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

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.


Confocal studies of Sau coincubated with Spn strains. (A) Sau, (B) SPJV09 (TIGR4), or (C) SPJV01 (D39), or mixtures of Sau and SPJV09 (D–F) or Sau and SPJV01 (G–I) was inoculated into an eight-well slide and incubated for 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). Spn strains were expressing the green fluorescent protein. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm. Gray arrows point out areas where Sau and TIGR4 are located.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037180&req=5

Figure 6: Confocal studies of Sau coincubated with Spn strains. (A) Sau, (B) SPJV09 (TIGR4), or (C) SPJV01 (D39), or mixtures of Sau and SPJV09 (D–F) or Sau and SPJV01 (G–I) was inoculated into an eight-well slide and incubated for 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). Spn strains were expressing the green fluorescent protein. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm. Gray arrows point out areas where Sau and TIGR4 are located.

Mentions: To gain insights on ultrastructural interactions between TIGR4, or D39, and Sau, we obtained confocal micrographs. At 4 h post-inoculation, control Sau biofilms were robust and covered ~90% of the abiotic substrate (Figure 6A) whereas in wells co-incubated with TIGR4, Sau biofilms were eradicated (Figure 6D). The few Sau cells attached to the substratum appeared to be in close proximity to TIGR4 bacteria suggesting physical interaction between the two species (Figure 6F, arrows). TIGR4 biofilms remained similar whether co-incubated with Sau or incubated alone (Figures 6B,E). Biofilms formed by Sau, when co-incubated with D39, were reduced to ~60% in comparison to control wells (Figures 6G,I). D39 biofilms were similarly observed whether incubated alone or with Sau (Figures 6C,H).


Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact
Confocal studies of Sau coincubated with Spn strains. (A) Sau, (B) SPJV09 (TIGR4), or (C) SPJV01 (D39), or mixtures of Sau and SPJV09 (D–F) or Sau and SPJV01 (G–I) was inoculated into an eight-well slide and incubated for 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). Spn strains were expressing the green fluorescent protein. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm. Gray arrows point out areas where Sau and TIGR4 are located.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037180&req=5

Figure 6: Confocal studies of Sau coincubated with Spn strains. (A) Sau, (B) SPJV09 (TIGR4), or (C) SPJV01 (D39), or mixtures of Sau and SPJV09 (D–F) or Sau and SPJV01 (G–I) was inoculated into an eight-well slide and incubated for 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). Spn strains were expressing the green fluorescent protein. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm. Gray arrows point out areas where Sau and TIGR4 are located.
Mentions: To gain insights on ultrastructural interactions between TIGR4, or D39, and Sau, we obtained confocal micrographs. At 4 h post-inoculation, control Sau biofilms were robust and covered ~90% of the abiotic substrate (Figure 6A) whereas in wells co-incubated with TIGR4, Sau biofilms were eradicated (Figure 6D). The few Sau cells attached to the substratum appeared to be in close proximity to TIGR4 bacteria suggesting physical interaction between the two species (Figure 6F, arrows). TIGR4 biofilms remained similar whether co-incubated with Sau or incubated alone (Figures 6B,E). Biofilms formed by Sau, when co-incubated with D39, were reduced to ~60% in comparison to control wells (Figures 6G,I). D39 biofilms were similarly observed whether incubated alone or with Sau (Figures 6C,H).

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.