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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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Related in: MedlinePlus

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.
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pone-0014786-g001: 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.

Mentions: The hybridization conditions were optimized for the target microorganism and the probes specificity was evaluated on pure cultures. For this, a mix of the two probes was prepared and applied to E. coli, S. enterica and L. monocytogenes pure culture smears. After the PNA FISH hybridization the samples were also stained with DAPI. Microscopic visualization showed that both PNA probes provided a strong fluorescent signal at 57°C, and no cross-hybridization was observed between the probes (Figure 1, rows 1 to 3). Next, the method was tested with a smear of the three species mixed together and results showed that it provided an accurate discrimination between the three species involved (Figure 1, fourth and fifth row). A red background on the green channel is slightly visible due to a small overlapping between the Alexa 594 absorption spectrum and the excitation filter (band pass [BP] from 470 to 490) used to visualize the LmPNA1253 probe. As such, and because the green barrier filters is a long pass filter (LP 516), the Alexa 594 fluorescence emission is still detected.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0014786-g001: 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.
Mentions: The hybridization conditions were optimized for the target microorganism and the probes specificity was evaluated on pure cultures. For this, a mix of the two probes was prepared and applied to E. coli, S. enterica and L. monocytogenes pure culture smears. After the PNA FISH hybridization the samples were also stained with DAPI. Microscopic visualization showed that both PNA probes provided a strong fluorescent signal at 57°C, and no cross-hybridization was observed between the probes (Figure 1, rows 1 to 3). Next, the method was tested with a smear of the three species mixed together and results showed that it provided an accurate discrimination between the three species involved (Figure 1, fourth and fifth row). A red background on the green channel is slightly visible due to a small overlapping between the Alexa 594 absorption spectrum and the excitation filter (band pass [BP] from 470 to 490) used to visualize the LmPNA1253 probe. As such, and because the green barrier filters is a long pass filter (LP 516), the Alexa 594 fluorescence emission is still detected.

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