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A Combined Immunofluorescence and Fluorescent Viability Cocktail Staining Procedure for Rapid Microscopic Detection and Enumeration of Live Legionella pneumophila

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Abstract

This report describes a combined immunofluorescence and fluorescence viability stain applied as one staining solution for rapid detection of live Legionella pneumophila in mixed bacterial populations. Instead of sequential viability staining with the Invitrogen BacLight LIVE/DEAD staining kit followed by antibody-Alexa Fluor (AF) 647 conjugate staining to identify live L. pneumophila, a combined single cocktail solution staining protocol was developed to simplify and accelerate the time to detection of viable L. pneumophila serogroup-1 (SG-1) in mixed species populations on a filter membrane. The stain cocktail will aid in accelerating fluorescence microscopic analysis of cooling tower, air conditioner and water fountain or other liquid samples for the presence of L. pneumophila and its viability status. Visibly red stained cells were identified as dead non-L. pneumophila SG-1 cells, while green fluorescing cells represented viable non-L. pneumophila SG-1 cells. Due to also staining red with antibody-AF 647, L. pneumophila SG-1 cells were pseudocolorized as blue to distinguish them from other dead cells. Fluorescence color emission mixing from the viability dyes (SYTO 9 and propidium iodide) with antibody-AF 647 stained L. pneumophila led to other fluorescent colors. For example, green plus pseudocolorized blue AF 647-antibody- labeled cells were identified as live cyan-colored L. pneumophila SG-1 cells. Magenta-colored cells resulted from dead L. pneumophila cells that combined red propidium iodide with blue pseudocolorized AF 647-antibody emissions. Analysis of measured RGB (red, green, blue) color values in microscopic images of mixed bacterial populations suggests the possibility of facile automated discrimination of subpopulations of live and dead L. pneumophila and non-L. pneumophila species by computers in 3-dimensional RGB color space after staining in the combined cocktail which will save time for more rapid microscopic detection of potential sources of Legionnaire’s disease.

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Availability of Data and Material

All data generated or analyzed during this study are included in this published article and the supplemental data file.

Code Availability

The authors can make custom code related to movement of the microscope stage, automated recognition, counting, outlining and RGB color image analysis of bacterial cells available to interested parties.

References

  1. Keserue HA, Cornillie N, Ehlert AK, Mills DC, Morger D, Piffaretti A et al (2020) Validation of the Legionella pneumophila SG1 DETECT kit for quantification of Legionella pneumophila serogroup 1 bacteria in potable waters, process waters, and surface waters: AOAC performance tested method 052002. J AOAC Int. https://doi.org/10.1093/jaoacint/qsaa126

    Article  Google Scholar 

  2. Palmer CJ, Bonilla GF, Roll B, Paszko-Kolva C, Sangermano LR, Fujioka RS (1995) Detection of Legionella species in reclaimed water and air with the EnviroAmp Legionella PCR kit and direct fluorescent antibody staining. Appl Environ Microbiol 61:407–412

    Article  CAS  Google Scholar 

  3. Fisher KE, Wickenberg LP, Leonidas LF, Ranz AA, Habib MA, Buford RM et al (2020) Next day Legionella PCR: a highly reliable negative screen for Legionella in the built environment. J Water Health 18:345–357

    Article  Google Scholar 

  4. Falzone L, Gattuso G, Lombardo C, Lupo G, Grillo CM, Spandidos DA et al (2020) Droplet digital PCR for the detection and monitoring of Legionella pneumophila. Int J Mol Med 46:1777–1782

    Article  CAS  Google Scholar 

  5. Sharaby Y, Rodríguez-Martínez S, Oks O, Pecellin M, Mizrahi H, Peretz A et al (2017) Temperature-dependent growth modeling of environmental and clinical Legionella pneumophila multilocus variable-number tandem-repeat analysis (MLVA) genotypes. Appl Environ Microbiol 83:e03295-e3316. https://doi.org/10.1128/AEM.03295-16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Delgado-Viscogliosi P, Simonart T, Parent V, Marchand G, Dobbelaere M, Pierlot E et al (2005) Rapid method for enumeration of viable Legionella pneumophila and other Legionella spp. in water. Appl Environ Microbiol 71:4086–4096

    Article  CAS  Google Scholar 

  7. Parthuisot N, Binet M, Touron-Bodilis A, Pougnard C, Lebaron P, Baudart J (2011) Total and viable Legionella pneumophila cells in hot and natural waters as measured by immunofluorescence-based assays and solid-phase cytometry. Appl Environ Microbiol 77:6225–6232

    Article  CAS  Google Scholar 

  8. Tenover FC, Carlson L, Goldstein L, Sturge J, Plorde JJ (1985) Confirmation of Legionella pneumophila cultures with a fluorescein-labeled monoclonal antibody. J Clin Microbiol 21:983–984

    Article  CAS  Google Scholar 

  9. Walker JT, McDermott P (2021) Confirming the presence of Legionella pneumophila in your water system: A review of current Legionella testing methods. J AOAC Int https://doi.org/10.1093/jaoacint/qsab003

  10. Yamaguchi N, Tokunaga Y, Goto S, Fujii Y, Banno F, Edagawa A (2017) Rapid on-site monitoring of Legionella pneumophila in cooling tower water using a portable microfluidic system. Sci Rep 7(1):1–8. https://doi.org/10.1038/s41598-017-03293-9

    Article  CAS  Google Scholar 

  11. Yamamoto H, Hashimoto Y, Ezaki T (1993) Comparison of detection methods for Legionella species in environmental water by colony isolation, fluorescent antibody staining, and polymerase chain reaction. Microbiol Immunol 37:617–622

    Article  CAS  Google Scholar 

  12. Barbesti S, Citterio S, Labra M, Baroni MD, Neri MG, Sgorbati S (2000) Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria. Cytometry 40:214–218

    Article  CAS  Google Scholar 

  13. Bruno JG, Mayo MW (1995) A color image analysis method for assessment of germination based on differential fluorescence staining of bacterial spores and vegetative cells using acridine orange. Biotech Histochem 70:175–184

    Article  CAS  Google Scholar 

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Funding

Work was funded by a Center for Disease Control (CDC) SBIR contract no. 75D30120P09235.

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Contributions

JGB conceived the overall experiments and wrote the manuscript, JJJ designed and conducted all bacterial fluorescence viability and immunofluorescence staining experiments and CJM provided assistance with microscopic image acquisition, RGB image stacking, automated bacterial recognition, counting and image analysis. All three authors edited the manuscript.

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Correspondence to John G. Bruno.

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The authors declare no conflicts of interest or competing interests.

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John, J.J., May, C.J. & Bruno, J.G. A Combined Immunofluorescence and Fluorescent Viability Cocktail Staining Procedure for Rapid Microscopic Detection and Enumeration of Live Legionella pneumophila. J Fluoresc 31, 1425–1432 (2021). https://doi.org/10.1007/s10895-021-02776-3

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  • DOI: https://doi.org/10.1007/s10895-021-02776-3

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