Abstract
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.
Highlights
According to Costerton et al [1], a biofilm is ‘‘a functional consortium of microorganisms attached to a surface and is embedded in the extracellular polymeric substances (EPS) produced by the microorganisms’’
L. monocytogenes and S. enterica are Grampositive and negative bacteria, respectively, and are important foodborne pathogens that persist on food contact surfaces due to biofilm formation [39,40,41]
A novel peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) probe was developed for L. monocytogenes, targeting the positions 1253 to 1267 of the 16S rRNA L. monocytogenes strain EDG-e (Figure S1)
Summary
According to Costerton et al [1], a biofilm is ‘‘a functional consortium of microorganisms attached to a surface and is embedded in the extracellular polymeric substances (EPS) produced by the microorganisms’’. To fully characterize and understand these systems, it is necessary to spatially discriminate between one or more populations, and as such, widely used methods in the biofilm area, such as cristal violet (CV), SYTO9/propidium iodide fluorochrome uptake and 49,6-diamidino-2-phenylindole (DAPI) staining, are insufficient due to their non-specific nature. To overcome this problem several researchers have been using different approaches, such as mutants expressing green fluorescent protein (GFP) [14,15,16], fluorescently labeled antibodies [17,18,19] and fluorescence in situ hybridization (FISH) [20,21,22]. 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
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