The Design of a PNA Probe to Improve Legionella pneumophila Monitoring

Ana Barbosa; Montserrat Nácher-Vázquez; Darla M. Goeres; Carina Almeida; Nuno F. Azevedo; Laura Cerqueira

doi:10.53941/jmhd.2025.100006

Abstract

Legionella is a significant public health threat in engineered water systems, requiring rapid and accurate environmental monitoring to prevent outbreaks. Traditional detection methods, such as culture-based assays and PCR, are limited by long processing times and the potential for false negatives due to viable but non-culturable (VBNC) cells. This study addresses these limitations by developing a novel Peptide Nucleic Acid-Fluorescence in situ Hybridization (PNA-FISH) method for the specific and sensitive detection of Legionella pneumophila. In silico analysis predicted high theoretical specificity (100.0%) and sensitivity (99.8%), results that were confirmed by experimental validation against 17 L. pneumophila strains and 37 non-target strains (including Pseudomonas, Acinetobacter, and other Legionella species), demonstrating strong fluorescence signals with no cross-reactivity. Furthermore, the method was successfully applied to artificially contaminated tap water, achieving a limit of detection of 10³ CFU mL⁻¹ directly on the filter membrane. This work highlights the potential of PNA-based probes to improve bacterial monitoring, offering fast, reliable, and field-adaptable detection of L. pneumophila. The findings support the integration of this probe into routine water system monitoring workflows, facilitating timely assessment of contamination and outbreak prevention.

References

1.
Glick, T.H.; Gregg, M.B.; Berman, B.; et al. Pontiac fever: An epidemic of unknown etiology in a health department: I. Clinical and epidemiologic aspects. Am. J. Epidemiol. 1978, 107, 149–160.
2.
McDade, J.E.; Shepard, C.C.; Fraser, D.W.; et al. Legionnaires’ disease: Isolation of a bacterium and demonstration of its role in other respiratory disease. N. Engl. J. Med. 1977, 297, 1197–1203.
3.
Diederen, B. Legionella spp. and Legionnaires’ disease. J. Infect. 2008, 56, 1–12.
4.
Hamilton, K.; Prussin, A.; Ahmed, W.; et al. Outbreaks of legionnaires’ disease and pontiac fever 2006–2017. Curr. Environ. Health Rep. 2018, 5, 263–271.
5.
Yao, X.H.; Shen, F.; Hao, J.; et al. A review of Legionella transmission risk in built environments: Sources, regulations, sampling, and detection. Front. Public Health 2024, 12, 1415157.
6.
Hammes, F.; Gabrielli, M.; Cavallaro, A.; et al. Foresight 2035: A perspective on the next decade of research on the management of Legionella spp. in engineered aquatic environments. FEMS Microbiol. Rev. 2025, 49, fuaf022.
7.
ISO 11731:2017; Water Quality–Enumeration of Legionella. ISO: Geneva, Switzerland, 2017.
8.
ISO/TS 12869; Water Quality—Detection and Quantification of Legionella spp. and/or Legionella pneumophila by Concentration and Genic Amplification by Quantitative Polymerase Chain Reaction (qPCR). ISO: Geneva, Switzerland, 2019.
9.
Yang, S.; Rothman, R.E. PCR-based diagnostics for infectious diseases: Uses, limitations, and future applications in acute-care settings. Lancet Infect. Dis. 2004, 4, 337–348.
10.
Shang, M.; Guo, J.; Guo, J. Point-of-care testing of infectious diseases: Recent advances. Sens. Diagn. 2023, 2, 1123–1144.
11.
Lotoux, A.; Milohanic, E.; Bierne, H. The viable but non-culturable state of listeria monocytogenes in the one-health continuum. Front. Cell. Infect. Microbiol. 2022, 12, 849915.
12.
Sannigrahi, A.; De, N.; Bhunia, D.; et al. Peptide nucleic acids: Recent advancements and future opportunities in biomedical applications. Bioorg. Chem. 2025, 155, 108146.
13.
Nacher-Vazquez, M.; Santos, B.; Azevedo, N.F.; et al. The role of Nucleic Acid Mimics (NAMs) on FISH-based techniques and applications for microbial detection. Microbiol. Res. 2022, 262, 127086. https://doi.org/10.1016/j.micres.2022.127086.
14.
Cerqueira, L.; Azevedo, N.F.; Almeida, C.; et al. DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH). Int. J. Mol. Sci. 2008, 9, 1944–1960. https://doi.org/10.3390/ijms9101944.
15.
Nacher-Vazquez, M.; Barbosa, A.; Armelim, I.; et al. Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples. Microorganisms 2022, 10, 1409. https://doi.org/10.3390/microorganisms10071409.
16.
Silva, A.R.; Melo, L.F.; Keevil, C.W.; et al. Legionella colonization and 3D spatial location within a Pseudomonas biofilm. Sci. Rep. 2024, 14, 16781.
17.
Rocha, R.; Sousa, J.M.; Cerqueira, L.; et al. Development and application of Peptide Nucleic Acid Fluorescence in situ Hybridization for the specific detection of Listeria monocytogenes. Food Microbiol. 2019, 80, 1–8. https://doi.org/10.1016/j.fm.2018.12.009.
18.
Cerqueira, L.; Moura, S.; Almeida, C.; et al. Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples. Microorganisms 2020, 8, 1950. https://doi.org/10.3390/microorganisms8121950.
19.
Rigby, S.; Procop, G.W.; Haase, G.; et al. Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles. J. Clin. Microbiol. 2002, 40, 2182–2186. https://doi.org/10.1128/JCM.40.6.2182-2186.2002.
20.
Gomez, A.; Miller, N.S.; Smolina, I. Visual detection of bacterial pathogens via PNA-based padlock probe assembly and isothermal amplification of DNAzymes. Anal. Chem. 2014, 86, 11992–11998. https://doi.org/10.1021/ac5018748.
21.
Noppakuadrittidej, P.; Charlermroj, R.; Makornwattana, M.; et al. Development of peptide nucleic acid-based bead array technology for Bacillus cereus detection. Sci. Rep. 2023, 13, 12482. https://doi.org/10.1038/s41598-023-38877-1.
22.
Zaid, M.H.M.; Abdullah, J.; Yusof, N.A.; et al. PNA biosensor based on reduced graphene oxide/water soluble quantum dots for the detection of Mycobacterium tuberculosis. Sens. Actuators B Chem. 2017, 241, 1024–1034.
23.
Kim, Y.; Mohanty, S.K. PNA Functionalized Gold Nanoparticles on TiO2 Nanotubes Biosensor for Electrochemical DNA Fragment Detection. Adv. Mater. Interfaces 2025, 12, 2400762. https://doi.org/10.1002/admi.202400762.
24.
Barbosa, V.B.; Rodrigues, C.F.; Cerqueira, L.; et al. Microfluidics combined with fluorescence in situ hybridization (FISH) for Candida spp. detection. Front. Bioeng. Biotechnol. 2022, 10, 987669.
25.
Ferreira, A.M.; Cruz-Moreira, D.; Cerqueira, L.; et al. Yeasts identification in microfluidic devices using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH). Biomed. Microdevices 2017, 19, 11.
26.
Rane, T.D.; Zec, H.C.; Puleo, C.; et al. Droplet microfluidics for amplification-free genetic detection of single cells. Lab Chip 2012, 12, 3341–3347.
27.
Mach, K.E.; Kaushik, A.M.; Hsieh, K.; et al. Optimizing peptide nucleic acid probes for hybridization-based detection and identification of bacterial pathogens. Analyst 2019, 144, 1565–1574.
28.
Wilks, S.A.; Keevil, C.W. Targeting species-specific low-affinity 16S rRNA binding sites by using peptide nucleic acids for detection of Legionellae in biofilms. Appl. Environ. Microbiol. 2006, 72, 5453–5462. https://doi.org/10.1128/AEM.02918-05.
29.
Simões, L.C.; Simões, M.; Oliveira, R.; et al. Potential of the adhesion of bacteria isolated from drinking water to materials. J. Basic Microbiol. 2007, 47, 174–183.
30.
Teixeira, H.; Sousa, A.L.; Azevedo, A.S. Bioinformatic tools and guidelines for the design of fluorescence in situ hybridization probes. In Fluorescence In-Situ Hybridization (FISH) for Microbial Cells: Methods and Concepts; Springer: Berlin/Heidelberg, Germany, 2021; pp. 35–50.
31.
Westram, R.; Bader, K.; Prüsse, E.; et al. ARB: A software environment for sequence data. In Handbook of Molecular Microbial Ecology I: Metagenomics and Complementary Approaches; Wiley-Blackwell: Hoboken, NJ, USA, 2011; pp. 399–406.
32.
Kumar, S.; Stecher, G.; Li, M.; et al. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549.
33.
Almeida, C.; Azevedo, N.F.; Fernandes, R.; et al. Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples. Appl. Environ. Microbiol. 2010, 76, 4476–4485.
34.
Oliveira, R.; Azevedo, A.S.; Mendes, L. Application of nucleic acid mimics in fluorescence in situ hybridization. In Fluorescence In-Situ Hybridization (FISH) for Microbial Cells: Methods and Concepts; Springer: Berlin/Heidelberg, Germany, 2021; pp. 69–86.
35.
Schneider, C.A.; Rasband, W.S.; Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 2012, 9, 671–675.
36.
Yilmaz, L.S.; Okten, H.E.; Noguera, D.R. Making all parts of the 16S rRNA of Escherichia coli accessible in situ to single DNA oligonucleotides. Appl. Environ. Microbiol. 2006, 72, 733–744. https://doi.org/10.1128/AEM.72.1.733-744.2006.
37.
Yilmaz, L.S.; Noguera, D.R. Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization. Appl. Environ. Microbiol. 2004, 70, 7126–7139. https://doi.org/10.1128/AEM.70.12.7126-7139.2004.
38.
Sousa, L.G.; Almeida, C.; Muzny, C.A.; et al. Development of a Prevotella bivia PNA probe and a multiplex approach to detect three relevant species in bacterial vaginosis-associated biofilms. NPJ Biofilms Microbiomes 2023, 9, 42.
39.
Whiley, H.; Taylor, M. Legionella detection by culture and qPCR: Comparing apples and oranges. Crit. Rev. Microbiol. 2016, 42, 65–74.
40.
Hassall, J.; Coxon, C.; Patel, V.C.; et al. Limitations of current techniques in clinical antimicrobial resistance diagnosis: Examples and future prospects. NPJ Antimicrob. Resist. 2024, 2, 16.
41.
Li, L.; Mendis, N.; Trigui, H.; et al. The importance of the viable but non-culturable state in human bacterial pathogens. Front. Microbiol. 2014, 5, 258.
42.
Almeida, C.; Azevedo, N.; Iversen, C.; et al. Development and application of a novel peptide nucleic acid probe for the specific detection of Cronobacter genomospecies (Enterobacter sakazakii) in powdered infant formula. Appl. Environ. Microbiol. 2009, 75, 2925–2930.
43.
Rohde, A.; Hammerl, J.A.; Appel, B.; et al. FISHing for bacteria in food–A promising tool for the reliable detection of pathogenic bacteria? Food Microbiol. 2015, 46, 395–407.
44.
Allegra, S.; Berger, F.; Berthelot, P.; et al. Use of flow cytometry to monitor Legionella viability. Appl. Environ. Microbiol. 2008, 74, 7813–7816.
45.
Nisar, M.A.; Ross, K.E.; Brown, M.H.; et al. Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR. Front. Microbiol. 2023, 14, 1094877.

Scilight Press

Author Information

Abstract

Keywords

References

About Scilight

Journals

Publishing Policies

Contact Us