|Tuesday, 17 August 2010 00:00|
Mª Adela Yáñez
The genus Legionella comprises gram-negative bacteria belonging to the gamma proteobacteria. The representative species of the genus is L. pneumophila, which was the first species to be described. L. pneumophila and related Legionella bacteria cause Legionellosis, a disease that emerged in the second half of the 20th century. The severity of Legionellosis varies from mild febrile illness (Pontiac fever) to a potentially fatal form of pneumonia (Legionnaires' disease) that can principally affect anyone, but in most cases affects people with increased susceptibility (due to age, illness, immunosuppression, or other risk factors such as smoking). The number of species, subspecies and serogroups of Legionellae continuously increases with accumulating knowledge. This genus currently comprises at least 50 species with 70 distinct serogroups ( Wullings and van der Kooij 2006; Benson and Fields 1998; La Scola et al. 2004). Although around half of them have been associated with disease, L. pneumophila (mainly serogroup 1) is the most common and prominent pathogen and is the causative agent of more than 70% of Legionellosis infections, 20-30% are caused by other serogroups, and 5-10% are caused by non-pneumophila species ( Joseph C 2002). The majority of human infections with species other than L. pneumophila are pneumonic (Fang and Vickers 1989). Of the reported non-pneumophila infection, the causes of infection are: L. micdadei (60%), L. bozemanii (15%), L. dumoffii (10%), L. longbeachae (5%) and other species (10%).
The name Legionnaire's disease originates from the fact that a pneumonia outbreak caused by L. pneumophila affected members of the Pennsylvania American Legion during a meeting in 1976 ( Brooks et al. 2004). The milder pneumonia referred to as Pontiac fever, on the other hand, is named after the town Pontiac in Michigan where employees and visitors of the health department suffered an acute respiratory disease. Both diseases are caused by aspiration of contaminated aerosols as produced by shower heads, air-conditioning systems, whirlpools, fountains, or dental devices ( Brooks et al. 2004; Bollin et al. 1985; Steinert et al. 2002). It has been shown by autopsy that L. pneumophila can spread from the respiratory system to the body. Legionellae have been detected in the spleen, liver, kidney, myocardium, bone and bone marrow, joints, inguinal and intrathoracic lymph nodes and digestive tract (Lowry and Tompkins 1993). Pathogenesis is mainly due to the ability of L. pneumophila to invade and multiply within human macrophages ( Steinert et al. 2002). The ability to invade other cells also has important implications for their lifecycle in the environment: in water, the majority of the Legionella cells are believed to live intracellularly as protozoan parasites. Infected protozoa might be an important vehicle of transmission to humans when inhaled ( Harb et al. 1998). At the same time the sheltered environment provided by the protozoan envelope protects Legionella and lowers its susceptibility to disinfection.
Outbreaks and legislation. The introduction of Legionella into several man-made aquatic environments has resulted in increased presence of the pathogen in bioaerosols, which presents a significant hazard for human health ( Pascual et al. 2001). Outbreaks of L. pneumophila occur throughout the world. Due to its high frequency Legionellosis has been classified as a statutory notifiable disease in most industrialized countries. Nevertheless the priority given to Legionellosis surveillance may need to be even greater than suggested by local morbidity and mortality due to its heavy impact on industrial, touristic, and social activities.
Due to differences in public health surveillance systems the provision of Legionella data is determined by each country's public health system ( WHO 1999). A country's national surveillance of Legionnaires' disease depends on factors such as: infrastructure and public health laws, adopted surveillance principles and standard operating procedures, notification law, data protection, patient confidentiality and freedom of information legislation. The cases of Legionellosis disease can be grouped into the following categories: community acquired, domestically acquired, nosocomial (i.e. health-care acquired), travel associated.
Some examples of Legionella outbreaks occurred are listed below:
Legionellae are ubiquitous in natural and anthropogenic water environments and have been found in lakes and rivers ( Fliermans et al. 1981), drinking water distribution systems ( Henke and Seidel 1986; Lawrence et al. 1999), potable hot water systems ( Yáñez et al. 2005), cooling towers ( Castilla et al. 2007; Ragull et al. 2007), WWTPs ( Palmer et al. 1993), whirlpools (Den Boer et al. 2002), and to a somewhat lesser extent in groundwater ( Brooks et al. 2004; Riffard et al. 2001, 2005). Legionella spp. are well known to colonize surface-attached biofilms ( Thomas et al. 2004) and have recently also been identified in floating biofilms. Under certain conditions the occurrence of biofilms might be associated with the abundance of Legionellae. Costa et al. hypothesized that Legionella concentrations in groundwater were lower than those found in man-made systems or surface waters because of the smaller amount of biofilm in groundwater compared to sometimes massive biofilm development in the other environments ( Costa et al. 2005). Nevertheless, groundwater sources have been reported as a cause of disease ( Fields et al. 2002; Riffard et al. 2005). Densities above 104 to 105 CFU/liter are considered a potential threat to human health and were associated to legionellosis ( Meenhorst et al. 1985; Patterson et al. 1994). A concentration of 103 CFU/liter was referred to as a first alert value ( Joly et al. 2006). In distribution systems, the presence of dead-end loops and water stagnation in the plumbing system are considered technical risk factors ( Steinert et al. 2002). Also the age of the plumbing system and the materials used were found to influence colonization (Leoni K 2003). Although Legionella can be abundant in wastewater ( Catalán et al. 1997; Palmer et al. 1993), they do not enter water through fecal pollution ( Steinert and Heuner 2005). Fecal indicators are thus no option for Legionella detection. However, elevated Legionella concentrations in sewage-contaminated coastal waters and in reclaimed water have been reported ( Ortiz-Roque and Hazen 1987; Palmer et al. 1993; Palmer et al. 1995).
Examples of studies on occurrence in water are:
Although disinfection seems to be successful in reduction of Legionella contamination or even short-term eradication, it has proven difficult to achieve sustainable long-term elimination from aquatic environments. Different cases of Legionella re-emergence after (hyper-)chlorination (e.g. on a cargo ship, in domestic hot water systems, or a hospital hot water distribution system) have been reported ( Caylà et al. 2001; García et al. 2007; Borella et al. 2004; Heimberger et al. 1991). Despite different treatments, the same Legionella strains persistently caused disease. The interaction of Legionella with free-living protozoa and other microorganisms might be a key factor in this respect ( García et al. 2007). Apart from obtaining nutrients and enabling them to survive under adverse environmental conditions, the association with protozoa and biofilms makes Legionella more resistant to disinfection ( Lemarchand et al. 2004). Resistance to 50 mg/L of chlorine has been reported when Legionella spp. were enclosed in cysts of amoeba ( Lemarchand et al. 2004). Significantly higher resistance against chlorine and monochloramine of L. pneumophila when associated with the protozoan Hartmannella vermiformis in a model multi-species biofilms was also reported by Donlan et al. ( Donlan et al. 2005). In addition to providing a sheltered environment to internalized Legionella, amoebae are suspected to be involved in the resuscitation of Legionella that have entered a VBNC state after disinfection ( García et al. 2007). Removal or reduction of biofilms and protozoa can therefore be considered essential when trying to reduce Legionella levels. Legionella log10 reductions were reported to be 2.07 and 2.11 (exposure to 0.5 mg/liter for 180 min) for chlorine and monochloramine in the absence of H. vermiformis, respectively, while reductions were only 0.67 and 0.81 in its presence. Monochloramine was considered a more efficient disinfectant than chlorine for Legionella eradication ( Fields et al. 2002). A reason might be their greater stability, which results in higher residual disinfectant concentrations over more extensive distribution systems ( Fields et al. 2002). In another study, chlorine dioxide was favored over chlorine for the same reason although both disinfectants were effective ( Loret et al. 2005). Disinfectants that combat biofilms more efficiently can be considered more sustainable for killing Legionella. Continuous treatment with chlorine dioxide was also identified as more efficient than other treatments for controlling L. pneumophila in a domestic water system with re-circulating loops and dead ends in the presence of biofilms in a study by Thomas et al. ( Thomas et al. 2004). A commonly used strategy in hot water systems for Legionella prevention is thermal disinfection. Maintaining the water temperatures above at least 50°C in hot water systems with occasionally increases to 65°C might be considered an effective control measure ( Blanc et al. 2005). The efficiency of heat treatment, however, also might depend on the presence of protozoa. The ingestion of Legionella by thermotolerant free-living amoebae was shown to increase heat tolerance of the intracellular pathogens ( Storey et al. 2004a). Protection from thermal disinfection might also derive to some extent from association with biofilms although the degree of protection would be expected to correlate with biofilms thickness ( Storey et al. 2004a; Storey et al. 2004b). A simplistic quantitative microbial risk assessment model presented by Storey et al. considered biofilms detachment and interaction with amoebae in water distribution systems as important ecological factors that significantly increase the risk of waterborne legionellosis ( Storey et al. 2004b).
The efficacy of various disinfection procedures has been reviewed in detail by Kim et al. ( Kim et al. 2002). The document includes thermal, UV, and chemical (metals, oxidizing and non-oxidizing agents) disinfection and concludes that thermal disinfection is effective at >60°C and among chemical disinfectants, oxidizing agents are in general more effective than non-oxidizing agents. UV irradiation by itself was considered insufficient for complete eradication of Legionella, although it can result in significant decrease of culturability within a short time.
The main drawbacks of the chemical disinfectants are that sometimes microorganism become resistant when subjected to a single disinfectant, and periodic alternation of disinfectants is often required to mitigate this effect. Moreover, depending on the disinfectant a high degree of corrosion in the facilities can occur. Non-chemical treatment methods are a desirable alternative, but any new treatment method must be carefully tested for its long-term anti-microbial efficacy potential adversely impacting scale formation, and corrosion or heat transfer efficiency of the overall system. In the case of treatment of Legionella new alternative methods are proposed such as cavitation and photocatalysis.
Cavitation. The cavitation is the formation, growth, and implosion of vapor bubbles in a liquid. It can be created by sound waves, lasers, or by fluctuations in fluid pressure (hydrodynamic cavitation). Cavitation has been reported to kill bacteria through chemical reactions, pressure pulses and micro-jets, and by localized high temperatures. In aqueous liquids, cavitation leads to the formation of hydroxyl radicals and hydrogen peroxide. These short-lived reactive radicals are capable of secondary oxidation and reduction reactions in the immediate vicinity of the bubble. This method has been applied to several cooling water systems ( Gaines et al. 2007) and results were very promising in terms of reducing heterotrophic plate counts and the presence of L. pneumophila. A corrosion study revealed no notable corrosion except for galvanized steel. The high variance in the heat transfer data indicates that further studies are required to fully verify a positive impact. Nevertheless, the implementation of physical disinfection systems offers a number of potential benefits, including lower operating costs, elimination of hazardous chemicals, the potential to "de-skill" the labor workforce, simplified equipment complexity and operation, reduced regulatory reporting requirement, and an enhanced corporate image by using a non-chemical "green" technology.
Photocatalysis. Heterogeneous photocatalysis using UV radiation and semiconductor oxide, which in most cases is the anatase form of TiO2, has emerged as an innovative method for water treatment. By using an aqueous oxygenated suspension of polycrystalline TiO2 and UV light it is possible to carry out the oxidation of organic species adsorbed at a solid suface. This oxidation process, which is started by photogeneration of active oxygenated radicals such as OH or O2-, can also be used to attack the cell membrane of microorganisms and to cause their inactivation ( Coronado et al. 2005). The method has been applied for the Legionella disinfection in different pilot facilities ( Cheng et al. 2007) and results indicated that the photocatalytic oxidation (PCO) has been efficacious in the inactivation of different strains of Legionella. A higher lethality of the photocatalytic method was observed towards Legionella in comparison to the other aerobic bacteria. This fact is explained by the hypothesis that the radicals attack the Legionella secretion system, which is highly active for virulence, and thus, promotes adhesion to the TiO2 surface. This attack would then be able to inactivate L. pneumophila without dispersing the outer membrane. Apart from this, the water flow through the catalyst fibres can facilitate the bacteria transport towards the TiO2 surface, and additionally the generated shear stress may help adhesion, at least for some bacteria as E. coli, contributing further to improve the photokilling efficiency; both factors may contribute to the efficiency of this photoreactor configuration ( Coronado et al. 2005).
Selected studies on disinfection are summarized in the following:
Legionellae are able to survive under a wide range of water conditions, including temperatures from 0 to 63°C and a pH range of at least 5.0-8.5 ( Health Canada 2006).
Temperature tolerance. Whereas growth is restricted to temperatures between 20 and app. 43°C, L. pneumophila has been isolated from water at temperatures below 10°C (surface water) up to 60°C (artificial water systems) ( Wullings and van der Kooij 2006). In a study examining Legionella spp. in hot water systems (with recirculation), colony counts were reported up to temperatures of about 56°C. The range between 55°C and 60°C seemed to be a critical temperature region above which survival in hot water systems is affected ( Darelid et al. 2002; Mathys et al. 2007). Exact temperature tolerance values vary between different studies as might be assumed due to strain variability, different exposure times, different bacterial 'history' and different experimental conditions. Although outbreaks caused by natural water were mainly associated with temperatures higher than 30°C ( Lemarchand et al. 2004), Legionella can survive for long periods of time at low temperatures. Paszko-Kolva et al. reported the potential of clinical and environmental L. pneumophila isolates to persist and survive up to 2.5 years under low nutrient conditions when seeded into drinking and creek water monitored by acridine orange direct counts and viable counts ( Paszko-Kolva et al. 1992). It was speculated that L. pneumophila detected in cold water systems are survivors from summer periods when water temperatures were above 20°C ( Wullings and van der Kooij 2006).
Nutrient and mineral requirements. Legionella proliferates in water when nutrients are available. The nutrients may be given in form of dissolved organic constituents or indirectly by the presence of other microorganisms which undergo decay resulting in nutrient release ( Grimes DJ 1991). The presence of iron is important as it promotes and assists the growth of Legionella. Studies performed in several facilities such as storage cisterns and service pipe works have shown a clear correlation between presence of iron oxide and growth of Legionella ( Borella et al. 2004). The iron is not only a determining factor for planktonic, but also for intracellular growth of L. pneumophila. Although virtually all pathogens require iron, L. pneumophila has relatively high metabolic requirements of this metal. The multiplication of L. pneumophila in human mononuclear phagocytes depends on the availability of labile iron that is obtained primarily from three proteins: transferrin, lactoferrin, and ferritin. It has been shown that agents that inhibit the availability of iron inhibit the intracellular multiplication of bacteria (Byrd and Horwitz 1989; Byrd and Horwitz 1990; Byrd and Horwitz 1991). Also the presence of other salts and minerals within the water have an effect on survival ( Heller et al. 1998; States et al. 1985). States et al. found that low levels of certain metals like zinc, or potassium (which are found in plumbing systems) enhance growth of L. pneumophila, although elevated concentrations are toxic ( States et al. 1985). Also low concentrations of sodium chloride (0.1 - 0.5%) were found beneficial for survival ( Heller et al. 1998). Under low-nutrient or adverse environmental conditions, survival might also be ensured by adapting a dormant state ( Steinert et al. 2002). Viable-but not culturable cells might represent a large portion of the environmental Legionellae population ( Steinert et al. 1997). This would correlate with the fact that higher cell numbers are typically obtained with molecular methods compared to culture isolation.
pH preference. Legionellae are acid-tolerant (they can withstand exposure to pH 2.0 for short periods) and have been isolated from environmental sources ranging from pH 2.7 to 8.3 ( Anand et al. 1983). Ohno et al. reported an optimal range between pH 6.0 and 8.0 in a microcosm study ( Ohno et al. 2003).
Association with other microorganisms. Although planktonic Legionella spp. have been reported to survive for extended time periods in the environment, their extraordinary ability to interact with other microorganisms is highly beneficial for survival. They attach to surfaces in aquatic environments and readily form biofilms together with other microorganisms. Apart from providing a protective environment and increasing survival of community members, biofilms allow interaction with other microorganisms. The beneficial impact of the presence of non-Legionella species is known to influence Legionella growth. Moreover, Legionella are well known to maintain parasitic relationships with protozoa like Hartmanella, Acanthamoeba, Valkampfia, Naegleria spp., and other protozoa like Tetrahymena ciliates ( Lemarchand et al. 2004; Steinert et al. 2002). As Legionella have the capacity to survive and replicate intracellularly in their hosts, the interaction with protozoa is considered a key factor in the Legionella amplification process ( Steinert et al. 1997). Indeed, although most Legionella species can be cultivated under laboratory conditions, it is not known to what extent they occur in the free planktonic state in water ( Brooks et al. 2004). In the water environment, Legionellae might live mainly or even exclusively within or associated with other organisms. It was suggested that Legionella only replicate within protozoan and infected mammalian cells or on laboratory media ( Kwaik YA 1998; Fields B 1996), but growth in the planktonic state or in biofilms cannot be excluded. Legionellae living intracellularly are believed to have increased resistance to harsh environmental conditions and better survival characteristics.
A summary of selected studies on survival is given in the following:
Although some studies have investigated the infective dose and the obtained results have indicated that Legionella spp. represent a health risk to humans when a concentration of 104 to 105 CFU per liter potable water is exceeded ( Delgado-Viscogliosi et al. 2005), the median infective dose (LD50) of Legionella is unknown for healthy adults. Outbreaks affecting mainly immunocompromised people can occur at low concentrations ( Patterson et al. 1994; Meenhorst et al. 1985). It is known that hospital patients have become infected after exposure of only a few minutes to the sources of some outbreaks, whereas the infection has also been reported at up to 3.2 km from the source of other outbreaks (WHO). Nevertheless, infection depends on many factors such as the bacterial load of the water/microbial quality, the degree of aerosol formation and the resulting dissemination of bacteria through air, host factors, the virulence of the particular strain of Legionella and their viability. Inhaled protozoa might serve as cofactors in pathogenicity: L. pneumophila-infected protozoa were more pathogenic in mice than the equivalent number of bacteria or a co-inoculum of bacteria and uninfected protozoa ( Brieland et al. 1997). Another study showed that L. pneumophila was at least 100-fold more invasive for human epithelial cells and 10-fold more invasive for mouse macrophages when grown in the presence of Acanthamoeba castellani compared to pure cultures ( Cirillo et al. 1994).
Many countries have developed guidelines or regulations for the control of Legionella in water systems and for the prevention of legionellosis. Guidelines are advisory, whereas regulations and codes of practice have a more formal standing and are supported by legislative enforcement (including, in the case of regulations, specific information on managerial responsibility and operator competency). The guidelines include NHMRC (1988), CDC (2003), Allegheny County Health Department, Pennsylvania (1997), ASHRAE (2000), Ehrlich, Steele & Sabatini (2000), Standards Association of Australia/Standard Association of New Zealand (2002), EWGLINET (2003), and WHO (2004, 2006). Regulations include HSC (2000), IEE (2001), Spanish RD 865 (2003), and Victorian Department of Human Services (2001). In general, each country has their own regulations and guidelines, but all serve to achieve efficient risk management. These regulations and guidelines include the necessity of testing programs and all of them include the culture isolation method based on norms such as ISO11731, the joint Australian/New Zealand Standard AS/NZS 3896, and AFNOR T90-431.
The "gold standard" isolation of Legionella is based on ISO11731 as a reference. This norm specifies how to proceed to isolate and enumerate Legionella from water samples: sample concentration by membrane filtration, different treatments aimed at decreasing the competing flora, plating onto Buffered Charcoal Yeast Extract (BCYE) supplemented with L-Cysteine and glycine, polymixin, vancomycin and cycloheximide (GVPC) agar plates, and incubation under proper conditions during up to 10 days.
In other fields distinct from the environmental testing, such as clinical diagnosis, culture isolation following ISO11731 is used in cases of severe respiratory failure, in compromised patients, nosocomial infections and in cases in which disease is caused by any Legionellae other than L. pneumophila serogroup 1. In all other situations, alternative methods are more frequently used due to the drawbacks of culture isolation of Legionella (slow growth-rate, inability to detect viable-and-non-culturable bacteria, and difficulty of isolating Legionella in samples with a high background of competing flora). In this sense, one of the most commonly employed methods is the urinary antigen detection by enzyme immunoassay (EIA). The outcome of the disease with Legionnaires' disease greatly depends on the speed with which a diagnosis can be performed and the initiation of appropriate therapy (Heath et al. 1996). Urinary antigen detection for the diagnosis of Legionnaires' disease has several advantages compared with other methods. It is a rapid, sensitive, and specific method for the detection of serum antibody levels. In addition to the EIA method described above, the market offers a variety of commercial kits with different characteristics and specifications ( Dominguez et al. 1998; Helbig et al. 2001).
Typing methodologies for epidemiological studies
Epidemiological linkage of clinical and environmental Legionella isolates is invaluable in locating the source and extent of infection, allowing implementation of corrective measures and treatment to prevent further infection. Previous studies have demonstrated the usefulness of differentiating isolates belonging to serogroup 1 of L. pneumophila in order to confirm or refute epidemiological associations in outbreak investigations ( Edelstein et al. 1986), but only in a local setting. Isolates of L. pneumophila sg 1 can be rapidly subtyped by using monoclonal antibody (MAb) subgrouping with panels based on the international MAb subgrouping panel (Harrison et al. 1998). However, MAb subgrouping lacks discrimination, dividing L. pneumophila sg 1 into only 8 to 10 phenons, depending on which MAb panels are used. Consequently, in addition to this phenotypic typing method, a number of molecular methods have been successfully employed locally for epidemiological purposes. These include ribotyping, amplified fragment length polymorphism (AFLP) analysis, pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP) analysis, restriction endonuclease analysis (REA), and arbitrarily primed PCR ( Fry et al. 1999). One of these methods, AFLP analysis ( Valsangiacomo et al. 1995), has been adopted as an international standard and it is now widely used by members of the European Working Group for Legionella Infections (EWGLI). Interlaboratory studies ( Fry et al. 1999) have assessed the reliability and discriminatory power of AFLP analysis for epidemiological typing of L. pneumophila sg 1 isolates. While these studies have demonstrated that the method is robust and rapid and allows the exchange of data between laboratories without the need for exchange of isolates, a program of proficiency testing showed that a significant proportion of laboratories could not correctly identify isolates 100% of the time (Fray et al. 2000). Incorrect identification was typically a result of data analysis issues (e.g., difficulties in comparing the AFLP banding patterns) rather than of the method itself. For this reason, a universal typing method was developed that would be simple, rapid, discriminatory, and truly "portable" and that might in the future be applied by all microbiology laboratories to aid in the investigation of legionellosis. The method is based on the sequence of housekeeping genes and named Multilocus Sequence Typing (MLST) (Enright and Spratt 1998). A modification of this method, "sequence-based typing" (SBT), includes, apart from housekeeping genes, genes under selective pressure, e.g. virulence genes, which offer more information about variability of closely related strains ( Gaia et al. 2003; Gaia et al. 2005). The results demonstrated the advantages of SBT of L. pneumophila sg 1 over other current methods. By using 5 loci, this methodology has the potential to become a new "gold standard" for the epidemiological typing of L. pneumophila ( Gaia et al. 2005).
The following pictures are part of the Public Health Image Library (PHIL) from the Centers for Disease Control and Prevention (CDC). The photos are in the public domain and thus free of copyright restrictions. We would like to express our appreciation for providing these images.
Under a moderately-high magnification of 8000X, this colorized scanning electron micrograph (SEM) depicted a large grouping of Gram-negative Legionella pneumophila bacteria. Of particular importance, is the presence of polar flagella, and pili, or long streamers, which due to their fragile nature, in some of these views seem to be dissociated from any of the bacteria.
Under a moderately-high magnification of 5011X, this 2002 scanning electron micrograph (SEM) revealed some of the ultrastructural morphology exhibited by small grouping of Hartmannella vermiformis amoebae trophozoites.
Links to useful external sites can be found in the following:
EWGLI (The European Working Group for Legionella Infections)
HEALTH AND SAFETY EXECUTIVE
|Last Updated on Tuesday, 03 April 2012 02:40|