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.


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