The Legionella bacterium is found in freshwater sources all over the world (1 ). The genus Legionella was not registered until 1979 – as a result of a major outbreak of Legionnaires’ disease among members of the American Legion (war veterans) in 1976, where Legionella pneumophila was found to be the cause (2 , 3 ). The first bacterium isolate which was subsequently thought to be Legionella , was isolated in 1943 in guinea pigs and appeared similar to the obligate intracellular bacterium Rickettsia . In 1954 a similar bacterium was described, which was found to infect free-living amoebae. This isolate was classified in 1995 as Legionella (4 ).
Today, we know Legionella as a small waterborne bacterium which can be found freely present in water. It is highly fastidious as regards the substrate it requires for growth, and therefore survives and multiplies in other organisms, especially in free-living freshwater amoebae (5 , 6 ). Legionella can cause respiratory disease in humans if a person inhales aerosolised water containing the bacterium. This exposure may occur daily if showering using water from contaminated water systems, although without it necessarily resulting in illness.
Infection with L. pneumophila is called Legionellosis. The infection usually presents as two distinct clinical entities: Legionnaires’ disease, a severe form of pneumonia with an approximately 30 % mortality rate; and Pontiac fever, an influenza-like illness of short duration (7 ). It has thus far not been demonstrated that these diseases can be transmitted from person to person (8 ).
To date, 53 different species of the Legionella bacterium have been identified (9 ), with some 20 of these being found in infections. The species L. pneumophila was found in more than 90 % of outbreaks and sporadic cases of Legionellosis, and more than 80 % of the L. pneumophila isolates belonged to serogroup 1 (10 ).
Legionella was registered in the 1980s and ’90s in outbreaks of disease with fatalities in Europe, but it was not until 2001 that the first outbreak occurred in Norway. It happened in Stavanger, where there were seven deaths (11 ), followed by an outbreak in Fredrikstad/Sarpsborg in 2005, with ten deaths (12 , 13 ). As a consequence of the latter outbreak the statutory microbiological control in Norway has been made much stricter, including a regulatory requirement designed to prevent the spread of Legionella from whirlpool spas and shower systems (14 ). New guidance has also been issued for the control and prevention of Legionella infection (8 ).
The Legionella regulations and guidance impose a high degree of responsibility on owners of devices and systems capable of spreading aerosolised water. In order to comply with the requirements, owners need to have a knowledge of which factors in the relevant water supply system may contribute to the growth and spread of the Legionella bacterium.
Where the Nordic fleet is concerned, the requirement is for all freshwater supplies on board ship, for both food and hygiene purposes, to be of drinking water quality. The freshwater supply is obtained either by filling fresh water in port (bunkering) or by freshwater production onboard ship via desalination of sea water using reverse osmosis (RO) or evaporation (EVA) (15 ). For ships, freshwater production from sea water will be the optimal choice, since L. pneumophila is a freshwater bacterium not naturally found in sea water.
The Royal Norwegian Navy has a large fleet of vessels which are subject to the Legionella regulations. A project was started in 2010 aimed at establishing the necessary knowledge for Legionella control on board naval vessels. It included surveys and risk assessments to identify the occurrence of Legionella in the water supply systems of all naval vessels, for the purpose of establishing a basis upon which the Navy could take the necessary measures to prevent the occurrence, growth and infection by the bacterium.
Material and method
The study comprised ten separate classes of vessel: minesweepers, submarines, coastal corvettes, frigates, defence logistics support and supply vessels, port utility vessels, coastguard vessels and others, making a total of 41 vessels.
Selection of water sampling points
The selection of water sampling points was based on an onboard inspection of one reference vessel from each class of vessel and on collected technical and procedural data. The following principles were applied:
For vessels with fewer than ten sampling points, all the points were analysed
For vessels with more than ten sampling points, up to 17 points were analysed
Out of a total of 3,807 possible sampling points, samples were taken from 439 (12 %). Samples were taken for analysis in two rounds separated by a one-year interval. In the second round of sampling, 36 of the 41 vessels were accessible for sampling.
Sample material
Table 1 contains an overview of water samples taken from vessels included in the study. In addition, samples were collected from 45 of the most used tapping points at the ten water filling (bunkering) stations; 43 of the tapping points were military and two civilian.
Table 1 Overview of sample material for analysis of L pneumophila during the study
Class of vessel
Number of vessels per class of vessel
Number of chosen sampling points
Minesweepers
6
9
Submarines
4
2
Coastal corvettes
2
6
Frigates
4
15
Defence logistics support & supply vessels
3
15
Port utility vessels
5
5
Coast guard vessels
13
15
Museum ships
1
8
Training ships
2
17
Specialised vessels
1
15
Total number
41
Water sampling
Water samples were collected in 1 L sterile flasks, dosed with sodium thiosulphate, from both the technical element of the water supply system (water tanks) and the distribution network (showers). The location of each sampling point was the same on every ship within the same class of vessel.
Microbiological analysis
Demonstration of presence of L. pneumophila . All the samples from the water supply systems were analysed with real-time PCR, as described previously (16 ). Positive samples were cultured on Legionella selective agar (buffered charcoal yeast extract, BCYE) plates to demonstrate live L. pneumophila , which is a prerequisite for infection. Where there was growth of L. pneumophila it was serotyped using MONOFLUO anti-Legionella Staining Reagent (Bio-Rad). All isolates of L. pneumophila serogroup 1 were genotyped using pulsed-field gel electrophoresis (PFGE) (17 ) in order to track possible collective routes of spread and contamination.
Demonstration of presence of free-living amoebae . As part of the effort to obtain systematic knowledge of the occurrence of free-living amoebae in water supply systems on board ships, parallel amoebic analyses and L. pneumophila analyses were carried out of all the samples from the water supply systems. The amoebic analyses were performed using cultivation and microscopic methods, in accordance with the methods described by Winiecka-Krusnell & Linder in 1999 (18 ).
Action upon findings of live L. pneumophila
Where L. pneumophila serogroup 1 was found, immediate action was taken to introduce disposable showerhead filters (0.2 μm) to prevent exposure to the Legionella bacterium from the shower water (using PALL water filters). Further follow-up was carried out in accordance with procedures described in the Legionella guidance issued by the Norwegian Institute of Public Health (8 ), that is to say mostly with respect to water heating systems.
Results
L. pneumophila
In the first round of sampling, analyses of 439 water samples from 41 naval vessels showed the presence of L. pneumophila DNA in 12 % of the samples. Positive samples were found in 20 out of 41 vessels. Live L. pneumophila serogroup 1 was isolated from seven of the vessels.
When the repeat survey was carried out one year later, the respective results of analysis were 13 positive vessels out of 36 surveyed and live L. pneumophila serogroup 1 in two of the vessels. These were vessels where no live L. pneumophila had been found in the first sampling round (Table 2).
Table 2 Occurrence of positive Legionella PCR, growth of Legionella and amoebae found on the first, second and both sampling rounds respectively
Positive sample first sampling round (N = 41)
Positive sample second sampling round (n = 36)
Positive sample both sampling rounds (n = 36)
Legionella PCR
20
13
9
Culturing for Legionella
7
2
0
Amoebae found
34
30
28
Of the 41 vessels, 31 had pressure tanks (hydrophore tanks) installed in the water supply system. Eight of the samples with live L. pneumophila derived from vessels with hydrophore tanks (26 % of vessels). Of the ten vessels without hydrophore tanks, live L. pneumophila was found in one sample (10 %).
From the 45 bunkering points surveyed, L. pneumophila was found in 16 % in the first sampling round, of which live L. pneumophila serogroup 1 was isolated from two points.
Free-living amoebae
Free-living amoebae were found in 39 out of 41 vessels. Acanthamoeba and Hartmannella were the most commonly occurring genus. Free-living amoebae were present in all the vessels in which L. pneumophila serogroup 1 was demonstrated. In the first sampling round free-living amoebae were found in samples from ten out of the 45 bunkering points surveyed in the study.
Intracellular L. pneumophila was isolated from the amoebae and found to have the same genotype pattern as in the water samples from the vessel concerned (data not shown).
Genotyping of L. pneumophila
In genotyping the Legionella isolates, we found a very limited number of genotypes, and three vessels were even contaminated with the same genotype (Fig. 1a). In two of the vessels we isolated genotypes that were also found in the bunkering station that each vessel had used, as exemplified in Fig. 1b.
Figure 1 a) Genotype pattern of L. pneumophila isolated from three different vessels in the same class. b) Genotype pattern of L. pneumophila from bunkering station and from the water supply system on one vessel (vessel D)
Discussion
L. pneumophila is, as demonstrated above, present in the freshwater supply systems on board Norwegian naval vessels. L. pneumophila DNA was found in 20 out of 41 vessels in the first sampling round. Live L. pneumophila serogroup 1 was isolated in seven of the vessels, and in these instances immediate action was taken to install disposable showerhead filters (0.2 μm) to prevent exposure to the Legionella bacterium from the shower water (using PALL water filters). Further follow-up was done in accordance with procedures described in the Legionella guidance issued by the Norwegian Institute of Public Health (8 ).
In the follow-up study a year later, no live bacteria were found on the seven vessels previously contaminated. However, infection was found on two other vessels – indicating that the actions taken after the first survey appear to have been inadequate. Free-living amoebae were found in the water supply system on most of the vessels, including all of the vessels with L. pneumophila . It was also possible to isolate L. pneumophila serogroup 1 from the amoebae, and these demonstrated the same genotype pattern otherwise found in the samples from the water supply system concerned. As it has been shown that the Legionella bacterium is able to survive and multiply in amoebae, this may explain its ability to survive for long periods in such water supply systems (19 ).
Samples were taken from 12 % of the relevant sampling points. The choice was based on inspections carried out on the reference vessel in each class and on whether the sampling point in question was representative of the exposure risk. Legionella can be difficult to detect, since the microbe colonises the walls in the water supply system and often only comes off in scale. However, on the basis of experience we considered an analysis of 12 % of possible sampling points as adequate for our purpose. The discovery of L. pneumophila with polymerase chain reaction (PCR) prior to culturing and genotyping is considered an appropriate strategy in view of the speed and sensitivity of the PCR method.
The fact that there is a tendency towards higher incidences of live L. pneumophila in vessels with hydrophore tanks, may indicate that these tanks play a certain role in terms of the growth of the bacterium. Hydrophore tanks are often sited in warm engine rooms, with resultant higher water temperatures than in the rest of the cold water system. In the light of recent knowledge about the intracellular growth of L. pneumophila , the water temperature may be decisive for the fate of the Legionella bacterium. Where temperatures are in excess of 20°C, the bacterium will be able to survive and multiply in the amoebae (20 ).
The same genotype of L. pneumophila was found in water from bunkering stations and in the water on board vessels that had filled water from the same bunkering station. This would appear to indicate that filling water from bunkering stations is one possible source of contamination.
Despite a number of outbreaks of Legionellosis being reported from passenger ships (21 – 23 ), few systematic studies have been made of Legionella colonisation in these ships’ water distribution systems. Studies carried out on cruise ships and ferries show that Legionella bacteria of different species are widespread in onboard water systems (24 ). No studies have been made of the possible transfer of L. pneumophila from fresh water supply systems. The results of our study are assumed to have a transfer value to fresh water supply systems in general (25 ).
