Diagnostic Bacteriology pp 137-146

Part of the Methods in Molecular Biology book series (MIMB, volume 1616)

Detection of Helicobacter pylori in the Gastric Mucosa by Fluorescence In Vivo Hybridization

  • Silvia Fontenete
  • Marina Leite
  • Ceu Figueiredo
  • Paul Cos
  • Nuno F. Azevedo
Protocol

Abstract

In this chapter, we describe a fluorescence in vivo hybridization (FIVH) protocol, using nucleic acid probes, for the detection of the bacterium Helicobacter pylori in the gastric mucosa of an infected C57BL/6 mouse model. This protocol should be easily extended to other microorganisms not only as a way to identify in vivo important microorganisms and their patterns of distribution within specific or at different anatomic sites, but also to better understand interaction mechanisms involving the microbiome and the human body.

Key words

Diagnostics Microbiology Nucleic acids In vivo Helicobacter pylori Fish FIVH 

References

  1. 1.
    DeLong EF, Wickham GS, Pace NR (1989) Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243(4896):1360–1363CrossRefPubMedGoogle Scholar
  2. 2.
    Guimaraes N, Azevedo NF, Figueiredo C, Keevil CW, Vieira MJ (2007) Development and application of a novel peptide nucleic acid probe for the specific detection of Helicobacter pylori in gastric biopsies. J Clin Microbiol 45(9):3089–3094CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Wagner M, Horn M, Daims H (2003) Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Curr Opin Microbiol 6(3):302–309CrossRefPubMedGoogle Scholar
  4. 4.
    Amann R, Fuchs BM (2008) Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol 6(5):339–348. doi:10.1038/nrmicro1888 CrossRefPubMedGoogle Scholar
  5. 5.
    Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59(1):143–169PubMedPubMedCentralGoogle Scholar
  6. 6.
    Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172(2):762–770CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Makristathis A, Riss S, Hirschl AM (2014) A novel fluorescence in situ hybridization test for rapid pathogen identification in positive blood cultures. Clin Microbiol Infect 20(10):O760–O763. doi:10.1111/1469-0691.12561 CrossRefPubMedGoogle Scholar
  8. 8.
    Harris DM, Hata DJ (2013) Rapid identification of bacteria and Candida using PNA-FISH from blood and peritoneal fluid cultures: a retrospective clinical study. Ann Clin Microbiol Antimicrob 12:2. doi:10.1186/1476-0711-12-2. 1476-0711-12-2 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glockner FO (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41(Database issue):D590–D596. doi:10.1093/nar/gks1219 CrossRefPubMedGoogle Scholar
  10. 10.
    Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM (2014) Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42(Database issue):D633–D642. doi:10.1093/nar/gkt1244. gkt1244 [pii]CrossRefPubMedGoogle Scholar
  11. 11.
    Zhang H (2003) Alignment of BLAST high-scoring segment pairs based on the longest increasing subsequence algorithm. Bioinformatics 19(11):1391–1396CrossRefPubMedGoogle Scholar
  12. 12.
    Berlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ, Telford WG, Yue S, Liu J, Cheung CY, Chang W, Hirsch JD, Beechem JM, Haugland RP, Haugland RP (2003) Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. J Histochem Cytochem 51(12):1699–1712CrossRefPubMedGoogle Scholar
  13. 13.
    Hayashi-Takanaka Y, Stasevich TJ, Kurumizaka H, Nozaki N, Kimura H (2014) Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging. PLoS One 9(9):e106271. doi:10.1371/journal.pone.0106271 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Hugenholtz P, Tyson GW, Blackall LL (2002) Design and evaluation of 16S rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization. Methods Mol Biol 179:29–42PubMedGoogle Scholar
  15. 15.
    Cerqueira L, Azevedo NF, Almeida C, Jardim T, Keevil CW, Vieira MJ (2008) DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH). Int J Mol Sci 9(10):1944–1960. doi:10.3390/ijms9101944 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kubota K, Ohashi A, Imachi H, Harada H (2006) Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes. Appl Environ Microbiol 72(8):5311–5317. doi:10.1128/aem.03039-05 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Robertson KL, Vora GJ (2012) Locked nucleic acid and flow cytometry-fluorescence in situ hybridization for the detection of bacterial small noncoding RNAs. Appl Environ Microbiol 78(1):14–20. doi:10.1128/aem.06399-11 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Fontenete S, Guimaraes N, Leite M, Figueiredo C, Wengel J, Filipe Azevedo N (2013) Hybridization-based detection of Helicobacter pylori at human body temperature using advanced locked nucleic acid (LNA) probes. PLoS One 8(11):e81230. doi:10.1371/journal.pone.0081230. PONE-D-13-15277 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Fontenete S, Leite M, Guimaraes N, Madureira P, Ferreira RM, Figueiredo C, Wengel J, Azevedo NF (2015) Towards Fluorescence In Vivo Hybridization (FIVH) detection of H. pylori in gastric mucosa using advanced LNA probes. PLoS One 10(4):e0125494. doi:10.1371/journal.pone.0125494. PONE-D-14-54901 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Fontenete SLM, Cappoen D, Santos R, Ginneken CV, Figueiredo C, Wengel J, Cos P, Azevedo NF (2016) Fluorescence in vivo hybridization (FIVH) for detection of Helicobacter pylori infection in a C57BL/6 mouse model. PLoS One 11(2):e0148353CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Marshall BJ (1988) The Campylobacter pylori story. Scand J Gastroenterol Suppl 146:58–66CrossRefPubMedGoogle Scholar
  22. 22.
    Lee A (1994) The microbiology and epidemiology of Helicobacter pylori infection. Scand J Gastroenterol Suppl 201:2–6CrossRefPubMedGoogle Scholar
  23. 23.
    Fontenete S, Leite M, Cappoen D, Santos R, Ginneken CV, Figueiredo C, Wengel J, Cos P, Azevedo NF (2016) Fluorescence In Vivo Hybridization (FIVH) for detection of Helicobacter pylori infection in a C57BL/6 mouse model. PLoS One 11(2):e0148353. doi:10.1371/journal.pone.0148353. PONE-D-15-52300 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Kumar R, Singh SK, Koshkin AA, Rajwanshi VK, Meldgaard M, Wengel J (1998) The first analogues of LNA (locked nucleic acids): phosphorothioate-LNA and 2′-thio-LNA. Bioorg Med Chem Lett 8(16):2219–2222. doi:S0960894X98003667 [pii]CrossRefPubMedGoogle Scholar
  25. 25.
    You Y, Moreira BG, Behlke MA, Owczarzy R (2006) Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res 34(8):e60. doi:10.1093/nar/gkl175. 34/8/e60 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Kierzek E, Ciesielska A, Pasternak K, Mathews DH, Turner DH, Kierzek R (2005) The influence of locked nucleic acid residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes. Nucleic Acids Res 33(16):5082–5093. doi:10.1093/nar/gki789. 33/16/5082 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Yan Y, Yan J, Piao X, Zhang T, Guan Y (2012) Effect of LNA- and OMeN-modified oligonucleotide probes on the stability and discrimination of mismatched base pairs of duplexes. J Biosci 37(2):233–241CrossRefPubMedGoogle Scholar
  28. 28.
    Maciaszek A, Krakowiak A, Janicka M, Tomaszewska-Antczak A, Sobczak M, Mikolajczyk B, Guga P (2015) LNA units present in the (2′-OMe)-RNA strand stabilize parallel duplexes (2′-OMe)-RNA/[All-RP-PS]-DNA and parallel triplexes (2′-OMe)-RNA/[All-RP-PS]-DNA/RNA. An improved tool for the inhibition of reverse transcription. Org Biomol Chem 13(8):2375–2384. doi:10.1039/c4ob02364a CrossRefPubMedGoogle Scholar
  29. 29.
    Fontenete S, Guimaraes N, Wengel J, Azevedo NF (2015) Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models. Crit Rev Biotechnol 36(3):566–577. doi:10.3109/07388551.2014.993589 PubMedGoogle Scholar
  30. 30.
    Adams AM, Harding PL, Iversen PL, Coleman C, Fletcher S, Wilton SD (2007) Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries. BMC Mol Biol 8:57. doi:10.1186/1471-2199-8-57 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Silvia Fontenete
    • 1
    • 2
    • 3
    • 4
    • 5
    • 6
  • Marina Leite
    • 2
    • 3
  • Ceu Figueiredo
    • 2
    • 3
    • 7
  • Paul Cos
    • 8
  • Nuno F. Azevedo
    • 1
  1. 1.LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of EngineeringUniversity of PortoPortoPortugal
  2. 2.i3s Instituto de Investigação e Inovação em SaúdeUniversidade do PortoPortoPortugal
  3. 3.IPATIMUP, Institute of Molecular Pathology and Immunology of the University of PortoPortoPortugal
  4. 4.Nucleic Acid Center, Department of Physics, Chemistry and PharmacyUniversity of Southern DenmarkOdense MDenmark
  5. 5.ICBAS, Institute of Biomedical Sciences Abel SalazarUniversity of PortoPortoPortugal
  6. 6.Epithelial Cell Biology Group, Cancer Cell Biology ProgramSpanish National Cancer Research CentreMadridSpain
  7. 7.FMUP, Faculty of Medicine of the University of PortoPortoPortugal
  8. 8.Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical, Biomedical and Veterinary SciencesUniversity of AntwerpAntwerpBelgium

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