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Pluronics-Formulated Farnesol Promotes Efficient Killing and Demonstrates Novel Interactions with Streptococcus mutans Biofilms.

Mogen AB, Chen F, Ahn SJ, Burne RA, Wang D, Rice KC - PLoS ONE (2015)

Bottom Line: In each tested S. mutans strain, biomass was significantly decreased (SNK test, p < 0.05) in the P85F and F biofilms relative to untreated biofilms.Parallel CFU/ml determinations revealed that biofilm growth in the presence of P85F resulted in a 3-log reduction in viability, whereas F decreased viability by less than 1-log.Collectively, these results suggest that Pluronics-formulated farnesol induces alterations in biofilm architecture, presumably via interaction with the sucrose-dependent biofilm matrix, and may be a viable treatment option in the prevention and treatment of pathogenic plaque biofilms.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, 32611, United States of America.

ABSTRACT
Streptococcus mutans is the primary causative agent of dental caries, one of the most prevalent diseases in the United States. Previously published studies have shown that Pluronic-based tooth-binding micelles carrying hydrophobic antimicrobials are extremely effective at inhibiting S. mutans biofilm growth on hydroxyapatite (HA). Interestingly, these studies also demonstrated that non-binding micelles (NBM) carrying antimicrobial also had an inhibitory effect, leading to the hypothesis that the Pluronic micelles themselves may interact with the biofilm. To explore this potential interaction, three different S. mutans strains were each grown as biofilm in tissue culture plates, either untreated or supplemented with NBM alone (P85), NBM containing farnesol (P85F), or farnesol alone (F). In each tested S. mutans strain, biomass was significantly decreased (SNK test, p < 0.05) in the P85F and F biofilms relative to untreated biofilms. Furthermore, the P85F biofilms formed large towers containing dead cells that were not observed in the other treatment conditions. Tower formation appeared to be specific to formulated farnesol, as this phenomenon was not observed in S. mutans biofilms grown with NBM containing triclosan. Parallel CFU/ml determinations revealed that biofilm growth in the presence of P85F resulted in a 3-log reduction in viability, whereas F decreased viability by less than 1-log. Wild-type biofilms grown in the absence of sucrose or gtfBC mutant biofilms grown in the presence of sucrose did not form towers. However, increased cell killing with P85F was still observed, suggesting that cell killing is independent of tower formation. Finally, repeated treatment of pre-formed biofilms with P85F was able to elicit a 2-log reduction in viability, whereas parallel treatment with F alone only reduced viability by 0.5-log. Collectively, these results suggest that Pluronics-formulated farnesol induces alterations in biofilm architecture, presumably via interaction with the sucrose-dependent biofilm matrix, and may be a viable treatment option in the prevention and treatment of pathogenic plaque biofilms.

No MeSH data available.


Related in: MedlinePlus

Farnesol-specific tower formation in S. mutans static biofilm.S. mutans UA159 static biofilms were grown in BM media containing 0.25% sucrose and 0.25% glucose, either with 2% P123 alone (A) or in the presence of 2% P123 formulated with farnesol (B) or triclosan (C). After 48 hours of growth, wells containing adherent biofilm were stained with LIVE/DEAD stain, and biofilm z-stacks were acquired at 400× magnification by CLSM. Representative orthogonal images of each biofilm are shown, representing 8–12 random fields of view. Scale bars = 20 μm.
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pone.0133886.g003: Farnesol-specific tower formation in S. mutans static biofilm.S. mutans UA159 static biofilms were grown in BM media containing 0.25% sucrose and 0.25% glucose, either with 2% P123 alone (A) or in the presence of 2% P123 formulated with farnesol (B) or triclosan (C). After 48 hours of growth, wells containing adherent biofilm were stained with LIVE/DEAD stain, and biofilm z-stacks were acquired at 400× magnification by CLSM. Representative orthogonal images of each biofilm are shown, representing 8–12 random fields of view. Scale bars = 20 μm.

Mentions: Although previous studies have demonstrated the effectiveness of antimicrobial-formulated Pluronic micelles in inhibiting growth of S. mutans biofilms [26–28], their contribution to biofilm architecture and physiology has not been investigated. Therefore, CLSM was employed in this study to observe changes in biofilm morphology associated with growth in the presence of farnesol-containing Pluronic micelles. Altered biofilm structure was observed in all three tested S. mutans strains when grown in media containing P85F (Fig 1B, 1F and 1J) or F alone (Fig 1C, 1G and 1K), whereas biofilms grown in the presence of P85 alone (Fig 1A, 1E and 1I) were similar to untreated biofilms (Fig 1D, 1H and 1L). Specifically, growth in the presence of P85F resulted in the formation of large towers containing a centralized area of dead or damaged cells surrounded by live viable bacteria, whereas growth with F alone resulted in a less densely-packed biofilm lacking tower formation, and contained a mixture of live and dead cells throughout the biofilm. S. mutans cells also tended to form longer chains in the F alone-treated biofilms. These qualitative biofilm characteristics were quantified using COMSTAT software, with both P85F and F biofilms of all three S. mutans strains displaying significant decreases in biomass (Fig 2A) and increases in roughness co-efficient (Fig 2B) compared to untreated and NBM biofilms. To determine if tower formation was dependent on the specific Pluronic (P85) and/or antimicrobial (farnesol) used, CLSM experiments were repeated on UA159 biofilms grown in the presence of P123 formulated with farnesol (P123F) as well as P123 formulated with triclosan (P123T). These results showed that tower formation occurred in biofilms grown with P123F (Fig 3B) but not in biofilms grown with P123T (Fig 3C) or P123 alone (Fig 3A), suggesting that tower formation is specific to farnesol-containing Pluronic micelles.


Pluronics-Formulated Farnesol Promotes Efficient Killing and Demonstrates Novel Interactions with Streptococcus mutans Biofilms.

Mogen AB, Chen F, Ahn SJ, Burne RA, Wang D, Rice KC - PLoS ONE (2015)

Farnesol-specific tower formation in S. mutans static biofilm.S. mutans UA159 static biofilms were grown in BM media containing 0.25% sucrose and 0.25% glucose, either with 2% P123 alone (A) or in the presence of 2% P123 formulated with farnesol (B) or triclosan (C). After 48 hours of growth, wells containing adherent biofilm were stained with LIVE/DEAD stain, and biofilm z-stacks were acquired at 400× magnification by CLSM. Representative orthogonal images of each biofilm are shown, representing 8–12 random fields of view. Scale bars = 20 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519314&req=5

pone.0133886.g003: Farnesol-specific tower formation in S. mutans static biofilm.S. mutans UA159 static biofilms were grown in BM media containing 0.25% sucrose and 0.25% glucose, either with 2% P123 alone (A) or in the presence of 2% P123 formulated with farnesol (B) or triclosan (C). After 48 hours of growth, wells containing adherent biofilm were stained with LIVE/DEAD stain, and biofilm z-stacks were acquired at 400× magnification by CLSM. Representative orthogonal images of each biofilm are shown, representing 8–12 random fields of view. Scale bars = 20 μm.
Mentions: Although previous studies have demonstrated the effectiveness of antimicrobial-formulated Pluronic micelles in inhibiting growth of S. mutans biofilms [26–28], their contribution to biofilm architecture and physiology has not been investigated. Therefore, CLSM was employed in this study to observe changes in biofilm morphology associated with growth in the presence of farnesol-containing Pluronic micelles. Altered biofilm structure was observed in all three tested S. mutans strains when grown in media containing P85F (Fig 1B, 1F and 1J) or F alone (Fig 1C, 1G and 1K), whereas biofilms grown in the presence of P85 alone (Fig 1A, 1E and 1I) were similar to untreated biofilms (Fig 1D, 1H and 1L). Specifically, growth in the presence of P85F resulted in the formation of large towers containing a centralized area of dead or damaged cells surrounded by live viable bacteria, whereas growth with F alone resulted in a less densely-packed biofilm lacking tower formation, and contained a mixture of live and dead cells throughout the biofilm. S. mutans cells also tended to form longer chains in the F alone-treated biofilms. These qualitative biofilm characteristics were quantified using COMSTAT software, with both P85F and F biofilms of all three S. mutans strains displaying significant decreases in biomass (Fig 2A) and increases in roughness co-efficient (Fig 2B) compared to untreated and NBM biofilms. To determine if tower formation was dependent on the specific Pluronic (P85) and/or antimicrobial (farnesol) used, CLSM experiments were repeated on UA159 biofilms grown in the presence of P123 formulated with farnesol (P123F) as well as P123 formulated with triclosan (P123T). These results showed that tower formation occurred in biofilms grown with P123F (Fig 3B) but not in biofilms grown with P123T (Fig 3C) or P123 alone (Fig 3A), suggesting that tower formation is specific to farnesol-containing Pluronic micelles.

Bottom Line: In each tested S. mutans strain, biomass was significantly decreased (SNK test, p < 0.05) in the P85F and F biofilms relative to untreated biofilms.Parallel CFU/ml determinations revealed that biofilm growth in the presence of P85F resulted in a 3-log reduction in viability, whereas F decreased viability by less than 1-log.Collectively, these results suggest that Pluronics-formulated farnesol induces alterations in biofilm architecture, presumably via interaction with the sucrose-dependent biofilm matrix, and may be a viable treatment option in the prevention and treatment of pathogenic plaque biofilms.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, 32611, United States of America.

ABSTRACT
Streptococcus mutans is the primary causative agent of dental caries, one of the most prevalent diseases in the United States. Previously published studies have shown that Pluronic-based tooth-binding micelles carrying hydrophobic antimicrobials are extremely effective at inhibiting S. mutans biofilm growth on hydroxyapatite (HA). Interestingly, these studies also demonstrated that non-binding micelles (NBM) carrying antimicrobial also had an inhibitory effect, leading to the hypothesis that the Pluronic micelles themselves may interact with the biofilm. To explore this potential interaction, three different S. mutans strains were each grown as biofilm in tissue culture plates, either untreated or supplemented with NBM alone (P85), NBM containing farnesol (P85F), or farnesol alone (F). In each tested S. mutans strain, biomass was significantly decreased (SNK test, p < 0.05) in the P85F and F biofilms relative to untreated biofilms. Furthermore, the P85F biofilms formed large towers containing dead cells that were not observed in the other treatment conditions. Tower formation appeared to be specific to formulated farnesol, as this phenomenon was not observed in S. mutans biofilms grown with NBM containing triclosan. Parallel CFU/ml determinations revealed that biofilm growth in the presence of P85F resulted in a 3-log reduction in viability, whereas F decreased viability by less than 1-log. Wild-type biofilms grown in the absence of sucrose or gtfBC mutant biofilms grown in the presence of sucrose did not form towers. However, increased cell killing with P85F was still observed, suggesting that cell killing is independent of tower formation. Finally, repeated treatment of pre-formed biofilms with P85F was able to elicit a 2-log reduction in viability, whereas parallel treatment with F alone only reduced viability by 0.5-log. Collectively, these results suggest that Pluronics-formulated farnesol induces alterations in biofilm architecture, presumably via interaction with the sucrose-dependent biofilm matrix, and may be a viable treatment option in the prevention and treatment of pathogenic plaque biofilms.

No MeSH data available.


Related in: MedlinePlus