BackgroundOur current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data.Methodology/Principal FindingsWe employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ∼56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm2) for 48 h biofilm: E. coli 2,1×108 (±2,4×107); L. monocytogenes 6,8×107 (±9,4×106); and S. enterica 1,4×106 (±4,1×105)]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica.SignificanceWhile PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment.

Proteus mirabilis is an enterobacterium that causes catheter-associated urinary tract infections (CAUTIs) due to its ability to colonize and form crystalline biofilms on the catheters surface. CAUTIs are very difficult to treat, since biofilm structures are highly tolerant to antibiotics. Phages have been used widely to control a diversity of bacterial species, however, a limited number of phages for P. mirabilis have been isolated and studied. Here we report the isolation of two novel virulent phages, the podovirus vB_PmiP_5460 and the myovirus vB_PmiM_5461, which are able to target, respectively, 16 of the 26 and all the Proteus strains tested in this study. Both phages have been characterized thoroughly and sequencing data revealed no traces of genes associated with lysogeny. To further evaluate the phages’ ability to prevent catheter’s colonization by Proteus, the phages adherence to silicone surfaces was assessed. Further tests in phage-coated catheters using a dynamic biofilm model simulating CAUTIs, have shown a significant reduction of P. mirabilis biofilm formation up to 168 h of catheterization. These results highlight the potential usefulness of the two isolated phages for the prevention of surface colonization by this bacterium.

After characterization of preferred conditions for Helicobacter pylori survival in the sessile state, it was observed that the bacterium transforms from spiral to coccoid under mild circumstances, whereas under extreme ones it is unable to undergo shape modification. This strongly supports the view that transformation into the coccoid form is an active, biologically led process, switched on by the bacterium as a protection mechanism.