What is Legionella?
This bacterium first got its name in 1976 following an outbreak of pneumonia at an American Legion conference at the Bellevue Stratford Hotel in Philadelphia which led to 221 cases with 34 fatalities – the causative agent came to be known as Legionella pneumophila. Today there are at least 50 species (with at least 16 of these associated with human infection) and 70 serogroups. Legionella is now know to be ubiquitous in the environment and is commonly found in environmental waters and potable mains water. Water supply companies do not test for Legionella and it does not feature in the Drinking Water Regulations, thought 5-10% of UK mains water is thought to contain Legionella.
Legionella causes respiratory illness which ranges from a mild flu (Pontiac Fever) through to a potential fatal pneumonia (Legionnaires Disease) but there have been not reported cases of person to person transmission of the disease. L.pneumophila causes about 90% cases with serogroup 1 being most commonly associated with Legionnaires diseases. The disease is contracted by inhaling aerosols typically of <5µm in size, but it can be readily treated with antibiotics. More common sources of infection are generally poorly maintained air conditioning units, cooling towers, spa baths and Jacuzzis etc
Following exposure there is a 2-10 day incubation period (usually 3-6 days) before the onset of the disease. Not everyone exposed will develop symptoms; some may only develop a mild ‘flu-like’ illness rather than full blown Legionnaire’s disease. Initial symptoms of Legionnaires disease include a high fever, chills, headache and muscle pain. A dry cough may also develop and most suffer difficulty with breathing. In addition about one third of patients will also develop diarrhoea or vomiting and half may become confused or delirious. Certain groups of people are known to be at higher risk of contracting Legionnaires’ disease than others. Men are between 2 and 5 times more susceptible than women, those over 45 years of age (older people are more likely to be infected and are more likely to die). Smokers, alcoholics, diabetics and those with cancer and chronic respiratory or kidney disease are also at a higher risk. Legionnaires’ disease is uncommon in younger people and is very uncommon under the age of 20. This disease can be fatal in about 10 – 15% of cases, with the fatality rate rising in susceptible people such as those with underlying disease or who are immunocompromised.
Employing the correct temperature regime within a water system is one of the most common methods for controlling Legionella and the key temperatures are detailed below:
- Above 70 °C (158 °F) – Legionella dies almost instantly
- At 60 °C (140 °F) – 90% die in 2 minutes
- At 50 °C (122 °F) – 90% die in 80–124 minutes, depending on strain
- 48 to 50 °C (118 to 122 °F) – Can survive but do not multiply
- 32 to 42 °C (90 to 108 °F) – Ideal growth range
- 25 to 45 °C (77 to 113 °F) – Growth range
- Below 20 °C (68 °F) – Can survive but are dormant, even below freezing
In order to multiply Legionella must have access to a source of IRON and the amino acid L-CYSTEINE, growth is also aided by nutrients which can be obtained from other bacteria in the water, algae, and amoebae. Sediment, sludge, scale and biofilms also play an important role in harbouring and providing suitable conditions in which Legionella may grow. Biofilms can be important in protecting Legionella from adverse temperatures and biocides within a system.
What does TVC mean?
This test is a general indication on the overall quality of the water and does not indicate if a sample will be harmful to drink or not. A raised TVC in a drinking water can impart a stale/ stagnant taste and/or odour to the water. Typically the TVC test is undertaken at 22oC and 37oC for drinking water and most other types of water, and at 30oC for cooling tower samples.
The UK water supply is governed by an EU directive on water supply and water quality. This directive has been introduced in the UK as the Water Supply (Water Quality) Regulations 2000, and is regulated by the Drinking Water Inspectorate.
When the regulations were first issued the TVCs were required as an indication of the overall bacteriological water quality. At this time the guide limits for bacterial levels were set at 10cfu/ml at 37oC and 100cfu/ml at 22oC. These were only ever guide limits; there has never been a statutory limit. Some mains water supplies will always have counts that exceed these limits, particularly surface derived waters. In 2005 revised guidance notes were issued for the 2000 Regulation where the above guide levels were removed and it was stated that counts should not differ from what is normally expected.
To quote section 6.25 paragraph (v) of Regulation 19 in the guidance:
“Colony counts (Standard: no abnormal change)
Colony counts are enumerations of the general population of heterotrophic bacteria present in water supply. In environmental waters these represent bacteria whose natural habitat is the water environment or those that may have been washed from soil or vegetation. It is well recognised that only a small fraction of the viable heterotrophic bacteria population is estimated by enumeration on nutrient rich media with incubation at 22oC and 37oC. However, monitoring of water supplies for colony count bacteria can be useful for monitoring trends in water quality and detecting potential sudden deterioration in water quality. The colony count at 22oC generally represents those bacteria naturally present in water and are not of sanitary significance. They may however be of greater relevance to the food and drinks industries where high numbers may impact on the quality of products. An increase in the colony count at 37oC can be a sensitive indicator of ingress and further investigations should be undertaken to establish the source. Colony counts may be useful in assessing the efficiency of water treatment and the cleanliness and integrity of the system. In all cases the value on monitoring is to establish data which characterises a water supply in terms of seasonal and longer term changes. Drinking water supplies derived from surface waters tend to support higher numbers of heterotrophic bacteria than those derived from ground water sources. The onset of significant changes in colony count results against the normal range established for that water supply is much more significant than the absolute values of individual results”.
What are coliform organisms and E.coli?
Coliforms are a group of bacteria that are widespread in the environment. They are found in the soil, in water, on plants and associated with animals. If these organisms are detected action should be taken to determine their source. Their presence indicates a failure of the system and that there is the potential for other more harmful organisms to be present.
E.coli (Escherichia coli) is a member of the coliform group. This organism is associated with the guts of warm-blooded animals – including humans. The significance of this organism is that it does not last very long outside the host, and therefore is an indicator of recent or current faecal contamination. E.coli is used as an indicator organism because it is generally non-pathogenic but indicates that faecal contamination is present, and hence there is the risk of enteric pathogens e.g. Salmonella, typhoid etc being present. Within a population the ratio of E.coli to pathogens is very high, so if action is taken when one E.coli is isolated the probability of a pathogen being present is very small.
If coliforms/E.coli are detected they may be derived from sewage contamination, but depending on the sample point they may also have come from animals or birds. If the sample point is a kitchen tap a coliform failure may be due to the tap being contaminated from soil following the preparation of vegetables. An unsatisfactory sample result from a toilet tap may be a result of the tap becoming contaminated by being touched with dirty hands.
What is the significance of Pseudomonas?
Many species of Pseudomonads occur naturally in ground and surface waters. Some of these may survive the treatment process or may be subsequently introduced into treated water as a result of contamination. In many cases this may not be a problem but if growth is sufficient the result may lead to deterioration in quality that may be reflected in an unacceptable taste and odour, without necessarily being a risk to health. In some mains water supplies low numbers of Pseudomonads are quite common and form part of the normal bacterial population.
The Pseudomonas group are widespread in nature occurring commonly in water and soil, and on damp or moist surfaces. When these organisms are present they may proliferate utilising nutrients either from the water or from plumbing materials used in the distribution system. Similarly they may growth in water contained in bottles (particularly plastic) and on surfaces such as plastic tubing and filters within drinks vending machines. These machines often contain a carbon filter the purpose of which is to remove any unwanted taste such as chlorine or the inherent taste of the water. However, these filters efficiently remove chlorine – the means of controlling bacterial numbers, as well as adsorbing bacterial nutrients onto the surface of the carbon. The net result is that these filters have the ability to support the growth of high bacterial number including Pseudomonads. In order to prevent this occurring, it is important that if filters are fitted, they are maintained or replaced in accordance with the manufacture’s recommendations. This group of organisms will readily grow on any damp or moist surfaces; they are also known to be coated in a slime-type layer comprised of polysaccharides (sugars). It is this coating that makes these organisms significant in the formation of slime layers within water distribution systems and the subsequent formation of a biofilm. The presence of Pseudomonads in mains water can be problematic if closed systems etc. are being charged with mains water containing these organisms, and the water treatment specialist is aiming for a Pseudomonas-free system.
Pseudomonas aeruginosa is ubiquitous in fresh water, sewage and soil and can also be derived from faeces of animals and humans. This organism can grow in low nutrient environments and can survive many months in water at ambient temperature. It is an important opportunistic pathogen and is particularly significant cause of hospital acquired infection. This is because of their resistance to many antibiotics and disinfectants and their ability to colonise aquatic low nutrient environments. However the vast majority of people exposed to this organism suffer no adverse health effects. Non- hospital acquired infections are often localised and are associated with contact with contaminated water. Although P.aeruginosa can be present in drinking water in small numbers the organism is not generally infectious if swallowed with the possible exception of profoundly immuno-compromised individuals. The numbers present in mains water are unlikely to be sufficient to cause infection unless they are allowed to multiply. However, taps can become locally contaminated with this and other organisms.
Routine testing of mains water for this organism is not recommended, but in view of its importance as an opportunistic pathogen testing may sometimes be required when water may be implicated as the cause of illness.
In order to minimise the growth of Pseudomonads within a system, filters etc should be maintained appropriately correct plumbing material employed, and the stagnation of systems and tanks should be avoided, and dead legs should be removed.
What are the ideal growth conditions for Legionella?
Ideal Growth Condition MUST be avoided
- Temperature of 37°C (Ideal Temp) avoid 20-45°C
- pH of 6.9 (5.5 – 8.1)
- Trace of Iron
- Bacterial Slime, Algae, Scale
- Oxygen, Iron, L-cysteine
What is the difference between a dead leg and a blind end?
Dead Leg: This is a section of a water system where there is no water movement and hence stagnation but CAN be flushed out. I.e. there is a tap or similar at the end from which the water can be flushed.
Blind End: This is a section of a water system where there is no water movement and hence stagnation but CANNOT be flushed out. I.e. may have been capped off etc.
There is no definitive length for either a dead leg or blind end; they may be millimetres or meters in length, both can allow the proliferation of unwanted bacteria. If there is no water movement biocides will not be effective as they do not come into contact with any bacteria present, and temperature control cannot be guaranteed if the water is not flowing.
How soon should I get my samples to the laboratory?
TVCs Coliforms, E.coli, Pseudomonads, Faecal Streptococci, Sulphite Reducing Bacteria (SRB) and Nitrite Reducing Bacteria (NRB) should be stored and transported at 2-10oC and in the dark to protect the samples from the sun’s harmful UV rays. The samples should be returned to the laboratory as soon as possible after sampling but within 24 hours.
Legionella should be stored at room temperature and in the dark. Again the samples should be returned to the laboratory as soon as possible but within 2 days.
Chemical samples should be returned to the laboratory ideally within 24 hours of sampling.
What are the acceptable limits for total viable counts (TVC)?
There are no statutory limits for the TVC in drinking water; instead the levels should not exceed what is normally expected. These levels can be obtained by trending results from locations or sample points that are regularly sampled, but otherwise it is hard to determine is a result is satisfactory or not.
If several samples have been taken from a single building and the majority of counts are approximately similar, this can be considered the norm, then if there are any sample points that yielded significantly higher counts they can be considered unsatisfactory and investigated further.
If a single sample only has been taken a judgement has to be made based on experience and knowledge of the site. Results of greater than 100cfu/ml may warrant investigation.
A raised TVC count does not mean the supply will be harmful to drink. This test is an overall indicator of quality but a raised TVC may impart a stale/stagnant taste and odour to the supply.
In cooling towers the TVC should not be greater than 10000cfu/ml. The higher the TVC the great the risk of biofouling and biofilm development.
What are the acceptable limits for Legionella?
In a Cooling Tower
With Legionella levels of <100cfu/litre, even if a not detected result is returned does not mean that Legionella is not present or that there is no risk. At these levels the systems is most likely under control, but the control measures should be maintained.
Levels of 100 – 1000cfu/litre are higher than would normally be expected indicating that the system is not fully under control. Low levels from several outlets can indicate low level contamination of the system. Current control measures should be checked and reviewed and biocide levels adjusted if appropriate. Resampling is recommended to double check the initial results and further resamples may be required after any remedial actions to assess their effectiveness.
At levels of >1000cfu/litre urgent action is required as the system is out of control and may well be a hazard to health. The system should be treated in a way to reduce these levels and resamples taken to confirm the actions have been effective. In addition the system and associated documentation should be reviewed to assess any further remedial actions that may be undertaken.
In a Hot & Cold Water System
Legionella levels between 100 – 1000cfu/litre, if most of the samples from a single site are negative the sample resamples should be taken and if similar results are returned review the control measures and a risk assessment should be undertaken to identify any remedial actions that may be required. However if most of the samples are positive the system may be colonised and a review of the control measures and a risk assessment should be undertaken immediately in order to identify any other remedial actions that should be taken. A system disinfection should also be considered.
Levels of >1000 cfu/litre – resamples should be taken and the control measures and risk assessment reviewed immediately in order to identify any remedial actions, which may include a system disinfection. Retesting should be undertaken a few days after disinfection and then at frequent intervals afterwards until it can be demonstrated that the system is back under control.
When interpreting the levels of Legionella present the species and serogroup of Legionella present is irrelevant. It ANY species of Legionella is present it shows that the system is capable of growing Legionella and therefore has the potential to allow the proliferation of the more pathogenic types.
What does a deviating sample mean?
This is a mandatory action which all accredited laboratories have been required to implement by UKAS.
Details relating to this requirement can be found by reviewing TPS63: http://www.ukas.com/technical-services/publications/technical-policy-statements-3
This policy requires a testing laboratory to clearly state on the final certificate of analysis any samples that may have been compromised in any way. The sample’s suitability for testing may be affected by the storage condition from sampling to delivery to the laboratory, the time between sampling and analysis, and the use of inappropriate sampling containers.
Once a sample has been received by our laboratory the sample will be maintained under appropriate conditions.
A guide to appropriate sample conditions is shown below:
Collect samples into a sterile bottle containing sodium thiosulphate
Store in the dark at room temperature
Return to the laboratory within 2 days of sampling.
All other microbiology:
Collect samples into a sterile bottle containing sodium thiosulphate
Store in the dark and at approximately 4oC (cool box will be satisfactory)
Return to the laboratory as soon as possible and within 24 hours.
Collected into an appropriate sample bottle
Store in the dark under appropriate temperature conditions
Return to the laboratory as soon as possible ideally within 24 hours.
Mercian Science will add the following comments when a sample is found to be deviating:
1. When there has been a delay between sampling and analysis: “This sample has exceeded the recommended time between sampling and receipt and therefore the results provided may be compromised”.
2. When the storage temperature cannot be assured: “This sample was received at an inappropriate storage temperature and therefore the results provided may be compromised”.
3. When samples are received in an inappropriate container: This sample was received in an inappropriate sample container and therefore the results provided may be compromised.
What does CFU mean?
CFU – Colony Forming Unit. This is an estimate of the number of bacteria in a sample e.g. 10cfu/ml means there are 10 bacteria in 1 ml. It is assumed that 1 bacteria will multiply to for 1 visible colony which is then counted, however, bacteria are able to clump together and 1 clump may still only produce 1 colony hence the term CFU is used rather than 10 bacteria/ml.
What does TVC mean?
TVC – Total Viable Count. This is an estimation of the total number of viable, living bacteria present that will grow on the standard bacterial growth media at specifically prescribed temperatures, usually 22oC 30oC, and 37oC. Sometime call heterotrophic bacterial counts. This not the total number of bacteria present but an estimate of the bacteria that are able to grow under defined conditions sure as incubation temperature and growth media.
TVC at 22oC (3 days) and 30oC (2 days) – This is an estimate of the total bacterial population present and is a useful tool for detecting significant changes in water quality, and for trending water quality if the same sample point is regularly visited.
TVC at 37oC. This test allows the growth of bacteria that will potentially grow at human body temperature and can be an early indicator of more serious contamination.
Why do we look for coliform bacteria?
Coliform bacteria are large group of bacteria comprising many species. This group are commonly found in the environment. They are found in the soil, in water, on plants and associated with animals. They tend not to commonly cause illness
The presence of coliform bacteria suggests ingress of a contaminant or a failure of the disinfection process. If these organisms are detected action should be taken to determine their source. Their presence indicates a failure of the system and hence the potential for other more harmful organisms to be present.
These bacteria can be present in domestic plumbing with kitchen sinks and taps being a recognised source.
Why do we look for Escherichia coli (E.coli)?
E.coli (Escherichia coli) is a member of the coliform group. This organism is associated with the guts of warm-blooded animals – including humans. The significance of this organism is that it does not last very long outside the host, and therefore is an indicator of recent or current faecal contamination.
E.coli is used as an indicator organism because it is generally non-pathogenic but indicates that faecal contamination is present, and hence there is the risk of enteric pathogens e.g. Salmonella, typhoid etc being present.
Within a population the ratio of E.coli to pathogens is very high; hence, if action is taken when 1 E.coli is isolated the probability of a pathogen being present is very small.
If coliforms/E.coli are detected they may be derived from sewage contamination but depending on the sample point, they may also have come from animals and birds. If the sample point is in a kitchen the failure may be due to the tap being contaminated from soil following the preparation of vegetables. If the sample point was a toilet tap, the tap may have been contaminated by being touched with dirty hands.
What is the significance of a dead leg or blind end?
A dead leg or blind end are parts of a water system that are not receiving a regular flow of water. These can range from centimetres of pipework to a large storage tank and both may have the same negative effects on the system. Regular flow may be prevented due to a valved off section of pipe (dead leg), a capped off end (blind end), a tank taken out of service but not drained etc
These areas will become stagnant and allow rapid proliferation of microorganisms. If present, Legionella will readily colonise dead legs and blind ends and either continually ‘seed’ a system over time, or if the dead leg is brought into service, may cause massive contamination.
Routine disinfection will NOT have any effect on a dead leg or blind end as the biocide MUST be in the water, and come into contact with the microorganisms in order to eradicate them.
Wherever possible blind ends and dead legs should be removed or engineered out. If this is not possible for a dead leg it should be put on a regular flushing schedule so that the water does not become stagnant.
What is Acanthamoeba and what is its significance in relation to Legionella?
Acanthamoeba is a microscopic, free-living amoeba that is relatively common in the environment. This amoeba has been isolated from water (including natural and treated water in pools or hot tubs), soil, air (in association with cooling towers, heating, ventilation and air conditioner [HVAC] systems), sewage systems, and drinking water systems (shower heads, taps).
Amoebae tend to be more resistant to biocides than bacteria, but significantly they form resistant spores under adverse condition. These spores are very resistant and will ‘germinate’ and release new amoebae when favourable conditions return.
Acanthamoeba is significant because it grazes on Legionella bacteria, but unlike other bacteria, Legionella remain viable and multiply within the amoebae. If a system is treated with a biocide it may cause the amoebae to encyst protecting the Legionella. When favorable conditions return the amoebae and the Legionella are released into the system
Acanthamoeba cysts are able to withstand free chlorine concentrations of over 40ppm for over 3 hours, i.e. a much high concentrations than those found in water systems and swimming pools. Thermal disinfection is more effective at killing Acanthamoeba than chlorine, but ingestion of Legionella by the amoebae will significantly protect them from raised temperatures. Acanthamoeba can be killed at 65oC for 30mins. Cysts will also benefit from added protection from disinfection if they are embedded within the depth of the biofilm.
Can bacteria cause corrosion?
Bacteria can play a very significant role in causing corrosion of system pipework, particularly if they are allowed to form a biofilm.
Acid-producing bacteria; Certain species of bacteria found in the biofilm are able to produce metabolites, such as organic or inorganic acids such as sulfuric acid or acetic acid. These acids significantly increase the rate of corrosion by dissolving oxides (protective film) from the metal surface and accelerating the cathodic reaction rate
Hydrogen-producing bacteria; Many microorganisms produce hydrogen gas as a product of carbohydrate (sugar) fermentation. Hydrogen gas can diffuse into metals and cause hydrogen embrittlement particularly in steel.
Iron bacteria Iron-oxidizing bacteria of which there are many species are aerobic and filamentous bacteria which oxidize iron from a soluble ferrous (Fe2+) form to an insoluble ferric (Fe3+) form. The dissolved ferrous iron could be from either the incoming water supply or the metal surface. The ferric iron these bacteria produce can attract chloride ions and produce ferric chloride deposits which can attack stainless steel. Brown or red-brown mounds on the internal surfaces are indicative of Iron Bacteria.