By: Paul S. Warden, Kristen S. Fallon, Ph.D., M.S.E.L., & Colin R. Fricker, Ph.D.
Part 1 of this article (January 2002) reviewed legionellosis (the diseases caused by Legionella infection), the sources and transmission of Legionella bacteria and conditions that tend to promote colonization. A discussion of the pros and cons of routine monitoring, as well as various recommended action limits, also was included.
In order for Legionella outbreaks to occur, a series of events needs to occur.1 These events are shown in the left column in Table 1. The factors that affect the likelihood of these events are shown in the center column, and those that represent opportunities for facilities managers to reduce the risk of legionellosis are identified in the right column.
Sample Collection
If monitoring is appropriate, samples should be collected at a variety of locations throughout the system, similar to coliform monitoring. The number and type of samples should be guided by the circumstances and objectives of monitoring, but for purposes of this discussion it will be assumed a facility is thought to be at risk for Legionella and a baseline of data is required. A thorough walk-through documenting the characteristics and features of the system along with the operational parameters and controls, should precede sample collection.
Sample Sites. Subsequent to the initial site survey, samples should be collected from some or all of the following sites.2,3,4
• Incoming water supply
• Each storage tank and water heater
• Representative hot and cold water faucets
• All cooling towers, evaporative condensers, humidifiers, spas, showers, etc.
• The water entering or leaving other equipment under suspicion (ice machines, misters, spray bottles, plastic injection molding equipment, etc.)
Sampling Procedures. Legionella samples may be collected as either swabs or water samples or preferably both. At faucets and showers, swab samples, first-draw water samples and post-flushing water samples all should be collected. It is critical that taps not be flushed prior to sampling as stagnated water data may be particularly revealing. In addition, biofilm research has shown that in most habitats, bacteria grow preferentially on surfaces rather than in the aqueous phase.5 Swabs collected from the inner walls of faucets have demonstrated equivalent sensitivity to bulk water samples, while resulting in higher recovery of Legionella.6 This is significant because outbreaks of Legionnaires’ disease have been linked to exposure to elevated levels of Legionella and suggested remedial actions have been based on Legionella concentration.7,8 Swab samples can be collected by removing the faucet or showerhead and thoroughly swabbing the interior surfaces. Swab samples should be collected using swabs with transport media or neutralizing buffer or site water to prevent desiccation.
Water samples should be collected in sterile, plastic bottles, with sodium thiosulfate added to neutralize chlorine or other oxidants. Typically, 250 milliliter (mL) to one liter (L) samples are collected for routine monitoring purposes, although up to 10 L samples may be warranted in some situations. Samples should not be refrigerated but should be protected from temperature extremes by shipment in an insulated cooler. Samples should be sent via overnight delivery to a qualified, experienced laboratory and analyzed within forty-eight hours of collection.9 The ISO method recommends that the time interval between sample collection and concentration be two days or less and should not exceed five days.10
When collecting bulk water samples, the initial temperature of the water should be recorded as the water runs into the sample bottle. Following collection of the “first draw” water sample, the water should be run continuously and temperature allowed to stabilize. The time to stabilize and final temperature also should be recorded, and then a post-flushing sample should be collected. Water heater samples should include water and sediment from the bottom drain and also water from the outlet (if possible). Humidifiers, spas, etc., should be sampled at their water reservoirs as well as the supply water. Cooling tower samples should include supply water, storage tanks, reservoirs, tower pond or basin (distant from make-up water entry and circulation system return) and any standing water in condensate trays or cooling coils. Samples should be collected from each tower (or each system if they are interconnected).
Legionella Methods. A variety of analytical methods is available for the detection of Legionella. Screening tests include Direct Fluorescent Antibody (DFA) techniques and Polymerase Chain Reaction (PCR) techniques, although these methods may not distinguish between viable and non-viable Legionella and DFA is subject to cross-reactivity with other organisms. Commercial PCR kits for the detection of Legionella at the genus level have been demonstrated to be inadequate due to lack of specificity.11
The traditional culture method for Legionella (still the “gold standard”) involves plating samples on selective agar and incubation in a carbon dioxide (CO2) enriched atmosphere for up to 10 days at approximately 37° C. Buffered Charcoal Yeast Extract (BCYE) supplemented with antibiotics such as glycine, polymixin, vancomycin and cycloheximide, (referred to as BCYE-DGVP or BYCE-GVPC) to retard other bacteria and fungal growth is used to selectively recover Legionella from mixed environmental matrices. Suspect colonies characteristic of Legionella then are harvested from the BCYE-GVPC plates (Figure 1) and patch plated to straight BCYE and BCYE-Cys plates for presumptive identification (Figure 2). (Legionella requires the presence of L-cystine for growth and positive isolates grow on BCYE-Cys but not on straight BCYE). Legionella speciation can be confirmed by DFA, PCR or cellular fatty acid (CFA) analysis. Acid or heat pretreatment of samples may be used to help clean up the sample and eliminate non-target bacteria. However, pretreatment may also kill some strains of Legionella, especially weakened isolates, and reduce the overall concentration of Legionella recovered.12 The culture procedure detects only viable, culturable organisms and is the generally accepted standard procedure for Legionella testing. The Centers for Disease Control and Prevention (CDC) method, Standard Methods #9260J, British Standard (BS) 6068-4.12-1998 and International Organization for Standardization (ISO) 11731 all are variations of this general procedure.
Supplemental Assays. In addition to Legionella-specific assays, monitoring programs should include standard heterotrophic (bacteria) plate count (HPC) analyses and microscopic examination of the water for amoeba and protozoa.
HPCs are frequently performed, but are commonly—and erroneously—interpreted as less expensive, surrogate tests for Legionella control. Managers should not regard general bacterial count data as replacements for Legionella monitoring. Research has demonstrated that HPCs do not adequately predict the presence or absence of Legionella in water systems or the risk of disease.13 However, control of Legionella by necessity includes controlling the populations of other microorganisms and shifts in HPC; Legionella ratios or a failure to control HPC levels may indicate a system at risk, even in the presence of relatively low levels of Legionella.14
Follow up Sampling. If periodic monitoring yields positive Legionella results, the analytical data and site risk factors should be reviewed by facility managers and/or safety personnel to determine the appropriate corrective actions. Various organizations worldwide have developed criteria for guidance or regulation that may be helpful to facility managers. However, considerable variation exists between recommended “action levels” (see Part 1 for discussion). If the results prompt a change in biocide treatment or operational practices, follow-up sampling should include Legionella-specific culture assays and not be limited to HPCs.
System Operation and Disinfection
Excellent operational guidance for facility potable and industrial water systems including cooling towers, humidifiers, spas and fountains are available from American Society of Heating, Refrigeration and Air-Conditioning Engineers, Inc. (ASHRAE), Occupational Safety and Health Administration (OSHA), CDC, Cooling Technology Institute (CTI) and others. The reader is directed to these sources for more information (Table 2).
A variety of disinfection techniques has been used in conjunction with Legionella-contaminated water systems including hyperchlorination, ultraviolet (UV) light, ozone, thermal eradication, instantaneous superheating systems, copper-silver ionization and other techniques. For these or other methods to be successful, all Legionella in the water column, inside commensal microbes and in biofilms must be killed. The effectiveness and costs (direct, financial and associated labor) can vary in practice, and information is readily available in the literature.8,15,16 A thorough review of disinfection techniques is beyond the scope of this article.
Well-designed water distribution and cooling systems, coupled with sound management and operational procedures, are essential to control Legionella in industrial facilities—and a monitoring program should not be considered as a replacement. However, most experts even those ill-disposed towards routine Legionella monitoring, would agree that monitoring should be considered if enough legionellosis risk factors apply to the system in question. Testing decisions should be based on a thorough review of the system, operations and population including items such as source water quality, system design, operational parameters and history, biofouling information, transmission sources, host susceptibility and Legionnaires’ Disease case history. No management program, regardless of its treatment, maintenance or monitoring components, can guarantee the absence of future legionellosis, but prudent operational practices combined with ongoing review of risk factors will allow facility managers to minimize exposure to Legionella and to its legal consequences.
About The Author: Paul Warden is the vice president of Analytical Services, Inc. (ASI). Dr. Kristen Fallon is the laboratory director of ASI. Dr. Colin Fricker is an independent water quality and treatment consultant affiliated with ASI for special projects and research. W
