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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.


Related in: MedlinePlus

TIGR4ΔspxB mutant eradicate Sau bacteria. Sau Newman strain was inoculated alone or with TIGR4ΔspxB and incubated for 4 h at 37°C. Planktonic cells or biofilms were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau counts (A) or blood agar plates with gentamicin to obtain TIGR4ΔspxB counts (B). 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 only with Sau.
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Figure 4: TIGR4ΔspxB mutant eradicate Sau bacteria. Sau Newman strain was inoculated alone or with TIGR4ΔspxB and incubated for 4 h at 37°C. Planktonic cells or biofilms were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau counts (A) or blood agar plates with gentamicin to obtain TIGR4ΔspxB counts (B). 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 only with Sau.

Mentions: Interference of planktonic cultures of Sau by Spn has been demonstrated to occur via hydrogen peroxide, a byproduct of the enzyme SpxB (Regev-Yochay et al., 2006). To investigate whether the observed physical contact-mediated killing requires hydrogen peroxide, we conducted experiments with an isogenic TIGR4ΔspxB mutant, which does not produce detectable levels of hydrogen peroxide (Regev-Yochay et al., 2006). As shown in Figure 4A, the hydrogen peroxide TIGR4ΔspxB mutant was able to eradicate Sau Newman strain within 4 h of incubation. The population of the isogenic mutant was not affected by co-incubation with Sau (Figure 4B).


Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact
TIGR4ΔspxB mutant eradicate Sau bacteria. Sau Newman strain was inoculated alone or with TIGR4ΔspxB and incubated for 4 h at 37°C. Planktonic cells or biofilms were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau counts (A) or blood agar plates with gentamicin to obtain TIGR4ΔspxB counts (B). 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 only with Sau.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: TIGR4ΔspxB mutant eradicate Sau bacteria. Sau Newman strain was inoculated alone or with TIGR4ΔspxB and incubated for 4 h at 37°C. Planktonic cells or biofilms were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau counts (A) or blood agar plates with gentamicin to obtain TIGR4ΔspxB counts (B). 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 only with Sau.
Mentions: Interference of planktonic cultures of Sau by Spn has been demonstrated to occur via hydrogen peroxide, a byproduct of the enzyme SpxB (Regev-Yochay et al., 2006). To investigate whether the observed physical contact-mediated killing requires hydrogen peroxide, we conducted experiments with an isogenic TIGR4ΔspxB mutant, which does not produce detectable levels of hydrogen peroxide (Regev-Yochay et al., 2006). As shown in Figure 4A, the hydrogen peroxide TIGR4ΔspxB mutant was able to eradicate Sau Newman strain within 4 h of incubation. The population of the isogenic mutant was not affected by co-incubation with Sau (Figure 4B).

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