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Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH).

Almeida C, Azevedo NF, Santos S, Keevil CW, Vieira MJ - PLoS ONE (2011)

Bottom Line: 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.While 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualta, Braga, Portugal.

ABSTRACT

Background: Our 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 findings: We 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/cm(2)) for 48 h biofilm: E. coli 2,1 × 10(8) (± 2,4 × 10(7)); L. monocytogenes 6,8 × 10(7) (± 9,4 × 10(6)); and S. enterica 1,4 × 10(6) (± 4,1 × 10(5))]. 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.

Significance: While 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.

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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.
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pone-0014786-g003: 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.

Mentions: Taking advantage of the robustness of the PNA FISH/DAPI method, we next investigated the strains ability to form biofilms in 7 different materials, and complemented the obtained results with two other well-known analytical methods: CFU counts and CV. This allows to determine how the behavior of each strain is affected in the presence of a different species in terms of cultivability, biofilm biomass and individual cell counts. A total of six biofilm experiments (three single-species and three dual-species biofilms - E. coli/S. enterica; E. coli/L. monocytogenes and S. enterica/L. monocytogenes) were performed in triplicate (Figures S2, S3, S4 and S5). Due to the large number of data obtained with the 3 methods, we determined the area under the cultivability, CV and PNA FISH curves by the trapezium rule, as described previously [46](Figure 3.A). Three areas were obtained for each method and biofilm experiment as exemplified in Figures 3B, C and D only for the glass support, from which the final average area was calculated. In order to normalized the areas, each value was divided for the higher value on each data serie, for instance, all CV areas were divided for the higher CV area, which was obtained for E. coli biofilm on glass. Results show that biofilm patterns are quite similar for six of the materials tested, regardless of the analytical method used (P>0,05 in all cases) (Figure 3.A). The exception was copper, which presented an inhibitory effect on biofilm formation for all three species (P<0,05).


Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH).

Almeida C, Azevedo NF, Santos S, Keevil CW, Vieira MJ - PLoS ONE (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3066202&req=5

pone-0014786-g003: 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.
Mentions: Taking advantage of the robustness of the PNA FISH/DAPI method, we next investigated the strains ability to form biofilms in 7 different materials, and complemented the obtained results with two other well-known analytical methods: CFU counts and CV. This allows to determine how the behavior of each strain is affected in the presence of a different species in terms of cultivability, biofilm biomass and individual cell counts. A total of six biofilm experiments (three single-species and three dual-species biofilms - E. coli/S. enterica; E. coli/L. monocytogenes and S. enterica/L. monocytogenes) were performed in triplicate (Figures S2, S3, S4 and S5). Due to the large number of data obtained with the 3 methods, we determined the area under the cultivability, CV and PNA FISH curves by the trapezium rule, as described previously [46](Figure 3.A). Three areas were obtained for each method and biofilm experiment as exemplified in Figures 3B, C and D only for the glass support, from which the final average area was calculated. In order to normalized the areas, each value was divided for the higher value on each data serie, for instance, all CV areas were divided for the higher CV area, which was obtained for E. coli biofilm on glass. Results show that biofilm patterns are quite similar for six of the materials tested, regardless of the analytical method used (P>0,05 in all cases) (Figure 3.A). The exception was copper, which presented an inhibitory effect on biofilm formation for all three species (P<0,05).

Bottom Line: 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.While 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualta, Braga, Portugal.

ABSTRACT

Background: Our 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 findings: We 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/cm(2)) for 48 h biofilm: E. coli 2,1 × 10(8) (± 2,4 × 10(7)); L. monocytogenes 6,8 × 10(7) (± 9,4 × 10(6)); and S. enterica 1,4 × 10(6) (± 4,1 × 10(5))]. 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.

Significance: While 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.

Show MeSH
Related in: MedlinePlus