Literature Review on Uptake of Legionella into Host Cells

Legionella Critical Review

Abstract

Legionella pneumophila (L. pneumophila) is of increasing concern due to its ability to cause Legionnaires’ disease, a severe pneumonia, and the difficulty in removing L. pneumophila from water systems. L. pneumophila thrives within the biofilm of premise plumbing systems, utilizing protozoan hosts for protection from disinfectants and other environmental stress. While there is a plethora of information regarding how L. pneumophila interacts with protozoa and human macrophages (causing Legionnaires’ disease), this information has yet to be used for a comprehensive quantitative microbial risk assessment (QMRA) model for L. pneumophila in drinking water systems. This review article seeks review the available information regarding how L. pneumophila invades host cells.

Background

Legionellosis, a respiratory disease caused by the inhalation of Legionella bacteria, incorporates both Pontiac Fever and Legionnaires’ disease (LD) a severe form of pneumonia. Pontiac Fever, a self-limiting flu-like illness, is characterized by a non-pneumonia respiratory infection lasting two to five days. Those affected may experience fever, chills, malaise, myalgia, headache, cough, or chest pain. The typical incubation period is one to two days. The disease was first discovered in Pontiac Michigan in 1968. At the time of discovery, the etiological agent of the disease was unable to be identified. It was not until the LD outbreak in 1976 that Legionella pneumophila (L. pneumophila) was identified as the causative agent 1,2.  LD is named so as it was discovered after an outbreak of pneumonia at a Legionnaires’ convention in Philadelphia. During this outbreak 29 of the 182 cases were fatal, and a gram-negative bacillus was isolated and determined to be the cause. It was also determined that the same bacterium was the previously unknown etiological agent of Pontiac Fever3–5.

LD has similar symptoms to Pontiac Fever with the addition of a severe pneumonia. Though LD may cause sub-clinical infections, more serious cases can cause long-term symptoms including easy fatigue, shortness of breath, muscle or joint pain, productive cough, and memory loss 6–8. LD can have an incubation time ranging from two days to two weeks, depending on the patient’s immuno-health and if antibiotic therapy was administered 8,9. There is an increased risk of contracting legionellosis in those with a non-municipal water supply, recent residential plumbing repair , electric water heaters (as opposed to gas), and those who work more than 40 hours a week 10–12. Elderly, males, those with nosocomial infections, renal disease or immunodeficiency have increased risk from Legionaries’ 10,13.

Legionellosis is the most common cause of all reported waterborne diseases in the US, and its incidence is steadily increasing 14,15.  The US average 10,000 – 15,000 cases year, and L. pneumophila was the cause of half of all reported waterborne disease outbreaks in 2005-20016–18. Due to the prevalence and seriousness of LD, L. pneumophila has been added to the United States Waterborne Disease Outbreak Surveillance System in 2001 and the US Environmental Protection Agency (EPA) Candidate Contaminant List (CLL) as an important pathogen. By 2006 Legionella spp. was the third most common etiological agent of waterborne disease reported 19,20.  It is believed that most cases are community acquired, though it is difficult to accurately report cases associated with travel, due to varying incubation times, and the possibility that symptoms do not require medical attention 21. Legionellosis can only be spread through a waterborne respiratory route, where contaminated water is aerosolized, and the bacteria is inhaled 22,23.  Sources of legionellosis outbreaks include contaminated hospital bathrooms 24, grocery-store mist machines 25, sprinklers 26, cooling towers 27–29, HVAC systems 30,31, humidifiers 32, large warm water systems 33, whirlpools 21,34, pools 21, and dentist offices or machines 35,36.

There are at least 51 species of Legionella and over 60 serogroups. The most common cause of Legionellosis is L. pneumophila, causing up to 90% of reported cases in the US 10,37,38.  L. pneumophila serogroup 1 is responsible for 84.2% of cases and serogroups 2-13 are responsible for 7.4%. Legionella longbeachae is responsible for 3.9% of cases and Legionella bozemanii 2.4% of cases in the US. These percentages remain similar in Europe, but change significantly in Australia and New Zealand where L. pneumophila serogroup 1 is responsible for 45.7% of cases, and L. longbeachae 30.4% of cases 38. L. pneumophila is more commonly found in water, where L. longbeachae is more common in soil and can be associated with gardening and potting soil 39. L. longbeachae has been shown to have enzyme systems that may assist in the degradation of plant material which L. pneumophila is lacking 40. Epidemiological patterns of legionellosis differ in these regions as well, studies have shown that more than 50% of confirmed cases in New Zealand and Australia were a result of contact with compost 41,42.

Legionella spp. are ubiquitous in aquatic environments with protozoan hosts 43. The bacteria are particularly difficult to kill due to the symbiotic relationship with protozoa within biofilms where Legionella tends to be present, such as premise plumbing in large distribution systems 16,44–46. It is estimated that 95% of the bacterial biomass in drinking water are found in biofilms on the walls of distribution systems 47. When temperature or water flow rate change the biofilms can slough off the pipes, releasing Legionella and allowing for it to be aerosolized when leaving a shower head, faucet, or other risk area discussed above 48. Outbreaks of legionellosis are still common due to the difficulty in treating these particular bacteria within the water system. Other review articles discuss health effects, pathology, treatment, transmission, etiology, epidemiology, and infectivity 9,20,22,23,49–53. Reviews that discuss growth and survival of Legionella either depend on a lab-based model, differing significantly from the biofilm environment, or the article focuses on a particular pathway, proteins, or genetics that affect Legionella’s lifecycle, however they do not focus on all of these aspects in one article 52,54–58.

This review focuses on the information needed to model the uptake of Legionella into host cells, emphasizing the physiological and genetic pathways allowing for this process. As combining the entire lifecycle of Legionella into one review would prove too long for one article, this review will focus on the uptake of Legionella into the host cells. The overall goal is to be able to combine this review with future reviews focusing on the survival and replication of Legionella within the host cell, Legionella’s expulsion from the original host cell and how the bacteria re-infects new hosts in order to have a clear picture of the entirety of Legionella’s lifecycle, including protein and gene interactions. The compilation of this information will allow for predictive microbiology and improved comprehensive quantitative microbial risk assessment (QMRA) model of Legionella’s lifecycle within the drinking water system.

L. pneumophila is challenging to remove from potable water in the drinking water system due to its interactions with host cells within biofilms. L. pneumophila typically infects a unicellular protozoan host, using it for protection 59,60. L.pneumophila living within amoeba are much less sensitive to chlorine than free living L. pneumophila, additionally, biofilms tend to form naturally during stressful events, such as chlorine dioxide or ultraviolet disinfection 61–63.  L. pneumophila does not only use the host cell for protection, but the endosymbiotic relationship can make the bacteria more capable of infecting mammalian cells, acting as a “unicellular incubator” 64. It is even possible that the bacterium is incapable of causing disease unless it has replicated within a host cell prior to infecting a mammalian macrophage 65,66.

L. pneumophila has high uptake rates in acanthamoeba, a genus of amoeba commonly found in freshwater and known as a routine host to the bacteria. Acanthamoeba polyphaga (A. polyphaga) has an uptake rate of 73 – 98% viable L. pneumophila cysts, while Acanthamoeba castellanni (A. castellanni) has an uptake rate of 96 – 100% 67. This review will focus on the pathways that permit the uptake of L. pneumophila into the host cell, with an emphasis on L. pneumophila and Acanthamoeba considering their prevalence, and macrophages considering their requirement for human infectivity.

Literature Review

In order to determine the mechanisms, proteins, and genes involved in the uptake of Legionella to the host cell an exhaustive literature review was conducted. Google Scholar, PubMed, Web of Science, Bioline International, and PLOS ONE were searched using the terms: “((Legionella) OR (Legionella pneumophila) OR (L. pneumophila) OR (Legionella longbeachae) OR (L. longbeachae) OR (Legionella bozemanii) OR (L. bozemanii)) AND ((Human Health Impacts) OR (Symptoms) OR (Infectivity) OR (Uptake) OR (Host Interaction) OR (Genetic Knockout) OR (Disinfection) OR (Acanthamoeba) OR (Acanthamoeba polyphaga) OR (Acanthamoeba castellanni) or (A. polyphaga) OR (A. castellanni) OR (Premise Plumbing) OR (Biofilms).” Relevant citations were forward and reverse citation searched and imported into a Zotero library. Over 1500 papers were reviewed for relevance and 146 papers were included in this review.

Legionella Life Cycle

Legionella pneumophila has adapted specialized communication with a variety of amoeba and macrophages in order to invade the host 68,69.  As opposed to other bacteria that are predated upon as a sole mechanism of uptake into amoeba, L. pneumophila can attract amoeba and initiates its own uptake as part of its lifecycle. This specialization causes L. pneumophila to have less competition with other bacteria common to L. pneumophila’s environment, such as Escherchia coli, Aeromonas hydrophilia, Flavobacterium breve, and Psuedomonas aeruginosa 70.  L. pneumophila is known to invade host cells in a variety of forms including traditional phagocytosis, coiling phagocytosis and pinocytosis, utilizing multiple receptor-mediated pathways. Not only will L. pneumophila utilize different methods to infect different species of host, in many cases the bacteria are capable of utilizing more than one method of invasion on the same host cell.

L. pneumophila can utilize either traditional or coiling phagocytosis in order to invade protozoan or macrophage host cells. Coiling phagocytosis is a rare form of phagocytosis resulting in the bacteria internalized within the host in a membrane-bound vacuole that does not fuse with lysosomes 71–73. The complement receptor 3 (CR3) is responsible in part for the mediation of coiling phagocytosis and persists in the phagosomal membrane 74.  CR3 is also responsible for L. pneumophila’s attachement to the surface of macrophages 75.Coiling phagocytosis is rare, with only about 10% of L. pneumophila infections occurring through this method, with traditional phagocytosis occurring more commonly 76.

Acanthoamoeba castellanii phagocytizes L. pneumophila through a receptor-mediated system, utilizing the mannose-associated mannose receptor or mannose-binding lectin (MBL)68.  L. pneumophila does not use the MBL solely for protozoan entry, it is also able to activate the MBL for quick entry to pulmonary macrophages in mammals. Interestingly, the MBL is the same receptor that A. castellanii uses to bind to the corneal epithelium, causing ocular infections 68. L. pneumophila has a high affinity for the α1-3d-mannobiose-binding site of the mannose receptor from A. castellanii, allowing for efficient uptake by the protozoa 68. L. pneumophila has a slightly lower affinity for the d-mannose binding receptor in A. castellani77. This specificity, while known for A. castellanii, is most likely inconsistent among the entire Acanthamoeba genus and other L. pneumophila will utilizing predominately different pathways when infecting other genus of protozoa.

The N-acetyl-D-galactosamin (Gal/GalNAc) lectin receptor pathway and galactose (gal) are utilized by L. pneumophila to invade Hartmannella vermiformis. It is shown that adding gal or GalNAc to a H. vermiformis culture will significantly impair L. pneumophila’s ability to invade the host cell 69.  It is believed that H. vermiformis relies on this pathway heavily, if not exclusively for receptor-mediated pinocytosis of L. pneumophila. Whereas other protozoa, such as A. castellani and A. polyphaga utilize multiple pathways fort L. pneumophila uptake75,78. It is uncertain what ligand from L. pneumophilia activates the Gal/GalNAc lectin receptor pathway, but it has been speculated that it is one of the competence and adherence-associated pili (CAP) 79.  CAP is one of two distinct pili on the surface of L. pneumophila. It is a Type IV pili involved in adherence to protozoan and mammalian cells 80. However, the CAP is not exclusively responsible for this attachment. In protozoa, the CAP communicates with the lectin protozoan receptor76. This attachment is not strong over the long term, necessitating the rapid uptake processes seen in the lectin pathway 72,75. There is redundancy in the genes responsible for pili production in L. pneumophila, pilD and pilE are both responsible for pre-pilin peptidase and result in the expression of Type IV pili81,82.

The outer membrane proteins and/or (lipopolysaccharide) LPS structures play an important role in adhesion and/or uptake of L. pneumophila by protozoa. Legionella LPS is able to access classical pathways for entering cells as described above. In humans that activation of the classical pathway can be explained by mediation by antibodies of the IgM class present in the normal human serum (NHS) 83. However, Legionella LPS is also able to enter cells via the alternate pathway if the antibodies needed to activate the classical pathway are not present 84.  The complement component C3, covalently binds to the major outer membrane protein (MOMP) of L. pneumophila via the alternate pathway of complement activation. C3 acts as a ligand for complement receptors CR1 and CR3 which mediate phagocytosis of L. pneumophila 85.

L. pneumophila relies on a 24-kDa macrophage infectivity proteniator (Mip) protein to efficiently infect both mammalian phagocytic cells and protozoan cells 86,87. This protein is coded for by the mip gene and is thought to be conserved throughout the Legionella genus 88–90. The Mip sequence from 35 Legionella species were conserved at the amino acid level 82 to 99% 90. Mip exhibits a peptidylprolyl cis/trans isomerase (PPIase) activity and belongs to the enzyme family of FK506-binding proteins (FKBP), also seen in Chlamydia trachomatis and Trypanosoma cruzi 91–93.  Amino acids involved in PPIase activity were found to be totally conserved 90. When the protein is transported through the cytoplasmic membrane the N-terminal signal sequence is cleaved off and the protein is found on the surface of the Legionella cell, allowing for its infectivity role 94,95. It has been shown that for Legionella to have its full virulence in higher organisms, such as guinea pigs, both the full-length Mip protein and PPIase activity are necessary 96. While the Mip protein is important for invasion and establishment within human macrophages and protozoa, it is not necessary for intracellular replication. The Mip protein is repressed directly after the invasion of a mononcytic human cell, and regains activity after 24 hours of intracellular replication. This allows for the bacteria to be infective again after replication 97.

L. pneumophila was shown to require actin polymerization and intact microtubule cytoskeleton in order to invade HeLa cells effectively 98. Similar results were shown in A. castallanii and H. vermiformis by utilizing cytocholasin D, a microfilament disrupter. However, cytocholasin D did not affect the uptake of L. pneumophila by A. polyphaga 64,68,.  This further evidences that L. pneumophila is capable of utilizing various and sometimes redundant pathways to infect host cells.

L. pneumophila which has been grown in A. castellanii culture is phenotypically different from L. pneumophila grown in standard media. Amoeba grown L. pneumophila cells were more invasive than media-grown L. pneumophila cells, 100-fold more invasive for epithelial cells and 10-fold for macrophages and amoeba 100.  The amoeba grown bacteria expressed new proteins which may be responsible for the phenotypic differences amongst the stains 100. Studies have shown that L. pneumophila can horizontally transfer genes to and from host cells 101,102.

Effects of Environmental Stress on Legionella

L. pneumophila thrive in warm, freshwater habitats, particularly where water is stagnant 60,103. The pathogen prospers where institutionalized hot water is kept under 50 ͦ C 44,104. For these reasons the premise plumbing of hospitals and hotels tend to be natural environments for the bacteria to live and proliferate. Unfortunately, the elderly and immunocompromised are at the highest risk for contracting LD, meaning L. pneumophila thriving within a hospital setting put the most vulnerable populations at higher risk. Furthermore, patients with ambulatory impediments may take longer to shower, causing a higher exposure time if L. pneumophila is present. Considering the rise in legionellosis incidence numerous attempts have been made to eradicate L. pneumophila from water systems, in particular from biofilms of premise plumbing where the bacteriais significantly more difficult to treat than free living (living outside a biofilm) L. pneumophila.

There have been a variety of water treatment options tested to eradicate L. pneumophila in water distribution systems including: oxidizing agents such as chlorine, monochloramine, chlorine dioxide, bromine, iodine, ozone, hydrogen peroxide, ultraviolet (UV) light, heat, and halogenated hydantions, silver and copper ions, non-oxidizing agents such as aldehydes, amines, heterocyclic ketones, guanidines, thiocyanates, organo-tin compounds, halogenated amides, halogenated glycols, thiocarbamates, and heat 56,105. While chlorine is commonly used and known to be generally effective, the continuous and rising number of legionellosis outbreaks causes concern that chlorine is not effective enough in removing L. pneumophila from water distribution systems106–108.  Considering the bacteria’s ability to grow within and use biofilms as protection from chlorination, complete eradication is considered the best course of action in preventing future outbreaks.

Chlorine exists as hypochlorous acid in water in either its neutral form of HOCl or as hypochlorite ion, OCl– in environments where pH > 7.6. The term “free chlorine” refers to HOCl and OCl–, though HOCl is known to be more biocidal. Free chlorine inactivates bacteria by adversely affecting their respiratory and transport activities, causing deleterious effects on bacterial membranes, and causing direct oxidative damage to proteins and nucleotide bases 109,110. Free-living L. pneumophila, were killed within 3 minutes of being exposed to an aqueous solution of 2 mg/L of chlorine, whereas L. pneumophila living within biofilms were much more difficult to treat utilizing chlorine 111. Well-developed biofilms, those 1-2 months old, were able to protect L. pneumophila even when exposed to free chlorine at 50 mg/L for 1 hour. The bacteria were able to survive treatment and continue to grow. Newer biofilms, developed in the past 72 hours, were unable to protect the bacteria to the same extent, but the treatment was not effective in eradicating L. pneumophila from the water distribution system. While no viable colonies were recovered, immediately after treatment colonies began growing again the next day 112.

The event of L. pneumophila being viable but non-cultureable (VBNC) is fairly common during water treatment processes, and has been documented after chlorination by multiple studies 63,109,113. It has been theorized the bacteria become VBNC either because the cell membrane is not permeable at low chlorine levels, and the cells are stressed or injured during treatment rather than inactived or the membrane is permeabilized early and the hole allows ethidium monoazide bromoide to enter the cell and prevent reproduction, but the PCR signal is not reduced 109. The occurrence of VBNC L. peumophila in water distribution systems may lead to false confidence that the bacteria have been eradicated from the system, if presence is determined by a culture method. Chlorination has been shown to have little to no effect in preventing L. pneumophila accumulation in some studies while other studies showed a complete eradication of the bacteria did not occur, chlorination did lower prevalence enough to prevent legionellosis outbreaks 114–116. Chlorine neutralization can allow for greater persistence of L. pneumophila in the biofilm, accelerate the development of the microbial community, and reduce the susceptibility of disinfection in the future 117. Therefore, it is important with regards to L. pneumophila, to ensure that premise plumbing systems do not go through regular periods of chlorine neutralization. Considering the decay of residual chlorine in large premise plumbing systems, these buildings are more likely to have sections of chlorine neutralization, which may also explain further why they are more at risk for a legionellosis outbreak.

It is possible that L. pneumophila are utilizing host cells that are resistant to chlorine to grow and thrive, or that chlorine is inefficient at penetrating the biofilm and therefore is not reaching the bacteria 115,118.  Some studies have effectively used shock hyperchlorination, (a short period of elevated chlorine dosage, 20-50 mg/L), however, maintaining high levels of chlorine in the water system can have both human health and structural effects 114,119. Chlorine can be corrosive to the pipes and its biproducts can cause adverse health effects for humans and the environment. The negative side effects of chlorine as a disinfectant lead to the use of monochloramine 120.

Monochloramine (NH2Cl), a derivative of ammonia, has been shown to be more effective than free chlorine as a residual disinfectant. Hospitals using free chlorine had an odds ratio of 10.2 of a LD outbreak in comparison to those using monochloramine 103. It has been suggested that monochloramine is more effective in penetrating the biofilm than chlorine 121,122. However, monochloramine showed similar issues to chlorine, while it was able to suppress L. pneumophila it proved incapable of eradicating the bacteria from the system and ineffective eradication could lead to monochloramine resistance 123–125.

Bromine and iodine have both been effective in disinfecting swimming pools and cooling water, however, bromine is not recommended for potable drinking water and there is controversy surrounding safe levels of consumption over an extended period of time with iodine 110,126.  While both are oxidizing agents similar to chlorine, neither is as effective as chlorine at inactivating L. pneumophila. Iodine was particularly inept at killing L. pneumophila within a biofilm 67,127,128.

Ozone can effectively inactivate bacteria by damaging DNA in both the gaseous and dissolved states 129,130. However, because it reacts so quickly it is unable to be used as a residual in the water treatment, but chlorine can be added after ozonization for the residual effect. Ozone is more effective than chlorine is controlling L. pneumophila, however, due to its quick dissipation and inability to be used as residual treatment, another form a treatment is required in conjunction with ozone in order to treat bacteria within biofilms in premise plumbing 131,132.

L. pneumophila is common to biofilms in premise plumbing and is known to have a commensal relationship with protozoa within the biofilms, which can act as protective reservoirs for the bacteria 115,133,134. As such, it is important that treatments are able to penetrate the extracellular polymeric substance (EPS) matrix of the biofilm and persist in large premise plumbing systems 135,136. Chlorine dioxide was shown to be most effective in reducing L. pneumophila levels in biofilms in copper piping, compared to chlorine, monochloramine, electro-chlorination, ozone, and copper-silver ionization, due to its longer residual activity and its ability to penetrate the biofilm 137.  However, the amoeba in the biofilm were resistant to all forms of treatment, and L. pneumophila was able to regrow after short periods of non-treatment 137,138.

Temperature may be the most effective means of controlling L. pneumophila in premise plumbing systems. L. pneumophila will survive in temperatures less than 20 ͦ C, however, they will not replicate 139.  L. pneumophila thrive in temperatures 20 – 50 ͦ C with little to no growth over 50 ͦ C, but have been isolated from hot water with temperatures as high as 66 ͦ C 140,141.  When temperatures reach 70 ͦ C the bacteria are killed almost instantly 142. Maintaining hot water systems over 50 ͦ C throughout the entirety of the premise plumbing is challenging from both an energy use and a hazard standpoint. Water stagnates for up to 23 hours a day within buildings leading to vast temperature fluctuations. Furthermore, maintaining high water temperatures from the heater to the tap can be costly, particularly if the premise plumbing system is large 143. Furthermore, facilities with large premise plumbing systems are concerned with the risk of scalding, specifically healthcare facilities who may have patients with decreased ambulatory abilities and other health concerns, such as dementia which may not allow them to recognize the water temperature or move from hot water in a timely manner. Maintaining temperatures high enough to retard the growth of or kill L. pneumophila could prove hazardous to the population 144.

High nutrient content, such as iron, organic carbon, nitrogen, and phosphorous support the growth and persistence of L. pneumophila, but the bacteria are also resistant against nutrient depletion 48,113,145,146. Similar to the bacteria’s defense against oxidative stress L. pneumophila can become VBNC when experiencing nutrient depletion 113. It appears that L. pneumophila can survive and thrive in any conventional pipe material, however the bacteria are most prolific in cast iron pipes and least productive in copper piping, with plastic piping as the middle ground 140,147. Piping materials can be taken into consideration for new construction projects, but tend to be a cost-prohibitive remediation or preventative factor in existing structures.

The laboratory and in-field experiments described above have shown that eradicating L. pneumophila within the biofilm is challenging if not unrealistic, and periods of treatment neutralization can lead to legionellosis outbreaks. Therefore, it is theorized that the prevention of legionellosis should focus around decreasing infectivity of L. pneumophila rather than eliminating the bacteria from premise plumbing.  Utilizing the lifecycle and genetic information discussed in this review and the following replication and egression reviews to create an effective QMRA model for L. pneumophila within the drinking water system is a first step in reducing the bacteria’s virulence and preventing legionellosis outbreaks.

Discussion

Legionellosis is a serious threat to human health and has been increasing in incidence throughout the US. Prevention of the disease is difficult due to the endosymbiotic relationship L. pneumophila has with protozoa within the biofilm of premise plumbing systems 45. L. pneumophila are particularly resistant to typical disinfection measures due to the ability to use protozoa as a reservoir 115. There is a good deal of information regarding L. pneumophila’s lifecycle and factors effecting the bacteria’s virulence and persistence. However, a comprehensive literature review providing information necessary to model the behavior of L. pneumophila within a biofilm was not available, necessitating this article. This literature review focuses on the uptake portion of the bacteria’s lifecycle. The mechanisms between L. pneumophila’s entry into a protozoa and a human macrophage are very similar, and the genetic knockdowns which prevent the bacteria from uptake into a protozoan typically prevent invasion of a macrophage as well 80,148. However, L. pneumophila has redundancies in the uptake pathway which allow the bacteria more versatility 69,76.

L. pneumophila utilizes multiple pathways in order to invade host cells, including traditional phagocytosis, coiling phagocytosis and pinocytosis, utilizing multiple receptor-mediated pathways 71–73. Not only will L. pneumophila utilize different methods to infect different species of host, in many cases the bacteria are capable of utilizing more than one method of invasion on the same host cell. Furthermore, L. pneumophila is unique in its ability to attract host cells and initiate invasion as part of its lifecycle 149.

Future literature reviews will detail L. pneumophila’s growth and replication within a host cell and the egression from a host cell and ability to reinfect the next host. Due to the amount of information currently available in the literature, the authors felt it necessary to write separate reviews for the main sections of the bacteria’s lifecycle.

Acknowlegments

1. Glick, T. H. et al. Pontiac Fever an Epidemic of Unknown Etiology in a Health Department: I. Clinical and Epidemiologic Aspects. Am. J. Epidemiol. 107, 149–160 (1978).

2. Kaufmann, A. F. et al. Pontiac Fever: Isolation of the Etiologic Agent (Legionella pneumophila) and Demonstration of its Mode of Transmission. Am. J. Epidemiol. 114, 337–347 (1981).

3. Fraser, D. W. et al. Legionnaires’ Disease. N. Engl. J. Med. 297, 1189–1197 (1977).

4. McDade, J. E. et al. Legionnaires’ Disease. http://dx.doi.org/10.1056/NEJM197712012972202 (1977). Available at: http://www.nejm.org/doi/full/10.1056/NEJM197712012972202. (Accessed: 19th December 2016)

5. Winn, W. C. Legionnaires disease: historical perspective. Clin. Microbiol. Rev. 1, 60–81 (1988).

6. Boshuizen, H. C. et al. Subclinical Legionella infection in workers near the source of a large outbreak of Legionnaires disease. J. Infect. Dis. 184, 515–518 (2001).

7. Kirby, B. D., Snyder, K. M., Meyer, R. D. & Finegold, S. M. Legionnaires’ disease: clinical features of 24 cases. Ann. Intern. Med. 89, 297–309 (1978).

8. Lattimer, G. L. et al. The Philadelphia epidemic of Legionnaire’s disease: clinical, pulmonary, and serologic findings two years later. Ann. Intern. Med. 90, 522–526 (1979).

9. Edelstein, P. H. & Meyer, R. D. Legionnaires’ Disease. Chest 85, 114–120 (1984).

10. Marston, B. J., Lipman, H. B. & Breiman, R. F. Surveillance for Legionnaires’ Disease: Risk Factors for Morbidity and Mortality. Arch. Intern. Med. 154, 2417–2422 (1994).

11. Matsunaga, K., Klein, T. W., Friedman, H. & Yamamoto, Y. Involvement of nicotinic acetylcholine receptors in suppression of antimicrobial activity and cytokine responses of alveolar macrophages to Legionella pneumophila infection by nicotine. J. Immunol. Baltim. Md 1950 167, 6518–6524 (2001).

12. Straus, W. L. et al. Risk factors for domestic acquisition of legionnaires disease. Ohio legionnaires Disease Group. Arch. Intern. Med. 156, 1685–1692 (1996).

13. Health, U. D. of, Services, H. & others. The health consequences of smoking: a report of the Surgeon General. Atlanta GA US Dep. Health Hum. Serv. Cent. Dis. Control Prev. Natl. Cent. Chronic Dis. Prev. Health Promot. Off. Smok. Health 62, (2004).

14. Neil, K. & Berkelman, R. Increasing incidence of legionellosis in the United States, 1990-2005: changing epidemiologic trends. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 47, 591–599 (2008).

15. (CDC) & Centers for Disease Control and Prevention. Legionellosis—United States, 2000-2009. MMWR Morb. Mortal. Wkly. Rep. 60, 1083 (2011).

16. Choffnes, E. R., Mack, A. & others. Global issues in water, sanitation, and health: workshop summary. (National Academies Press, 2009).

17. Yoder, J. et al. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking–United States, 2005-2006. Morb. Mortal. Wkly. Rep. Surveill. Summ. Wash. DC 2002 57, 39–62 (2008).

18. National Research Council (US) Committee on Drinking Water Contaminants. Review of Methods for Assessing Microbial Pathogens. (National Academies Press (US), 1999).

19. US EPA, O. Contaminant Candidate List 3 – CCL 3. Available at: https://www.epa.gov/ccl/contaminant-candidate-list-3-ccl-3. (Accessed: 13th January 2017)

20. Craun, G. F. et al. Causes of Outbreaks Associated with Drinking Water in the United States from 1971 to 2006. Clin. Microbiol. Rev. 23, 507–528 (2010).

21. Benin, A. L. et al. An outbreak of travel-associated Legionnaires disease and Pontiac fever: the need for enhanced surveillance of travel-associated legionellosis in the United States. J. Infect. Dis. 185, 237–243 (2002).

22. Rose, J. B. Future health assessment and risk-management integration for infectious diseases and biological weapons for deployed US forces. Strateg. Prot. Health Deployed Assess. Health Risks Deployed US Forces 59–112 (2000).

23. Muder, R. R., Victor, L. Y. & Woo, A. H. Mode of transmission of Legionella pneumophila: a critical review. Arch. Intern. Med. 146, 1607–1612 (1986).

24. Osterholm, M. T. et al. A 1957 Outbreak of Legionnaires’ Disease Associated with a Meat Packing Plant. Am. J. Epidemiol. 117, 60–67 (1983).

25. Mahoney, F. J. et al. Communitywide outbreak of Legionnaires’ disease associated with a grocery store mist machine. J. Infect. Dis. 165, 736–739 (1992).

26. Boer, J. den et al. A large outbreak of Legionnaires’ disease at a flower show, the Netherlands, 1999. Emerg. Infect. Dis. 8, 37–43 (2002).

27. Pastoris, M. C. et al. Molecular epidemiology of an outbreak of Legionnaires’ disease associated with a cooling tower in Genova-Sestri Ponente, Italy. Eur. J. Clin. Microbiol. Infect. Dis. 16, 883–892 (1997).

28. Brown, C. M. et al. A community outbreak of Legionnaires’ disease linked to hospital cooling towers: an epidemiological method to calculate dose of exposure. Int. J. Epidemiol. 28, 353–359 (1999).

29. Bhopal, R. S., Fallon, R. J., Buist, E. C., Black, R. J. & Urquhart, J. D. Proximity of the home to a cooling tower and risk of non-outbreak Legionnaires’ disease. BMJ 302, 378–383 (1991).

30. Schwarzenbach, R. P., Egli, T., Hofstetter, T. B., Gunten, U. von & Wehrli, B. Global Water Pollution and Human Health. Annu. Rev. Environ. Resour. 35, 109–136 (2010).

31. Dondero, T. J. et al. An Outbreak of Legionnaires’ Disease Associated with a Contaminated Air-Conditioning Cooling Tower. N. Engl. J. Med. 302, 365–370 (1980).

32. Berglund, B. et al. Effects of indoor air pollution on human health. Indoor Air 2, 2–25 (1992).

33. Armstrong, T. W. & Haas, C. N. Legionnaires’ disease: evaluation of a quantitative microbial risk assessment model. J. Water Health 6, 149–166 (2008).

34. Jernigan, D. et al. Outbreak of Legionnaires’ disease among cruise ship passengers exposed to a contaminated whirlpool spa. The Lancet 347, 494–499 (1996).

35. Lück, P. C. et al. Prevalence of Legionella species, serogroups, and monoclonal subgroups in hot water systems in south-eastern Germany. Zentralblatt Hyg. Umweltmed. Int. J. Hyg. Environ. Med. 193, 450–460 (1993).

36. Lück, P., Lau, B., Seidel, S. & Postl, U. Legionellae in dental units–a hygienic risk? Dtsch. Zahn Mund Kieferheilkd. Zentralbl. 80, 341–346 (1991).

37. Fields, B. S., Benson, R. F. & Besser, R. E. Legionella and Legionnaires’ Disease: 25 Years of Investigation. Clin. Microbiol. Rev. 15, 506–526 (2002).

38. Yu, V. L. et al. Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey. J. Infect. Dis. 186, 127–128 (2002).

39. O’CONNOR, B. A. et al. Does using potting mix make you sick? Results from a Legionella longbeachae case-control study in South Australia. Epidemiol. Infect. 135, 34–39 (2007).

40. Cazalet, C. et al. Analysis of the Legionella longbeachae genome and transcriptome uncovers unique strategies to cause Legionnaires’ disease. PLoS Genet. 6, e1000851 (2010).

41. Centers for Disease Control and Prevention (CDC). Legionnaires’ Disease associated with potting soil–California, Oregon, and Washington, May-June 2000. MMWR Morb. Mortal. Wkly. Rep. 49, 777–778 (2000).

42. Graham, F., White, P., Harte, D. & Kingham, S. Changing epidemiological trends of legionellosis in New Zealand, 1979–2009. Epidemiol. Infect. 140, 1481–1496 (2012).

43. Fliermans, C. B. et al. Ecological distribution of Legionella pneumophila. Appl. Environ. Microbiol. 41, 9–16 (1981).

44. Blackburn, B. G. et al. Surveillance for waterborne-disease outbreaks associated with drinking water–United States, 2001-2002. Morb. Mortal. Wkly. Rep. Surveill. Summ. Wash. DC 2002 53, 23–45 (2004).

45. National Research Council (US) Committee on Indicators for Waterborne. Ecology and Evolution of Waterborne Pathogens and Indicator Organisms. (National Academies Press (US), 2004).

46. World Health Organization. Guide to Ship Sanitation. 3rd edition. (2011).

47. Flemming, H.-C. Biofilms in Drinking Water Systems-Part I: Overview. GAS WASSERFACH WASSER ABWASSER 139, S65–S72 (1998).

48. Jjemba, P. K., Johnson, W., Bukhari, Z. & LeChevallier, M. W. Occurrence and Control of Legionella in Recycled Water Systems. Pathogens 4, 470–502 (2015).

49. CARRINGTON, C. B. Pathology of Legionnaires’ disease. Ann. Intern. Med. 90, 496–499 (1979).

50. Balows, A., Fraser, D. & others. International symposium on Legionnaires’ disease, 13-15 November 1978, Atlanta, Georgia. Ann. Intern. Med. 90, 489–714 (1979).

51. SWARTZ, M. N. Clinical aspects of Legionnaires’ disease. Ann. Intern. Med. 90, 492–495 (1979).

52. Isenberg, H. D. Microbiology of Legionnaires’ Disease Bacterium. Ann. Intern. Med. 90, 499 (1979).

53. Eickhoff, T. C. Epidemiology of Legionnaires’ Disease. Ann. Intern. Med. 90, 499 (1979).

54. Hubber, A. & Roy, C. R. Modulation of host cell function by Legionella pneumophila type IV effectors. Annu. Rev. Cell Dev. Biol. 26, 261–283 (2010).

55. Isberg, R. R., O’Connor, T. & Heidtman, M. The Legionella pneumophila replication vacuole: making a cozy niche inside host cells. Nat. Rev. Microbiol. 7, 13–24 (2009).

56. Kim, B. R., Anderson, J. E., Mueller, S. A., Gaines, W. A. & Kendall, A. M. Literature review—efficacy of various disinfectants against Legionella in water systems. Water Res. 36, 4433–4444 (2002).

57. Abdel-Nour, M., Duncan, C., Low, D. E. & Guyard, C. Biofilms: The Stronghold of Legionella pneumophila. Int. J. Mol. Sci. 14, 21660–21675 (2013).

58. Taylor, M., Ross, K. & Bentham, R. Legionella, Protozoa, and Biofilms: Interactions Within Complex Microbial Systems. Microb. Ecol. 58, 538–547 (2009).

59. Rowbotham, T. J. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J. Clin. Pathol. 33, 1179–1183 (1980).

60. Barbaree, J. M., Fields, B. S., Feeley, J. C., Gorman, G. W. & Martin, W. T. Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila. Appl. Environ. Microbiol. 51, 422–424 (1986).

61. Dupuy, M. et al. Efficiency of water disinfectants against Legionella pneumophila and Acanthamoeba. Water Res. 45, 1087–1094 (2011).

62. Schwartz, T., Hoffmann, S. & Obst, U. Formation of natural biofilms during chlorine dioxide and u.v. disinfection in a public drinking water distribution system. J. Appl. Microbiol. 95, 591–601 (2003).

63. Gião, M. S., Wilks, S., Azevedo, N. F., Vieira, M. J. & Keevil, C. W. Incorporation of natural uncultivable Legionella pneumophila into potable water biofilms provides a protective niche against chlorination stress. Biofouling 25, 345–351 (2009).

64. Harb, O. S., Gao, L. Y. & Abu Kwaik, Y. From protozoa to mammalian cells: a new paradigm in the life cycle of intracellular bacterial pathogens. Environ. Microbiol. 2, 251–265 (2000).

65. Horwitz, M. A. & Silverstein, S. C. Legionnaires’ Disease Bacterium (Legionella pneumophila) Multiplies Intracellularly in Human Monocytes. J. Clin. Invest. 66, 441–450 (1980).

66. Nash, T., Libby, D. & Horwitz, M. Interaction between the legionnaires’ disease bacterium (Legionella pneumophila) and human alveolar macrophages. Influence of antibody, lymphokines, and hydrocortisone. J. Clin. Invest. 74, 771 (1984).

67. Berk, S. G., Ting, R. S., Turner, G. W. & Ashburn, R. J. Production of Respirable Vesicles Containing Live Legionella pneumophila Cells by Two Acanthamoeba spp. Appl. Environ. Microbiol. 64, 279–286 (1998).

68. Declerck, P., Behets, J., De Keersmaecker, B. & Ollevier, F. Receptor-mediated uptake of Legionella pneumophila by Acanthamoeba castellanii and Naegleria lovaniensis. J. Appl. Microbiol. 103, 2697–2703 (2007).

69. Harb, O. S., Venkataraman, C., Haack, B. J., Gao, L. Y. & Kwaik, Y. A. Heterogeneity in the attachment and uptake mechanisms of the Legionnaires’ disease bacterium, Legionella pneumophila, by protozoan hosts. Appl. Environ. Microbiol. 64, 126–132 (1998).

70. Declerck, P. et al. Impact of non-Legionella bacteria on the uptake and intracellular replication of Legionella pneumophila in Acanthamoeba castellanii and Naegleria lovaniensis. Microb. Ecol. 50, 536–549 (2005).

71. Rittig, M. G. et al. Coiling phagocytosis is the preferential phagocytic mechanism for Borrelia burgdorferi. Infect. Immun. 60, 4205–4212 (1992).

72. Horwitz, M. A. The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J. Exp. Med. 158, 2108–2126 (1983).

73. Bozue, J. A. & Johnson, W. Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion. Infect. Immun. 64, 668–673 (1996).

74. Clemens, D. L. & Horwitz, M. A. Membrane sorting during phagocytosis: selective exclusion of major histocompatibility complex molecules but not complement receptor CR3 during conventional and coiling phagocytosis. J. Exp. Med. 175, 1317–1326 (1992).

75. Steinert, M., Ott, M., Lück, P. C., Tannich, E. & Hacker, J. Studies on the uptake and intracellular replication of Legionella pneumophila in protozoa and in macrophage-like cells. FEMS Microbiol. Ecol. 15, 299–307 (1994).

76. Kwaik, Y. A., Gao, L.-Y., Stone, B. J., Venkataraman, C. & Harb, O. S. Invasion of Protozoa by Legionella pneumophila and Its Role in Bacterial Ecology and Pathogenesis. Appl. Environ. Microbiol. 64, 3127–3133 (1998).

77. Cao, Z., Jefferson, D. M. & Panjwani, N. Role of Carbohydrate-mediated Adherence in Cytopathogenic Mechanisms of Acanthamoeba. J. Biol. Chem. 273, 15838–15845 (1998).

78. Köhler, R. et al. Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila. Mol. Gen. Genet. MGG 262, 1060–1069 (2000).

79. Abu Kwaik, Y., Venkataraman, C., Harb, O. S. & Gao, L.-Y. Signal Transduction in the Protozoan Host Hartmannella vermiformis upon Attachment and Invasion by Legionella micdadei. Appl. Environ. Microbiol. 64, 3134–3139 (1998).

80. Stone, B. J. & Abu Kwaik, Y. Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells. Infect. Immun. 66, 1768–1775 (1998).

81. Lucas, C. E., Brown, E. & Fields, B. S. Type IV pili and type II secretion play a limited role in Legionella pneumophila biofilm colonization and retention. Microbiol. Read. Engl. 152, 3569–3573 (2006).

82. Mampel, J. et al. Planktonic Replication Is Essential for Biofilm Formation by Legionella pneumophila in a Complex Medium under Static and Dynamic Flow Conditions. Appl. Environ. Microbiol. 72, 2885–2895 (2006).

83. Mintz, C. S., Arnold, P. I., Johnson, W. & Schultz, D. R. Antibody-independent binding of complement component C1q by Legionella pneumophila. Infect. Immun. 63, 4939–4943 (1995).

84. Mintz, C. S., Schultz, D. R., Arnold, P. I. & Johnson, W. Legionella pneumophila lipopolysaccharide activates the classical complement pathway. Infect. Immun. 60, 2769–2776 (1992).

85. Bellinger-Kawahara, C. & Horwitz, M. Legionella pneumophila fixes complement component C3 to its surface-demonstration by ELISA. in Program of the 1987 Annual Meeting of the American Society of Microbiology, Atlanta, GA, March 1–6 (1987).

86. Cianciotto, N. P. & Fields, B. S. Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages. Proc. Natl. Acad. Sci. 89, 5188–5191 (1992).

87. Fields, B. S. The molecular ecology of legionellae. Trends Microbiol. 4, 286–290 (1996).

88. Cianciotto, N. P., Eisenstein, B. I., Mody, C. H. & Engleberg, N. C. A mutation in the mip gene results in an attenuation of Legionella pneumophila virulence. J. Infect. Dis. 162, 121–126 (1990).

89. Cianciotto, N. P., Eisenstein, B. I., Mody, C. H., Toews, G. B. & Engleberg, N. C. A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection. Infect. Immun. 57, 1255–1262 (1989).

90. Ratcliff, R. M., Lanser, J. A., Manning, P. A. & Heuzenroeder, M. W. Sequence-Based Classification Scheme for the GenusLegionella Targeting the mip Gene. J. Clin. Microbiol. 36, 1560–1567 (1998).

91. Fischer, G., Bang, H., Ludwig, B., Mann, K. & Hacker, J. Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPlase) activity. Mol. Microbiol. 6, 1375–1383 (1992).

92. Lundemose, A. G., Kay, J. E. & Pearce, J. H. Chlamydia trachomatis Mip-like protein has peptidyl-prolyl cis/trans isomerase activity that is inhibited by FK506 and rapamycin and is implicated in initiation of chlamydial infection. Mol. Microbiol. 7, 777–783 (1993).

93. Moro, A., Ruiz-Cabello, F., Fernández-Cano, A., Stock, R. P. & González, A. Secretion by Trypanosoma cruzi of a peptidyl-prolyl cis-trans isomerase involved in cell infection. EMBO J. 14, 2483–2490 (1995).

94. Wintermeyer, E. et al. Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells. Infect. Immun. 63, 4576–4583 (1995).

95. Helbig, J. H., Kurtz, J. B., Pastoris, M. C., Pelaz, C. & Lück, P. C. Antigenic lipopolysaccharide components of Legionella pneumophila recognized by monoclonal antibodies: Possibilities and limitations for division of the species into serogroups. ResearchGate 35, 2841–5 (1997).

96. Köhler, R. et al. Biochemical and Functional Analyses of the Mip Protein: Influence of the N-Terminal Half and of Peptidylprolyl Isomerase Activity on the Virulence of Legionella pneumophila. Infect. Immun. 71, 4389–4397 (2003).

97. Wieland, H., Faigle, M., Lang, F., Northoff, H. & Neumeister, B. Regulation of the Legionella mip-promotor during infection of human monocytes. FEMS Microbiol. Lett. 212, 127–132 (2002).

98. Garduño, R. A., Quinn, F. D. & Hoffman, P. S. HeLa cells as a model to study the invasiveness and biology of Legionella pneumophila. Can. J. Microbiol. 44, 430–440 (1998).

99. Moffat, J. F. & Tompkins, L. A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. Infect. Immun. 60, 296–301 (1992).

100. Cirillo, J. D., Falkow, S. & Tompkins, L. S. Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion. Infect. Immun. 62, 3254–3261 (1994).

101. Felipe, K. S. de et al. Evidence for Acquisition of Legionella Type IV Secretion Substrates via Interdomain Horizontal Gene Transfer. J. Bacteriol. 187, 7716–7726 (2005).

102. Gomez-Valero, L., Rusniok, C. & Buchrieser, C. Legionella pneumophila: population genetics, phylogeny and genomics. Infect. Genet. Evol. 9, 727–739 (2009).

103. Kool, J. L., Carpenter, J. C. & Fields, B. S. Effect of monochloramine disinfection of municipal drinking water on risk of nosocomial Legionnaires’ disease. Lancet Lond. Engl. 353, 272–277 (1999).

104. Ashbolt, N. J. Microbial contamination of drinking water and disease outcomes in developing regions. Toxicology 198, 229–238 (2004).

105. Atlas, R. M. Legionella: from environmental habitats to disease pathology, detection and control. Environ. Microbiol. 1, 283–293 (1999).

106. Berjeaud, J.-M. et al. Legionella pneumophila: The Paradox of a Highly Sensitive Opportunistic Waterborne Pathogen Able to Persist in the Environment. Front. Microbiol. 7, (2016).

107. Declerck, P. Biofilms: the environmental playground of Legionella pneumophila. Environ. Microbiol. 12, 557–566 (2010).

108. Emtiazi, F., Schwartz, T., Marten, S. M., Krolla-Sidenstein, P. & Obst, U. Investigation of natural biofilms formed during the production of drinking water from surface water embankment filtration. Water Res. 38, 1197–1206 (2004).

109. Delgado-Viscogliosi, P., Solignac, L. & Delattre, J.-M. Viability PCR, a culture-independent method for rapid and selective quantification of viable Legionella pneumophila cells in environmental water samples. Appl. Environ. Microbiol. 75, 3502–3512 (2009).

110. Haas, C. N. Disinfection. in Water quality and treatment, A handbook of community water supplies (ed. Pontius, F.) (McGraw Hill, 1990).

111. Miyamoto, M., Yamaguchi, Y. & Sasatu, M. Disinfectant effects of hot water, ultraviolet light, silver ions and chlorine on strains of Legionella and nontuberculous mycobacteria. Microbios 101, 7–13 (1999).

112. Cooper, I. R. & Hanlon, G. W. Resistance of Legionella pneumophila serotype 1 biofilms to chlorine-based disinfection. J. Hosp. Infect. 74, 152–159 (2010).

113. Murga, R. et al. Role of biofilms in the survival of Legionella pneumophila in a model potable-water system. Microbiology 147, 3121–3126 (2001).

114. Casini, B. et al. Molecular epidemiology of Legionella pneumophila serogroup 1 isolates following long-term chlorine dioxide treatment in a university hospital water system. J. Hosp. Infect. 69, 141–147 (2008).

115. Kilvington, S. & Price, J. Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure. J. Appl. Bacteriol. 68, 519–525 (1990).

116. Långmark, J., Storey, M. V., Ashbolt, N. J. & Stenström, T.-A. Accumulation and Fate of Microorganisms and Microspheres in Biofilms Formed in a Pilot-Scale Water Distribution System. Appl. Environ. Microbiol. 71, 706–712 (2005).

117. Codony, F., Morató, J. & Mas, J. Role of discontinuous chlorination on microbial production by drinking water biofilms. Water Res. 39, 1896–1906 (2005).

118. De Beer, D., Srinivasan, R. & Stewart, P. S. Direct measurement of chlorine penetration into biofilms during disinfection. Appl. Environ. Microbiol. 60, 4339–4344 (1994).

119. Yu-sen, E. L., Vidic, R. D., Stout, J. E. & Victor, L. Y. Legionella in water distribution systems. Am. Water Works Assoc. J. 90, 112 (1998).

120. White, G. C. Handbook of chlorination for potable water, wastewater, cooling water, industrial processes, and swimming pools. in Handbook of chlorination for potable water, wastewater, cooling water, industrial processes, and swimming pools (Van Nostrand Reinhold, 1972).

121. Chen, X. & Stewart, P. S. Chlorine penetration into artificial biofilm is limited by a reaction- diffusion interaction. Environ. Sci. Technol. 30, 2078–2083 (1996).

122. LeChevallier, M. W., Cawthon, C. D. & Lee, R. G. Inactivation of biofilm bacteria. Appl. Environ. Microbiol. 54, 2492–2499 (1988).

123. Shen, Y. et al. Effect of Disinfectant Exposure on Legionella pneumophila Associated with Simulated Drinking Water Biofilms: Release, Inactivation, and Infectivity. Environ. Sci. Technol. 51, 2087–2095 (2017).

124. Williams, M. M. & Braun-Howland, E. B. Growth of Escherichia coli in Model Distribution System Biofilms Exposed to Hypochlorous Acid or Monochloramine. Appl. Environ. Microbiol. 69, 5463–5471 (2003).

125. Chiao, T.-H., Clancy, T. M., Pinto, A., Xi, C. & Raskin, L. Differential Resistance of Drinking Water Bacterial Populations to Monochloramine Disinfection. Environ. Sci. Technol. 48, 4038–4047 (2014).

126. Backer, H. & Hollowell, J. Use of iodine for water disinfection: iodine toxicity and maximum recommended dose. Environ. Health Perspect. 108, 679–684 (2000).

127. Cargill, K. L., Pyle, B. H., Sauer, R. L. & McFeters, G. A. Effects of culture conditions and biofilm formation on the iodine susceptibility of Legionella pneumophila. Can. J. Microbiol. 38, 423–429 (1992).

128. Thomas, W. M., Eccles, J. & Fricker, C. Laboratory observations of biocide efficiency against Legionella in model cooling tower systems. ASHRAE Trans. 105, 607 (1999).

129. Farooq, S., Chian, E. & Engelbrecht, R. Basic concepts in disinfection with ozone. J. Water Pollut. Control Fed. 1818–1831 (1977).

130. Hamelin, C. Production of single-and double-strand breaks in plasmid DNA by ozone. Int. J. Radiat. Oncol. Biol. Phys. 11, 253–257 (1985).

131. Domingue, E. L., Tyndall, R., Mayberry, W. & Pancorbo, O. Effects of three oxidizing biocides on Legionella pneumophila serogroup 1. Appl. Environ. Microbiol. 54, 741–747 (1988).

132. McGrane, W. Ozone, a study of the effects of biocides on Legionella pneumophila. Ind. Water Treat. 27, 28–32 (1995).

133. King, C. H., Shotts, E. B., Wooley, R. E. & Porter, K. G. Survival of coliforms and bacterial pathogens within protozoa during chlorination. Appl. Environ. Microbiol. 54, 3023–3033 (1988).

134. Storey, M., Ashbolt, N. & Stenström, T. Biofilms, thermophilic amoebae and Legionella pneumophila-a quantitative risk assessment for distributed water. Water Sci. Technol. 50, 77–82 (2004).

135. Szewzyk, U., Szewzyk, R., Manz, W. & Schleifer, K.-H. Microbiological Safety of Drinking Water. Annu. Rev. Microbiol. 54, 81–127 (2000).

136. Wimpenny, J., Manz, W. & Szewzyk, U. Heterogeneity in biofilms. FEMS Microbiol. Rev. 24, 661–671 (2000).

137. Thomas, V. et al. Amoebae in domestic water systems: resistance to disinfection treatments and implication in Legionella persistence. J. Appl. Microbiol. 97, 950–963 (2004).

138. Srinivasan, A. et al. A 17‐Month Evaluation of a Chlorine Dioxide Water Treatment System to Control Legionella Species in a Hospital Water Supply. Infect. Control Hosp. Epidemiol. 24, 575–579 (2003).

139. Schwake, D. O., Alum, A. & Abbaszadegan, M. Impact of Environmental Factors on Legionella Populations in Drinking Water. Pathogens 4, 269–282 (2015).

140. Rogers, J., Dowsett, A., Dennis, P., Lee, J. & Keevil, C. Influence of temperature and plumbing material selection on biofilm formation and growth of Legionella pneumophila in a model potable water system containing complex microbial flora. Appl. Environ. Microbiol. 60, 1585–1592 (1994).

141. Dennis, R. Legionnaires’ disease–preventative maintenance. J. Inst. Hosp. Eng. 42, 14 (1988).

142. Dennis, P. Environmental factors affecting the survival and pathogenicity of Legionella pneumophila. (CNAA, 1986).

143. Proctor, C. R. & Hammes, F. Drinking water microbiology — from measurement to management. Curr. Opin. Biotechnol. 33, 87–94 (2015).

144. Murray, J. A study of the prevention of hot tapwater burns. Burns 14, 185–193 (1988).

145. Cianciotto, N. P. Iron Acquisition by Legionella pneumophila. BioMetals 20, 323–331 (2007).

146. Orsi, N. The antimicrobial activity of lactoferrin: Current status and perspectives. BioMetals 17, 189–196 (2004).

147. Gagnon, G. A. et al. Disinfectant efficacy of chlorite and chlorine dioxide in drinking water biofilms. Water Res. 39, 1809–1817 (2005).

148. Tiaden, A. et al. The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila. Environ. Microbiol. 12, 1243–1259 (2010).

149. Shevchuk, O., Jäger, J. & Steinert, M. Virulence Properties of the Legionella Pneumophila Cell Envelope. Front. Microbiol. 2, (2011).