An Overview of Legionella Analyses

By Christopher Goulah, Ph. D, EMSL Analytical, Inc.
© March 2021

Background

The first recognized outbreak of Legionnaires’ disease occurred in the US at the American Legion Convention in Philadelphia during the summer of 1976. Of the 182 people who were stricken, twenty nine died from the disease. As a result of the efforts of the US Centers for Disease Control (CDC), this was the first time the bacteria was cultured and identified. Earlier outbreaks of the disease went undiagnosed. Since that time, there have been many identified outbreaks in this country and abroad prompting professional organizations and health departments worldwide to implement guidelines for diagnosing and reporting the disease, monitoring the organism, and remediation action levels. After a significant outbreak in New York City in the summer of 2015, both the New York State Department of Health (NYS-DOH) and the New York City Department of Health and Mental Hygiene (NYC-DOHMH) passed groundbreaking legislation for cooling towers with NYS-DOH expanding their legislation to include potable water in healthcare facilities.

Transmission and Epidemiology

Ubiquitous in all aquatic environments, Legionella bacteria are found in groundwater, municipal water as well as fresh and marine surface waters. The bacteria enter our plumbing systems, whirlpool spas, and cooling towers via these water sources. Unless control measures are conducted properly and routinely, the biofilm, scale, and corrosion that builds up over time in these systems will protect the organism and allow it to multiply.

Contaminated aerosolized water from cooling towers, whirlpool baths, nebulizers, faucets, and showerheads can become airborne. When a susceptible host inhales the contaminated aerosol, Legionellosis can occur. Aspiration (inhalation of liquid water) of contaminated water or melted ice chips can also cause the disease. Legionella can cause a severe form of pneumonia (Legionnaires’ Disease) often accompanied by serious long term health effects, or the mild flu-like illness called Pontiac Fever. Other organs, native and prosthetic heart valves, prosthetic hip and knee replacements may become infected and asymptomatic infections may also occur.

Risk factors include age, gender, compromised immune systems, and pre-existing medical conditions such as chronic obstructive pulmonary disease, cancer, diabetes, kidney failure and the use of rheumatoid arthritis drugs and chemotherapy. Men over 50 years of age who are heavy smokers and drinkers are at greatest risk. However, there have been cases of the disease in healthy, younger people. Pre-mature, immuno-compromised, or ventilated neonates are also at risk from hospital acquired infection. For these reasons, many state and federal health departments have guidelines that recommend routine monitoring of Legionella in critical care hospitals and nursing home facilities.

Although the disease is generally under-reported, travel (cruise ships), hotel, and resort related outbreaks are reported each year. These are mostly associated with the use of whirlpool spas. While community-acquired outbreaks involving cooling towers and whirlpool spas receive the most media attention, studies indicate that building potable water sources account for most of the infections. This is particularly true in hospitals and nursing homes where there are large numbers of immunosuppressed or critically ill people.

For these reasons many state health departments have guidelines that recommend routine monitoring for Legionella. In August, 2014 the Veterans Administration (VA) developed a directive (VHA 1061) that requires all VA locations that have inpatient services to implement monitoring of their potable water systems. There are also draft guidelines for the prevention of Legionellosis in building water systems that were created by the American Society of Heating, Refrigeration and Air Conditioning Engineers (ASHRAE).

Choosing Sampling Methods

Proper methods for collecting and analyzing samples are necessary and should follow generally agreed to “best practices”. Since the bacteria in water are present in very low levels, 1000 mL potable water samples are recommended by the US Public Health Centers for Disease Control (CDC). This sample size allows for the bacteria in the water to be concentrated, allowing for a more sensitive detection and quantitation limit. Many professional guidelines recommend semi-annual sampling for potable water sources.

In non-potable water sources such as cooling tower water, a 250 ml sample size is sufficient. Professional guidelines suggest these sources be monitored quarterly.

Sampling should be conducted in a way that maximizes recovery of the organism and identifies the route(s) of exposure. Legionella samples should be collected wherever water aerosolization may occur. In addition, points of limited flow (or stagnation) should be identified as these are locations where biofilm can develop.

Biofilm is a slimy extracellular matrix that is composed of extracellular polymeric substances (EPS) and can contain bacteria, blue green algae, amoeba, and protozoans. Biofilm protects these organisms, including Legionella, from direct exposure to ultra-violet (UV) light, desiccation, and the chemicals used to control its growth. In addition, the Legionella bacteria are ingested by the protozoans and amoeba and will continue to multiply inside protected vesicles within these organisms. Once these organisms die large numbers of Legionella bacteria can be released into the surrounding environment.

Because biofilm protects the organism, and enhances Legionella multiplication, incorporating swabs in your sampling protocol is can be an important addition to a sampling protocol. Swab sampling of biofilm found in cooling tower sumps, potable water faucets, showerheads, and whirlpool spa filters can help obtain a better picture of the presence of Legionella. Very often, biofilm swab samples demonstrate the presence of Legionella that may go undetected by water sampling alone. Taking samples of drinking water and ice machines may reveal Legionella contamination due to stagnating water.

While collecting air samples for Legionella directly mimics the route of exposure, it is generally not recommended for routine monitoring purposes. Legionella are unlikely to survive the exposure to UV light and desiccation for long periods of time. During air sampling, the bacteria experience high attrition (death) from the impaction of the bacterial cells on the collection media.

Only sterile, appropriately preserved bottles and swabs obtained from your lab should be used to collect samples. Potable water bottles should be preserved with sodium thiosulfate to neutralize residual chlorine in the water sample. These preserved bottles should also be used when collecting water from cooling towers that have been treated with chlorine or bromine compounds.

When collecting samples, proper personal protection equipment is recommended: N95 or HEPA particulate respirator, face shield or splash googles, and waterproof gloves should be worn. When taking samples of hot water storage tanks it is prudent to wear a face shield, thermal resistant apron, and thermal, water resistant gloves. Take care to minimize the generation of any aerosols when collecting the samples.

It is recommended to collect what is known as a “first-draw” sample. This is the first water that comes out of the outlet when the faucet or valve is turned on. This is in contrast to a “flush-draw” sample where the water is run for several minutes prior to collection. The importance of the first-draw sample is that it collects the water specific to a particular outlet along with any loose biofilm that may be present. A flush-draw sample is collecting the water from further up the line. As an example, if you were to collect (10) hot water samples and waited for the water to heat up before collecting the sample all (10) samples would be identical as they would represent the water from the hot water tank, NOT the water specific to the outlets.

After collecting a water sample, be sure to leave an air space in the bottle. Since Legionella require oxygen for their survival, an air space in the bottle will ensure that aerobic conditions are maintained during shipment to the lab. Samples should be packed and shipped to minimize amplification of any bacteria present. Using an insulated cooler with freezer packs is recommended to avoid temperature extremes during shipping. Samples should be shipped overnight to the lab.

Analytical Methods – Culture Test

Legionella are aerobic, fastidious bacteria; they have very strict requirements for growth. Two of these requirements are iron and L. cysteine. Because Legionella grow so slowly, they are often overgrown by faster growing bacteria and can even be inhibited by metabolites released by other organisms. Legionella will not grow on standard microbiological media used for aerobic plates or for detecting other bacteria. For this reason labs use methods that are selective for isolating and identifying the organism.

Currently the most recognized method in the US for identifying and enumerating Legionella is the culture method. This method uses an improved procedure developed by the CDC when it first isolated the organism after the American Legion outbreak in Philadelphia in 1976. The method uses buffered charcoal yeast extract agar (BCYE) as the base formulation. While the CDC-method remained tried and true for many years, the updated method released by the International Organization of Standards (ISO) incorporates the CDC-method along with expanded treatments to enhance the recovery of Legionella from environmental samples. This new method (ISO 11731:2017) is the standard by which most regulatory bodies require their certified labs to perform under.

Water samples are generally concentrated in order to enhance the ability to detect very low levels of bacteria. This is usually accomplished by filtering the sample through a sterile membrane filter. The filter-bound bacteria are then resuspended in 5 mL of sterile buffer. Aliquots of this concentrated samples are then pre-treated prior to plating onto selective media.

Most bacteria are highly susceptible to either heat or highly acidic conditions whereas Legionella are largely resistant to both. By incubating aliquots of the concentrated sample in either high heat and/or low pH conditions much of the non Legionella background bacteria can be eliminated allowing the slower growing Legionella to flourish uninhibited.

The BCYE plates are incubated at 35-370 F. Because the Legionella bacteria from environmental samples grow slowly, turnaround times for releasing results are typically after 7 to 10 days of incubation.

After 72 to 96 hours, the colonies are examined using a dissecting microscope with UV light. Legionella colonies appear as convex, circular white colonies having edges that resembles ground glass with the colonies often exhibiting a blue, green, purple or red iridescence. Certain species of “blue-white” Legionella (including anisa, bozemanii, gormanii, and dumofii) can exhibit an auto-fluorescence and glow bright blue under certain UV light.

Any suspect Legionella colonies are streaked onto separate BCYE plates that either contain (+) or are absent (-) for iron and cysteine. If the colonies grow on the BCYE (+) plates but not on the BYCE (-) plates, they are presumptively identified as Legionella.

The presumptive colonies are then analyzed using Direct Fluorescence Antibody (DFA) to directly visualize, and subsequently confirm, the identification of species and serotypes. Since Legionella in environmental samples grow slowly, a confirmed negative sample result should be provided only after the 10th incubation day.

While it is estimated that 85% of the outbreaks in the US are caused by L. pneumophila serotype 1, there are other serotypes of L. pneumophila and even other Legionella species that can cause the disease. Not all labs employ the same method for isolating and identifying the organism. Ascertain whether your lab uses the method to give you the level of identification and quantitation you need. The results for the culturable method are expressed as Colony Forming Units (CFU)/ml. While it is standard microbiological practice to express results as CFU/ml, this can sometimes be confusing to non-microbiologists.

Analytical Methods – Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a genetic test which looks for the deoxyribonucleic acid (DNA) that is specific for Legionella. While PCR is not considered the “gold standard” for Legionella analysis, it may be very useful for quickly determining the presence or absence of Legionella in a sample. Since same day qualitative results can be obtained, the quick turnaround time can be useful for confirming the presence of Legionella during an outbreak when time is critical.

PCR results are reproducible, accurate, precise, and very sensitive. The detection limit is theoretically a single DNA fragment. However, because PCR measures the DNA associated from both live and dead Legionella, viability information is not available. A positive result by PCR should always be followed by culture analysis to determine the presence and quantity of any viable Legionella.

The primary disadvantage of PCR is the potential for sample matrix effects. The presence of common divalent cations in the sample such as calcium, magnesium, or silver, and the divalent form of copper will cause false negative results unless the samples are processed properly. This requires that the lab have a strict Quality Assurance program that includes positive, negative, and sample matrix controls.

Other disadvantages of PCR are it cannot individually identify the 50 species known to cause disease and it cannot identify serotypes. While most PCR labs can identify L. pneumophila, there may be other species or serotypes colonizing your water system that can cause disease that you would like determined.

Analytical Methods – Whole Genome Sequencing (WGS)

Non-microbiologists often confuse the terms genus, species, serotype and strain. These are independent terms for the identification of organisms and each is used to reach a successively more specific level of identification. (i.e., Legionella pneumophila (serotype 1 – Philadelphia) is the identification of the bacteria that caused the 1976 outbreak in Philadelphia.)

Whole Genome Sequencing (WGS) can be used to analyze the sequence of the DNA to determine the strain of an individual isolate. During an epidemiological investigation, this information can then be used to compare to known strains in the public record or can be used to compare a clinical isolate from a known case of Legionnaires disease to an isolate of an environmental sample from a potential dissemination site to establish a cause-effect relationship.

Intent of the Risk Assessment

The intent of your Legionella risk assessment will determine the type of data you need. Proactive monitoring is conducted to determine baseline conditions and concentrations of existing Legionella or to validate the effectiveness of a maintenance program in the absence of suspected cases of Legionellosis. With this type of monitoring a qualitative, present/absent result or a quantitative result of Legionella spp. is sufficient. Species and serotype identification is optional. Strain identification or subtyping is unnecessary.

Reactive monitoring is conducted when a suspected or confirmed case of Legionnaires’ disease occurs. In this situation, individual species and serotype quantification is required. For an epidemiological investigation of an outbreak, whole genome sequencing or SBT can be used to determine if the genetic “fingerprints” of the environmental and clinical samples are related.

The actual concentration of Legionella provides useful information concerning the degree of contamination. However it should be understood that the concentrations are relative and that an actual infectious level in CFU/mL has not been determined so the goal should always be to achieve non-detectable (ND) levels. Bacterial populations are always in a state of flux with bacterial cells multiplying, dying, or remaining dormant. Since bacteria can multiply exponentially, an order of magnitude difference (10x) in the results should be considered significant. A difference of a few CFUs or a low single digit multiplication of results is not. The goal is to demonstrate a history of non-detectable results over time.

To recap, the analytical method used determines the type and accuracy of the results. While using BCYE agars to isolate Legionella is the recognized “gold standard” worldwide, there are still some labs using other methods. Also, the reagents and methods used for Legionella identification are not standardized. This makes comparing lab results very difficult. Be sure to identify the isolation method the lab is using as well as the identification methods used. This will ensure you obtain the information you need to maintain and comply with your established water management plan (WMP).

About the Author:

Dr. Goulah has more than 20 years experience in the areas of Microbiology, Laboratory Management, and Infectious Disease with a focus on human disease. He is currently the National Program Director of Legionella for EMSL Analytical, Inc., Buffalo, NY. He may be reached at (716) 989-5138 or cgoulah@emsl.com.

References:

American Society of Heating, Refrigerating and Air-Conditioning Engineers (ASHRAE) (2000), Atlanta, GA. Minimizing the Risk of Legionellosis Associated with Building Water Systems. ASHRAE Guideline 12-2000, February 2000.

International Standards Organization (1998). Water Quality Detection and Enumeration of Legionella, ISO 11731. Geneva, Switzerland.

American Society of Heating, Refrigerating and Air-Conditioning Engineers (ASHRAE) (2015). Legionellosis: Risk Management for Building Water Systems. ANSI/ASHRAE Standard 188-2015, June 2015.

Fields, Barry S., Robert F. Benson, Richard E. Besser, and “Legionella and Legionnaires’ Disease: 25 Years of Investigation.” Clinical Microbiology Reviews. 15(2002): 506-526.

“Legionella 2019: A Position Statement and Guidance Document” Association of Water Technologies. 2019.

“Legionella: Human Health Criteria Document.” U.S. Environmental Protection Agency, Office of Ground Water and Drinking Water. 1999.

Centers for Medicare and Medicaid Services (2017). Legionella Risk.

“Legionella: Risk for Infants and Children.” U.S. Environmental Protection Agency, Office of Ground Water and Drinking Water. 1999.

CDC (2016 and 2017) (Toolkit and VitalSigns). Developing a Water Management Program to Reduce Legionella Growth and Spread in Buildings: A Practical Guide to Implementing Industry Standards. Atlanta: Centers for Disease Control and Prevention.

OSHA Technical Manual, Section III, Chapter 7: Legionnaires’ Disease. Occupational Safety and Health Administration, Department of Labor, Washington, D.C.

“Procedures for the Recovery of Legionella from the Environment.” U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control. 2005.

Ratcliff, Rodney M., Janice A. Lanser, Paul A. Manning, and Michael W. Heuzenroeder. “Sequence Based Classification Scheme for the Genus Legionella Targeting the mip Gene .” Journal of Clinical Microbiology. 36(1998): 1560-1567.

Scaturro, M., M. Losardo, G. De Ponte, and M. L. Ricci. “Comparison of Three Molecular Methods Used for Serotyping of Legionella pneumophila Strains during an Epidemic of Legionellosis in Rome.” Journal of Clinical Microbiology. 43(2005): 5348-5350.

“Water Quality-Detection and Enumeration of Legionella.” International Standards Organization. 11731-2. 2004

World Health Organization (2018). Legionellosis. https://www.who.int/en/news-room/fact-sheets/detail/legionellosis

An Overview of Legionella Analyses