pna-fish

Relevant information

The understanding of multispecies interactions in biofilms is still in its infancy, partly due to the lack of suitable methods for localizing specific populations within these structures (Elias and Banin 2012). Based on the know-how of elements of this team, PNA probes were used to differentiate populations within biofilms using state-of-the-art PNA-FISH.

In the literature, there were PNA probes described both for C. albicans and P. aeruginosa (Perry-O’Keefe et al. 2001; Rigby et al. 2002). Nonetheless, these probes were not part of a multiplex method (i.e. were used simultaneously in one sample) and hence need to work at similar temperatures. This is particularly important in the application of probes in biofilms because the ribosome content of the cells might be quite low, which means that deviations from optimal melting temperatures might prevent the observation of both populations (Almeida et al. 2011). The melting temperature of the DNA/PNA duplex for each probe was estimated according to the nearest-neighbour method described by Santa-Lucia et al. with the respective correction for PNA described in Giesen et al. (1998).

All newly designed probes were tested in the laboratory to confirm their specificity and sensitivity at the desired temperature, according to protocols already developed by us (Guimarães et al. 2007). This part employed culture collection strains and clinical isolates isolated previously by the Team or acquired from partner labs. When applied to biofilms exposed to antibiotics, PNA-FISH demonstrated the co-localization of P. aeruginosa and C. albicans (Figure 1), indicating that both microorganisms might be conjugating efforts in order to fend off a previously successful antibiotic treatment.

References

  • Almeida C, Azevedo NF, Santos S, Keevil CW, Vieira MJ (2011) Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH). PLoS One 6:e14786. doi: 10.1371/journal.pone.0014786
  • Elias S, Banin E (2012) Multi-species biofilms: living with friendly neighbors. FEMS Microbiol Rev. doi: 10.1111/j.1574-6976.2012.00325.x
  • Giesen U, Kleider W, Berding C, Geiger a, Orum H, Nielsen PE (1998) A formula for thermal stability (Tm) prediction of PNA/DNA duplexes. Nucleic Acids Res 26:5004–6.
  • Guimarães 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 biopsy specimens. J Clin Microbiol 45:3089–94. doi: 10.1128/JCM.00858-07
  • Perry-O’Keefe H, Rigby S, Oliveira K, Sørensen D, Stender H, James Coull, Hyldig-Nielsen JJ (2001) Identification of indicator microorganisms using a standardized PNA FISH method. J Microbiol Methods 47:281–292.
  • Rigby S, Procop GW, Haase G, Wilson D, Hall G, Kurtzman C, Oliveira K, Oy S Von, Hyldig-nielsen JJ, Coull J, Stender H (2002) Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles. 40:2182–2186. doi: 10.1128/JCM.40.6.2182