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Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact

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ABSTRACT

Staphylococcus aureus (Sau) strains are a main cause of disease, including nosocomial infections which have been linked to the production of biofilms and the propagation of antibiotic resistance strains such as methicillin-resistant Staphylococcus aureus (MRSA). A previous study found that Streptococcus pneumoniae (Spn) strains kill planktonic cultures of Sau strains. In this work, we have further evaluated in detail the eradication of Sau biofilms and investigated ultrastructural interactions of the biofilmicidal effect. Spn strain D39, which produces the competence stimulating peptide 1 (CSP1), reduced Sau biofilms within 8 h of inoculation, while TIGR4, producing CSP2, eradicated Sau biofilms and planktonic cells within 4 h. Differences were not attributed to pherotypes as other Spn strains producing different pheromones eradicated Sau within 4 h. Experiments using Transwell devices, which physically separated both species growing in the same well, demonstrated that direct contact between Spn and Sau was required to efficiently eradicate Sau biofilms and biofilm-released planktonic cells. Physical contact-mediated killing of Sau was not related to production of hydrogen peroxide as an isogenic TIGR4ΔspxB mutant eradicated Sau bacteria within 4 h. Confocal micrographs confirmed eradication of Sau biofilms by TIGR4 and allowed us to visualize ultrastructural point of contacts between Sau and Spn. A time-course study further demonstrated spatial colocalization of Spn chains and Sau tetrads as early as 30 min post-inoculation (Pearson's coefficient >0.72). Finally, precolonized biofilms produced by Sau strain Newman, or MRSA strain USA300, were eradicated by mid-log phase cultures of washed TIGR4 bacteria within 2 h post-inoculation. In conclusion, Spn strains rapidly eradicate pre-colonized Sau aureus biofilms, including those formed by MRSA strains, by a mechanism(s) requiring bacterium-bacterium contact, but independent from the production of hydrogen peroxide.

No MeSH data available.


Colocalization between Sau and Spn. Sau and TIGR4 (A–F) or Sau and D39 (G–L) were inoculated together into an eight-well slide and incubated for 1 h (A–C, G–I) or 2 h (D–F, J–L) at 37°C. Biofilms were fixed with 2% PFA and stained with an anti-Sau antibody followed by an Alexa 555-labeled anti-rabbit secondary antibody (red) and then an anti-Spn antibody labeled with Alexa 488 (green). Bacterial DNA was stained by DAPI (blue). Micrographs were taken by confocal microscopy and analyzed using Imaris software. Panels show representative xy optical sections (~0.4 μm each). Bar = 10 μm at right panels and is valid for its corresponding horizontal panels. Vertical panels show specific channels. Arrows point out areas of colocalization between Sau and Spn. (M) Sau colocalized with Spn after 1 h of co-incubation, or free Sau bacteria, were counted in 30 different micrographs. Means were plotted and error bars represent the standard errors. (*), statistical significance (p < 0.001).
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Figure 8: Colocalization between Sau and Spn. Sau and TIGR4 (A–F) or Sau and D39 (G–L) were inoculated together into an eight-well slide and incubated for 1 h (A–C, G–I) or 2 h (D–F, J–L) at 37°C. Biofilms were fixed with 2% PFA and stained with an anti-Sau antibody followed by an Alexa 555-labeled anti-rabbit secondary antibody (red) and then an anti-Spn antibody labeled with Alexa 488 (green). Bacterial DNA was stained by DAPI (blue). Micrographs were taken by confocal microscopy and analyzed using Imaris software. Panels show representative xy optical sections (~0.4 μm each). Bar = 10 μm at right panels and is valid for its corresponding horizontal panels. Vertical panels show specific channels. Arrows point out areas of colocalization between Sau and Spn. (M) Sau colocalized with Spn after 1 h of co-incubation, or free Sau bacteria, were counted in 30 different micrographs. Means were plotted and error bars represent the standard errors. (*), statistical significance (p < 0.001).

Mentions: Experiments showed above suggested that Sau and Spn colocalize; to further confirm physical colocalization, we stained the pneumococcal capsule and Sau capsule by fluorescence, and confocal micrographs were analyzed using the Imaris software. As shown in Figure 8, there was a spatial colocalization between Sau and TIGR4 bacteria as early as 1 h post-inoculation. The Pearson's coefficient (PC) of colocalized volume was 0.78, which statistically confirmed true spatial colocalization. TIGR4 surrounded Sau making contact with individual bacterium and those Sau bacteria forming tetrads (Figures 8A–C). Removing the channel of the Spn capsule (green), or Sau capsule (red), allowed us to better visualize specific points of contact (Figure 8, arrows in Sau+DNA and Spn+DNA). Colocalization between Sau and TIGR4 was also observed at 2 h post-inoculation (PC = 0.72) indicating bacteria remained joint (Figures 8D–F). Further analysis of more than 30 confocal micrographs demonstrated that most Sau bacteria are in contact with Spn (mean = 5.16, median = 4), in comparison to those Sau bacteria observed alone (mean = 1.2, median = 0.0; Figure 8M). Whereas, Spn strain D39 did not eradicate Sau biofilms, D39 bacteria were observed colocalizing with Sau at 1 h (PC = 0.73) or 2 h (PC = 0.89) post-inoculation (Figures 8G–L). In most cases a long chain of Spn made contact with tetrads or aggregates of Sau bacteria.


Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact
Colocalization between Sau and Spn. Sau and TIGR4 (A–F) or Sau and D39 (G–L) were inoculated together into an eight-well slide and incubated for 1 h (A–C, G–I) or 2 h (D–F, J–L) at 37°C. Biofilms were fixed with 2% PFA and stained with an anti-Sau antibody followed by an Alexa 555-labeled anti-rabbit secondary antibody (red) and then an anti-Spn antibody labeled with Alexa 488 (green). Bacterial DNA was stained by DAPI (blue). Micrographs were taken by confocal microscopy and analyzed using Imaris software. Panels show representative xy optical sections (~0.4 μm each). Bar = 10 μm at right panels and is valid for its corresponding horizontal panels. Vertical panels show specific channels. Arrows point out areas of colocalization between Sau and Spn. (M) Sau colocalized with Spn after 1 h of co-incubation, or free Sau bacteria, were counted in 30 different micrographs. Means were plotted and error bars represent the standard errors. (*), statistical significance (p < 0.001).
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Figure 8: Colocalization between Sau and Spn. Sau and TIGR4 (A–F) or Sau and D39 (G–L) were inoculated together into an eight-well slide and incubated for 1 h (A–C, G–I) or 2 h (D–F, J–L) at 37°C. Biofilms were fixed with 2% PFA and stained with an anti-Sau antibody followed by an Alexa 555-labeled anti-rabbit secondary antibody (red) and then an anti-Spn antibody labeled with Alexa 488 (green). Bacterial DNA was stained by DAPI (blue). Micrographs were taken by confocal microscopy and analyzed using Imaris software. Panels show representative xy optical sections (~0.4 μm each). Bar = 10 μm at right panels and is valid for its corresponding horizontal panels. Vertical panels show specific channels. Arrows point out areas of colocalization between Sau and Spn. (M) Sau colocalized with Spn after 1 h of co-incubation, or free Sau bacteria, were counted in 30 different micrographs. Means were plotted and error bars represent the standard errors. (*), statistical significance (p < 0.001).
Mentions: Experiments showed above suggested that Sau and Spn colocalize; to further confirm physical colocalization, we stained the pneumococcal capsule and Sau capsule by fluorescence, and confocal micrographs were analyzed using the Imaris software. As shown in Figure 8, there was a spatial colocalization between Sau and TIGR4 bacteria as early as 1 h post-inoculation. The Pearson's coefficient (PC) of colocalized volume was 0.78, which statistically confirmed true spatial colocalization. TIGR4 surrounded Sau making contact with individual bacterium and those Sau bacteria forming tetrads (Figures 8A–C). Removing the channel of the Spn capsule (green), or Sau capsule (red), allowed us to better visualize specific points of contact (Figure 8, arrows in Sau+DNA and Spn+DNA). Colocalization between Sau and TIGR4 was also observed at 2 h post-inoculation (PC = 0.72) indicating bacteria remained joint (Figures 8D–F). Further analysis of more than 30 confocal micrographs demonstrated that most Sau bacteria are in contact with Spn (mean = 5.16, median = 4), in comparison to those Sau bacteria observed alone (mean = 1.2, median = 0.0; Figure 8M). Whereas, Spn strain D39 did not eradicate Sau biofilms, D39 bacteria were observed colocalizing with Sau at 1 h (PC = 0.73) or 2 h (PC = 0.89) post-inoculation (Figures 8G–L). In most cases a long chain of Spn made contact with tetrads or aggregates of Sau bacteria.

View Article: PubMed Central - PubMed

ABSTRACT

Staphylococcus aureus (Sau) strains are a main cause of disease, including nosocomial infections which have been linked to the production of biofilms and the propagation of antibiotic resistance strains such as methicillin-resistant Staphylococcus aureus (MRSA). A previous study found that Streptococcus pneumoniae (Spn) strains kill planktonic cultures of Sau strains. In this work, we have further evaluated in detail the eradication of Sau biofilms and investigated ultrastructural interactions of the biofilmicidal effect. Spn strain D39, which produces the competence stimulating peptide 1 (CSP1), reduced Sau biofilms within 8 h of inoculation, while TIGR4, producing CSP2, eradicated Sau biofilms and planktonic cells within 4 h. Differences were not attributed to pherotypes as other Spn strains producing different pheromones eradicated Sau within 4 h. Experiments using Transwell devices, which physically separated both species growing in the same well, demonstrated that direct contact between Spn and Sau was required to efficiently eradicate Sau biofilms and biofilm-released planktonic cells. Physical contact-mediated killing of Sau was not related to production of hydrogen peroxide as an isogenic TIGR4&Delta;spxB mutant eradicated Sau bacteria within 4 h. Confocal micrographs confirmed eradication of Sau biofilms by TIGR4 and allowed us to visualize ultrastructural point of contacts between Sau and Spn. A time-course study further demonstrated spatial colocalization of Spn chains and Sau tetrads as early as 30 min post-inoculation (Pearson's coefficient &gt;0.72). Finally, precolonized biofilms produced by Sau strain Newman, or MRSA strain USA300, were eradicated by mid-log phase cultures of washed TIGR4 bacteria within 2 h post-inoculation. In conclusion, Spn strains rapidly eradicate pre-colonized Sau aureus biofilms, including those formed by MRSA strains, by a mechanism(s) requiring bacterium-bacterium contact, but independent from the production of hydrogen peroxide.

No MeSH data available.