophilic Gramnegative bacillus (Figure 1). It contains branched-chain fatty acids, has a nonfermentative metabolism, and requires L-cysteine-HCl and iron salts for growth. Chemoorganotrophic, amino acids rather than carbohydrates are used as an energy source (Edelstein and Cianciotto, 2006). L. pneumophila strains are facultative parasites of some eukaryotic cells including free-living protozoa and human macrophages. L. pneumophila reaches to lungs and subsequently macrophages mainly by inhalation of contaminated aerosol droplets (Fields, 1996).

A. Uzel. Figure 1. Gram staining of a L. pneumophila serogroup 1 strain isolated from a building water distribution system.

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Introduction

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The length of the bacterium depends on growth conditions, stage of life cycle and whether the bacterium is grown in intracellular or extracellular environments. Planktonic cells are rod-shaped, 0.3–0.9 m in width and approximately 1.3 m in length. Bacterium grown in solid culture length can reach up to 40 m but when grown in amoebae, macrophages, or macrophage-like cell lines morphology transform into short coccobacilli and become highly motile by means of one or more polar or lateral flagella. The growth temperature in environment is estimated among 10-45 C with a 35-37 C optimum in vitro temperature. The pH optima for in vitro growth are 6.8-7.0. The G+C content of the L. pneumophila is 39 mol% and DNA relatedness between strains of L. pneumophila is 75–100%. L. pneumophila contains plasmids differing in size between 21-95 MDa belonging to multiple incompatible groups (Mintz, 1999; Edelstein and Cianciotto, 2006). This book will mainly focus on habitats, diseases, pathogenicity and analysis of L. pneumophila.

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Chapter II

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Microbial Ecology Legionellae are well suited to aquatic environments and freshwater is the primary reservoir for legionellae worldwide (Fliermans, 1981). The natural habitat for L. pneumophila appears to be freshwater environments including rivers, lakes, streams and thermally polluted waters. Although it has been isolated from riparian soil, mud and excavations, it has not been isolated from dry soil (Yu, 1990). Natural water sources contain only small number of L. pneumophila cells (10 cells per liter) but this bacterium can survive in a wide range of environmental conditions: temperature, 0 - 63 C; pH, 5.0 – 8.5; and dissolved oxygen, 0.2 – 15.0 mg/liter (Fliermans and Harvey, 1984; Yu, 1990). Legionellae have been detected in as many as 40% of freshwater environments by culture and in up to 80% of freshwater sites by PCR (Fields, 1996). Although L. pneumophila can be found in waters ranging from cold to very hot, its multiplication is limited to temperatures between 25 and 42°C with an optimal growth at 35±1°C. Since legionellae are relatively more tolerant to chlorine, the organism survives through the water treatment process and passes into the water distribution system. Furthermore, in aquatic habitats L. pneumophila can infect and multiply in different species of free-living fresh water protozoa. In 1980, Rowbotham described the ability of L. pneumophila to infect Acanthamoeba and Naegleria (Rowbotham, 1980). Presence of the protozoa in the environment provides a suitable and protected niche for L. pneumophila against unfavorable physical and chemical conditions. Legionellae have been reported to multiply in 14 species of amoebae, two species of ciliated protozoa and one species of slime mold (Rowbotham, 1980; Hägele et al., 2000; Solomon and Isberg, 2000).

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These natural host cells provide nutrients, protect the bacteria from harsh conditions and serve as a vehicle for the colonization of new habitats (Steinert et al., 2007). Once the L. pneumophila enters the water distribution system, subsequent growth and proliferation occurs in man-made habitats such as cooling towers (Shelton et al., 1994), potable water systems of big buildings especially in hospitals (Stout et al., 1992), hotels, (Uzel et al., 2005), cruise ships (CastellaniPastoris et al., 1999) hot water tanks, heated spas (Jernigan et al., 1996), architectural fountains, waterfall systems (Hlady et al., 1993), evaporative air coolers and humidifiers (Mastro et al., 1991). The main source of the L. pneumophila contaminating the water distribution systems appears to be the municipal water supply network. When the L. pneumophila cells enter to the manmade systems that acts effective bacterial amplifiers such as hot water tanks, can reach as much as 106 cfu liter-1 (States et al., 1989). The major factors for the multiplication of L. pneumophila in such systems are the water temperature, the input of nutrients and the growth of biofilms throughout the system (Yu, 1990). Presence of the sediment and commensal microflora may provide the essential nutrients required for multiplication (Wadowsky and Yee, 1985). Temperature seems to be a particularly critical parameter. L. pneumophila strains may remain viable and dormant in cold water at temperatures below 20°C, only multiplying when the temperature reaches to an appropriate level. Water temperatures between 25 and 42°C with an optimal growth at 35 C promote L. pneumophila multiplication (Ohno et al., 2003). L. pneumophila has been isolated from natural water at temperatures ranging from 0-65 C. Carvalho et al. (2008) showed the presence of legionellae at polar lakes of Antarctic Peninsula by culturing and environmental DNA analysis. These authors suggested that the symbiotic association of Legionella species and amoebas could allow for survival and resistance of the bacteria at low-temperature environments. In relatively high temperatures L. pneumophila cells are much more heat resistant comparing with other Gram negative mesophilic heterotrophic bacteria. However, in pure culture above 50 C cells start to die rapidly. In a previous study, it has been reported that cell growth and metabolic activity decreased considerably in all strains of L. pneumophila at temperatures above 45 C. Nevertheless, metabolic activity was retained at 51.6 C beyond the maximum temperature for cell growth; L. pneumophila could survive planktonically, retaining its metabolic activity, although its cultivability was lost in an environment with a high temperature such as hot spring water (Kusnetsov et al., 1996; Ohno et al., 2003) In addition to the temperature, design, installation, management and maintenance of these water systems play an important role for the colonization of

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Microbial Ecology

7

L. pneumophila, for example presence of dead ends which contains stagnant water (Koneman et al., 1992). Stagnant conditions support L. pneumophila growth. The type of material used in the construction of plumbing systems also has been shown to impact the ability of L. pneumophila to colonize on these surfaces. L. pneumophila appears to be more abundant on elastomeric surfaces than copper or stainless steel (Rogers et al. 1994; Van der Kooij et al. 2005).

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Biofilms and L. Pneumophila Microbial biofilms are extremely complex heterogeneous microbial ecosystems and may consist of bacteria, algae and grazing protozoa. Biofilm formation occurs worldwide in natural and artificial environments including plumbing fixtures, heating, ventilating and air-conditioning equipments, medical and dental devices. Biofilms facilitates nutrient and gaseous exchange, and protects microorganisms not only from biocides but also from periodic increases in temperature and attempts at physical removal, especially in areas where surfaces are scaled or corroded (Hornei et al., 2007). Biofilms, in water distribution systems harbors many Legionella species and protect them from harsh environmental conditions (Rogers et al., 1994; Walker and Marsh, 2007). Biofilms are considered as ecological niches in which L. pneumophila not only survives but proliferates and infects their susceptible hosts (Barbeau et al., 1998). Quite a few studies focused on L. pneumophila association with biofilms. Biofilms can provide harbor and a gradient of nutrients to colonizing microorganisms. Some authors suggested that L. pneumophila can exist in biofilms for long periods but their multiplication needs protozoan hosts. Murga et al. (2001) developed a model biofilm system to determine the ability of L. pneumophila to grow in a potable-water biofilm without an association with Hartmanella vermiformis. They showed that the L. pneumophila can persist in biofilm without H. vermiformis but did not develop microcolonies in the absence of H. vermiformis. However, higher numbers of L. pneumophila were recovered from the biofilm matrix and from bulk liquid indicating the planktonic state of L. pneumophila in the presence of H. vermiformis. On the contrary, other authors suggested that the biofilms support the survival and multiplication of legionellae outside a host cell (Rogers and Keevil 1992). L. pneumophila is able to form biofilm under laboratory condition (Storey at al., 2004) and in building water systems the majority of the legionellae are biofilm associated, as they are more

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easily detected from swab samples than from flowing water (Rogers et al., 1994). Moreover, legionellae growth, within a biofilm composed of naturally occurring waterborne microorganisms in the absence of protozoa has been shown in a model system study by using cycloheximide, an eukaryotic protein synthesis inhibitor (Surman et al., 2002). The materials of the water distribution system also affect the growth of biofilms and L. pneumophila. Some plumbing materials support or enhance the proliferation of microorganisms, including Legionella spp.. In water piping systems, L. pneumophila has been found to be most abundant in biofilms on plastics at 40 C, where it accounted for up to 50% of the total biofilm flora; in contrast, pipes with copper surfaces were inhibitory to total biofouling and included only low numbers of L. pneumophila (Rogers et al., 1994). Van der Kooij et al. (2005) also showed that the biofilm formation on copper surfaces was similar to those on stainless steel surfaces, but significantly lower concentrations of legionellae were observed in water from the copper pipes and on copper surfaces.

Produced by E. E. Hames-Kocabas Figure 2. Diagram of a biofilm formation in the pipeline. Biofilms are complex microbial communities and consist of many microbial species. They provide nutrient and forms a shelter to microorganisms against harsh environmental conditions and antimicrobial substances.

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Microbial Ecology

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Accompanying microorganisms may have positive or negative effects to the presence of L. pneumophila in biofilms. Recently Guerrieri et al. (2008) showed that while some aquatic bacteria like Pseudomonas fluorescens have antagonistic effect against biofilm formation and stability of L. pneumophila sg 1, Acinetobacter lwoffii enhances its biofilm counts. In another study, Mampel et al. (2006) reported that under flow conditions L. pneumophila adhered to biofilms formed by monocultures of Empedobacter breve, Microbacterium spp., and Acinetobacter baumanii, but it did not attach to Pseudomonas spp., Corynebacterium glutamicum, or Klebsiella pneumoniae biofilms. Previous research showed that sudden changes in water pressure within plumbing pipes may cause rupture and distribution of the biofilm layer. Shands et al., (1985) reported a dramatic increase in L. pneumophila recovery after a pressure shock of a plumbing system. Eventually biofilms have a significant impact on L. pneumophila existence and multiplication in natural and man-made habitats. Biofilms are not only colonization or dissemination ways for L. pneumophila but also provides dormancy to unfavorable environmental conditions. Legionellae grown in biofilms are more resistant than the same bacterial species in the water systems (Cargill et al., 1992; Santegoeds et al., 1998).

Free Living Protozoa and L. pneumophila The association between L. pneumophila and free-living amoebae was first reported by Rowbotham (1980), who revealed the infection and intracellular multiplication of L. pneumophila in free-living amoeba. After conformation of Rowbotham’s observations by other researchers (Tyndall and Dominigue, 1982; Newsome et al., 1985), Barbaree et al. (1986) showed an association between outbreaks of Legionnaires’ disease and the presence of amoebae. Legionella species can multiply in 14 species of amoebae, including Acanthamoeba, Naegleria and Hartmanella spp., Saccamoeba, Vexillifera and Platyamoeba, the ciliates Tetrahymena pyriformis and T. vorax (Rowbotham, 1980; Tyndall and Domingue, 1982; Rowbotham, 1986; Wadowsky et al., 1991; Fields et al., 2002; Shadrach et al., 2005) and one species of slime moulds (Fields et al., 2002). Interaction with free-living protozoa has some benefits in the life cycle of L. pneumophila such as growth in natural and man-made habitats, resistance to environmental conditions and pathogenicity.

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The natural habitats of L. pneumophila generally have low nutrient content (Steinert et al. 2002). Since L. pneumophila has cysteine and iron salts dependency for growth, there is a discrepancy between the fastidious nature of L. pneumophila and its widespread distribution in nutritionally poor environment in which the bacterium is often detected (Steinert et al. 2002; Devos et al., 2005). It is assumed that intracellular life cycle in protozoa can provide the necessary growth requirements for L. pneumophila. Protozoa are an important factor for the environmental survival and growth of L. pneumophila and have been detected in environments implicated as sources of legionellosis (Fields et al., 1989; Breiman et al., 1990). It is now widely accepted that L. pneumophila, and probably most other Legionella species are facultative parasites of free-living amoeba, and intracellular form within the amoeba is the major, if not sole form (Edelstein and Cianciotto, 2006). Protozoa do not only provide nutrients for the intracellular legionellae, but also provides a shield against harsh environmental conditions (Steinert et al., 2002). Intracellularly grown L. pneumophila have been shown to be highly resistant to chemical disinfectants and to treatment with biocides compared to in vitro-grown cells (Barker et al., 1992). Abu Kwaik et al., (1997) reported that the amoebae-grown L. pneumophila showed a dramatic increase in their resistance to harsh environmental conditions such as fluctuation in temperature, osmolarity, pH, and exposure to oxidizing agents. Another researcher also reported the release of the L. pneumophila containing biocide resistant vesicles from protozoa (Berk et al., 1998). This increased resistance against environmental conditions should also be considered in eradication studies. An additional aspect of the intracellular growth of L. pneumophila within amoebae is the phenotypic changes and enhances of the virulence of the bacterium for macrophages and immunocompromised mice (Brieland et al., 1996; Cirillo et al., 1994). The susceptibility of the L. pneumophila cells to biocides is also greatly affected by the phenotype induced by intra-amoebic growth and results in a ca. 1,000-fold increase in the level of resistance compared with that of cells grown in vitro (Barker et al., 1992). In a further study, researchers found that in vitro grown cells were larger than their intracellularly grown counterparts and resistance characteristics were lost (Barker et al., 1995). Furthermore, presence of the L. pneumophila cells within the amoebae at high concentrations may protect the bacterium from the harmful effects of aerosolization suggesting that the path of infection may be the packet of bacteria contained within an amoebal vacuole or cyst (Edelstein and Cianciotto, 2006).

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Microbial Ecology

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L. pneumophila has also a viable but not cultivable (VBNC) “dormant” state like some other pathogenic bacteria (Paszko-Kolva et al., 1992; Yamamoto et al., 1996). Several studies suggest that L. pneumophila isolates were noncultivatable because they were in amoebae, and required prolonged incubation to amplify the small numbers present (Hussong et al., 1987; Sanden et al., 1992). It is also not clear VNBC L. pneumophila cells are pathogenic or not for humans. These interactions between protozoa and L. pneumophila show that the protozoa have important role in the life cycle and pathogenicity of the L. pneumophila.

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Chapter III

Environmental Analysis

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Sampling Sites and Sample Types Since there are lots of variable factors for each water system, the number and types of samples for L. pneumophila analysis should be determined on an individual system basis. There are many different system types such as domestic, nosocomial and community environments (Edagawa et al., 2008; Martinelli et al, 2000; Anaissie et al., 2002), hotels (Uzel et al., 2005), industrial facilities (Ishimatsu et al., et al., 2001), swimming pools (Leoni et al., 2001), dental unit waterlines (Singh and Coogan, 2005) and even cruise ships (Castellani-Pastoris et al., 1999). Recently L. pneumophila have been identified in aeration ponds of a biological treatment plant at high rate (Blatny et al., 2008). Depending to the system type there may be various types of plumbing, heating, ventilation and airconditioning installations that will be sampled. System design and material effect the colonization of L. pneumophila and therefore an appropriate environmental sampling method must be determined for each individual facility. Guidelines would be useful for detecting the sampling sites (ACHD, 1997; APHA, 1992; ASHRAE, 2000). A person who takes environmental samples must be aware of the contamination risk and should be able to select the appropriate sampling sites (Hornei et al, 2007). Although water and swabs samples are the standard ways of environmental sampling, air samples are also useful for detecting the contamination source in outbreaks (Shelton et al., 2000; Breiman et al., 1990). Swab samples should be taken from sites where biofilms are likely to form at

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critical points and water samples should be taken from both cold water and hot water lines. Areas those are difficult to reach, such as within the jets of hot tubs, thermostatic mixer valves, faucets or showers heads are considered appropriate swaps sites. According to Ta et al. (1995), the optimum L. pneumophila recovery from a hospital water system was obtained by swabs rather than the water samples. The swabs can be submerged in a small volume of water taken from the same system or in appropriate solution to prevent drying during transportation to the laboratory. For water samples it is recommended to take at least 1 liter of water so it can be concentrated if necessary. If there is any information about the use of oxidizing agents such as chlorine, appropriate amount of sodium thiosulfate should be added to the sample. All kinds of samples should be immediately transported to the laboratory in dark colored containers to protect them from extreme temperatures and from light. The water and swabs are the main types of samples for analyzing environmental legionellae. However, since L. pneumophila transmission primarily occurs via infective aerosols, detection of legionellae by air sampling may be important in epidemiological investigations of Legionnaires’ disease (Ishimatsu et al., 2001). Breiman et al. (1990) showed that the air sampling could be used as a useful tool for detecting and tracking L. pneumophila related with outbreaks. In another study, researchers detected the L. pneumophila in air at various positions within the biological treatment plant originating from the aeration ponds (Blatny et al., 2008). Selection of sampling sites and types depends on the reason for sampling; the sample may be taken either before or after disinfection. For routine monitoring or before disinfection the first flush will be an appropriate water sample and represent the worst condition. After disinfection, the sample taken from a running outlet will represent the efficiency of the procedure (Hornei et al., 2007). Samples should be collected from areas within the system that represent potential exposure. ASHRAE published a guideline which describes the legionellae harboring systems able to produce infectious aerosols (ASHRAE, 2000). These systems include the following structures: potable water systems; emergency water systems such as safety showers, eye wash stations and fire sprinkler systems; heated spas such as whirlpool spa, hot tub, whirlpool, whirlpool bath; architectural fountains and waterfall systems; cooling towers including fluid coolers and evaporative condensers; direct evaporative air coolers, misters (atomizers), air washers and humidifiers, indirect evaporative coolers and metal working systems.

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Environmental Analysis

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Selective Isolation and Culture

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Clinical and environmental samples differ for isolation of L. pneumophila. The bronchoalveolar lavage (BAL), lung tissue, pleural fluid or extrapulmonary sites such as pericardial fluid, skin lesions, brain tissue and other organs comprise clinical specimens (Pasculle, 2000). Subsequent procedures for isolation of L. pneumophila may differ significantly because of differences in contaminating bacterial and fungal flora in clinical and environmental specimens (Edelstein and Cianciotto, 2006). This section will mainly focus on recovery from environmental samples.

Figure 3. Isolation of L. pneumophila from environmental samples.

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Since Legionella bacteria are usually establish a small portion of the total bacterial population in environmental samples and rarely present in high numbers, it is usually necessary to first concentrate the microfloras. Therefore it is important to eliminate or suppress the competing background flora which may differ significantly during primary culture (Hornei et al, 2007). There are alternative selective isolation procedures that all have their own advantages and disadvantages. Generally, direct inoculum of non-treated water sample on selective media containing L-cysteine has poor sensitivity and specificity. L. pneumophila and background microorganisms found in water samples can be concentrated by using centrifugation or membrane filtration (Boulanger and Edelstein 1995). There is no consensus on one of the concentration methods until now. While some researchers centrifuging the water samples at 6000 g for 10 minutes, others did at 3000 g for 30 min. However, when analyzing too many samples, centrifuging is generally used as an alternative method against filtration since it is time consuming (Brindle, et. al. 1987). Besides, Ta et al., (1995) found the filtration was more effective than the centrifugation for recovery of L. pneumophila in environmental water samples. In concentration with filtration one should take into account some factors like filter type, pore size and re-suspension method (APHA, 1992). The concentration of the sample may increase the sensitivity but not the specificity (Ta et al., 1995; Leoni and Legnani, 2001). The recovery of the L. pneumophila from background microflora in concentrated water samples can be further increased by heat or acid pretreatment because legionellae are relatively more tolerant to these factors than many other background bacteria (Tiefenbrunner, et. al 1993; Ta et al., 1995; Türetgen et al., 2008). Heat pretreatment is made by holding the sample at 50 C for 30 min or 60°C for 1–2 min (Leoni and Legnani, 2001; Edelstein et al., 1982). Acid pretreatment is made by acidification of the 1 ml concentrated sample by 9 ml buffered HCl-KCl solution (pH 2.2) for 5-15 min and after addition of 1 ml neutralization buffer. In some cases acid treatment may also inhibit the growth of legionellae (Ta et al., 1995). These pretreatment methods facilitate the inhibition of contaminant microflora on isolation media (Edelstein and Edelstein, 1996). It is known that pretreatment of concentrated water samples with heat or acid increase the recovery rate, but like in the sample concentration case, there is no absolute consensus on which pretreatment method is superior to the other. The efficacy of these two methods probably depends on the number and diversity of the microorganisms consisting of the background microflora (Pasculle, 1992; Ta, et.

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al. 1995). In a previous study, researchers have reported a decrease in the recovery rate when heating has been used according to acid pretreatment. Nevertheless, this decrease has been attributed to the increased heat sensitivity of the laboratory strains (Bartie et al., 2001). In other previous reports, some preferred acid pretreatment (Verran, et. al. 1995; Brindle, et. al. 1987), while others preferred heat pretreatment (Jousimies-Somer, et. al. 1993). According to Koneman, et al. (1992), heat pretreatment is more suitable for clinical specimens and acid pretreatment is better for environmental samples. The general isolation procedure was summarized in Figure 3. L. pneumophila and many other species generally do not grow on standard microbiological media such as sheep blood agar or chocolate agar because of their L-cysteine dependency (Edelstein and Cianciotto, 2006). Many culture media are available for primary isolation of L. pneumophila. However, they may differ by their selective and growth supplementary contents. It is possible to use nonselective medium such as BCYE agar (ACES Buffered Charcoal Yeast Extract medium supplemented with cysteine, ferric pyrophosphate, and alpha-ketoglutarate) Figure 4) for isolation from clinical specimens but more selective medium should be used for isolation from environmental samples which have more diverse and high numbers of contaminating bacteria. The more selective MWY agar is a modified BCYE agar by addition of glycine, vancomycin, polymyxin B and natamycin (Feeley et al., 1979; Wadowsky and Yee, 1981; Edelstein, 1982). Another modified BCYEα medium is CCVC agar (polymyxin E, cephalothin, vancomycin and cycloheximide), which is more successively suppress the background microflora and suitable for heavily contaminated samples (Bopp et al., 1981). GVPC agar (BCYE agar with the addition of glycine, vancomycin, polymyxin B, cycloheximide) is another frequently used medium (Leoni and Legnani, 2001). Another most commonly used medium GVPA (BCYE agar supplemented with glycine, anisomycin, vancomycin and polymyxin B) is less inhibitory to some Legionella species (APHA, 1992). Differential dyes; Bromothymol blue and bromocresol purple were also used but not found useful (Wadowsky and Yee, 1981; Vickers et al., 1981). In another study, researchers reported an increase of the recovery of other pathogenic Legionella species L. micdadei and L. bozemanii with the addition of albumin to BCYE agar (Morill et al., 1990). In a comparative study, DGVP medium containing bromocresol blue, bromocresol purple, vancomycin, and polymyxin B gave optimal results for recovery of L. pneumophila (Ta et al., 1995). Combination of two different medium may enhance the isolation of different Legionella species.

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A. Uzel.

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Figure 4. Colony appearance of L. pneumophila Serogroup 1 on BCYE agar.

Agar plates inoculated with 0.1-0.2 ml sample by using pour plate technique and incubated at 36 ± 1 C in humidified air plus 2.5% carbon dioxide containing atmosphere. Plates are incubated up to 10 days with examination under a dissecting microscope in every 2 or 3 days. Legionella colonies start to appear on agar media in 2-3 days but may take several more days to develop colonies especially if a few organisms are present. The shapes of the Legionella colonies may vary from punctuate to smooth and size can reach up to 3-4 mm. When examined under a dissecting microscope, Legionella colonies show crystalline internal structures and speckled opalescent appearance are common. L. bozemanii, L. dumoffii and L. gormanii colonies exhibit a characteristic bluewhite fluorescence under long-wave U.V. light (Koneman et al., 1992). Suspected Legionella colonies are subcultured onto cysteine-free BCYE agar or an ordinary unsupplemented 5% sheep blood agar plate for examining their cysteine requirement. The colonies grown in cysteine-free media are most probably not belonging to Legionella genus. The Gram negative bacilli isolates that characteristically grow on selective media but not grow on cysteine deficient media should be further characterized. When using cysteine dependence for confirmation it should be kept in mind that some bacteria are able to produce extracellular cysteine that can support the growth of Legionella colonies

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(Wadowsky and Yee, 1983). Confirmation of the presumptive colonies may be done with immunologic methods. The most used laboratory test for species determination is direct immunofluorescence. Commercially available latex agglutination kits may also be used for species and serotype identification. It is possible to obtain non-reacting strains with available antisera. This may be due to non-Legionella species that require cysteine and fail to grow on blood agar and may resemble Legionella colonies on selective media. For further characterization of Legionella species, several tests should be done (Edelstein and Cianciotto, 2006; Koneman et al., 1992; Brenner et al., 1984). Although the culture test is the most common and preferred method for environmental analysis of L. pneumophila, some alternative techniques have been developed based on the detection of specific proteins or nucleic acids. Direct Fluorescent Antibody (DFA) is used commonly to detect the environmental legionellae (Vickers et al. 1990; Palmer et al. 1993). The use of PCR (polymerase chain reaction) for the environmental detection of L. pneumophila has been found valuable in some investigations of legionellosis outbreaks and is particularly useful for eliminating epidemiologically and geographically implicated sources (Shelton et al, 2000; Ballard et al., 2000). Environmental detection of L. pneumophila is possible by using fluorescent antibodies, in situ hybridization, probe hybridization, PCR, Real time PCR and DNA sequencing (Table 1). Generally, PCR gave higher detection rates up to 81.1% comparing to cultural methods with only 20-40% recovery rate (Palmer et al., 1995; Lye et al., 1997). This discrepancy could be due to VNBC cells. Steinert et al. (1997) have shown that VNBC cells of L. pneumophila may become cultivable again by cocultivation with axenic Acanthamoeba castellani. While PCR seems superior to culture, PCR inhibitors (particularly iron) found in environmental samples may cause false negative results (Miyamoto et al., 1997). Besides, each method used for environmental detection of L. pneumophila seems to have own advantages and disadvantages. DFA is a useful method but cross contaminations with nonLegionella species is a major problem and interpretation of the results is often subjective (Bartie et al., 2003). Thus, culture technique continues to be the most commonly used method for environmental analysis of L. pneumophila and accepted as “gold standard”. Although several standards are available for cultural analysis there is no consensus on single standard cultural method. As within any test, whether a cultural or PCR based Legionella test gives the result of L. pneumophila contamination at the time of the assay (Shelton et al., 2000). Legionella bacteria are widespread in natural environment as well as manmade environments.

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Table 1. Examples of the methods used for direct detection of Legionella spp., L. pneumophila and its serogroups from environmental samples excluding culture method Method

Target molecule

Environmental source

Reference

DFA

Species specific antigens

Plant waters

Bartie et al., 2003

Epifluorescence microscopy

Species specific antigens

Natural water

DelgadoViscogliosi et al., 2005

In situ hybridization and immunogold staining

Leg 705 probe

Amoeba and water

Desai et al., 1999

Fluorescent in situ hybridization (FISH)

16Sr DNA

Amoeba

Grimm et al., 1998

PCR-gene probe

mip gene

Water

Bej, et al., 1991

PCR, DNA probe

23S rDNA, 16S rDNA

Biofilm

Schwartz et al., 1998

PCR

5S rDNA, mip gene

Cooling tower water

Koide et al., 1993

PCR (Enviro AmpTM Legionella kits), DFA staining

5Sr DNA, mip gene, surface antigens

Ground, surface and potable water

Lye et al., 1997

Seminested PCR

16S rDNA

Hospital cooling tower water

Miyamoto et al., 1997

Nested PCR

16S rDNA

Shower, industrial,

Devos et al., 2005

natural and tap water DGGE

16S rDNA

Activated sludge

Nielsen et al., 1999

Real time PCR

16S rDNA

Hospital water

Wellinghausen et al., 2001

PCR, Real time PCR

16S rDNA

Hot water system

Edagawa et al., 2008

Real time PCR

mip gene

Hospital water

Morio et al., 2008

Duplex real time PCR

mip gene

Building water

Behets et al., 2007

FISH, PCR, Real time PCR, DNA Sequencing

mip gene, 18S DNA

Natural floating biofilms

Declerck et al., 2007

mip; macrophage infectivity potentiator. FISH; Fluorescence in situ hybridization. DGGE; Denaturing gradient gel electrophoresis. DFA; Direct fluorescent antibody.

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The value of a positive L. pneumophila test in a water distribution system must be questioned. What is the importance of the L. pneumophila existence in a water distribution system? Should a positive finding trigger a search for clinical cases or is it unimportant in the absence of any proven cases. It is difficult to predict the risk of LD based on the concentration and prevalence of L. pneumophila in water (Fiore et al., 1998). It is estimated that if all institutional water sources and cooling tower waters in the United States were samples during summer season, as many as 40% would test positive for L. pneumophila. It is obvious that, despite the undiagnosed LD cases, confirmed cases are not that much. Multiple variables contribute to an outbreak. In a previous study it is estimated that 10 4-105 cfu l-1 concentrations of L. pneumophila presents a health risk for humans and LD outbreaks occur at these concentrations (Meenhorst et al. 1985; Patterson et al. 1994; Leoni and Legnani 2001). In addition, presence of susceptible hosts such as elderly people and immunocompromised patients, adequate aerosolization of the bacterium, viability of the bacterium in aerosols, and virulence of the bacteria are important factors for occurrence of the LD. However, it is reasonable that high concentrations of L. pneumophila in water sources pose a higher risk than low concentrations and several methods have been developed eradication of L. pneumophila from water systems.

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Chapter IV

Identification

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Legionellae can be identified and differentiated by using different methods including; phenotypic characteristics, growth requirements, fatty acid and carbohydrate analysis, ubiquinones, serology and various molecular techniques. The most useful methods for bacterial identification seem to be L-cysteine growth dependence, serotyping, cellular fatty acids, ubiquinones, 16S rDNA sequence and mip gene sequence (Ratcliff et al., 1998).

Conventional Methods Culture continues being a valuable tool for isolation of L. pneumophila from environmental samples. Not only growth rate and colonial morphology is important for preliminary identification of L. pneumophila but also L-cysteine dependency is important for presumptive identification. The preparation of the culture medium and pretreatment of samples is mentioned above. BCYE- agar is usually supplemented with antibiotics for eliminating background bacteria. The colonies of Legionella sp. grown on BCYE- agar can be screen with dissecting microscope for preliminary suspicion. A presumptive identification of L. pneumophila can be made onto a cysteine deficient BCYE- agar or other cysteine deficient media such as sheep blood agar. The Legionella spp. isolate will not grow on BCYE- agar without cysteine or on sheep blood agar but on BCYE- agar supplemented cysteine. L-cysteine dependency disappears after several passage of the bacterium (Maiwald et al., 1998; Edelstein and Cianciotto,

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2006). Smith, (1982) has been reported L-cysteine containing paper disk can be place on cysteine-deficient BCYE agar will result formation of satellite colonies around the disk. The cellular morphology of Legionella spp. is variable depending upon conditions and phases of growth. Post-exponential phase of L. pneumophila is flagellated and coccoidal shaped in intra-amoebal growth, this bacterium transform to very long and nonmotile form in the stationary phase on solid media. In general filamentous forms turn into coccoidal form in the late stationary phase of growth (Pine et al., 1979; Byrne and Swanson, 1998). In addition, the production of single polar flagellum of L. pneumophila influenced by growth conditions such as temperature, the growth phase, the viscosity and the osmolarity and amino acids content of the medium (Heuner et al., 1999). Biochemical characteristic of L. pneumophila is not sufficient for identification due to general biochemical inertness of the bacterium. Thus correct identification of L. pneumophila using only traditional biochemical test can not useful. Nitrate reductase, urease and carbohydrate use are negative. L. pneumophila produces lipase, phosphatase and beta-lactamase (Thorpe and Miller, 1981). The -lactamase that is inactivating the cephalosporine are produced by L. pneumophila and can be detect using nitrocefin (Marre et al., 1982). While oxidase reaction gives variable results, peroxidase, gelatinase and hippurate hydrolysis show positive reaction. Catalase activity is also positive but extremely weak (Pine et al., 1979; Hebert, 1981; Edelstein and Cianciotto, 2006). Autofluorescent reaction of L. pneumophila which may disappear along with serial passage is yellow-green but this characteristic depends on growth conditions and colony age. The browning medium appears when the bacterium is cultured on tyrosine-supplemented agar (tyrosine-containing buffered yeastextract medium without charcoal) (Edelstein and Cianciotto, 2006). Analyses of fatty acids or unusual lipopolysaccharides of the cell wall can be performing successfully using gas liquid chromatography-mass spectroscopy (Diogo et al., 1999; Lambert and Moss, 1989). Legionellae have shown relatively unreactive characteristics in traditional biochemical tests. Serologic methods which are widely used in diagnosis have some limitation for specificity due to cross-reactions. Therefore more complex identification methods have been developed for legionellae (Ratcliff et al., 1998). Lambert and Moss (1989) have suggested the placing of 23 Legionella species into three groups according to the most abundant cellular fatty acids. Wilkinson et al. (1990) identified legionellaelike organisms, which were isolated from water samples during the outbreak of legionellosis in South Australia, using cellular fatty acid analysis, ubiquinone

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analysis, and DNA hybridization techniques and the results represented 6 possible new species. Diogo et al (1999) also reported that numerical analysis of fatty acid methyl ester (FAME) profiles is useful in identifying Legionella species. The most frequently technique used for the identification of Legionella species is serologic methods. Several commercial antisera to L. pneumophila sg 1 and to L. pneumophila can be used for clinical isolates, because L. pneumophila particularly L. pneumophila sg1 is prevalent in most clinical isolates. The existence of cross-reactions in these serologic methods is not allowing a differentiation among all species. This is especially true for countries where legionellosis caused by species other than L. pneumophila is common or in studies to find the contamination source of the outbreak from the environmental samples (Ractliff et al., 1998; Wilkinson et al., 1990; Benson et al., 1987). Through absorption with bacterial suspensions of heterologous serogroups, crossreacting antibodies should be removed from the polyclonal immune sera because many of the serogroups have common antigens. Due to the existence of crossreacting serogroups, serologic methods do not discriminate to a sufficient level for definitive species identification (Benson et al. 1987; Edelstein and Edelstein 1989; Helbig et al., 1997; Edelstein and Cianciotto, 2006). In another study Walter et al. (2008) flow-through chemiluminescence sandwich immunoassay on antibody microarray platforms for detection of L. pneumophila together with E. coli O157:H7 and S. typhimurium in water samples. They produced antibody microarrays on poly(ethylene glycol)- modified glass substrates by means of a contact arrayer and used the species-specific biotinylated antibodies for cell detection. By recording the chemiluminescence reaction with a charge coupled device (CCD) camera they completed the overall assay in 13 minutes.

Nucleic Acid Based Methods Various molecular techniques have also been developed to identify and differentiate at the species level of the legionellae such as DNA–DNA hybridization and sequence analysis of specific genes (16S rRNA, the mip gene) (Miyamoto et al., 1997; Cloud et. al., 2000; Hayden et al., 2001; Leskelä et al., 2005; Grimm et al., 1998; Lindsay et al., 1994; Wilson et al., 2003; Behets et al., 2007). Nucleic acid based methods have a great advantage for identification of the bacterium because they are not affected by growth or chromatographic

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conditions (Ratcliff et al., 1998) although most of these methods are not standardized and their discriminative ability rather variable (Atlas, 1999). DNA-DNA hybridization is the best method for identification of Legionella spp. or a new species. This method is technically demanding for routine application, for its procedure requires DNA from the test strain being hybridized with DNA from all known species of Legionella. For this reason its use is limited to laboratories where taxonomic studies are done (Fields et al., 2002). Kuroki et al. (2007) have used quantitative DNA-DNA hybridization and agglutination methods for identification of legionellae as a novel species from soils contaminated with industrial wastes. Various target genes and region of chromosomal DNA have been used in PCR tests for Legionella detection including the rRNA (5S, 16S) genes (Pinar et. al., 1997; Miyamoto et al., 1997; Cloud et. al., 2000; Hayden et al., 2001; Leskelä et al., 2005; Grimm et al., 1998) their intergenic spacer regions (Riffard et al., 1998; Herpers et al., 2003), the heat-shock protein (dnaJ) (Liu et al., 2003), the polymerase gene (rpoB) (Ko et al., 2002; Ko et al., 2006), the macrophage infectivity potentiator (mip) gene (Lindsay et al., 1994; Hayden et al., 2001; Wilson et al., 2003; Behets et al., 2007; Casini et al., 2008), and random DNA sequences (Whitney et al., 1997; Grattard et al., 1996; Pruckler et al., 1995; Wilson et al., 2003; Jaulhac et al., 1992; Koide and Saito 1995; Lindsay et al., 1994). While the 16S and 5S rRNA genes are specific for L. pneumophila, the mip gene is genus specific, (Den Boer and Yzerman, 2004). Sequence analysis of specific genes particularly mip gene is the most promising method for taxonomic studies of Legionella spp. Mip protein, an outer membrane protein, is required for infection of macrophages and protozoa. Cianciotto and Fields (1992) have shown the association of this gene in efficient infection using mip- stains. Ractliff et al. (1997) have phylogenetically compared most Legionella species using both the 16S rRNA and mip genes. The result showed that at the DNA level species variation in the mip gene were more than twofold. Ractliff et al. (1998) then have developed the first proposed sequence-based phylogenic (genotypic) scheme for legionellae targeting this virulence associated mip gene. Detection of L. pneumophila targeting the mip gene using real-time PCR assay have been reported as 100% sensitive and 100% specific (Wilson et al, 2003). Analysis of 16S rRNA and 5S rRNA sequencing as well as random amplified polymorphic DNA analysis (RAPD) has been reported for detection and differentiation of L. pneumophila (Pinar et al., 1997; Lo Presti et al., 1998; Salloum et al., 2002; Reischl et al., 2002; Herpers et al., 2003; Uzel, et al., 2005;

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Cooper et al., 2008). As 16S rRNA sequence can be amplified and sequenced with universal primers without difficulty, the variation in the 16S rRNA sequence are used in many genotypic schemes. Wilson et al. (2007) have used partial 16S rRNA gene sequencing for identification of L. pneumophila and nonpneumophila Legionella spp. and tested 49 Legionella species accurately identified. Discriminative ability of 5S rRNA gene for genotyping in all species of Legionella has been found inadequate but this genotypic scheme may be useful for the differentiation of L. pneumophila from other species (Murdoch et al., 1999). Over the past few years, assays for Legionella and L. pneumophila using direct (real-time) monitoring the generation of PCR fragments have also been documented in clinical and environmental samples (Ballard et al., 2000; Hayden et al., 2001; Rantakokko-Jalava and Jalava, 2001; Wilson et al., 2003; Behets et al., 2007; Reischl et al., 2002; Herpers et al., 2003; Morio et al., 2008; Templeton et al., 2003; Shannon et al., 2007). Real-time PCR reduces the risk of crosscontamination, minimizes manual time for the PCR and eliminates the post PCR analysis (Hayden et al., 2001; Rantakokko-Jalava and Jalava 2001; Fields et al., 2002; Reischl et al., 2002; Wellinghausen et al., 2001). Nucleic acid based methods, such as PCR is the solution for some problems faced while using conventional methods for the detection of pathogen. However there are difficulties in sensitivity and specificity in post PCR analysis, quantification, and low efficiency as well as in the distinction between live and dead cells. Some of these problems can be surpassed with the help of real-time quantitative PCR technology. When using DNA specific probes for the quantification of amplified PCR products all the problems of post PCR processing can be averted (Guy et al., 2003; MacKay 2004; Shannon et al., 2007). Diederen et al. (2007) have evaluated the utility of real-time PCR for diagnosis of LD using the 16S rRNA gene and mip genes. Specific mip gene based PCR is useful together with urinary antigen test in patients with suspected LD who produce sputum and might allow the early detection of a significant number of extra patients. In the detection of L. pneumophila and Legionella spp., Templeton et al (2003), used multiplex real-time PCR assay with internal control, without the need for post amplification analysis to distinguish the targets of 2.5 cfu/ml in sensitivity. In the result of this study, there was a similarity of 100% among the 10 culture positive samples, however, four additional samples of 74 culture negative samples showed positive results.

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In another study with environmental water samples using real-time PCR, the mip gene was selected as a target and the results were compared with standard culture methods. The results did not show any correlation between the two methods, but it was reported that, due to the sensitivity, simplicity and reproducibility of real-time PCR, a rapid screening of an important amount of samples in order to find the contaminated environmental water source can help to determine the situation during an outbreak (Morio et al., 2008). Nevertheless, this method does not replace a reference method itself, because clinical and environmental samples should be verified with cultural methods. Evaluating from this aspect, real-time PCR is a complementary method to reference methods (Ballard et al., 2000; Morio et al., 2008). Real-time PCR studies targeting 23S-5S spacer for direct detection and differentiation of Legionella spp. and L. pneumophila from environmental samples were performed in 2.5 cfu/reaction sensitivity with no false-positive result in comparison to culture results. However, Ballard et al. (2000) reported that, PCR reactions can be inhibited due to the iron content of environmental samples. Hayden et al. (2001) have compared conventional and nucleic acid based methods for the direct detection of Legionella species in clinical samples. They reported that there are significant advantages of real-time PCR compared with conventional PCR and cultural methods. However they also concluded the best method for detecting multiple Legionella species in lung tissue as the cultural method. Moreover, useful rapid tests for the detection of L. pneumophila in lung tissue are also Warthin-Starry (WS) staining, Legionella genus LightCycler-PCR (LC-PCR), and L. pneumophila species-specific in situ hybridization assay. Reischl et al. (2002) targeted the 16S rDNA gene of Legionella spp. by using two pairs of hybridization probes and established a dual-color LC-PCR assay that can detect and differentiate at the same time Legionella spp. and Legionella pneumophila. A 100% sensitivity and specificity for L. pneumophila was monitored in a study where 26 culture-positive and 42 culture-negative respiratory samples from patients were applied. Reischl et al. (2002), stated that, LC-PCR assay characteristics like speed, single-capillary format, dual-color hybridization probe detection, species information from melting curve analysis add a value to direct methods such as DFA or urinary antigen detection methods and is moreover an alternative method to the conventional PCR for the detection of Legionella spp.

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Subtyping Several typing methods have been used to find environmental contamination source of L. pneumophila. The most widely used one was subtyping of L. pneumophila sg 1 using a monoclonal antibody typing panel and also monoclonal typing schemes for other L. pneumophila serogroups being developed (Edelstein and Cianciotto, 2006). However serotyping seems to be insufficient to identify the contamination source of L. pneumophila sg 1 in epidemiologic investigations; whereas sequence based subtyping methods have been used for this purpose (Fields et al., 2002). L. pneumophila sg 1 can be divided in to a number of subtypes by various sequence based methods but they are not standardized and their discriminative ability is rather variable (Lück et al., 2007; Fields et al., 2002). Pruckler et al. (1995) have been compared different subtyping methods; pulsed-field gel electrophoresis (PFGE), arbitrarily primed PCR (AP-PCR) and monoclonal antibody (MAb) analysis for discriminating clinical and environmental isolates from seven unrelated outbreaks of LD. The authors concluded that investigation of outbreaks of legionellosis, MAb analysis should be used in combination with either AP-PCR or PFGE for better discrimination among the strains. PFGE has been used for subtyping of L. pneumophila sg 1 strains isolated from a hospital water supply and the authors reported that this method has great potential for epidemiological studies (Casini et al., 2008). There are also numbers of studies showing the usefulness of AP-PCR for identifying sources of LD outbreaks (Bansal and McDonell, 1997; Gomez-Lus et al., 1993; Grattard et al., 1996; Whitney et al., 1997). Valsangiacomo et al. (1995) have used the Amplified Fragment Length Polymorphism (AFLP) in molecular typing of L. pneumophila and reported this method as a fast, efficient and reproducible for typing strains of Legionella pneumophila isolated from both humans and the environment. A research evaluated macrorestriction analysis (MRA) by using PFGE as a valuable method for epidemiological studies of infections caused by L. pneumophila serogroups 3 and 4 (Lück et al., 1995). A further study of Jonas et al. (2000) has evaluated the performance and convenience of SfiI MRA, AFLP, and AP-PCR for nosocomial outbreaks of LD in hospitals. The authors concluded that AFLP is the most promising typing method for application in hospital for epidemiological studies, in terms of technical demands, reproducibility and simplicity of the patterns.

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The infrequent-restriction-site PCR (IRS-PCR) and repetitive element PCR (rep-PCR) have been reported useful tools for typing Legionella species (Georghiou et al., 1994; Riffard et al., 1998; van Belkum et al., 1993). Bangsborg et al. (1995) have also used ribotyping for typing of 58 clinical and environmental L. pneumophila strains. Ribotyping might be useful for species identification within Legionella genus (Bangsborg et al., 1995; Salloum et al., 2002).

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Chapter V

Environmental Control

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Prevention Since L. pneumophila prevail among natural aquatic environments it is impossible to eradicate the organism from nature. However, it can be controlled in its man-made reservoirs and prevent Legionella colonization. For effective prevention of LD, risk assessments and control measures must be implemented proactively not after arising of the disease. There are many guidelines describing the prevention and control strategies for L. pneumophila in man-made systems in national or international manner (EWGLI, 2005; CTI, 2000; ASHRAE, 2000). A successful prevention strategy should start with good engineering design and management of related water systems (Edelstein and Cianciotto, 2006). Antibacterial agents used for disinfection of L. pneumophila do not penetrate to dead ends and other parts where stagnant water is present (Hoebe and Cool, 2000). Some guidelines recommend the routine cultural monitoring of water systems for a successful prevention and control (ACDH, 1997). However, this approach is not recommended by others in the absence of established cases (Sehulster and Chinn, 2003). Monitoring L. pneumophila by culture may be necessary in some cases: to evaluate the effectiveness of a treatment method, tracing the source of an outbreak or protect the patients that have very high risk for LD such as organ transplant recipients who stay at the clinic (Butler et al., 1997; ASHRAE, 2000). Besides, nosocomial LD can easily escape from diagnosis, especially in hospitals lacking specialized methods for detecting Legionella and positive environmental samples may cause more attentive and

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easy diagnose (Yu, 1990). On the other hand, a great number of people are exposed to L. pneumophila in natural and urban environment. It seems some strains are either not highly virulent or most people are not susceptible (Koneman et al., 1992). Besides, the infective degree of exposure to pathogen is unknown. Efficiency of aerosolization is another important factor for L. pneumophila pathogenicity. A negative culture result does not guaranty a L. pneumophila free system since the presence of VNBC cells. It represents the present time and any variable such as nutrient entry and temperature change can amplify the bacterial number instantly. Even if a culture test has performed, the interpretation of the results is often complicated because of the different culture methods in various laboratories, variable culture results among different sampling sites and sample types in a system and fluctuations in the L. pneumophila concentration in the same system. So, presence of the organism can not be linked directly to the LD. L. pneumophila present frequently in water systems without any diagnosed disease at least in public places. Some factors resulting from the unique life cycle of L. pneumophila can affect the disinfection procedures in negative manner: biofilm formation and symbiosis with amoeba. The presence of the L. pneumophila in biofilms dramatically decreases the efficiency of the disinfectant agent. Similarly, it is difficult to kill L. pneumophila in its intracellular life cycle within an amoeba and even it is more difficult when associated with amoeba cyst (Barker et al., 1995; Kim et al., 2002). Symbiosis with amoeba transparently increases the resistance of L. pneumophila to harsh environmental conditions such as fluctuation in temperature, osmolarity, pH, and exposure to oxidizing agents (Abu Kwaik et al., 1997). L. pneumophila containing vesicles can be released from protozoa and these vesicles are extremely resistant to biocides (Berk et al., 1998).

Eradication Several methods have been developed for eradication of L. pneumophila from contaminated water systems; thermal eradication, UV irradiation and use of various biocides including metal ions, oxidizing agents, non-oxidizing agents (Yu, 1990; Lin et al., 1998a; Kim et al., 2002; Hoebe and Kool, 2000). These disinfection procedures can be used either singly or in combination according to the type and conditions of the water system. Most of these methods are mainly developed for disinfection of L. pneumophila from water distribution systems and

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cooling towers which are the main source of infection Lin et al., 1998a). While some methods can be used locally, other are suitable for systemic use (Lin et al., 1998b) but all methods for preventing the growth of Legionella in water systems have drawbacks and none is 100% effective (Hoebe and Kool, 2000).

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Hyperchlorination Hyperchlorination is one of the first methods used for Legionella disinfection. Municipal chlorination of the water (1ppm ) is inadequate for L. pneumophila, because of its multiplication in biofilms and protozoa (Yu, 1990). Planktonic L. pneumophila cells are also more resistant to chlorine and 2.0-6.0 ppm needs to control bacterium (Koneman et al., 1992). Hyperchlorination can be used in two modes; shock and continuous hyperchlorination. In shock hyperchlorination a high chlorine dose is introduced to water system to ensure the 20 - 50 ppm free chlorine throughout the system. After 2 hours system discharged and refilled again with 0.5 - 1.0 ppm chlorine containing water (Lin et al, 1998a). Continuous hyperchlorination made by maintain the 2.0 - 6.0 ppm chlorine concentration by continuous injection of calcium hypochlorite, sodium hypochlorite, chlorine dioxide or gaseous chlorination. If L. pneumophila associate with Acanthamoeba cysts 50 ppm free chlorine is required for disinfection (Kilvington and Price, 1990). Chlorine also may not penetrate to biofilm and dead peripheral areas of the system (Hoebe and Kool, 2000). Therefore chlorine usually suppresses legionellae rather than kill the bacteria. Levin et al., (1995) reported the recolonization in five moths after shock hyperchlorination in one hospital. Another drawback of the chlorination is the corrosion. Chlorine is known as highly corrosive and cause serious damage at the plumbing system and equipment. Furthermore, chlorine may interact with organic matter and can cause the production of trihalomethans, which is a known carcinogen (Pasculle, 2000; Lin et al., 1998a). Helms et al., (1988) reported an increase in the trihalomethane levels ( 100 micrograms/L) and corrosion damage after 5 year continuous chlorination. Monochloramine treatment presents an alternative to chlorine. Some evidence indicates that, monochloramine may be considerable more effective than free chlorine in municipal water plants and hospitals (Flannery, 2006).

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Thermal Eradication

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Heat treatment seems more readily eradicate legionellae from water systems. L. pneumophila cannot multiply above 50 C and water temperatures greater than 60 C are inhibitory to the bacterium (Kusnetsov et al., 1996). Thermal eradication (superheat-flush method) of legionellae is possible if the hot water temperature reaches to 70 C minimally and than all water outlets are flushed for 30 min. During the flushing the distal outlet temperature of the water should be maintained at 60 C (Lin et al., 1998b; Yu, 1993). This method is especially useful to terminate the ongoing outbreaks related with the water distribution systems of the buildings. However, thermal eradication is a temporary disinfection method. Re-colonization of the system with legionellae occurs within weeks. For long term prevention it is recommended that the water temperature should be kept at a baseline of 55 - 60 C (Stout et al., 1998; Darelid, 2002). Sometimes it is possible to combine the chlorination with superheating. Snyder et al., (1990) reported a reduction in L. pneumophila through heat flushing fallowed by continuous supplemental chlorination of hospital hot water. This method has also some disadvantages as others; time consuming, scalding can occur and can only be applied to hot water systems. Other water systems such as cold water pipelines remains contaminated.

UV Irradiation UV light irradiation is another alternative for legionellae control. UV units can be installed near peripheral outlets and eliminates the Legionella without addition of chemicals to the water (Kim et al., 2002). Despite its availability and easy usage, UV irradiation is a focal solution and has not a systemic effect like heat or chlorination. Usually prefiltration of the water is required, leaves no residual in water and it is not suitable for big buildings (Liu et al., 1995). Besides, Muraca et al (1987) reported a 5-log decrease at the L. pneumophila levels but not a total eradication when using a model plumbing system with continuous UV irradiation. The photocatalytic activity of TiO2 on L. pneumophila was also tested by some investigators (Dadjour et al., 2006; Cheng et al., 2007). Accordingly, the presence of TiO2 significantly enhanced the disinfection of L. pneumophila and environmental isolates were more susceptible than the type strain.

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Ozone Treatment Ozone is also used to eradication of legionellae and found useful (Edelstein et al., 1982). Previous studies showed that ozone kills L. pneumophila more effectively than either chlorine or hydrogen peroxide (Edelsteine et al., 1982; Domingue et al., 1988). Although ozone is more effective than chlorine it has many drawbacks. Ozone is an unstable molecule and does not stay in water enough to provide a residual effect against a potential contamination which must be produced at the point of application. Usually chlorine can be added after ozone treatment to provide the residual effect (Kim et al., 2002).

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Copper-Silver Ionization One of the newest methods developed for Legionella control is the coppersilver ionization. Metal ions are well known bactericidal agents and it has been shown that Copper and silver ions synergistically kill L. pneumophila in vitro (Miuetzner et al., 1997; Chen et al., 2008). These ions are generated electrolytically from a flow cell containing electrodes made of a copper/silver metal alloy and introduced to water. Final concentrations of the copper and silver ions are adjusted to 0.2-0.4 ppm – 0.02-0.04 ppm respectively. These levels are under the maximum contaminant levels for drinking water regulated by the US Environmental protection agency (Lin et al., 1998a). This method has also its own advantages and disadvantages among the other control methods developed for legionellae, but it seems a little bit more reliable. This method has a systemic effect; metal ions kill the legionellae rather than suppress and eliminate it from the system. It is easy to install and maintain (Campos et al., 2003). High temperature does not reduce the effectiveness of the metal ions unlike the chlorine; conversely a synergistic effect of temperature and chlorine has been observed (Yahya et al., 1992). Recolonization of the system occurs later when comparing the other disinfection methods because of the residual effect of the metal ions. Liu et al. (1994) reported a 6 - 12 weeks delayed recolonization even after inactivating the ionization system. However, hard water can cause scalding of the electrodes. In addition, ionizing process is affected by pH and it is difficult to provide the necessary copper and silver ion concentration at above pH 7.6 (Campos et al., 2003). The metal ion levels should be monitored by atomic absorption. Resistance development against metal ions in long term use is possible (Rohr et al., 1999).

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Copper and silver ionization offers a good alternative to other disinfection methods. Several reports support the elimination of the Legionella from water systems. Many researchers reported efficient elimination of L. pneumophila from water distribution systems by copper-silver ionization (Colville et al., 1993; Miuetzner et al., 1997; Chen et al., 2008). However, some opposite reports are confusing. In some hospitals legionellae have been recovered from water systems and cases of LD have occurred despite copper-silver ionization (Hoebe and Kool 2000).

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Chapter VI

Epidemiology

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Legionellosis Legionellosis presents in two different forms; Legionnaries’ Disease (LD, a severe pneumonia) and Pontiac Fever (a self-limited flu-like illness), but opportunistic extrapulmonary syndromes may also occur in rare cases particularly in immunocompromised patients (Shelton et al., 2000; Lowry and Tompkins, 1993). In addition, many persons who are infected with Legionellae, as proven by seroconversion, remain asymptomatic (Boshuizen et al., 2001). Most legionellosis cases are associated with L. pneumophila, in particular, strains belonging to serogroup 1 among over 50 Legionella species. L. pneumophila sg 1 is the cause of approximately 90% of all cases of LD from which a bacterial strain was isolated (Benin et al., 2002; Yu et al., 2002). However, in Australia and New Zealand, L. pneumophila sg 1 accounts for only approximately 50% of cases of community-acquired legionellosis, while L. longbeachae accounts for approximately 30% of cases (Yu et al., 2002). It is possible to further subtyping the L. pneumophila sg1 strains using various phenotyping or genotyping techniques. In a previous study, Helbig et al., (2002) showed that while L. pneumophila sg1 was responsible 85% of the total LD cases, one of the specific subtype of L. pneumophila sg1caused 67% - 90% of these cases. LD can be seen in a broad spectrum of illness from a mild cough and slight fever to coma and death (Yu, 1990). The attack rate is usually low; below 1%. (Den Boer et al., 2002). However, the case-mortality rate of adequately treated LD varies from 7% to 24%, with elderly and immuno-compromised patients

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being most susceptible (Steinert et al., 2002). The differentiation of the LD from pneumococcal pneumonia is not clinically possible (Diederen, 2008). Nonspecific symptoms may include fever, malaise, lethargy, chest pain, hyponatremia, non-productive cough, dyspnea and diarrhea (Shelton et al., 2000). The chest radiography commonly indicates alveolar filling, focal infiltrates, and lung consolidation occasionally with pleural exudation. However, LD can not be separated from other types of pneumonia with the chest radiography findings (Edelstein and Cianciotto, 2006). Accurate medical diagnosis depends to microbiological testing when a patient is in a high-risk category. LD may be classified in thee groups consisting of travel associated, community acquired and hospital acquired (nosocomial) cases. In every cases LD can be seen in sporadic cases or as outbreaks (Diederen 2008). Community acquired LD shows more severe symptoms than the nosocomial cases probably due to delayed diagnosis. Additionally neurological and gastrointestinal symptoms are seen more frequently in community acquired than hospital acquired LD cases (Sabria and Yu, 2002). The second form of Legionellosis is Pontiac Fever. This disease is non-fatal, non-pneumonic and is accompanied by symptoms similar to a mild flu. Contrary to LD, Pontiac fever has a high attack rate and it can affect up to 95% of exposed individuals (Glick et al., 1978). It is a self-limiting disease of short duration and does not need any therapy. The main symptoms are tiredness, high fever and chills, myalgia, headache and arthralgia and generally appear from a few hours to a few days after exposure (Diederen 2007). Since L. pneumophila has not been isolated from Pontiac fever patients it has been speculated that Pontiac fever is caused by VBNC cells of Legionella (Steinert et al., 1997). However, others explain Pontiac fever by changes in virulence factors, toxic or hypersensitivity reactions (Kaufmann et al, 1981). LD may occur as sporadic cases or outbreaks and in both instances the disease can be acquired from community or nosocomially. While sporadic cases are dispersed over the year, outbreaks become frequent in the summer and autumn seasons (EWGLI, 2005). This may be attributed to warmer conditions promotes the multiplication of L. pneumophila in reservoirs. Especially in temperate countries many cases or clusters of LD may be associated with travelling (Hutchinson et al., 1996). EWGLI describes travel associated cases as “Cases who in the ten days before onset of illness stayed at or visited an accommodation site that has not been associated with any other cases of Legionnaires’ disease, or cases that stayed at an accommodation site linked to other cases of Legionnaires ’ disease but more than two years previously” (EWGLI, 2005).

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Retrospective studies have revealed the origins of the confirmed LD cases in Europe and US. In a European wide study, the distribution of the 1.334 unrelated clinical L. pneumophila isolates collected between 1991 and 2000 according to the category of LD was as follows; 44.9% community acquired 29.01% nosocomial and 26.01% travel associated. According to the same study the distribution of L. pneumophila serogroups 1, 4 and 5 that have virulence associated epitope recognized by the Dresden panel were higher in nosocomial strains (53.5%) than in community acquired cases (27.3%) and travel-associated strains (14.2%) (Helbig et al., 2002). Benin et al. (2002) also analyzed the 6757 confirmed cases of LD between 1980 and 1998 in US and found that 21% of the cases were travel associated and 35% were nosocomial. They also revealed that of the nosocomial cases for which outbreak information was available, 28% were associated with an outbreak. Most of the LD cases occur sporadically (Marston et al., 1997). Most of the reported cases of LD between 1980 and 1989 were occurred as sporadic cases in US notwithstanding they are nosocomial or community acquired. Only 11% of the confirmed cases of LD were found outbreak related (Marston et al., 1994). The estimation of the true incidence of Legionellosis worldwide is difficult, because countries differ greatly in their surveillance system. Also clinicians and microbiology laboratories may overlook the disease since clinical manifestations of LD are not distinguishable from other bacterial pneumonias (Koneman et al., 1992; Fields et al., 2002). A lack of clinical awareness among clinicians is another reason for the failure to diagnose. In addition, L. pneumophila strains are fastidious and could not easily be detected by conventional methods (Fields et al., 2002). Even if the LD is truly diagnosed in a hospital, that may not get reported to the responsible department because of the lack of the awareness. It is estimated that less than 5% of cases may eventually be reported to public health authorities through passive surveillance (EWGLI, 2005). Moreover, incidence may be change between different populations. While the incidence was found 1% in volunteer groups, it can increase up to 40% in hospitalized community acquired patients. The incidence of the LD among all community acquired pneumonias is between 2-5% (Yu, 1990; Pasculle, 1992; Marston et. al. 1994). LD can be seen in all ages (7 moths-84 years) but most cases tend to occur at over 50 years in people who have impaired respiratory and cardiac function, heavy smokers or immunocompromised patients, hematologic malignancies, AIDS and lung cancer patients and in male gender (Franzin et al., 2001; England, et. al. 1981; Marston et al, 1994; Diederen, 2008). Interestingly LD seems extremely rare among children. The mortality rate of LD changes between 5-40%.

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It can be higher (50-80%) in nosocomial cases (Sullivan, 2001). In a previous study mortality rate was found 14% for nosocomial infections and 5-10% for community acquired infections in the US and Australia (Benin et al., 2002; Howden et al., 2003).

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Treatment L. pneumophila is not sensitive against penicillin and cephalosporin since it produces a wide spektrum of - Lactamases (Pasculle, 1992; Koneman, et. al. 1992). Together with all ß-lactam agents and penems, aminoglycosides, glycopeptides and chloramphenicols are also ineffective for treatment of LD (Edelstein and Cianciotto, 2006). Erythromycin was the first drug of choice for LD. The recommended daily dose for erythromycin is quite high (2.0-4.0g) and can cause ototoxicity (Yu, 1990). L. pneumophila found intracellularly in humans and the antibiotic should reach the effective concentration in the host cells not in the blood plasma. Rifampicin was also found effective against L. pneumophila but a combination with erythromycin is recommended (Yu, 1990; Edelstein, 1995). However, intracellularly grown L. pneumophila is resistant to as high as 8mg/ml Erythromycin (Barker et al., 1995) and the number of therapeutic failures with erythromycin, as well as the side-effects and drug interactions, led to the consideration of other drugs such as the new macrolides and quinolones for the treatment of LD (Stout and Yu, 1997; Pedro-Botet and Yu, 2006). Azithromycin and many fluoroquinolone agents have greater in vitro activity against Legionella species and better intracellular penetration. In addition, these agents have fewer side effects than erythromycin. Five to 10 days of azithromycin therapy is recommended for most of LD cases (Stout et al., 1997; Edelstein, 1998). The patient’s response to treatment depends on many factors including the age, other diseases, severity of the infection and the immune status. Delay in treatment leads to increased mortality. Based on the above factors the untreated disease causes 580% fatality.

Mode of Transmission LD is transmitted from the environment to man primarily via the inhalation of infectious aerosols (Fraser, 1980; Muder et al., 1986; Blatt et al., 1993).

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Generally, any water source that may produce aerosols between 1-5 m droplet sizes should be considered a potential source for the transmission of L. pneumophila. L. pneumophila has a low attack rate which is 1-5% among who’s exposed for an airborne pathogen (Yu, 1993). This is partly explained by aerosols being infectious only if they are within a certain size range (Rowbotham, 1992). Particles smaller than 5 m can be easily reach the lower respiratory tract and cause infection in susceptible people. Many aerosol producing devices have been associated with LD outbreaks including cooling towers (Dondero et al, 1980; Isozumi et al, 2005; Kirrage et al., 2007) evaporative condensers (Breiman et al., 1990), showers (Cordes et al., 1981), water taps (Ott et al., 1991), whirlpool spas (Den Boer et al., 2002; Armstrong and Haas, 2007), humidifiers (Arnow et al., 1982), decorative fountains (Hlady et al., 1993) and misters (Mahoney et al., 1992). However in some cases, microaspiration or direct instillation of contaminated water into the lungs could be the mode of transmission (Marrie et al., 1991; Yu, 1993; Venezia et al., 1994). In addition to aerosolization and aspiration, ingestion is also described as a mode of transmission in rare cases (White et al., 1980). Essentially, all modes of transmission may play a role in LD; with airborne transmission more likely in cases of travel associated illness and outbreaks, and aspiration in nosocomial cases (Maiwald et al., 1998). Interestingly, there is no evidence for person-to-person transmission of LD (Yu et al., 1983; EWGLI, 2005).

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Chapter VII

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Pathogenity Interaction of the L. pneumophila with the protozoa is considered to be at the central importance of pathogenesis of L. pneumophila. Abu Kwaik et al. (1998) and Harb et al. (2000) reviewed the role of protozoa in LD intensively. Protozoan host support intracellular bacterial replication and increase in resistance to harsh conditions such as high temperature, acidity and high osmolarity and also the bacteria exhibit a dramatically enhanced ability to infect mammalian cells in vitro. In addition, it has been demonstrated that intracellular bacteria within Hartmannella vermiformis are more infectious and also nonculturable L. pneumophila can be achieved by coculture with protozoa. It can explain the failure of isolating the bacteria from environmental sources of infection on artificial media. The strategies of L. pneumophila to survive and replicate are remarkably similar in protozoon and mammalian cell. During long-standing evolutionary interaction between aquatic protozoa and L. pneumophila it has been contemplated that this relation may generate the bacterium’s virulence traits which adapted this pathogen to infect human cells (Steinert et al., 2002; Molmeret et al., 2005; Brüggemann et al., 2006). Furthermore, L. pneumophila reflects its coevolution and intimate relationship with its eukaryotic hosts by possessing many eukaryotic-like genes in its genome (Khemiri et al., 2008; Brüggemann et al., 2006; Jules and Buchrieser 2007; Steinert et al., 2007; Albert-Weissenberger et al., 2007). Steinert et al., (2007) reviewed thoroughly the role of horizontal gene transfer during the evolution of legionellae and its importance for the intracellular lifestyle of this pathogen. The complete genome analysis of legionellae showed that the bacterium has certain protein-encoding genes which

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are of foreign origin. In contrast to the whole genome G+C ratio of legionellae, there are large regions where different existing G+C content in genome have been found. Recently, Glökner et al. (2008) have identified and analyzed a new type of conjugation/type IVA secretion system (trb/tra) of L. pneumophila. They concluded that this new type secretion system may explain the horizontal gene transfer of chromosomal DNA. While the mechanism of intracellular replication of L. pneumophila is similar, attachment mechanism and receptors of the host cell are different within amoebae and alveolar macrophage (Bozue and Johnson, 1996; Horwitz and Silverstein, 1981). L. pneumophila attached to H. vermiformis mediated by Gal/GalNAc (galactose/N-acetyl-D-galactosamine) lectin receptors (Venkataraman et al., 1997). Whereas, in the attachment mechanisms of L. pneumophila to entry into Acanthamoeba polyphaga there is no host protein synthesis required (Harb et al., 1998). The importance of the protozoan-bacterium interaction in transmission of L. pneumophila has been shown (Berk et al., 1998). Authors reported that the Acanthamoeba species secreted vesicles surrounded by membrane filled with viable L. pneumophila, can easily spread to air as an aerosol. During the course of infection, L. pneumophila is phagocytosed by alveolar macrophages through conventional or coiling phagocytosis (Horwitz, 1984; Rittig et al., 1999; Steinert et al., 2002; Molmeret et al., 2004). Complement component C3 and subsequent binding to CR1 and CR3 receptors enhance the conventional phagocytosis of the bacteria into macrophages. Entry of L. pneumophila into monocytes by coiling phagocytosis mostly appears when the legionellae are grown in amoebae (Cirillo et al., 1999). During this mechanism, engulfment takes place by the extension of a unilateral pseudopod, which coils around the bacterium (Rittig et al., 1999). The bacteria reprogram the endocytic pathway within minutes of internalization by altering the endosomal-lysosomal degradation pathway of the phagocytic cell. Phagosome traffic is modified by preventing phagosomelysosome fusion and as a result creates a suitable niche for intracellular replication of L. pneumophila (Horwitz, 1983a; Horwitz, 1983b; Hilbi et al., 2001; Scott et al., 2003; Brüggemann et al., 2006). The ability to avoid phagosome–lysosome fusion is the key virulence factor of L. pneumophila and in case of any mutation in this trait, infection of the host cell is prevented (Horwitz, 1987; Horwitz, 1983b). Intracellular replication takes place in this specialized phagosome which is surrounded by rough and smooth endoplasmic reticulum (ER) and mitochondria, called replicative phagosome or legionellae-containing vacuole (LCV) (Swanson and Isberg, 1994; Tilney et al., 2001; Cianciotto, 2001;

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Pathogenity

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Kagan and Roy, 2002; Steinert et al., 2007; Ragaz et al., 2008). The morphology of LCV is similar in protozoan and mammalian cells and operates with Dot/Icm type IV (defective organelle trafficking/ intracellular multiplication) secretion system that is encoded by 25 genes located on two locus called dot and icm (Steinert et al. 2007; Sadosky, et al. 1993; Edelstein, et al. 1999; AlbertWeissenberger et al., 2007). Tilney et al. (2001) found that phagosomes containing L. pneumophila dotA mutants did not have ER vesicles or mitochondria attached to their surface. The virulence of L. pneumophila is mainly associated with the Dot/Icm type-IV secretion system and the lsp type II (Legionella secretion pathway) secretion system. Dot/Icm type-IV secretion system which is functionally homologous to conjugation systems secreted virulence factors that inhibit phagosome-lysosome fusion (Cianciotto, 2001; Albert-Weissenberger et al., 2007). Lsp type II protein secretion system secretes many degradative enzymes and this system is dependent upon the type IV prepilin peptidase (PilD). Rossier and Cianciotto (2001) studies demonstrated that a mutation within the PilD peptidase gene is the reason for a sharp reduction in virulence and an even more remarkable reduction in intracellular infection within amoebae and macrophages than the effects of a mutation in type II secretion system or type IV pilus genes. These findings suggest the existence of another potentially novel pilD-dependent mechanism for promoting L. pneumophila intracellular infection of human cells. Besides, Lsp secretion system is also critical to infect both protozoan and macrophage hosts so mutations within the lps genes reduce the infectivity of L. pneumophila (Rossier and Cianciotto, 2001; Cianciotto, 2001). During the intracellular replication within protected LCV, L. pneumophila converts to a replicative form (exponential phase) that is sodium resistant, unflagellated and show reduced virulence factors (Byrne and Swanson, 1998; Hammer et al., 2002; Swanson and Fernandez-Moreira, 2002; Molofsky and Swanson 2004; Steinert et al., 2007). As a consequence, the Legionella containing vacuole fuses with lysosome where L. pneumophila replicates until nutrients become exhaust. Nutrient limitation then leads to the transition to transmissive form (stationary phase) that express many virulence-associated factors allowing the pathogen to escape from its host cell for transmission and survive in the new host cells (Byrne and Swanson 1998; Molofsky and Swanson, 2004). This cellular differentiation creates legionellae biphasic life cycle and the entry into the transmissive phase is probably initiated by stringent response via (p)ppGpp alarmones (Hammer and Swanson 1999; Hammer et. al., 2002; Steinert et al., 2007). L. pneumophila infection results in the death of the macrophage by

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Atac Uzel and E. Esin Hames-Kocabas

apoptosis and late necrosis which is mediated by a pore-forming activity (Gao and Abu Kwaik, 1999; Alli et al., 2000; Molmeret and Abu Kwaik, 2002; Molofsky and Swanson, 2004). However in protozoan hosts, Lep proteins (LepA and LepB) that are the effector proteins of the Dot/Icm system have a role in the release of intracellular pathogens (Ninio and Roy, 2007). Isberg et al. (2009) have recently published a detailed review concerning the internalization and intracellular replication of the L. pneumophila. A number of surface structures of L. pneumophila are important in its pathogenesis. Lipid A which is located in lypopolysaccharide layer (LPS), has a moderately weak endotoxic activity; on the other hand some studies showed a correlation between the LPS expression and virulence. Another surface structure is a porin, major outer membrane protein (MOMP) which is encoded by ompS binds the complement components and mediates the uptake of L. pneumophila via the CR1 and CR3 receptors of the macrophage (Steinert et al., 2002; Edelstein and Cianciotto, 2006). L. pneumophila produces hairlike-projections known as type IV pilus which are promote attachment to host cell and biofilms as well as the 60-kDa heat shock protein Hsp60 (Stone and Abu Kwaik 1998; Liles et al., 1998; Steinert et al., 2002). Flagella do not play a role in intracellular replication of L. pneumophila however, independent of adherence; they increase the invasion ability of the pathogen into host cells (Dietrich et al., 2001). One of the first characterized virulence factors of Legionella is the macrophage infectivity potentiator protein (Mip) (surface-exposed homodimeric membrane protein) which is encoded by the mip gene. The Mip protein that exhibit propyl-proline isomerase (PPIase) activity is required for the early stages of intracellular infection of macrophages, protozoa, and lung epithelia (Fischer et al., 1992; Cianciotto et al., 1990; Cianciotto, 2001; Albert-Weissenberger et al., 2007). Secreted factors of L. pneumophila have been reviewed by Edelstein and Cianciotto (2006) in detail. Variety pigments, degradative enzymes and putative toxins are produced in L. pneumophila when grown in bacteriological media. Mutations within the IIy gene which requires the production of brown pigment, do not effect on intracellular infection. Partially purified heat-stable peptide, putative toxins of L. pneumophila reduce the oxidative burst in neutrophils, but its importance can not be evaluated thoroughly by reason of inability for purifying and defining its genetic bases. In recent years, microarray analyses have been used to reveal L. pneumophila virulence traits. During its life cycle L. pneumophila can shift its phenotype between infectious and replicative form that grows intracellularly in macrophages or amoebae. Recently, Edwards et al. (2009) have identified 22 inducers in cell

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Pathogenity

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differentiation of L. pneumophila by using Phenotype Microarrays. They found that the short chain fatty acid excess triggers the stringent response by using SpoT and eventually shift its phenotype. Tiaden et al. (2007) used DNA microarrays for analysing the transcriptome profiles of wild type L. pneumophila and lqsR mutant strains. They showed that in the stationary growth phase lqsR regulates a number of virulence or transmission factors potentially involved in cellular interactions between L. pneumophila and host cells.

Produced by E.E. Hames-Kocabas and A. Uzel. Figure 5. Life cycle of L. pneumophila. A. L. pneumophila inhabits natural freshwater environments such as lakes, rivers, streams and may enter to man-made environments by municipal water. These environments such as water distribution systems and cooling towers serve as effective bacterial amplifiers. B. L. pneumophila involves in biofilm formation and associate with fresh water protozoa both in natural and man-made environments. This relationships increase the resistance of L. pneumophila against harsh environmental conditions. C. Transmission to humans primarily occurs by inhalation of the contaminated aerosols. L. pneumophila enters the macrophage by conventional or coiling phagocytosis then inhibits phagosome-lysosome fusion. It replicates intracellularly in the Legionella containing vacuole (LCV) and leaves the host cell.

There is a variable and relatively minor effect of growth temperature on L. pneumophila virulence and intracellular replication (Edelstein et al., 1987). L.

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pneumophila produce two catalase-peroxidases to eliminate the reverse effect of H2O2 by converting to water and oxygen (Bandyopadhyay and Steinman, 1998). The iron acquisition functions of L. pneumophila also play a key role in pathogenesis as well as a number of other loci, including the pts (phosphoenolpyruvate phosphotransferase), eml (early stage macrophage induced locus), milA (macrophage-specific infectivity loci), enh (enhanced entry) rib (release of intracellular bacteria), rtxA (repeats in toxin) genes (Harb et al., 2000; Gao et al., 1997; Gao et al., 1998; Harb and Abu Kwaik, 2000; Alli et al., 2000; Cianciotto, 2001; Edelstein and Cianciotto 2006). The overall life cycle of L. pneumophila was shown in Figure 5.

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Chapter VIII

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Diagnose For the specific laboratory diagnosis of Legionella infections, four standardized methods are used. These are culture of legionellae, direct immunofluorescence assay (DFA), detection of antigens in urine and detection of specific antibodies. Currently the other method for diagnosis of Legionella, using PCR, is being used as a research tool. The methods have improved since the first documented outbreak of legionellosis in 1976. However there is currently no available test able to diagnose all Legionella spp in the desired quality in respect with sensitivity and specificity. In addition, diagnosis is generally effective only for L. pneumophila sg 1 (Diederen, 2008; Lück et al., 2002). Although, a number of diagnostic methods have been developed during the last three decades, diagnostic tests for legionellosis have changed significantly since 1995. The methods at present used for diagnosing LD are culture, detecting Legionella antigens (urinary antigen detection), DFA, detection of specific antibodies in serum samples and detection of bacterial DNA (Den Boer and Yzerman, 2004; Hornei et al., 2007). Legionellae can be isolated from a number of specimens such as blood, lung tissue, lung biopsy specimen, respiratory secretions (sputum, BAL and bronchial aspirates) and stool (Fields et al., 2002).

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Culture

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Culture is still considered as the “gold standard” for diagnosis of legionellosis among the diagnostic methods (Edelstein et al., 1982; Edelstein 1993; Fields et al., 2002; Van der Kooij et al., 2005). Generally, the specificity is near 100% and the sensitivity is near 60% (30-80%) (Maiwald et al., 1998). The culture for diagnosis of L. pneumophila is important especially when the specimens are collected from immunocompromised patient, hospitalized patients or suspicion of etiologic agent is other than L. pneumophila sg 1 and in severe pneumonial diseases (Hornei et al., 2007). Due to the fact that some Legionella-like amoebal pathogens cannot be grown on routine Legionella culture media, amoebae have been used to recover an uncultured bacterium by co-cultivating (Fry et al., 1991). Identification and confirmation procedures of Legionella colonies are the same whether the isolates are from clinical or environmental samples (Hornei et al., 2007). L. pneumophila colonies have cut-glass appearance and are speckled green, blue or pink-purple iridescence (Barker et al., 1986; Maiwald et al., 1998). In addition, presumptive identification of L. pneumophila should be confirmed on the basis of L-cysteine dependency using cysteine-free agar (Barker et al., 1986).

Direct Immunofluorescence Assay (DFA) DFA was the first and rapid microscopic examination method for detection of infections from lung tissue and respiratory secretions even if non-culturable or non-viable legionellae are present (Maiwald et al., 1998; Fields et al., 2002). During outbreaks of the disease, this assay is particularly useful for rapid diagnosis of Legionella (Maiwald et al., 1998). DFA assay has also been used for serologic identification of Legionella isolates from clinical or environment samples. Fluorescein-conjugated antibody (antisera) can be used for the rapid identification of isolates from sputum or lung biopsy specimens (Edelstein and Edelstein, 1989). Staining procedure takes 2-3 hours and should not typically be used for other than L. pneumophila species (Lück et al., 2002). A monoclonal, highly specific fluorescein-isothiocyanate-conjugated antibody to an outer membrane protein of L. pneumophila is commercially available. Monoclonal antibodies (MAbs) are highly specific for only L. pneumophila and reacts with all serogroups of this species (Maiwald et al., 1998).

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The sensitivity of DFA assay has been documented to range from 25% to 75% and is highly specific >95 % (Edelstein, 1987). The difference in the sensitivity rate is due to the type of specimen, technical equipment and laboratory experience (Edelstein, 1993). Fluorescein-conjugated serogroup specific polyclonal antisera may cause cross-reaction with a variety of other bacteria including Pseudomonas spp. (Edelstein and Edelstein, 1989), Corynebacterium spp. (Summersgill and Snyder, 1990), Francisella tularensis (Roy et al., 1989), Bacteroides fragilis (Edelstein and Edelstein, 1989), and Bordetella spp. (Benson et al.,1987). This cross-reaction does not affect the results critically when isolated colonies on artificial media are used because the result can be evaluated with colony morphologies and cysteine dependency (Maiwald et al., 1998). However fluorescein-conjugated polyclonal antisera may cause significant problems in the case of direct detection of infectious agent from specimen.

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Detection of Antigens in Urine The detection of Legionella antigenuria has been used since shortly after the first outbreak in Philadelphia (Tilton, 1979; Berdal et al., 1979). The urinary antigen of L. pneumophila can be detected by using enzyme immunoassays (EIA) (Chang, et al., 1996; Hackman et al., 1996; Kazandjian et al., 1997) and immunochromatographic assays (Helbig et al., 2001). The early studies for antigen detection in urine specimens resulted in the development of commercial radioimmunoassay have similar performance with EIA but due to the difficulty involved in the manipulation of radioactive isotopes and requirement of special licenses to perform, RIA test was replaced by an ELISA in the mid-1980s (Hackman et al., 1996; Dominguez et al., 1997). EIA is more easily applied and its results are on a comparable level to RIA. The EIA urine antigen test has 8085% sensitivity, which shows no significant difference to the results obtained by culture or serology and its specificity is ca 99.5% (Hackman et al., 1996; Kazandjian et al., 1997). Concentration of the antigen present in urine specimen improves the sensitivity of both methods without decreasing their specificity (Dominguez et al, 1996; Dominguez et al, 1997). Ramirez and Summersgill (1994) compared urinary antigen testing, DFA and culture and concluded that urinary antigen detection was the most useful one. On the other hand in some cases such as patients with mild pneumonia, there may be

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under-diagnosis if urine antigen test alone was used (Yzerman et al., 2002; Kohler et al., 1984). Since urine samples can be obtained easily and permit early diagnosis of Legionella antigenuria, this assay allowed a rapid public health response with reasonable sensitivity and high specificity even after the initiation of the specific legionellosis antibiotic therapy (Berdal et al., 1979; Tilton, 1979; Kashuba and Ballow, 1996). The urinary antigen detection test is substantially more sensitive in the majority of community acquired and travel-associated LD when Pontiac L. pneumophila sg 1 is the causative agent. In cases of nosocomial disease, when L. pneumophila serogroups other than sg 1 are frequent, this assay has limitations because the test uses monoclonal antibodies (MAb) MAb2 or MAb3/1 (Lück et al., 2002; Hornei et al., 2007). The target antigen of this assay is a portion of the lipopolysaccharides (LPS) of the Legionella cell wall which is a determinant of serogroup specificity has been used for diagnosis of LD (Neumeister et al., 1998; Ciesielski et al., 1986; Brenner et al., 1988). Rapid antigen detection based on immunochromatographic (ICT) membrane assays for L. pneumophila sg 1 in urine is also available (Dominguez et al., 1999; Wever et al., 2000; Diederen and Peeters, 2006). As the test considerably reduces the time needed for detection and does not require specialized laboratory equipment, it can be recommended for the detection of L. pneumophila sg 1. In addition, ICT assays (the Binax NOW legionellae urinary antigen tests) have been developed in order to provide levels of sensitivity and specificity similar to those of the EIA (Dominguez et al., 1999; Helbig et al., 2003; den Boer and Yzerman. 2004). Diederen and Peeters, (2006) compared two new ICT assays with Binax NOW and concluded that the performances of these new tests are below the acceptable level for diagnostic assay.

Detection of Specific Antibodies The indirect immunofluorescence assay (IFA) was initially developed to detect antibodies after the 1976 outbreak (McDade et al., 1977). Since then, a number of serologic tests have been developed to detect antibodies to different species and serovars of Legionella such as the indirect haemagglutination (IHA) test (Yonke et al., 1981), ELISA, microagglutination test (Farshy et al., 1979; Farshy et al., 1978; Klein et al., 1979) and the immunoblot analysis (Sampson et al., 1986). However, among these methods, IFA and ELISA have found

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widespread application (Malan et al., 2003). Only IFA is well standardized for measurement of antibody to L. pneumophila sg 1 and ELISA can then be confirmed with the L. pneumophila sg 1 IFA. The sensitivity of antibody detection is generally limited by the time required for seroconversion. The required fourfold rise in antibody titer can be detected by IFA (den Boer and Yzerman, 2004). Seroconversion takes about two weeks but in some cases, this may take more than 10 weeks. Therefore an antibody titer does not allow the diagnosis during the acute illness. On the other hand, some patients do not develop sufficient increase in antibody titer during the prolonged monitoring (Monforte et al., 1988; Maiwald et al., 1998; Diederen 2008). The sensitivity of commercial kits for antibody to L. pneumophila is ranging from 41% to 94%. The specificity of L. pneumophila sg 1 antigen is at least 99% (Wilkinson et al., 1981; Diederen, 2008; Edelstein and Cianciotto 2006).

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Detection of Legionella Nucleic Acids Nucleic acid base methods such as PCR are candidate to be part of routine tests of L. pneumophila due to the long culture times, non-viable and/or nonculturable strains and cross-reaction in serological tests. The first use of PCR for clinical samples was reported by Jaulhac et al. (1992). Urinary antigen assay and nucleic acid based PCR analysis are useful for rapid diagnosis of the LD. On the other hand, although most diagnostic rapid and simple test diagnose the LD caused by L. pneumophila sg 1, PCR has a potential to detect all serogroups of L. pneumophila therefore it is useful in the diagnosis of nosocomial infections (Fields et al., 2002). While PCR techniques provide highly sensitive and rapid diagnostic tests for all Legionella species, there are existing limitations in current techniques making validation difficult (Waterer et al., 2001). A commercial first radiolabeled DNA probe design for environmental samples has been used for the detection of L. pneumophila rRNA, is no longer produced due to varying sensitivity and specificity (Fields et al., 2002). The detection of Legionella by PCR has been used experimentally in environmental and clinical samples, but still needs further validation for widely adoption (Edelstein and Cianciotto, 2006). In addition, most clinical studies which are to detect Legionella by using PCR have revealed more number of positive results compared to the conventional methods such as DFA and culture (Cloud et al., 2000; She et al., 2007; Lück et al. 2002). PCR is not an appropriate method for

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non-specific screening of clinical specimens because the evaluation of a positive PCR result should be confirmed by other methods in order to demonstrate if there is a false positive result or a real increase of sensitivity. AP-PCR method has been used mostly for intraspecies discrimination for identify sources of disease-causing strains in epidemiological investigations. Hayden et al. (2001) have compared LightCycler PCR (LC-PCR), in situ hybridization (ISH), DFA, and culture methods for direct detection of legionellae species from 43 specimens (BAL and open lung biopsy specimens). The result of this comparative study has shown that culture was the best method for detecting Legionella species in lung tissue and Legionella genus LC-PCR, and L. pneumophila-specific ISH were useful as a rapid test. Comparison of the diagnostic methods is presented at Table 2.

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Table 2. Diagnostic methods for Legionnaires’ disease Method

Sample type

Sensitivity (%)

Specificity (%)

Comments

Culture

Respiratory tract secretion, blood

30 - 80

100

Gold standard, require average 7 days

DFA testing

Respiratory tract secretion

25 - 75

>95

Urinary antigen

Urine

80 - 85

99.5

Serolog ical tests

Serum

41 - 94

>99

Very rapid, procedure takes 2–3 hours, not useful for nonpneumophila species Limited with L. pneumophila sg 1, very rapid (within hours) Time required for seroconversion (2-10 weeks) (L. pneumophila sg 1 antigen)

DFA; Direct immunofluoresence assay.

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Conclusion Since the discovery of the L. pneumophila, many aspects of its biology have been revealed. Currently, we know that L. pneumophila is an opportunistic intracellular pathogen and can infect very diverse hosts including protozoa and mammalian cells. Humans are considered as accidental hosts of this bacterium. L. pneumophila is able to survive and multiply in human macrophages by altering the endocytic pathway. Type IV secretion system, encoded by the dot/icm genes is the center for pathogenesis. There may be other hosts for intracellular multiplication yet to be discovered. Legionellae can multiply in man-made environments such as water distribution systems and cooling towers. Transmission from these reservoirs to man is airborne and primarily occurs via the infective aerosols. It is worth to investigate why no person to person transmission has been reported until now. Association with biofilms and free living protozoa protects L. pneumophila from harsh conditions and it is relatively more resistant than other pathogenic bacteria to chemical and physical disinfection. Although development of several control techniques it is still difficult to eradicate legionellae from water systems. From the viewpoint of public health perspective legionellae-free drinking water remains a challenge.

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Bartie, C., Venter, S.N., Nel, L.H. (2003). Identification methods for Legionella from environmental samples. Water Res, 37, 1362-70. Bartie, C., Venter, S.N., Nel, L.H., (2001). Evaluation of Detection Methods for Legionella Species Using Seeded Water Samples. Water SA, 27, 523–527. Behets, J., Declerck, P., Delaedt, Y., Creemers, B., Ollevier, F. (2007). Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples. J. Microbiol. Methods, 68, 137-44. Bej, A.K., Mahbubani, M.H., Atlas, R.M., (1991). Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods. Appl. Environ. Microb, 57, 597-600. Benin, A.L., Benson, R.F., Besser, R.E. (2002). Trends in legionnaires disease, 1980–1998: declining mortality and new patterns of diagnosis. Clin. Infect. Dis, 35, 1039-46. Benson, R.F., Thacker, W.L., Plikaytis, B.B., Wilkinson, H.W. (1987). Crossreactions in Legionella Antisera with Bordetella pertussis Strains. J. Clin. Microbiol, 25, 594-596. Berdal, B.P., Farshy, C.E., Feely, J.C. (1979). Detection of Legionella pneumophila antigen in urine by enzyme-linked immunospecific assay. J. Clin. Microbiol, 9, 575-8. Berk, S.G., Ting, R.S., Turner, G.W., Ashburn, R.J. (1998). Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl. Environ. Microbiol. 64, 279-86. Blatny, J.M., Reif, B.A.P., Skogan, G., Andreassen, O., Høiby, E.A., Ask, E., Waagen, V., Aanonsen, D., Aaberge, I.S., Caugant, D.A. (2008). Tracking Airborne Legionella and Legionella pneumophila at a Biological Treatment Plant. Environ. Sci. Technol, 42, 7360-7. Blatt, S.P., Parkinson, M.D., Pace, E., Hoffman, P., Dolan, D., Lauderdale, P., Zajac, R.A., Melcher, G.P. (1993). Nosocomial Legionnaires’ disease: aspiration as a primary mode of disease acquisition. Am. J. Med, 95, 16-22. Bopp, C.A., Sumner, J.W., Morris, G.K., Wells, J.G. (1981). Isolation of Legionella spp. from environmental water samples by low-pH treatment and use of a selective medium. J. Clin. Microbiol, 13, 714-9. Boshuizen, H.C., Neppelenbroek, S.E., van Vliet, H., Schellekens, J.F., Boer, J.W., Peeters, M.F., Spaendonck, M.A. (2001). Subclinical Legionella infection in workers near the source of a large outbreak of legionnaires disease. J. Infect. Dis, 184, 515-8.

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Index

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A absorption, 25, 35 accidental, xi, 55 accommodation, 38 accuracy, 60 acid, xii, 16, 23, 24, 25, 27, 28, 47, 53, 65, 72 acidification, 16 acidity, 43 Acinetobacter, 9 acute, 53 adaptation, 71 aerobic, 2 aerosols, xii, 2, 14, 21, 40, 41, 44, 47, 55, 60 agar, 17, 18, 23, 24, 50, 65, 70, 76 age, 24, 40 agents, 1, 10, 14, 31, 32, 35, 40, 50, 51, 52, 58, 60, 64, 71, 80 agglutination, 19, 26 AIDS, 39 air, 6, 7, 13, 14, 18, 44, 60, 64, 77 albumin, 17, 76 algae, 7 alpha, 17 alternative, 16, 19, 28, 33, 34, 36 alveolar macrophage, 44 amino, 2, 24 amino acids, 2, 24 aminoglycosides, 40

antagonistic, 9 Antarctic, 6, 61 antibiotic, 1, 23, 40, 52, 58, 71 antibody, xii, 20, 25, 29, 50, 52, 53, 67, 77, 79, 80 antigen, xii, 27, 28, 49, 51, 52, 53, 54, 59, 61, 63, 64, 67, 68, 71, 72, 82, 85 Antisera, 59 apoptosis, 46 application, xii, 26, 29, 35, 53 aquatic habitat, xii, 5 arthralgia, 38 aspiration, 41, 59, 83 assessment, 76 asymptomatic, 37 atlas, 26, 58, 59 atmosphere, 18 attachment, 44, 46, 68, 83 atypical pneumonia, xi availability, 34 awareness, 39

B bacilli, 18 bacillus, 2 bacteria, xi, 6, 7, 9, 10, 11, 16, 17, 18, 19, 21, 23, 33, 43, 44, 48, 51, 55, 80, 84

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Index

88

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bacterial, 2, 6, 9, 15, 16, 23, 25, 32, 37, 39, 43, 47, 49, 57, 68, 75, 79, 80 bacterium, xii, 1, 3, 5, 10, 21, 23, 24, 25, 33, 34, 43, 44, 50, 55, 60, 65, 68, 69, 70, 75, 82, 83 BAL, 15, 49, 54 benefits, 9 best practice, 62 binding, 44, 69, 78 biofilm formation, 8, 9, 32, 47, 61, 74, 78, 84 biofilms, xii, 6, 7, 8, 9, 13, 20, 32, 33, 46, 55, 76, 79, 80, 81 biogenesis, 73 biopsy, 49, 50, 54, 68 birth, 66 blood, 1, 17, 18, 23, 40, 49, 54 blood plasma, 40 brain, 15 broad spectrum, 37 bronchoalveolar lavage, 15, 68, 70 buffer, 16 buildings, 6, 34, 64

C calcium, 33 capillary, 28 carbohydrate, 2, 23, 24 carbon, 18 carbon dioxide, 18 carcinogen, 33 cardiac function, 39 catalase, 24, 48, 58 CDC, 80 cell, xii, 3, 6, 7, 24, 25, 35, 43, 44, 45, 46, 47, 52, 75, 81, 82 cell differentiation, 47 cell growth, 6 cell line, 3 cephalosporin, 40 charcoal, 24, 76, 83 charge coupled device, 25

chemicals, 34 chemiluminescence, 25 chemotherapy, 64 chest radiograph, 38 children, 39 chlorination, 33, 34, 62, 81 chlorine, xii, 5, 14, 33, 35, 71, 76, 85 chocolate, 17 chromium, iii cis, 66 classification, 76, 78 clinical assessment, 81 clinician, 84 cloning, 58 clusters, 38 coil, 69 colonization, 6, 9, 13, 31, 34, 66, 73 coma, 37 community, xi, xii, 8, 13, 37, 38, 39, 40, 52, 69, 74, 75, 84, 85 complement components, 46 components, 46, 68 composition, 80 concentration, xii, 16, 21, 32, 33, 35, 40 conditioning, 7, 13, 58, 64 congress, vi conjugation, 44, 45, 67 consensus, 16, 19 consolidation, 38 construction, 7 contaminant, 16, 35 contamination, 13, 19, 25, 27, 29, 35, 77 control, xi, 27, 31, 33, 34, 35, 55, 58, 60, 62, 63, 65, 73, 79, 80, 81, 84 conversion, 82 cooling, xii, 6, 14, 20, 21, 33, 41, 47, 55, 64, 66, 70, 72, 80 copper, xii, 7, 8, 35, 36, 73, 75, 79, 81, 83, 85 correlation, 28, 46 corrosion, 33 corrosive, 33 corynebacterium, 9, 51 cough, 37

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Index couples, 65 critical points, 14 crystalline, 18 culture, xii, 3, 5, 6, 16, 17, 19, 20, 23, 27, 28, 31, 49, 50, 51, 53, 54, 61, 68, 69, 70, 74, 79, 82, 85 culture conditions, 61 culture media, 17, 50 cycles, 62 cycloheximide, 8, 17 cysteine, 1, 2, 10, 16, 17, 18, 23, 50, 51 cysts, 10, 32, 33, 71

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D dating, 1 death, 37, 45 degradation, 44 degradation pathway, 44 density, xii detachment, 81 detection, xii, 14, 19, 20, 25, 26, 27, 28, 49, 50, 51, 52, 53, 54, 58, 61, 63, 64, 67, 68, 69, 72, 73, 74, 75, 76, 78, 79, 80 diarrhea, 38 differentiation, 25, 26, 28, 38, 45, 47, 65, 69, 78, 83 discovery, 65 discrimination, 29, 54 diseases, xi, 50 disinfection, xii, 14, 31, 32, 33, 34, 35, 36, 55 dissolved oxygen, 5 distribution, xii, 2, 5, 6, 7, 8, 9, 10, 21, 32, 34, 36, 39, 47, 55, 66, 69, 73, 78, 81, 83 diversity, 16, 72 division, 68 DNA, xii, 3, 6, 19, 20, 25, 26, 27, 44, 47, 49, 53, 58, 63, 66, 70, 73, 74, 78, 83 DNA sequencing, xii, 19 drinking water, 35, 55, 69, 80 drug interaction, 40 drugs, 40

89

drying, 14 duration, 38, 72 dyes, 17 dyspnea, 38

E E. coli, 25 ecological, 7 ecology, 57, 65 ecosystems, 7 elderly, 21, 37 electrodes, 35 electrophoresis, 20, 29, 78 ELISA, 51, 52 encephalitis, 80 encoding, 43 endoplasmic reticulum, 44, 71, 78 energy, 2 environment, vi, xi, 3, 5, 6, 10, 19, 29, 32, 40, 50, 60, 72, 84 environmental conditions, 5, 7, 8, 9, 10, 32, 47 environmental contamination, 29 environmental factors, 69 enzyme immunoassay, 51, 61, 64, 71 enzyme-linked immunosorbent assay, 74, 82 enzymes, 45, 46, 82 epidemic, 67, 78, 84 epidemiology, 62, 69 epithelia, 46 epithelial cell, 57, 67 epitope, 39 Escherichia coli, 85 ester, 25 ethylene, 25 ethylene glycol, 25 etiologic agent, 50, 71 etiology, 67 eukaryotic cell, 2 Euro, 70 Europe, 39

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Index

90 evolution, 43 excretion, 72 exposure, ii, 10, 14, 32, 38, 60, 71

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F failure, 39, 43, 76 false negative, 19 false positive, 54 family, vii, 1, 58, 60, 83 fatty acids, 2, 23, 24 fever, xii, 37, 38, 67, 71 filtration, 16, 60 fingerprinting, 58, 67, 83 fire, 14 FISH, 20 flagellum, 24, 63, 69 floating, 20 flora, 8, 15, 16, 78 flow, 9, 25, 35, 74, 77 fluctuations, 32 fluid, 14, 15 fluorescence, 18, 68 flushing, 34, 81 fresh water, xi, xii, 5, 47, 57, 79 fungal, 15 fusion, xii, 44, 47, 60, 70

G gas, 24 gastrointestinal, 38 gel, 20, 29, 77 gender, 39 gene transfer, 43 generation, 27 genes, 20, 23, 25, 26, 27, 28, 43, 45, 46, 48, 55, 57, 58, 59, 61, 62, 65, 73, 76, 77, 78, 79, 81, 84 genome, 43 genomic, 67, 71 glass, 25, 50

glycine, 17 glycopeptides, 40 gold, 19, 50 gold standard, 19, 50 gram negative, 1, 2 grazing, 7 groups, 3, 24, 38, 39 growth, 1, 2, 3, 5, 6, 7, 8, 9, 10, 16, 17, 18, 23, 24, 25, 33, 47, 58, 61, 62, 64, 65, 77, 78, 79, 84 growth rate, 23 growth temperature, 3, 47, 65 guidelines, 31

H habitat, 5 hands, 62 harmful effects, 10 headache, 38 health, 21, 67, 80 heat, 6, 16, 26, 34, 46, 76, 81 heat shock protein, 46 heating, 7, 13, 17 hematologic, 39 heterogeneous, 7 heterotrophic, 6 high risk, 31, 38, 74 high temperature, 6, 43, 72 horizontal gene transfer, 43 hospital, 6, 14, 29, 31, 33, 34, 36, 38, 39, 57, 61, 62, 65, 66, 69, 71, 73, 75, 77, 79, 81, 82, 84 hospitalization, 74 hospitalized, 39, 50, 70 host, xi, 6, 7, 40, 43, 44, 45, 46, 47, 60, 63, 66, 68, 70, 71, 75, 80, 81, 82 hot spring, 6, 75, 76 hot water, xii, 6, 14, 20, 34, 62, 64, 66, 72, 75, 79, 81 hotels, 6, 13

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Index human, xi, 2, 11, 21, 29, 40, 43, 45, 47, 55, 57, 61, 69, 70, 78, 79 human exposure, 57 hybridization, xii, 19, 20, 25, 26, 28, 54, 58, 63, 68, 73 hydrogen, 35 hydrogen peroxide, 35 hydrolysis, 24 hypersensitivity reactions, 38 hyponatremia, 38

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I ICT, 52 identification, 1, 19, 23, 24, 25, 26, 27, 30, 50, 73, 78, 80, 84 IFA, 52, 53 immunoassays, 51 immunocompromised, 10, 21, 37, 39, 50 immunofluorescence, 19, 49, 52, 74, 84 in situ hybridization, 19, 20, 28, 54, 63, 68 in vitro, 3, 10, 35, 40, 43 inactivation, 58, 81 incidence, 39 incubation, 11 industrial, 13, 20, 26, 72 industrial wastes, 26, 72 inertness, 24 infection, 1, 2, 9, 10, 26, 29, 33, 40, 41, 43, 44, 45, 46, 49, 50, 53, 57, 59, 60, 61, 63, 67, 71, 72, 73, 74, 75, 78, 79, 80 infectious, 1, 14, 40, 43, 46, 51, 58 influenza, xii ingestion, 41 inhalation, xii, 2, 40, 47 inhibition, 16, 60 inhibitor, 8, 19 inhibitory, 8, 17, 34 initiation, 52 injection, 33 injury, vi inoculation, 65

91

inoculum, 16 interaction, 11, 40, 43, 44, 47, 76, 81, 82 interference, 79 internalization, 44, 46 iodine, 61 ionization, xii, 35, 36, 75, 79, 81 ions, 32, 35 iron, 1, 2, 10, 19, 28, 48 irradiation, xii, 32, 34 IRS, 30 isolation, 1, 15, 16, 17, 23, 64, 65, 71, 72, 75, 83, 84 isoprenoid, 72 isotopes, 51

J JAMA, 69, 80

K killing, 76, 79

L lakes, 5, 6, 47 latex, 19 lectin, 44, 83 Legionella pneumophila, vi, xi, 1, 28, 29, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 Legionella spp, 8, 20, 23, 24, 26, 27, 28, 49, 59, 63, 64, 69, 70, 71, 72, 73, 78, 83, 84 Legionellaceae, 1, 58, 60, 61, 83, 84 lesions, 15 lethargy, 38 licenses, 51 life cycle, xi, 3, 9, 10, 11, 32, 45, 46, 48, 68, 76 lifestyle, 43 limitations, 24, 45, 52, 53, 68 lipase, 24

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Index

92 Lipid, 46 lipopolysaccharides, 24, 52, 62, 68, 76 liquid chromatography, 24 locus, 45, 48, 68 long period, 7 low-temperature, 6 LPS, 46, 52 lung, 1, 2, 15, 28, 38, 39, 41, 46, 49, 50, 54, 60, 68 lung cancer, 39 lysosome, xii, 44, 45, 47, 60, 70

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M macrophage, 2, 3, 10, 20, 26, 44, 45, 46, 47, 48, 55, 57, 61, 66, 67, 68, 79, 82 magnetic, vi maintenance, 6 malaise, 38 mammalian cell, xii, 43, 45, 55, 68 management, 6, 31 manipulation, 51 man-made, xi, xii, 6, 9, 19, 31, 47, 55 matrix, 7 measurement, 53, 81 measures, 31 media, 1, 16, 17, 18, 23, 24, 43, 46, 50, 51, 58, 64, 65, 70 medication, 75 melting, 28, 78 memory, vii metabolic, 6 metabolism, 2 metal ions, 32, 35 mice, 10 microarray, 25, 46, 47 microbial, xii, 5, 7, 8, 57, 65, 78, 82, 84 microbial communities, xii, 8 microflora, 6, 16, 17 micrograms, 33 microorganisms, 7, 8, 9, 16, 63, 78 microscope, 18, 23

microscopy, 20, 79 mimicking, 79 mitochondria, 44 mitochondrial, 71 model system, 8, 68 mold, 5 monoclonal, 29, 50, 52, 67, 68, 69, 83 monoclonal antibodies, 29, 50, 52, 67, 68 monocytes, 44, 62, 69, 70, 76 morphological, 77 morphology, 3, 23, 24, 45 mortality, 37, 39, 40, 59 mortality rate, 37, 39 moths, 33, 39 MRA, 29 multiplication, 5, 6, 7, 9, 33, 38, 45, 55, 58, 66, 69, 83, 84 murine model, 60 mutagenesis, 65 mutant, 45, 47, 57, 70 mutation, 44, 45, 61 myalgia, 38

N N-acety, 44 natural, xii, 5, 6, 7, 9, 10, 19, 20, 31, 32, 47, 80 natural environment, 19 natural habitats, 10 necrosis, 46 nested PCR, 63 network, 6, 82 neutralization, 16 neutrophils, 46 normal, 72 nosocomial pneumonia, 75, 77 nucleic acid, xii, 19, 28, 53 numerical analysis, 25 nutrient, 6, 7, 8, 10, 32, 45, 77, 85

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Index

O observations, 9 open lung biopsy, 54, 68 organ, 31 organelle, 45 organic, 33 organic matter, 33 organism, 5, 31, 32, 65 ototoxicity, 40 oxidation, xii, 61 oxidative, 46 oxygen, 5, 48 ozone, xii, 35, 76

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P pain, 38 parameter, 6 parasites, xii, 2, 10 Parkinson, 59 passive, 39 pathogenesis, 43, 46, 48, 55, 57 pathogenic, 11, 17, 55, 80 pathogens, 46, 50, 68, 75, 80 pathology, 58 patients, 21, 27, 28, 31, 37, 38, 39, 50, 51, 53, 70, 74, 77, 79, 85 PCR, 5, 19, 20, 26, 27, 28, 29, 30, 49, 53, 54, 58, 59, 62, 63, 67, 68, 69, 73, 74, 75, 76, 77, 78, 80, 82, 84 penicillin, 40 peptidase, 45 peptide, 46 pericardial, 15 periodic, 7 permeability, 71 permit, 52 pertussis, 59 phagocytic, xii, 44 phagocytosis, xii, 44, 47, 60, 69 phenotype, 10, 46, 58, 68

93

phenotypic, 10, 23, 57 phosphate, 78 physiological, 58 pig, 1, 60, 65 pigments, 46 pipelines, 34 plants, 33 plasma, 40, 82 plasma membrane, 82 plasmids, 3 plastics, 8 platforms, 25 play, 6, 41, 46, 48 pleural, 15, 38 pneumonia, xi, 1, 37, 38, 51, 60, 63, 65, 66, 70, 74, 75, 78, 79, 84 poliovirus, 85 polyethylene, 83 polymerase, 19, 26, 59, 67, 72, 77, 83 polymerase chain reaction, 19, 59, 67, 72, 77, 83 polymorphism, 58 pools, 13 poor, 10, 16 population, 16, 74 pore, 16, 46, 57, 71 pregnant, 82 pressure, 9 prevention, xi, 31, 34, 65, 69 probe, 19, 20, 28, 53, 59 production, 24, 33, 46 proliferation, 6, 8, 81 property, vi protection, 35 protein, xii, 8, 19, 26, 43, 44, 45, 46, 50, 60, 66, 73, 76, 78, 79 protein synthesis, 8, 44 proteobacteria, xii protozoa, xii, 2, 5, 7, 8, 9, 10, 11, 26, 32, 33, 43, 46, 47, 55, 57, 58, 61, 66, 68 Pseudomonas, 9, 51 Pseudomonas spp, 9, 51 public, xi, 32, 39, 52, 55, 61, 64, 80

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Index

94 public health, xi, 39, 52, 55, 61 pyrophosphate, 17

Q quinone, 72

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R radioactive isotopes, 51 radiography, 38 radiolabeled, 53 random, 26, 73 Random Amplified Polymorphic DNA (RAPD), 26, 58, 73 range, 5, 41, 51, 63 reactivity, 81 reagent, 65, 83 real time, 20 receptors, 44, 46 recognition, 1 recolonization, 33, 35 recovery, 9, 14, 15, 16, 17, 19, 60, 76 regulation, 57, 73 relationships, 43, 47, 79 relevance, 77 renal, 73 replication, 43, 44, 45, 46, 47, 70, 74, 78, 81 reservoirs, 5, 31, 38, 55 residential, 81 resistance, 6, 9, 10, 32, 43, 47 respiration, 72 respiratory, 1, 28, 39, 41, 49, 50, 58, 62, 73, 75, 78 reticulum, 44, 71, 78 ribosomal, 66, 78 risk, 13, 21, 27, 31, 38, 57, 74 risk assessment, 31, 57 rivers, 5, 47 RNA, 72

S safety, 14 salmonella, 85 salts, 1, 2, 10 sample, 14, 16, 18, 32 sampling, 13, 14, 32, 60 satellite, 24 search, 1, 21 secretion, 44, 45, 54, 55, 60, 67, 73, 79 sediment, 6 sensitivity, 16, 27, 28, 49, 50, 51, 52, 53 sequencing, 26, 62, 66, 84 serologic test, 52 serology, 23, 51 serum, 49, 63 services, vi severity, 40, 85 sewage, 67, 77 sheep, 17, 18, 23 shelter, 8 shock, xii, 9, 26, 33, 46 side effects, 40 silver, xii, 35, 36, 73, 75, 79, 81, 85 similarity, 27 sites, 5, 13, 14, 15, 32, 71 skin, 15 sludge, 20 smokers, 39 sodium, 14, 33, 45 soils, 5, 26, 72, 79 species, xi, 1, 5, 6, 7, 8, 9, 10, 17, 19, 24, 25, 26, 27, 28, 30, 37, 40, 44, 50, 52, 53, 54, 60, 62, 64, 65, 66, 67, 68, 72, 73, 74, 77, 78, 79, 80, 82, 84, 85 specificity, 16, 24, 27, 28, 49, 50, 51, 52, 53, 62, 65 spectroscopy, 24 spectrum, 37 speed, 28 sporadic, 38, 39, 75, 85 spore, 2

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Index sputum, 27, 49, 50, 66 stability, 9 stages, 46, 67 stainless steel, 7, 8, 83 standardization, 82 starvation, 85 steel, 7, 8, 83 sterile, 74 strains, 2, 3, 6, 17, 19, 26, 29, 30, 32, 34, 37, 39, 47, 53, 54, 58, 61, 62, 67, 74, 83 strategies, 31, 43 streams, 5, 47 strength, 76 subgroups, 69 subjective, 19 substances, 8 substrates, 25 summer, 21, 38 supplemental, 34, 81 supply, 6, 29, 57, 61, 77 surface structure, 46 surveillance, 39, 62, 70, 74, 81 survival, 6, 7, 10, 72, 76, 77 susceptibility, 10, 58, 61 suspensions, 25 symbiosis, 32 symbiotic, 6 symptoms, 38 synergistic effect, 35 synthesis, 8, 44 systems, xi, xii, 6, 7, 8, 9, 14, 21, 31, 32, 34, 36, 45, 47, 55, 61, 64, 66, 69, 71, 72, 73, 75, 80, 81, 82, 83

T tanks, xii, 6, 66, 75 targets, 27, 69 taxonomic, 26 technology, 27 temperature, 3, 5, 6, 7, 10, 24, 32, 34, 35, 43, 62, 73, 77, 78

95

therapy, 38, 40, 52, 71 thermal treatment, 75 time, 16, 19, 20, 26, 27, 28, 32, 34, 52, 53, 59, 63, 64, 67, 74, 76, 78, 80, 84 time consuming, 16, 34 tissue, 15, 28, 49, 50, 54 toxic, 38 toxins, 46, 48 tracking, 14 traffic, 44, 71 training, 75 traits, 43, 46, 61 trans, 66 transcriptomics, 71 transfer, xii, 44, 75 transition, 45, 71 transmission, 14, 41, 44, 45, 47, 55, 66, 68, 71, 85 transplant, 31, 73 transplant recipients, 31 transportation, 14 travel, xi, 38, 39, 41, 52, 65, 69, 70 triggers, 47 tyrosine, 24

U ultraviolet light, 73 urease, 24 urinary, xii, 27, 28, 49, 51, 52, 68, 71, 72, 85 urine, 49, 51, 52, 59, 61, 63, 64, 68 UV irradiation, xii, 32, 34 UV light, 34, 76

V vacuole, 10, 44, 45, 47, 70, 78 validation, 53 vancomycin, 17 variable factor, 13 variables, 21 variation, 26, 27

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Index

96 VBNC, 11, 38 ventilation, 13 virulence, xii, 10, 21, 26, 38, 39, 43, 44, 45, 46, 47, 61, 62, 65, 68, 82 viscosity, 24

W wastes, 72 wastewater, 57, 79, 80 wastewater treatment, 80 water supplies, 81 wild type, 47 workers, 59 World Health Organization, 69

Y

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yeast, 24, 65, 76, 83

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