Flow Cytometry Quantifying Legionella in Shorter Time

Identifying the source of infection is essential for taking action in outbreaks of Legionnaire's disease. The combination of immunomagnetic separation and flow cytometry delivers precise results in less than two hours and knows virtually no limits to the variety of applications.

Legionella were not discovered and described until 1976. At that time, a mysterious pneumonia affected numerous parti­cipants during a congress of the American Legion, a veteran's association. After a feverish search, doctors microbiologists identified the infectious agent causing for this “Legionnaire’s disease” and named it Legionella [1].

In contrast to other bacteria, Legionella are comparatively heat-resistant and prefer to multiply at temperatures between 25 °C and 45 °C. Through the establishment of hot water systems, air conditioners or whirlpools, they seem to have found an ecological niche that renders them a serious health hazard. Infection occurs by inhaling aerosols, which are produced in the shower or in air-conditioning systems. But also decorative fountains, for example, have already been identified as a source of infection. It is estimated that 10,000 cases of life-threatening Legionnaires' pneumonia occur annually in Europe. The lethality rate averages 10% [2]. Outbreaks are particularly serious if they occur in hospitals, retirement homes or hotels.

The largest outbreak of Legionnaires' disease in Germany to date occurred in Warstein in 2013. Investigations have shown that the activated sludge basin of a wastewater treatment plant was highly contaminated with Legionella. Via a river, the bacteria reached industrial recooling plants and from there were distributed into the environment via aerosols. A total of 165 cases of illness and suspected cases had been reported [6]. In Switzerland the number of cases of Legionnaire's disease increased by 35% since 2016. The Swiss government wants to stop the epidemic with a task force [18]. In the US it is the state of New York that leads the nation a 38% increase in cases compared with 2016. Of the state total, New York City recorded 441 cases — a 65% increase over 2016.

The fact that the source of an outbreak can often only be identified after about two weeks, if at all, since the analysis is time-consuming and prone to errors, contributes to the fact that so many cases have occurred.

Questioning the standard procedure

The established standard method for Legionella detection, ISO 11731 [7], is based on the cultivation of bacteria on agar plates, which leads to variable results and takes up to 14 days [8-10]. An extensive round robin test between different laboratories in the USA has also shown that the concentration of Legionella can be dramatically underestimated using plating methods [11]. One reason for this is that not all bacterial cells present and alive in a given sample will grow on agar medium [12]. Therefore, the standard method does not detect these cells. Especially after stagnation and chemical or thermal disinfection, such cells, called VBNC — viable but non-culturable cells — occur more frequently. Since the efficiency of disinfection measures is assessed using the standard method, the microbiological results are difficult to interpret [13, 14]. It is therefore not surprising that very high levels of Legionella are often measured weeks after disinfection measures have been carried out [4, 15].

It is questionable that the vast majority of information on the spread and behavior of pathogens in water systems has been compiled using these conventional, cultivation-dependent methods. To the best of our knowledge, this means that the accuracy and reliability of the data is neither satisfactory nor sufficient for the assessment of drinking water hygiene.

The new method – faster and more precise

The rapid analysis of the total cell count of bacterial cells in water samples by flow cytometry is already established in water laboratories worldwide [16, 17]. rqmicro, a spin-off of the Federal Institute of Technology (ETH) Zurich, set itself the objective of optimizing this method for various pathogens and matrices. Thanks to this ingenious sample preparation process developed by the interdisciplinary team at rqmicro, it is possible to isolate, purify and quantify Legionella from water samples in record time.

The method is based on the immunomagnetic separation (IMS) of target cells using magnetic particles and subsequent flow cytometric detection — in less than two hours from sampling to result. In contrast to the established methods, the new method detects all potentially infectious Legionella — including cells that do not grow on agar plates, the VBNC cells.

Magnetic particles bind to the surface of Legionella via antibodies specifically developed for the immunomagnetic separation. The application of a magnetic field is used to separate the target cells from the sample matrix. This step is necessary as one liter of drinking water commonly contains about 100 million bacterial cells. The detection of only one hundred Legionella that might be present in a sample, which corresponds to a concentration of 0.0001%, is equivalent to the proverbial search for the needle in a haystack.

In order to quantify the target cells by flow cytometry after the separation step, they are labeled with antibodies bound to a fluorescent dye. In addition to the accurate evaluation and faster quantification, the flow cytometric detection reveals the physiological state of cells. Membrane-damaged and thus mostly dead cells can be distinguished from viable cells. This is achieved by using a viability dye that only penetrates and stains damaged cells.

The ability to assess not only the presence but also the physiological conditions of cells is particularly useful for disinfection measures. It creates the basis for reassessing and improving today's disinfection and remediation measures, which unfortunately do not always lead to sustainable problem solving [4, 15]. New test methods also enable the optimization of sanitary and building technology, for example by improving piping systems and critical sampling points.

Automation using microfluidic technology

The rqmicro Cell-Stream device performs immunomagnetic cell separation automatically and efficiently. The integration of microfluidics in disposable cartridges notably reduces application errors and working time. The workflow is simple. After filtration of a water sample of up to one liter, the cells are resuspended by vortexing the filter in three mL buffer solution. Antibodies bound to magnetic particles or fluorophores respectively, are added to this cell suspension. After incubation, the samples are transferred onto the cartridge and processed automatically by the Cell-Stream device.

The fluid in the cartridge moves through the microfluidic channels by means of a patented microair pressure process — the fluid always remains in the cartridge and does not come into contact with the instrument.

There will be other kits launched in 2018

The Cell-Stream has been commercially available since mid-2016 with a kit for Legionella pneumophila SG1. In the first half of 2018, kits for L. p. SG1-15 and L. spp. will be launched. Further kits for the detection of Pseudomonas aeruginosa, E. coli, Salmonella, Giardia lamblia and Cryptosporidium parvum are under development.

After sample preparation with the Cell-Stream the samples can also be used for today's cultivation methods — with significant advantages. The separation and purification eliminates more than 95% of the competing flora that accompanies Legionella in surface waters, wastewater and cooling towers.

In addition, the acid and heat treatments required for the standard process but potentially detrimental to Legionella, become obsolete thanks to the sample preparation. This fact favours that Legionella present in a sample actually are able to grow on agar plates. Internal and external validation studies are underway and already show promising results. Alternatively, after sample preparation, target cells can also be used for PCR, MALDI-TOF or fluorescence microscopy.

References

[1] D. J. Brenner, A. G. Steigerwalt, and J. E. McDade, “Classification of the Legionnaires’ disease bacterium: Legionella pneumophila, genus novum, species nova, of the family Legionellaceae, familia nova,” Ann. Intern. Med., vol. 90, no. 4, pp. 656–658, Apr. 1979.

[2] ECDC, “Legionnaires’ disease in Europe, 2010,” European Centre for Disease Prevention and Control, Stockholm, 2012.

[3] T. Kistemann, “Hygienisch-mikrobiologische Risiken von Großgebäude-Wasserinstallationen,” Bonn, Germany, 2004.

[4] S. Völker, C. Schreiber, and T. Kistemann, “Drinking water quality in household supply infrastructure--A survey of the current situation in Germany,” Int. J. Hyg. Environ. Health, vol. 213, no. 3, pp. 204–209, Jun. 2010.

[5] “List of Legionnaires’ disease outbreaks,” Wikipedia, the free encyclopedia. 25-Jan-2014.

[6] Wikipedia, “Legionellose-Ausbruch in Warstein 2013,” Wikipedia. 10-Jan-2014.

[7] ISO, “ISO 11731. Water quality - detection and enumeration of Legionella,” International Organization for Standardization, Geneva, Switzerland, 2004.

[8] C. A. Boulanger and P. H. Edelstein, “Precision and accuracy of recovery of Legionella pneumophila from seeded tap water by filtration and centrifugation.,” Appl. Environ. Microbiol., vol. 61, no. 5, pp. 1805–1809, May 1995.

[9] R. H. Bentham, “Routine sampling and the control of Legionella spp. in cooling tower water systems.,” Curr. Microbiol., vol. 41, no. 4, pp. 271–275, Oct. 2000.

[10] C. Napoli, R. Iatta, F. Fasano, T. Marsico, and M. T. Montagna, “Variable bacterial load of Legionella spp. in a hospital water system,” Sci. Total Environ., vol. 408, no. 2, pp. 242–244, Dec. 2009.

[11] C. E. Lucas, T. H. Taylor Jr, and B. S. Fields, “Accuracy and precision of Legionella isolation by US laboratories in the ELITE program pilot study,” Water Res., vol. 45, no. 15, pp. 4428–4436, Oct. 2011.

[12] J. D. Oliver, “Recent findings on the viable but nonculturable state in pathogenic bacteria,” FEMS Microbiol. Rev., vol. 34, no. 4, pp. 415–425, Jul. 2010.

[13] S. Allegra et al., “Evaluation of an immunomagnetic separation assay in combination with cultivation to improve Legionella pneumophila serogroup 1 recovery from environmental samples,” J. Appl. Microbiol., Jan. 2011.

[14] S. Allegra, F. Berger, P. Berthelot, F. Grattard, B. Pozzetto, and S. Riffard, “Use of flow cytometry to monitor Legionella viability,” Appl. Environ. Microbiol., vol. 74, no. 24, pp. 7813–7816, Dec. 2008.

[15] B. Bendinger and J. Benölken, “Prexisnahe Untersuchungen zur Kontamination von Trinkwasser in halbtechnischen Trinkwasser-Installationen,” presented at the 24. Mühlheimer Wassertechnisches Seminar, Mühlheim, 19-May-2010.

[16] F. Hammes, M. Berney, Y. Wang, M. Vital, O. Köster, and T. Egli, “Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes,” Water Res., vol. 42, no. 1–2, pp. 269–277, Jan. 2008.

[17] F. Hammes and T. Egli, “Cytometric methods for measuring bacteria in water: advantages, pitfalls and applications,” Anal. Bioanal. Chem., vol. 397, no. 3, pp. 1083–1095, Jun. 2010.

[18] https://www.blick.ch/news/schweiz/bund-gibt-zu-wir-haben-die-lage-nicht-mehr-im-griff-lungenkrankheit-breitet-sich-in-der-schweiz-aus-id7902027.htmlh

[19]http://markets.businessinsider.com/news/stocks/Record-Year-for-Legionnaires-Disease-Across-New-York-692553

* Dr. H.-A. Keserue, A.-K. Ehlert & Dr. D. Schaffhauser: rqmicro AG, 8952 Schlieren/ Switzerland

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