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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.


Time course study of physical interaction between Sau and Spn strains. Sau (A–D), TIGR4 (E–H), or Sau and TIGR4 (I–P) were inoculated into an eight-well slide and incubated for 1, 2, 3, or 4 h 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. Panels show representative xy optical sections (~0.4 μm each). Bar at the right panel is valid for its corresponding horizontal panels. Panels (I–L) show the red and green channels while panels (M–P) the red and blue channels. Bars = 10 μm, except were indicated (7 μm).
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Figure 7: Time course study of physical interaction between Sau and Spn strains. Sau (A–D), TIGR4 (E–H), or Sau and TIGR4 (I–P) were inoculated into an eight-well slide and incubated for 1, 2, 3, or 4 h 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. Panels show representative xy optical sections (~0.4 μm each). Bar at the right panel is valid for its corresponding horizontal panels. Panels (I–L) show the red and green channels while panels (M–P) the red and blue channels. Bars = 10 μm, except were indicated (7 μm).

Mentions: Given that TIGR4 killed Sau and MRSA strain USA300, a time course study was conducted to evaluate physical interactions in detail. As shown in Figures 7A–D, Sau rapidly formed aggregates, i.e., tetrads, at 1 h post-inoculation, which continued growing until forming a bacterial lawn 4 h later. TIGR4 formed chains that aggregated on the bottom of the well (Figures 7E–H) but did not produce, at this time-point, the robust bacterial lawn observed with Sau. When incubated with TIGR4, Sau biofilms were not produced. The few bacteria attached to the bottom were surrounded by TIGR4 (Figures 7I–L).


Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact
Time course study of physical interaction between Sau and Spn strains. Sau (A–D), TIGR4 (E–H), or Sau and TIGR4 (I–P) were inoculated into an eight-well slide and incubated for 1, 2, 3, or 4 h 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. Panels show representative xy optical sections (~0.4 μm each). Bar at the right panel is valid for its corresponding horizontal panels. Panels (I–L) show the red and green channels while panels (M–P) the red and blue channels. Bars = 10 μm, except were indicated (7 μm).
© Copyright Policy
Related In: Results  -  Collection

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Figure 7: Time course study of physical interaction between Sau and Spn strains. Sau (A–D), TIGR4 (E–H), or Sau and TIGR4 (I–P) were inoculated into an eight-well slide and incubated for 1, 2, 3, or 4 h 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. Panels show representative xy optical sections (~0.4 μm each). Bar at the right panel is valid for its corresponding horizontal panels. Panels (I–L) show the red and green channels while panels (M–P) the red and blue channels. Bars = 10 μm, except were indicated (7 μm).
Mentions: Given that TIGR4 killed Sau and MRSA strain USA300, a time course study was conducted to evaluate physical interactions in detail. As shown in Figures 7A–D, Sau rapidly formed aggregates, i.e., tetrads, at 1 h post-inoculation, which continued growing until forming a bacterial lawn 4 h later. TIGR4 formed chains that aggregated on the bottom of the well (Figures 7E–H) but did not produce, at this time-point, the robust bacterial lawn observed with Sau. When incubated with TIGR4, Sau biofilms were not produced. The few bacteria attached to the bottom were surrounded by TIGR4 (Figures 7I–L).

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 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.