1. Session 3: Legionella Ecology and the Bases of Control
Facilitator notes:
Legionnaires’ disease: Risk assessment, outbreak investigation and control
Session 3: Legionella Ecology and the Bases of Control
ECDC, 2013

2. Objectives Specific objective of this session:
Discuss how natural and artificial environments favour the growth of Legionella
Review the common settings and sources of Legionnaires’ outbreaks
Discuss the factors influencing control processes
Related to the course objectives:
To know the common settings and sources of LD outbreaks, especially how manmade environments favour growth of Legionella.
Facilitator notes:

3. Outline This session consists of the following elements Presentation:
Transmission of Legionella to humans
Factors influencing the occurrence, survival and growth of Legionella
Natural and artificial environment
role of temperature, protozoa and bio-films
Common settings and sources of Legionella
Factors leading to proliferation of Legionella and the key control interventions.
Exercise
Case scenarios for group exercise and plenary feedback
Facilitator notes:

4. Background/Context It is now 35+ years since Legionella was discovered
Since then we have learnt a lot about Legionella ecology and control.
High risk systems - all systems which contain
non-sterile water
operate within the range of ºC
have the potential to be aerosolised
Risk Factors - increase the risk of Legionella growth:
Stagnation
The presence of nutrients
The presence of other microorganisms
Lack of controls:
hot temp <50 ºC - cold > 20 ºC
lack of appropriate biocide concentrations
Convention of the American Legion in July 1976 at the Bellevue-Stratford Hotel in Philadelphia, where 234 people became ill and 34 died.
Bacterium identified in Jan 1977.
Source was id as an air-conditioning ventilator in the hotel lobby 4

5. Where are the bugs?
Dept of Health survey: Hospitals - 70% Hotels - 53% Businesses - 75% Cooling towers - 54%
Lower Saxony study
(Habicht 1988) 1241 samples:
Hospitals 70%
Hotels 18%
Temp range 35-45ºC most frequently positive
Highest 66 ºC
Homes (BRE Study) 10%-25%

6. Infection via an Aerosol
An aerosol is not a spray although it can be formed from a spray by small droplets drying to leave suspended droplet nuclei.
Aerosols are formed by bubbles released at a water surface (concentration effect).
An aerosol is not visible.
Small particles <5µm can remain in suspension in air for prolonged periods and can be inhaled deep into the lungs.
Aerosols can travel long distances.

7. Aspiration
Hospitals and healthcare premises – need to consider aspiration as a potential source
Drinking contaminated water
In a very small number of cases Legionnaires’ disease can be acquired through aspiration of contaminated water i.e. swallowing water and it going down the wrong way into the lungs). Those particularly at risk are patients in hospital such as post surgery patients (drinking whilst prone), with swallowing defects or elderly.
Sucking ice made from contaminated water

8. Aerosol formation from running a bath
Air 500L/min by cyclone with liquid capture followed by culture on R2A and nutrient agar
The slightest illustrates the formation aerosol by running a bath is very old data we gathered while investigating an outbreak in a hotel in Brighton in The aerosol referred to the air is not anti-legionella but just total organisms in the air but it does illustrate quite clearly the generation of the aerosol and at any time you remain sitting in the bath they are so only gradually fades away.
From Dennis PJL 1986

9. Sources of aerosol Water drops falling onto a hard surface
Bubbles rising to the water surface and bursting
Rain
Running a tap
Running shower
Flushing a toilet
Spraying plants
Humidifiers
Water running over pack of cooling towers
Wave formation

10. Aerosol survival of L. pneumophila
Pontiac (MAb2+)
Olda (MAb2-)
Bellingham (MAb2-)
Indoors
Survival higher at high humidity.
Strains responsible for most community outbreaks (MAb2+) survive best.
Survival improved by extracts of some co-existing organisms.
Outdoors
No experimental evidence but likely to be reduced by:
Sunlight
Open Air Factor (OAF, ozone olefines from internal combustion)
The Pontiac MaB2 (3:1) strain causes most outbreaks and community cases- laboratory studies that it survives the longest in the atmosphere
Humidity is an important factor in the ability of legionella to survive.
Figures from Dennis and Lee, J. Appl. Bacteriol., 1988, 65, 135–141
Aerosol survival of monoclonal subtypes of L. pneumophila sg. 1
Experiment performed at 60% rh and 20°C
Effect of humidity on aerosol survival of L. pneumophila sg 1 at 20°C
Figures from Dennis and Lee, J. Appl. Bacteriol., 1988, 65, 135–141

11. Ecology of Legionella species
Widespread in natural aquatic environment
Legionellae have been found in natural freshwater systems including:
Thermal waters (Fliermans et al., 1981; Verissimo et al., 1991)
Sewage-contaminated coastal waters (Ortiz-Roque & Hazen,1987)
Wells to a depth of 1170 metres (Fliermans, 1996)
Groundwater (low concentrations) (Frahm & Obst, 1994; Lye et al., 1997)
Potting mixes isolated from composted wood wastes L. longbeachae, L. bozemanii, and L. dumoffi (Hughes & Steele, 1994;Steele & McLennan, 1996)
L. longbeachae can survive in damp potting soils for several months

12. Factors for growth from environmental sources
Within pH range 2.7 to 8.3 (Anand et al 1983)
Multiply in temperatures 25ºC - 45 ºC
Optimum range 32 ºC -42 ºC
Greatest 37 ºC -42 ºC
Association with other aquatic species
Amoebae
Cyanobacteria
Ciliates
Algae
Other bacteria

13. Legionella and temperature
Decimal reduction times (D, minutes) of L. pneumophila sg. 1 at different temperatures
50oC – min
60oC – 2min.
Detectable in waters up to ~55oC
Basis of control in hot and cold water systems
Circulate hot water at 60°C so that water at tap reaches at least 50°C within 1 minute.
Cold should be less than 20°C within 2 minutes of turning on tap
D = time to kill 90% of a population
Data from Dennis et al J. Appl. Bacteriol. 56:349 – 350 and Schulze-Robbecke et al Schriftenr. Ver Wasser Boden. Lufthyg. 72: 86 – 89

14. Common problems identified with temperature control
Lack of insulation
Common problems identified with temperature control
Warm cold supply
Lack of use / stagnation
Oversupply
Cross connections
Lack of insulation
Warm overflows
Shower hose with residual warm water 14

15. Representative temperature monitoring is essential
This shows how important it is to make sure that sufficient temperature monitoring is carried out- otherwise false conclusions could be drawn about the status of control and risk of the system

16. Effect of the presence of nutrients in sediment
Hot water tank water
Pure water
 Filtered tank water
FIG. 2. Hot-water-tank water support of the growth and survival
of L. pneumophila. Nonsterile hot-water-tank water in which microflora
and sediment were present (0) supported the survival of L.
pneumophila. The growth-promoting effect of dissolved organic
nutrients was minimal as evidenced by the decline in viable L.
pneumophila in both sterile supernatant (O), which contained
dissolved organic nutrients, and sterile high-pressure liquid chromatography
water (A), which did not contain dissolved organic
nutrients .
Stout et al.1985 Ecology of Legionella pneumophila within Water Distribution Systems AEM 49:

17. Legionellae require other aquatic organisms for growth
Photo courtesy of Barry Fields CDC Atlanta
Legionellae require other aquatic organisms for growth
Associated with 14 species of amoebae, including Acanthamoebae spp; Hartmanella vermiformis; Tetrahymena pyriformis; Naegleria spp 2 species of ciliated protozoa, and one species of slime mold
Tetrahymen pyriformis infected with L. pneumophila
Legionella will not grow in sterile water (but can survive) they need other miroorganisms such as suitable protozoan hosts and or biofilms to grow within water systems.
Host specificity.
Once inside amoeba, survival depend on temperature of water;
22oC – digested by amoeba,
35oC – proliferate inside amoeba.
Impact on flagella – pathogenicity (linked to its capacity to proliferate in humans):
30oC – more flagellated bacteria
37oC – less flagellated bacteria. B A
Acanthamoeba polyphaga
A: uninfected exposed to avirulent Pontiac strain
B: infected exposed to virulent Pontiac strain
Electronmicrographs , DIC and DAPI photos by permission Dr S Surman

18. Virulence is important for legionellae to affect both amoebae and human monocytes and macrophages
To replicate inside the host Legionella resists/evades the hosts’ natural defence mechanism.
Protozoan host specificity- if legionellae is ingested by unsuitable amoebal host then the legionella would be either egested or digested
Host acceptability may be temperature dependent:
At 35 ºC legionellae proliferate inside suitable host
At 22 ºC amoebae digest legionellae
Can infect macrophages of guinea pigs, rats, gerbils and some mice
Not all animals affected - challenges of some birds (pigeons; leghorn chickens, quails and most mouse strains have been unsuccessful.
There is evidence of host specificity:- not all protozoa make suitable hosts for legionellae
Virulence is important – avirulent strains do not have the capability to infect cells

19. Intracellular growth can be protective
Protozoan vegetative cells and especially cysts are protective.
L. pneumophila have been shown to survive inside amoebal cysts following hyperchlorination eg 50ppm chlorine overnight (Kilvington).
Intra-amoebal growth patterns vary compared to those grown in vitro – modifications of lipopolysaccharide and fatty acid content of the cell envelope.
May explain increased biocide resistance expressed following intracellular growth.
Cysts also protect against drying and heat
Windborne cysts may be a means of dissemination and infection of new systems

20. Biofilms support legionellae survival
What is a biofilm?
A consortium of micro-organisms growing at interfaces:
Air / water
Surface / water
Stages of biofilm formation:
Non specific binding on surfaces.
Colonisation - hydrophobicity and electrochemical nature of substratum is important.
Gylcocalyx formation – protect microcolonies.
Stages:
Non-specific binding on film surfaces – conditioning phase.
Colonisation phase- multiplication to form microcolonies/stacks.
Glycocalyxlayer provide protection for microcolonies.

21. © Dr Susanne Surman-Lee
What are Biofilms?
Extremely complex heterogenenous microbila ecosystems; bacteria, algae and grazing protozoa.
© Dr Susanne Surman-Lee

22. Biofilm features
Rougher surfaces - preferentially colonised and form microniches.
Protection from shear stresses; turbulent flow and biocide activity.
Different physiological state .
Nutritional advantage.
Gylocalyx – hydrated polyanionic polysaccharide matrix.

23. Benefits of growth in biofilms
Niches for bacteria with differing metabolic needs e.g. both aerobic and anaerobic bacteria can be isolated from the same biofilm.
Ionic exchange.
Traps metal ions and nutrients transported by permeases into cells.
Biocide resistance.
Can take up to 1000 x greater exposure to a given biocide concentration to kill an organism in a biofilm as it would to kill the same organism in water.

24. Oxygen gradients in corrosion pits

25. Presence of scale & corrosion increases the risk of biofilm formation
Rougher materials preferentially colonised.
Microniches.
Protection from biocides.
Protection from flow factors.
Inhibits flow- increases stagnation.
Iron = growth factor. 25

26. Where are the biofilms?
Outlets, washers, seals, flexible hoses, plastic shower heads, thermostatic mixer valves
Corrosion / scale
Dead legs / blind ends
Areas of stagnation e.g. oversized header tanks
Interfaces in aquatic systems
e.g. cutting oil/ water

27. Factors Encouraging Biofilm Formation in artificial-made Water Systems
Presence of nutrients for microbial growth.
Assimilable (useable) Organic Carbon (AOC) in supply water.
Accumulation of dirt and debris in storage tanks.

28. Choice of materials
Increased use of polymeric materials in plumbing which can leach organic compounds from surfaces - these may provide nutrients to support microbial growth e.g. polyvinyl chloride.
Bezanson (1991) showed PVC significantly more susceptible to colonisation than copper or brass.
Habicht (1988) showed copper had an inhibitory effect in the first 5 years; no significant reduction in isolation rates after.

29. Growth on different plumbing materials relative to glass (=1)
Legionella and supporting flora were grown in continuous culture model systems.
Counts are expressed as a ratio relative to the counts on glass (=1)
e.g. there was approximately 10 X the growth of aerobic heterotrophs on cPVC as there was on glass.
Conclusion:
Higher flexibility of plastic generally correlates with increased support for growth

30. Use materials which are less susceptible to colonisation to reduce likelihood of legionellae growth. Assisted baths - have different types of plastic hoses. The white hose feeding the shower head contained very much higher counts of L.pneumophila than did the black.

31. Beware of Flexible hoses
Non-compliant hoses imported from China.
Example of damage sustain to hose during installation.
Damage/distortion was only identified after the hoses were removed.

32. Legionella Risk factors
Poor design of water system/ pipework - difficult to clean and remove biofilms.
Stagnation
Areas of low or no flow
Tanks too large
Pressure vessels
Dead / blind ends
Cold Water Tanks with inlets and outlets on same side leading to short circuiting and stagnation.

33. Where are the risks? Industrial sites with water cooling systems
Large buildings with water cooled air conditioning
Hot and cold water systems used in for example:-
Hospitals /nursing homes
Hotels / holiday accommodation / offices / factories
Whirlpool spas
Respiratory therapy equipment
Construction sites etc 33

34. In artificial systems
Potential for growth in all artificial water sources if conditions allow:
in wet cooling systems, hot and cold water systems, cutting fluids, manufacturing processes.
grows in non-sterile tap water and can survive indefinitely if the water conditions are suitable.
long term survival in sterile distilled and sterile tap water but not growth.

35. References Dennis and Lee, J. Appl. Bacteriol., 1988, 65, 135–141
Dennis et al J. Appl. Bacteriol. 56:349 – 350
Schulze-Robbecke et al Schriftenr. Ver Wasser Boden. Lufthyg. 72: 86 – 89

36. Facilitator notes:
Acknowledgements The creation of this training material was commissioned in 2010 by ECDC to Health Protection Agency (UK) and the University of Chester (UK) with the direct involvement of Louise Brown, Janice Gidman, Emma Gilgunn-Jones, Ian Hall (on behalf of the ECDC Legionnaires Disease Outbreak Toolbox Development Group), Tim Harrison, Rob Johnston, Carol Joseph, Sandra Lai, John Lee, Falguni Naik, Nick Phin, Michelle Rivett, and Susanne Surman-Lee. The revision and update of this training material was commissioned in 2017 by ECDC to Transmissible (NL) with the direct involvement of Arnold Bosman and Kassiani Mellou.