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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.


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Spn TIGR4 kills preformed Sau biofilms. Sau was inoculated in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Sau biofilms were left uninoculated (Sau) or co-inoculated with Spn strain D39 (Sau/D39) or TIGR4 (Sau/TIGR4) and incubated for 4 h at 37°C. Biofilms (A) or planktonic cells (B) were harvested, serially diluted and plated onto salt mannitol agar plates to obtain bacterial counts. (C) Dilutions were also plated onto blood agar plates with gentamicin to obtain Spn planktonic and biofilm counts. Error bars represent the standard errors of the means calculated using data from at least three independent experiments; the median (cfu/ml) is shown inside bars. Statistical significance (p < 0.05) in comparison to wells inoculated with Sau (*), Sau/D39 (♦) or D39 (#).
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Figure 9: Spn TIGR4 kills preformed Sau biofilms. Sau was inoculated in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Sau biofilms were left uninoculated (Sau) or co-inoculated with Spn strain D39 (Sau/D39) or TIGR4 (Sau/TIGR4) and incubated for 4 h at 37°C. Biofilms (A) or planktonic cells (B) were harvested, serially diluted and plated onto salt mannitol agar plates to obtain bacterial counts. (C) Dilutions were also plated onto blood agar plates with gentamicin to obtain Spn planktonic and biofilm counts. Error bars represent the standard errors of the means calculated using data from at least three independent experiments; the median (cfu/ml) is shown inside bars. Statistical significance (p < 0.05) in comparison to wells inoculated with Sau (*), Sau/D39 (♦) or D39 (#).

Mentions: We then tested whether pre-colonized Sau biofilms could be eradicated by Spn. To assess this, Sau was incubated for 4 h to form biofilms, after which planktonic cells were removed. Early log-phase cultures of D39, or TIGR4 cells, (~1 × 106 cfu/ml) were inoculated into preformed Sau biofilms and then incubated for an additional 4 h period. As seen in Figure 9, pre-colonized Sau biofilms were significantly reduced by incubating with D39, or TIGR4. Furthermore, reduction of pre-formed Sau biofilms by TIGR4 (~1.5 × 103 cfu/ml) was significantly different than Sau reduction produced by incubating with D39 (~3.2 × 105 cfu/ml).


Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact
Spn TIGR4 kills preformed Sau biofilms. Sau was inoculated in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Sau biofilms were left uninoculated (Sau) or co-inoculated with Spn strain D39 (Sau/D39) or TIGR4 (Sau/TIGR4) and incubated for 4 h at 37°C. Biofilms (A) or planktonic cells (B) were harvested, serially diluted and plated onto salt mannitol agar plates to obtain bacterial counts. (C) Dilutions were also plated onto blood agar plates with gentamicin to obtain Spn planktonic and biofilm counts. Error bars represent the standard errors of the means calculated using data from at least three independent experiments; the median (cfu/ml) is shown inside bars. Statistical significance (p < 0.05) in comparison to wells inoculated with Sau (*), Sau/D39 (♦) or D39 (#).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037180&req=5

Figure 9: Spn TIGR4 kills preformed Sau biofilms. Sau was inoculated in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Sau biofilms were left uninoculated (Sau) or co-inoculated with Spn strain D39 (Sau/D39) or TIGR4 (Sau/TIGR4) and incubated for 4 h at 37°C. Biofilms (A) or planktonic cells (B) were harvested, serially diluted and plated onto salt mannitol agar plates to obtain bacterial counts. (C) Dilutions were also plated onto blood agar plates with gentamicin to obtain Spn planktonic and biofilm counts. Error bars represent the standard errors of the means calculated using data from at least three independent experiments; the median (cfu/ml) is shown inside bars. Statistical significance (p < 0.05) in comparison to wells inoculated with Sau (*), Sau/D39 (♦) or D39 (#).
Mentions: We then tested whether pre-colonized Sau biofilms could be eradicated by Spn. To assess this, Sau was incubated for 4 h to form biofilms, after which planktonic cells were removed. Early log-phase cultures of D39, or TIGR4 cells, (~1 × 106 cfu/ml) were inoculated into preformed Sau biofilms and then incubated for an additional 4 h period. As seen in Figure 9, pre-colonized Sau biofilms were significantly reduced by incubating with D39, or TIGR4. Furthermore, reduction of pre-formed Sau biofilms by TIGR4 (~1.5 × 103 cfu/ml) was significantly different than Sau reduction produced by incubating with D39 (~3.2 × 105 cfu/ml).

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.


Related in: MedlinePlus