Assessment of flow cytometry for microbial water quality monitoring in cooling tower water and oxidizing biocide treatment efficiency
Introduction
The conditions encountered in cooling tower systems are ideal for Legionella development and proliferation in water circuits and high proportions of occurrence are reported (Li et al., 2015; Rafiee et al., 2014). During this process, water droplets may release these pathogenic bacteria to the surroundings and can lead to human exposure and thus to outbreaks of legionellosis (Ambrose et al., 2014; Lévesque et al., 2014; Scaturro et al., 2015). In France, the management and monitoring of cooling towers as part of Legionella risk prevention is regulated, specifying characteristics of installations subject to declaration or registration. Common methods for environmental monitoring of Legionella are the standard culture-based method (NF T90 431, 2017). Health risk management is based on Legionella investigation to verify that the level remains below alert thresholds. Indeed, a level greater than or equal to 103 colony forming units per liter (CFU l−1) must lead to risk analysis and corrective actions; a level higher or equal to 105 CFU l−1 imposes installation emptying, cleaning and disinfection. Chemical biocide control in cooling towers is a critical part of the overall prevention strategy against Legionella. Various biocide treatment programs, by volumetric injection or regulated and alternating shocks are commonly used to limit microbial growth in the systems. As a result, count of culturable microorganisms (ISO 6222, 1999) is used as bacterial indicator related to the biocide treatment effectiveness. The advantage of such monitoring lies on the fact that the quantification of the total culturable flora is easier and faster (48 h) than Legionella detection, showing a time to result of >10 days. However, this method has also some drawbacks in terms of active risk management. Even if the time to result is shorter than for Legionella, a 2-day period is still long. Furthermore, this culture-based method enables to visualize only the microbial cells able to grow on a culture medium. In fact, <1% of bacteria found in the environment are culturable (Hammes et al., 2008; Wang et al., 2010). Indeed, a major part of microbial population, that does not have or have lost the ability to form colonies on agar plates under stress conditions, is called viable but not culturable (VBNC). These bacteria may nevertheless still have active cellular machinery, which makes possible their potential pathogenicity for humans (Ramamurthy et al., 2014; Zhang et al., 2015). In addition, a culture-based method requires a sample to be sent to the laboratory and cannot be performed directly in the field. The ATP measurement is also frequently used in the field for cooling tower disinfection monitoring (Duda et al., 2015; Mueller et al., 2009) and gives information on global microbial activity. However, this method does not provide precise data related to the real bacterial concentration and to the impact of biocidal treatments employed.
Flow cytometry is an alternative technique enabling to overcome the aforementioned constraints. Its principle allows individual qualitative and quantitative characterization of cells suspended in a liquid medium. Besides to a rapid quantification, flow cytometry provides in a short time information related the physiological state of cells through the use of adapted fluorochromes and has been adapted to different matrices such as seawater and drinking water (Grégori et al., 2001; Hammes and Egli, 2010; Helmi et al., 2014). However, few studies have been carried out using flow cytometry in the field of disinfection and Legionella issues in cooling tower (Fuchslin et al., 2010; Keserue et al., 2012). Moreover, these works have been mainly conducted using only pure Legionella strains. The detection approaches are based on the use of antibodies to obtain a specific detection (Fuchslin et al., 2010; Tyndall et al., 1985) or even to demonstrate their viability using fluoregenic compounds (Keserue et al., 2012). These approaches present considerable cost in terms of consumables due to the use of antibodies for immunomagnetic separation and for specific detection and require some expertise in sample preparation. In view of these observations, these methods are difficult to apply in real conditions. Moreover, these approaches do not provide information related to the impact of disinfection on the entire microbial population, potentially having the ability to generate a biofilm. Other tests in order to evaluate the impact of different biocidal treatments have been also carried out directly on pure Legionella cultures, namely without the use of antibodies, using double staining with SYTO9 and PI (Allegra et al., 2008; Mustapha et al., 2015). A team studied the resistance of different strains of Legionella to chlorine dioxide, ClO2, (Mustapha et al., 2015). The authors stated that switch to the VBNC stage, measured using flow cytometry, takes place between 4 and 5 mg l−1 of ClO2. The VBNC status can be reversible and some VBNC cells can regain their ability to grow afterwards. Another study (Allegra et al., 2008) demonstrated that Legionella cells can lose their ability to grow after heat treatment and then recover it by passing an intermediate VBNC state that is not detectable by conventional cultivation methods. This point emphasizes the fact that the measurement of VBNC cells is an important aspect in terms of safety.
The present work has been carried out on the quantification of active microbial cells, a category that may include VBNC and culturable bacteria, irrespective of genus or species. Compared with the approaches described above, the advantages are related to the fact that the measurement of the impact of a biocidal treatment is evaluated on the whole natural indigenous population, which may behave differently from pure strains. The relevance of flow cytometry has been investigated in order to highlight the gain related to cooling tower monitoring compared to usual methods and to define to what extent the performance would allow to adapt biocide treatment rates. For this purpose, the first part of the study was dedicated to real cooling tower water sample analysis to compare the detection performance of flow cytometry, ATP measurements, and the standard culture-based method. The second part of the study was based on a kinetic assessment in order to compare biocide way of action on cooling tower indigenous flora.
Section snippets
Sample collection
A total of 27 tertiary cooling tower samples were collected over a 4-month period (from 20 different sites). Water was sampled in 1-l plastic bottles containing 20 mg of thiosulfate and delivered the same day to the laboratory. Samples were kept at 4 °C until analysis, including flow cytometry and culture methods, with the addition of ATP measurement only for the last 10 samples due to collection limitation. Free chlorine potentially remaining after neutralization was measured in the samples
Cooling tower monitoring
The analysis of 27 cooling tower samples was performed in order to compare the performance and quantitative response of flow cytometry using ATP measurement and the culture-based standard method. The presence of culturable Legionella was observed for 1 sample out of 27 with a concentration of 1.2.105 CFU l−1 (data not shown). All data related to bacterial quantification and physico-chemical characterization of water samples are listed in Table 1.
For sample 1 (Table 1), the measured residual
Discussion
The environmental monitoring of microbial populations in water of cooling towers is essential to minimize health risks for the population. This study evaluated the use of flow cytometry for the control of bacterial populations in water circuit of cooling towers. Comparative tests were carried out on real and controlled water samples before and after disinfection treatments, with various methods, including laboratory and fieldable flow cytometry systems using stains for membrane integrity and
Conclusions
This work aimed to evaluate the efficiency of flow cytometry for regular cooling tower monitoring and disinfection efficiency control in comparison with current monitoring methods. Moreover, the use of markers for cell integrity and activity brought information about the oxidizing biocides mechanisms of action on bacterial cells. The main conclusions based on our observations are as follows:
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Culture-based method and ATP measurement, commonly used for cooling tower disinfection monitoring, give
Acknowledgments
This work was funded by Veolia Environnement. The authors thank all the laboratory team for their technical contribution and Oliver Keserue for language help. The authors acknowledge Veolia Water Solutions, especially Hydrex division, who provided the Hydrex biocide products applied by Veolia and related technical information concerning them. Hydrex is the global water treatment additives brand name from Veolia.
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