N.86 – LEGIONELLA PNEUMOPHILA QUANTIFICATION IN BIOAEROSOL

TRIO

The disease

Legionnaires’ disease was first recognised as a result of an outbreak of acute pneumonia that occurredat the Convention of the American Legion in Philadelphia USA in 1976.

The route of infection has been recognised as the inhalation of small aerosol containing bacteria of the Legionella sp into the lungs of the host animal. The bacterium primarily responsible for this disease is Legionella pneumophila sero-group 1 Pontiac. It can result in typical Legionnaires’ disease an acute pneumonia with low attack rate and relatively high fatality rate or Pontiac fever, a mild non-pneumoniainfection with a high attack rate.

Epidemiological studies have shown outbreaks to be mainly associated with cooling towers and hotwater systems but also with whirlpool baths, clinical humidifier in respiratory equipment, supermarket vegetable sprays, natural spa baths, fountains and potting composts.

Middle aged males, smokers and immuno-compromised patients are the most likely to succumb to this disease due to their reduced immune defence systems. The female population and children have a very low rate of incidence.

Monitoring of Legionella is often carried out as part of a planned programme of maintenance of a water system or cooling tower.

 

Air Sampling

In certain instances, it is therefore beneficial to demonstrate the presence of Legionellae in aerosol droplets associated with suspected reservoirs of the bacterium. Although not essential for the identification of reservoirs of disease-causing strains of Legionella, air sampling can better define the role of certain devices in disease transmission.

Sampling for biological contaminants in air can be used to determine the presence of a particular organism in an air sample, the number of viable organisms, the number of particles bearing organisms and the size distribution of particles. Before, initiating a sampling protocol, an investigator should determine which information will be collected, For each determination, the investigator should consider the type of sampler to be used, the probable concentration of organisms, sampling time, and environmental factors affecting the aerosol.

When obtaining air samples for Legionella, a primary objective is establishing the presence of the bacteria in aerosol droplets. The following protocol is directed toward this purpose. Although some information regarding particle size and numbers of viable bacteria can be calculated from these procedures, this information should be considered approximate.

 

Air Sampler Protocol

  1. Air sampler preparation

Be sure that the battery of the instrument is charged.

The aspirating head of the sampler should be sterilised by dry heating (160°C for 2 hours) or steam heating (autoclave at 121°C for 20 minutes) or isopropylic alcohol. An alternative is to use gamma irradiated aspirating head ready to be used.

  1. Air sampler positioning

2.1. HVAC (Heating Ventilation Air Conditioning) exit air-ducts

The head of the air sampler should be positioned at about 20-30 cm from the exit of the air duct.

2.2. Shower, tap water, faucet, etc

The head of the air sampler should be positioned at about 30 cm from the water source.

  1. Volume of air

It is suggested to aspirate 1000 litres of air for each cycle. The final result should be reported as Colony Forming Units (CFU) per 1000 litres of air (1 cubic meter).

TRIO.BAS-MONO-GUANTO

Microbiological Media

Buffered charcoal yeast extract (BCY Ea) agar.

 

Quality Control of Media

Each batch of medium should be tested for pH and the ability to support the growth of L. pneumophila. Ideally, all media should be tested with a standardised tissue inoculum. However, since this material is not generally available, stock culture inoculum is recommended.

  1. The pH of the medium must be 6.9 +/- 0.05. Measure with a pH meter. To check solid media, use a surface electrode (if available), or emulsify the agar from one plate in distilled water, and measure the pH of the emulsion. The pH is critical. If given lots of ingredients do not conform to the acceptable pH range adjust the pH of the completed liquid medium by adding 1,0 N HCl or 1,0 N KOH.
  1. Check for support of growth in the following manner:
    1. Prepare a standard stock culture inoculum by suspending L. pneumophila (log phase) in sterile distilled water to a concentration of 109 cells/ml.
    2. Inoculate the medium with 0,05 mI of the standard inoculurn. Streak the plates to obtain isolated colonies, and incubate at 35°C in air plus 2.5% C02.
    3. Examine the media daily. On agar Petri dishes, growth should be present in the heavily inoculated area after 1 day. Isolated colonies should be macroscopically visible in 2 to 3 days. If cells in standard inoculum have been refrigerated or frozen, growth will be slower.
  1. lf possible, selective media should be checked using an environmental water specimen containing a known concentration of legionellae and other bacteria.

 

Examination of Cultures for Legionellae

  1. Examine all cultures after 72 to 96 hours incubation. Macroscopic examination should be done with a dissecting microscope and oblique lighting to detect bacterial colonies resembling Legionella. These colonies are convex and round with entire edges. The centre of the colony is usually bright white in colour with a textured appearance that has been described as “cut-glass like or speckled. The white centre of the colony is often bordered with blue, purple, green, or rod iridescence. Some species of legionellae produce colonies that exhibit blue-white or red autofluorescence. Further examination of primary isolation plates with long-wave UV light can easily detect these fluorescent colonies.
  1. Aseptically pick each suspect colony onto (a) BYCEa agar and (b) BCYEa agar plate without L-cysteine [BCYEa (-)] or a BCYEa / BCYEa (-) biplate. Pick to – 4 representative colonies per sample.
  1. Streak the inoculated portion of each plate with a sterile loop to provide areas of heavy growth, and incubate the plates for 24 hours.
  1. Bi-plates without growth are re-incubated an additional 24 hours. Colony picks that grow on BCYEa agar, but not BCYEa (-) agar, are presumptive Legionella species and must be examined further for definite identification using the direct fluorescent antibody (DFA) or slide agglutination test (SAT).
  1. Since Legionella is a relatively slow-growing bacterium, negative plates should be re-incubated untiI 7days post-inoculation and then re-examined for Legionella colonies. Discard plates after the seventh day.

 

References

Center for Disease Control – Laboratory – Atlanta – USA.