Review by: Stine Skotte Bjerregaard, M.Sc. Biotech. Eng., Adept Water Technologies, September 2014.
About the Legionella bacterium
Legionella is a facultative, rod-shaped, and gram-negative bacterium of the bacterial class Gammaproteobacteria and most Legionellae are motile due to one or more polar or subpolar unsheathed flagella [1; 2; 4]. It can be found in aquatic environments and can exist in free-living planktonic forms or more often as intracellular parasites of protozoa . The most well known and common pathogen of the Legionella genus is Legionella pneumophila (L. pneumophila), and L. pneumophila serogroup 1 is accountable for more than 70% of diagnosed cases of Legionella-associated disease . This review is focused on Legionella pneumophila, however other Legionella species are associated with a health risk as well, when present in dental unit waterlines.
L. pneumophila is an opportunistic pathogen, which can cause severe illness in immunocompromised individuals . It can cause severe pneumonia with a relative high mortality rate, when transmitted through aerosols. L.pneumophila can also cause the less severe illness Pontiac fever, which gives flu-like symptoms . Pneumonia caused by L.pneumophila is also known as Legionnaires’ disease, which was named after the first diagnosed incidence of L.pneumophila caused pneumonia; an outbreak in 1976 in Philadelphia, USA, where members of the American legion got infected, and several of them died .
The pathogenicity and virulence of L. pneumophila depends on several factors including the state of the host´s immune system and dose of inhaled cells . The precise mechanisms and interplay between virulence factors of L. pneumophila are many and are still not completely clear, but the main factor is the ability to utilize cells of the immune system for protection and proliferation . Macrophages which are immune cells that can take up bacteria by coiling and kill them inside the cell with antibacterial substances, can also uptake L.pneumophila, however L.pneumophila forms a surrounding vacuole and lives protected inside the macrophage until the L.pneumophila growth is so substantial that the macrophage will undergo cell lysis, and L.pneumophila escapes out in the lung . L.pneumophila can damage lung tissue by producing exotoxins and enzymes, which facilitate coagulative necrosis, congestion, hemorrhage and abscess formation . L.pneumophila cannot transmit from person to person .
Legionella pneumophila in dental unit waterlines (DUWLs)
L.pneumophila is a known microbiological risk in cooling towers, air conditioning tanks, hot water systems, storage tanks, showers and spas, where they can transmit to humans from water aerosols [9; 10]. It has also been known for more than 30 years that L.pneumophila can be found in DUWLs, and due to some of the instruments such as the 3-in-1 air/water syringe, turbine and highspeed drill, the aerosols with L.pneumophila from the dental unit water can be sprayed into the patients mouth and possibly be inhaled, or spread into the room where dentists and dental staff can inhale the aerosols .
Many studies since have elucidated the extent of the presence of L.pneumophila in DUWLs, and even in DUWLs where disinfection procedures are used [12;13]. The width of the currency of L.pneumophila in DUWLs is further emphasized by a study showing that dentists and dental staff have a higher level of anti-Legionella antibodies in their blood than the general population, stating that they have a far higher exposure to Legionella [12;14;15]. Furthermore dentists and dental staff also have higher rates of respiratory infections compared to the general population [12;16-18]. The turbine handpieces spread small aerosols in the dental clinic, and these aerosols can contain L.pneumophila, hence it is not only the patients whom are in risk for obtaining L.pneumophila infection –the dentists and dental staff are definitely in a risk zone as well . In fact there are strong indications that an elderly Californian dentist died from Legionella at his own dental clinic .
It has been difficult enough to link fatal L.pneumophila infection to L.pneumophila from DUWLs, but it is even more difficult to link the more mild form of Legionellosis “Pontiac fever” with DUWLs, since Pontiac fever easily can be confused with a common flu and is often undiagnosed . The number of less severe L.pneumophila infections is probably underestimated, since it is difficult to obtain specific laboratory evidence of sporadic cases of Pontiac fever, and most people would not connect illness to DUWLs as a possible infection source 
It has been discussed whether the Legionella in DUWLs is a regular health risk at all, since documentation of strong-evidenced-diagnosed cases of severe illness or mortality due to Legionella from DUWLs is sparse . It has been difficult to prove that the infection was caused by L.pneumophila originating from DUWLs. In recent years there have been two fatal disease-cases caused by L.pneumophila, where evidence of the infection being caused by water from DUWLs was very strong: one incidence with an elderly but healthy Italian woman and another incidence with a 60-year old man with underlying disease [22;23].
The recommendations and guidelines from dental associations in most countries and from the Center for Disease Control regarding heterotrophic aerobic bacteria (total viable count) in water of DUWLs vary among the different authorities from 100 CFU/ml, 200 CFU/ml to 500 CFU/ml. There are guidelines for the maximal concentration of Legionella as well; some authorities recommend less than 1000 CFU/l Legionella species, while other authorities only have recommendations for the species Legionella pneumophila and here the limit is 100 CFU/l.
The development in molecular microbiological testing in recent years, have made it plausible to sequence genomes of L.pneumophila isolated from a patient and compare them to L.pneumophila genomes from suspected DUWLs and thereby have very strong evidence of the source of infection, and this could lead to lawsuits against dentists if a patient is infected by L. pneumophila from DUWLs.
Legionella pneumophila lifestyle
L.pneumophila can enter DUWLs from the water supply, and theoretically, L.pneumophila can grow in aquatic environments in temperatures from 5-55°C with an optimum at 25- 42°C, however L.pneumophila appears to be more prone to just persist in water and wait for an opportunity to meet a host within whom it can proliferate. L.pneumophila can persist intracellular in several eukaryotic cells e.g. in amoebae and macrophages (cells of the immune systems) [1;26].
Amoebae live in water and can also be found in dental unit waterlines where they graze on biofilm, and can uptake bacteria by coiling [1;26]. While most bacteria get killed within the amoeba and are used as nutrition source, L.pneumophila can persist within the amoeba inside a vesicle [8;25]. L.pneumophila can then live in the amoeba where it is sheltered from disinfectants [8;26]. The intracellular environment is perfect for proliferation for the L.pneumophila cell, and there can be hundreds of L.pneumophila cells within the amoeba before it will burst and the L.pneumophila cells will escape out in the environment where they can spread and find new host cells [25;26].
L.pneumophila in DUWLs which have been proliferated within an amoeba is more pathogenic to humans than L.pneumophila cells that have lived a long time as planktonic cells in the water or in biofilms, since the environment within the amoeba promotes expression of genes, which are beneficial for an intracellular lifestyle, hence these L.pneumophila cells are trained specialists in living within macrophages, which will uptake L.pneumophila cells present in the lungs [3;8]. Inside the macrophages, L.pneumophila cells are protected against most antibiotics when living as an intracellular parasite [1;3; 8].L. pneumophila is both capable of forming a single-species biofilm and to colonize multispecies biofilms . Some studies have shown that L.pneumophila can grow in biofilm in contrast to planktonic cells, in the waterlines, and in general Legionella benefits from living sheltered within a biofilm [1;8;28]. A biofilm within the DUWLs acts as a reservoir for L.pneumophila, since it will prohibit L.pneumophila from being flushed out of the system and instead contribute to upconcentration and proliferation of L.pneumophila in the DUWLs [29;30]. What is measured in water from DUWLs is then closer to the number of L.pneumophila cells, which are shedded of a mature biofilm rather than the actual number of L.pneumophila, and a combination with a biofilm swab would give a more exact picture of how many L.pneumophila cells that are present in the DUWLs .
Figure 1 Legionella pneumophila can enter DUWLs from the main water and can use dead microorganisms, organic material, limestone and particles on the waterlines as nutrients. (2A) Mature multispecies biofilm shedding of bacteria, and L. pneumophila enters the biofilm where it can live protected and be supplied with nutrients (2B) Some of the L. pneumophila cells which are shedded of the mature biofilm can attach to new biofilm and proliferate (3) Amoebae graze on biofilm and uptake L. pneumophila by coiling. (4) L. pneumophila can persist and proliferate inside a vesicle in the amoeba. (5) When L.pneumophila has proliferated to high cells numbers, the amoeba undergo cell lysis and L. pneumophila cells are spread out in the waterlines. (6A) L. pneumophila cells released from the amoeba can enter new biofilm and possibly become a parasite of a new amoeba (6B) L. pneumophila cells will be passed out to the instruments and cupfiller, where they could risk to enter lungs of patients and dental staff, when spread through aerosols.
Whether L.pneumophila will stay and proliferate in DUWLs depends on the temperature, the amount of nutrients in form of organic material and the already existing microbiota . Some microorganisms will fight L.pneumophila and will not allow L.pneumophila to enter a biofilm, while other bacteria promote attachment of L.pneumophila to the biofilm . Legionella species can also compete against each other e.g. L.pneumophila pneumophila will send out surfactants to kill other Legionella species . L.pneumophila is a necrotrophic bacterium which means that it can use some dead bacterial species as nutrients, which is why it can be problematic to heat up water systems if not all of the rather heat-resistant L.pneumophila are killed. The other dead bacteria will act as a huge nutrition source and there will be no competition for surviving L.pneumophila [8; 28].
Besides living in biofilms L. pneumophila is also associated with microbial hotspots – dense microbial communities without an extracellular matrix, in general L. pneumophila thrives better, when other (Legionella-friendly) microorganisms are present .
Notes to avoid Legionella in dental unit waterlines
It is important to take measurements to prevent Legionella from entering the dental unit. The actions to eliminate Legionella in the DUWLs should not only focus on which disinfection method is able to kill Legionella effectively. It is just as important to find a method that can control heterogenic biofilm and microbiological hotspots within the DUWLs at all times, as well as keeping amoeba out of the system, and thereby preventing Legionella persistence and growth, .
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