Figure 1.
Epifluorescence microscopy pictures of a multiplex assay for mono-species and a three-species smear, using two PNA probes (SalPNA1873 and LmPNA1253) and DAPI staining.
In the columns we have the microscopy filter used to visualize each fluorochrome (from left to right, Alexa 594, Alexa 488 and DAPI). The first three rows present the pure smears for each species used. No cross-hybridization was observed between the two PNA probes. The fourth row shows a smear with the three species mixed. The bottom image represents the bands superposition discriminating the cells of the three populations.
Figure 2.
PNA FISH validation for biofilm samples.
(A) Percentage of cells detected by PNA FISH for 24 and 48 h biofilms, in comparison with the total cells counts by DAPI. (B) Correlation between the PNA FISH counts and the DAPI counts for 24 and 48 h S. enterica e L. monocytogenes pure- culture biofilms. A high correlation between the two methods was observed and up to 48 hours at least 90% of the populations is detected by PNA FISH.
Figure 3.
Biofilm formation for single- and dual-species biofilms.
On panel A it is possible observe the normalized areas for each biofilm on each adhesion material for cultivability, CV and PNA FISH/DAPI graphs (A). Panels B, C and D are shown as examples of CV, PNA FISH/DAPI and cultivability graphs, respectively, on the glass support. Similar graphs for the remaining supports are provided in the Figures S2, S3, S4 and S5.
Figure 4.
Biofilm formation profiles for each species on single- and dual-species biofilms.
Cultivability (A) and PNA FISH/DAPI (B) areas showing the populations variations when co-cultured with a different species. (C) CV areas showing two typical CV profiles, the E. coli profile (at grey) suggesting a high production of exopolymers, and the L. monocytogenes and S. enterica profile (at pink) showing a reduced ability to produce exoplimers. The CV profile for E. coli/S. enterica biofilm suggests that Salmonella affected the E. coli ability to produce exopolymers.
Figure 5.
Dual-species biofilms spatial organization for 48 h.
(A) Epifluorescence images showing an homogeneous distribution of the species. (B) CLSM transversal images showing that dual-species biofilms with E. coli presented two well defined layers. For Salmonella/Listeria biofim, it was not observed the formation of two layers.
Figure 6.
Comparison between PNA FISH/DAPI and cultivability measurements.
Viable and cultivable bacteria adhered to the different material for S. enterica (AI) and L. monocytogenes (AII) pure culture biofilm and Salmonella/Listeria dual-especie biofilm (AII). Percentages of cells detected by cultivability for each specie, on single and dual-specie biofilm, adhered to copper (BII) and the remaining six material (BI- average values determined for the six materials together). Correlation between the PNA FISH counts and the CFU counts for 24 and 48 h biofilms (C) (all the 6 biofilm experiments included).
Figure 7.
Tri-species biofilm formation.
(A) Biofilm populations for 24 and 48 hours on each support material. (B) CLSM images distinguishing each bacteria and the superposition of the three fields. (D) CLSM showing the biofilm three-dimensional spatial distribution. A frontal quadrant (red rectangle) was removed to show the existence of an upper layer exclusively formed by E. coli, over a mixed Salmonella and Listeria layer. The bottom blank rectangle shows a transversal biofilm image showing the well defined layers.
Figure 8.
Schematic representation of the tri-species biofilm formation showing the main steps and the key factors involved on the two layers appearing.