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What Is Legionella? How Do I Sample Legionella?

What Is Legionella?

The genus Legionella is an assemblage of bacterial species that are ubiquitous and reside as intracellular parasites of protozoa and amoebae in moist environments. Legionella was first identified, both as an organism, and as a health concern, after an epidemic outbreak of pneumonia among members of the Pennsylvania American Legion during a bicentennial celebration at a hotel in Philadelphia. Prior to this convention a gentleman named Gram had developed a bacterial stain, now called the “Gram stain,” which was felt to stain all bacteria either red or blue, called “Gram positive” or “Gram negative.” Initially, when the cause of the pneumonia outbreak was investigated, bacterial causes were initially eliminated because nothing associated with the pneumonia was responding to this stain very well. As it turns out, Legionella just Gram stains poorly. Arguably, the genus Legionella should probably be considered Gram variable even though much of the literature characterizes them as Gram negative rods. The genus was given its name since it was the cause of the outbreak.

Why Is Legionella Significant?

Some Legionella species are potentially pathogenic to humans and cause illnesses ranging from self-limiting infections, with mild fever and flu-like symptoms, to severe and possibly fatal pneumonia, as occurred at the American Legion conference. The most common transmission route for Legionella infections in humans is through aerosolized water particles. Growth and transmission are often promoted by modern technologies, including air conditioning systems, heating systems, plumbing systems, and medical devices. Because Legionella live as intracellular parasites of free-living protozoa and amoebae, the organisms can occasionally avoid disinfection by conventional techniques.

How Do I Sample Legionella?
Samples can be collected to determine environmental reservoirs and potential transmission routes for the organisms; however, since it is ubiquitous, the mere presence of Legionella in an environment does not predict the potential for an outbreak of Legionellosis. Environmental monitoring is most beneficial when trying to identify the source of an outbreak, or the efficacy of treatment regimens and other prevention measures.

Sampling for Legionella species typically involves collecting water samples and swabs from potential sources. Collection sites range from taps and faucets to water storage reservoirs. Ideally, samples should be taken from a water source or other moist environment. Air sampling generally results in a low recovery rate of Legionella. Also, since it requires moist environments, samples collected from sites that demonstrate periodic drying will also generally result in a low recovery rate. Importantly, samples should be shipped for overnight delivery as they should be set up for analysis within 24 hours.

How Are Legionella Samples Analyzed?
Upon receipt in our laboratory, samples are checked against the Chain of Custody, logged into our database, LabServe™, and prepared for analysis by a trained technician following specific detailed procedures. Legionella samples are prepared for plating using a variety of methods that are dependent upon the sample collection source. Potable and other “clean” water sources are concentrated by filtration. Samples from environments containing a high microbial load are treated with a mild acid solution, or treated with heat, to select for Legionella species. Prepared samples are then plated onto differential and selective agars for quantitative analysis.

Plates are incubated and examined daily for a minimum of five days by a qualified, trained analyst. The plates are examined for general colonial morphological characteristics. Presumptive colonies are confirmed by various identifying characteristics recommended in the training we obtained from the Centers for Disease Control and Prevention (CDC). Many Legionella species are further confirmed using antigenic tests and are speciated and serotyped accordingly. The final colony densities are calculated and entered into LabServe™. Data are reported in colony forming units (cfu) / unit of sample (e.g. milliliters, grams, swab, etc.).Follow up: In the last issue of the Environmental Reporter we discussed sewage analysis and received a some good questions: “Don’t swab samples dry out?” and “I’ve attended a seminar where Endotoxin was suggested as a better way to go. Is that true?”

Swab's that are used for bacterial sampling are held in a moist transporting media, which helps prevent desiccation of the bacterial organisms. In reference to the endotoxin, it is known that endotoxins are present in the outer membrane of all viable and non-viable gram-negative bacteria. The measurement of endotoxin is usually indicative of the amount of viable and non-viable gram-negative bacteria that could have been present at the site of sampling and usually has nothing to do with testing for sewage contamination. Sewage can contain gram-negative bacteria that can also occur in nonenteric environments and can be isolated from environmental samples in the apparent absence of fecal contamination. The presence of some of these gram-negative bacterial organisms is not indicative of fecal contamination and so could provide false positives if endotoxin is used as a method for testing sewage clearance. For further information on Endotoxin and their application, the chapter “Endotoxin and other bacterial cell-wall components” by Donald K. Milton in the ACGIH “Bioaerosols: Assessment and Control” is a good reference.

This article was originally published on February 2004.