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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.


Washed TIGR4 bacteria rapidly kill preformed Sau biofilms. (A) Sau was inoculated (Sau) in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Another set of wells were inoculated with TIGR4 and incubated for 4 h at 37°C. Planktonic cells, biofilms, or supernatants from this TIGR4 4 h culture were separated as specified in Material and Methods. Preformed Sau biofilms were left uninoculated (Sau), or inoculated with ~1 × 106 cfu/ml of an early-log phase culture of planktonic TIGR4 cells (+Spn), or 4 h cultures of washed bacteria (+Plank/Bio), washed planktonic bacteria (+Plank), washed biofilms (+Bios) or supernatant (+Sup) and incubated for 2 h at 37°C. Cultures were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau (cfu/ml). Error bars represent the standard errors of the means calculated using data from at least three independent experiments. Statistical significance in comparison to wells inoculated with (*, p < 0.004) Sau or (♦, p < 0.001) +Plank/Bio. (B–E) Sau was inoculated into an eight-well slide and incubated for 4 h at 37°C. Sau Biofilms were challenged with 4 h cultures of washed TIGR4 bacteria and incubated for 30 min (B), 1 h (C), 1.5 h (D), and 2 h (E). At the end of incubation, 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). DNA was stained with DAPI. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm.
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Figure 10: Washed TIGR4 bacteria rapidly kill preformed Sau biofilms. (A) Sau was inoculated (Sau) in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Another set of wells were inoculated with TIGR4 and incubated for 4 h at 37°C. Planktonic cells, biofilms, or supernatants from this TIGR4 4 h culture were separated as specified in Material and Methods. Preformed Sau biofilms were left uninoculated (Sau), or inoculated with ~1 × 106 cfu/ml of an early-log phase culture of planktonic TIGR4 cells (+Spn), or 4 h cultures of washed bacteria (+Plank/Bio), washed planktonic bacteria (+Plank), washed biofilms (+Bios) or supernatant (+Sup) and incubated for 2 h at 37°C. Cultures were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau (cfu/ml). Error bars represent the standard errors of the means calculated using data from at least three independent experiments. Statistical significance in comparison to wells inoculated with (*, p < 0.004) Sau or (♦, p < 0.001) +Plank/Bio. (B–E) Sau was inoculated into an eight-well slide and incubated for 4 h at 37°C. Sau Biofilms were challenged with 4 h cultures of washed TIGR4 bacteria and incubated for 30 min (B), 1 h (C), 1.5 h (D), and 2 h (E). At the end of incubation, 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). DNA was stained with DAPI. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm.

Mentions: Our next experiments fractionated TIGR4 cultures into planktonic cells, biofilms and supernatants and evaluated killing of Sau by these fractions. Since inoculating TIGR4 with Sau at the same time eradicated Sau biofilms in 4 h, cultures of TIGR4 were grown for 4 h and then planktonic cells, biofilms and culture supernatant were separated and incubated with preformed Sau biofilms. We hypothesized that Spn from 4 h cultures (i.e., activated cultures) would kill Sau biofilms faster and therefore preformed biofilms were incubated for 2 h. As expected, inoculating preformed Sau biofilms with early log-phase TIGR4 cultures reduced, but did not eradicate, preformed biofilms within 2 h (Figure 10A). However, washed Spn (planktonic+biofilms), planktonic, or biofilms harvested from 4 h cultures eradicated Sau biofilms (Figure 10A). Sterile supernatant from this 4 h culture was only able to reduce Sau biofilms (5.6 × 103 cfu/ml) in comparison with the non-inoculated control (1.1 × 106 cfu/ml).


Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact
Washed TIGR4 bacteria rapidly kill preformed Sau biofilms. (A) Sau was inoculated (Sau) in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Another set of wells were inoculated with TIGR4 and incubated for 4 h at 37°C. Planktonic cells, biofilms, or supernatants from this TIGR4 4 h culture were separated as specified in Material and Methods. Preformed Sau biofilms were left uninoculated (Sau), or inoculated with ~1 × 106 cfu/ml of an early-log phase culture of planktonic TIGR4 cells (+Spn), or 4 h cultures of washed bacteria (+Plank/Bio), washed planktonic bacteria (+Plank), washed biofilms (+Bios) or supernatant (+Sup) and incubated for 2 h at 37°C. Cultures were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau (cfu/ml). Error bars represent the standard errors of the means calculated using data from at least three independent experiments. Statistical significance in comparison to wells inoculated with (*, p < 0.004) Sau or (♦, p < 0.001) +Plank/Bio. (B–E) Sau was inoculated into an eight-well slide and incubated for 4 h at 37°C. Sau Biofilms were challenged with 4 h cultures of washed TIGR4 bacteria and incubated for 30 min (B), 1 h (C), 1.5 h (D), and 2 h (E). At the end of incubation, 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). DNA was stained with DAPI. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm.
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Related In: Results  -  Collection

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Figure 10: Washed TIGR4 bacteria rapidly kill preformed Sau biofilms. (A) Sau was inoculated (Sau) in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Another set of wells were inoculated with TIGR4 and incubated for 4 h at 37°C. Planktonic cells, biofilms, or supernatants from this TIGR4 4 h culture were separated as specified in Material and Methods. Preformed Sau biofilms were left uninoculated (Sau), or inoculated with ~1 × 106 cfu/ml of an early-log phase culture of planktonic TIGR4 cells (+Spn), or 4 h cultures of washed bacteria (+Plank/Bio), washed planktonic bacteria (+Plank), washed biofilms (+Bios) or supernatant (+Sup) and incubated for 2 h at 37°C. Cultures were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau (cfu/ml). Error bars represent the standard errors of the means calculated using data from at least three independent experiments. Statistical significance in comparison to wells inoculated with (*, p < 0.004) Sau or (♦, p < 0.001) +Plank/Bio. (B–E) Sau was inoculated into an eight-well slide and incubated for 4 h at 37°C. Sau Biofilms were challenged with 4 h cultures of washed TIGR4 bacteria and incubated for 30 min (B), 1 h (C), 1.5 h (D), and 2 h (E). At the end of incubation, 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). DNA was stained with DAPI. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm.
Mentions: Our next experiments fractionated TIGR4 cultures into planktonic cells, biofilms and supernatants and evaluated killing of Sau by these fractions. Since inoculating TIGR4 with Sau at the same time eradicated Sau biofilms in 4 h, cultures of TIGR4 were grown for 4 h and then planktonic cells, biofilms and culture supernatant were separated and incubated with preformed Sau biofilms. We hypothesized that Spn from 4 h cultures (i.e., activated cultures) would kill Sau biofilms faster and therefore preformed biofilms were incubated for 2 h. As expected, inoculating preformed Sau biofilms with early log-phase TIGR4 cultures reduced, but did not eradicate, preformed biofilms within 2 h (Figure 10A). However, washed Spn (planktonic+biofilms), planktonic, or biofilms harvested from 4 h cultures eradicated Sau biofilms (Figure 10A). Sterile supernatant from this 4 h culture was only able to reduce Sau biofilms (5.6 × 103 cfu/ml) in comparison with the non-inoculated control (1.1 × 106 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.