Automatic Early Warning System to Detect and Quantify <em>Legionella</em> Species in Cooling Towers

Research Article

J Bacteriol Mycol. 2018; 5(3): 1071.

Automatic Early Warning System to Detect and Quantify Legionella Species in Cooling Towers

Rodríguez G1*, Solís I1, Jiménez M1, Sabater M1, Martínez MA1, Bedrina B1, Lázaro M1, Ceña S2, Puig A2, Davide D2, Fisac C2 and Rodríguez J2

¹Biótica SL, Scientific Park of University Jaime I, Castellón, Spain

²Idneo Technologies, Barcelona, Spain

*Corresponding author: Rodríguez G, R&D Department, Biótica SL, Scientific Parc of University Jaume I, Campus Rius Sec, Espaitec 2, Ground Floor, Laboratory 2, Castellón, Spain

Received: May 16, 2018; Accepted: June 21, 2018; Published: June 28, 2018


Early determination of Legionella spp concentrations is essential to avoid inadequate or unnecessary disinfection treatments. Culture confirmation takes 10 to 12 days, delaying the identification of potential infection sources. Faster, but accurate alternatives are needed. On site measurement of the Legionella spp concentration will greatly facilitate the timely steps and management of potentially risk sources. We developed a rapid method based on the immunomagnetic separation combined with enzyme-immunoassay for the quantitative determination of Legionella spp in water samples (Legipid®). The aim of this work was to adapt this method to develop a completely automated device able to perform on site. Use of this method for Legionella spp quantification as a fully-automated system could provide dramatic improvement in time-to-result to shortening decision-making process. We describe an automated immunosensor including filtering module and disposable reagents cartridges that allows for rapid determining of Legionella levels. Automated filtering module allow for rapid and efficient water samples concentration. Magnetic immuno-beads provide the separation of the whole cell target from the rest of the sample and their concentration. An optical reader provides easily accessible digital readouts of Legionella concentration measurements. The study evaluated this device as a reasonable approach for Legionella quantification and could produce results in as few as 2 hours with no downstream workup. Performance parameter was also comparable (sensitivity 100%; specificity 92.6%; accuracy 96.7%). The performance of the completely automated analyser allows on-site analysis of Legionella levels without hands-on steps prone to human error.

Keywords: Legionella; Automation; Immunosensing; On-Site; Prevention


Legionnaires’ disease (LD) is an environmental worldwide disease comprising pneumonia symptoms ranging from slight fever to lung infiltrates and multisystem failure [1]. The majority of infections are caused by strains belonging to different Legionella pneumophila serogroups [2-4]. This pathogen is a thin, rod-like (0.5-0.7 µm of thickness and 2-20 µm of length) aerobic gram-negative bacterium that inhabits a wide variety of naturally occurring and anthropogenic aquatic zones, where it is essentially an obligate intracellular parasite of free-living protozoa and a secondary colonizer of biofilms [3-4]. Manmade water systems with aerosol generation have been identified as potential reservoirs of this bacterium, such as those found in hotels or resorts, hospitals, long-term care facilities, and cruise ships. Transmission to human lungs can occur by inhalation of aerosolized droplets of water contaminated with the bacterium Legionella (bioaerosols). Among others, showerheads, cooling towers, hot tubs, and decorative fountains have been categorized as risk facilities. Thus, the control of Legionella in these settings has been considered the most effective strategy for prevention of LD [5]. LD is a higher public health priority for research and policy development. LD has around 10-15% fatality rate, ranging 5-30%, and many others who survive only do so after extensive hospital treatment, with an average length of hospital stay of 10.3 days. Total cost of all hospitalizations is over $433,000,000 in United States and $1,359,000,000 in Europe, with a total cost per patient exceeding $24,000–$34,000 [6-8].

Bioaerosols from cooling towers are often suspected to cause community-acquired LD outbreaks. Cooling towers are designed to remove heat from a building or facility by spraying water down through the tower. To prevent and control Legionella contamination in cooling towers, maintenance actions should focus on lowemission cleaning procedures of cooling towers combined with control measurements of water and air samples. An inadequate water management program, dense biofilm within the cooling towers, and high ambient temperatures can promote Legionella spp. proliferation. Management and control strategies should be supported by an improved Legionella detection method that provides reliable, rapid and valuable information related to the public health risk. Moreover, procedures allowing rapid detection and risk assessment in the potential sources of infection, such as cooling towers, are essential for adequate public health measures.

Considerable effort has been focused on the determination of Legionella spp because species of Legionella other than Legionella pneumophila are also causing health issues. The traditional method for Legionella detection in environmental samples is based on cultivation in selective artificial media [9,10]. Although recovery of a bacterial isolate by culture is the standard for identification of Legionella in the environment, well-known limitations of this method could compromise their utility in preventive or rapid control action. Among these drawbacks, we can highlight (a) long time to confirm results (from 2 to 28 days are required), (b) changes on environmental water samples have been identified during their transport to laboratory, which might take up to 1-2 days, (c) likely presence of viable and infective but no-cultivable cells, (d) poor sensitivity, and (e) a rate of inconclusive results up to 20% by interfering microbiota [11,12]. In this scenario, routine testing based on rapid Legionella detection method could be combined with risk assessment and control measures. The use of alternative not-growth based methods to detecting or quantifying Legionella in water samples may permit to complement current epidemiological purposes of the culture method enabling a prevention strategy based on a comprehensive risk assessment. PCR or PMA-PCR are very sensible but they are inappropriate methods for discriminating between live and dead cells, being unreliable to detect Legionella for regulatory purposes [13,14]. Moreover, the lack of correspondence between the results of this PCR techniques and the culture method makes difficult the understanding of the result and its application to take timely steps on the risk facilities.

Current shortcomings in the quantification of Legionella have been reported as a barrier to Legionella control [15], encouraging the development and approval of novel rapid test methods for quantifying live Legionella in water samples. In this context, a method based on immunomagnetic separation (IMS) for Legionella spp monitoring based on the use of anti-Legionella antibodies immobilized on magnetic beads have proved its efficacy [16]. IMS combines specific, whole-cell antibody recognition with magnetic bead-based purification for bacterial concentration. This method has proved to be more sensitive than the culture method, and provides the possibility to selectively quantify viable Legionella cells with an established equivalence between the results using this technique and those obtained using culture results [17]. This test has already been evaluated by Public Health laboratories by comparing this test method with q-PCR and conventional culture [18]. We developed a rapid method based on the immuno-magnetic separation combined with enzyme-immunoassay for the quantitative determination of Legionella spp in water samples (Legipid®). The aim of this work was to adapt this method to develop a completely automated device able to perform on site.

Materials and Methods

Legipid® test method

Legipid® is a test based on immunomagnetic separation (IMS) by anti-Legionella immuno-modified magnetic beads, combined with an enzyme-linked colorimetric detection for a rapid 1 h test. This IMS method is certified by the Research Institute of the Association of Official Analytical Chemist (AOAC-RI). Briefly, original water sample is concentrated by filtration or similar, and this prepared sample is eluted and dispensed into the test cuvette. A suspension of Legionella binding magnetic beads is added. Legionella cells present in the prepared sample will bind to the antibodies immobilized onto the surface of the beads, to form bacteria/bead complexes. Antibodies bind to antigens expressed on the surface of Legionella cells. As these complexes can be separated by a magnet, they can be easily washed and resuspended. Next, complexes are incubated with an enzymeconjugated anti-Legionella antibody to form labelled complexes. After washing steps, the Legionella/magnetic bead complexes are visualized by the colorimetric reaction when enzyme substrates are added. A control (without target) can be tested in parallel in another control cuvette. The results are reported as equivalent colony forming units (CFUeq), i.e. the amount of colony forming units (CFU) that would have been obtained by using the culture method in absence of interfering microbiota and being all Legionella cells available to the antibodies.

Automated analyzer

The analyzer includes a fluidic circuit that performs the following functionalities: sample intake, sample filtering, filter elution, reagents pipetting, liquid waste disposal and circuit sterilizing and cleaning. This fluidic circuit is composed of: two peristaltic pumps, one membrane pump, 5 electro valves, 3 bottles for liquid consumables, an intermediate measurement tank, a buffering manifold, a level sensor, a tube sensor for liquid presence, and an automated coupling system for disposable filters (Figure 1). The automated analyzer includes modules in which discrete aspects of the complete Legionella assay are performed using a plurality of reagents contained in different wells on a disposable polypropylene cartridge (Figure 2). The analyzer has bar code readers so that the instrument can identify each cartridge from the bar code. Then, information regarding all the reagents is specified on the cartridge. All the cartridges (up to 10) are loaded into a refrigerated module at 5±3°C until ready to use.