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Experimental Therapeutics

Triclosan Is an Aminoglycoside Adjuvant for Eradication of Pseudomonas aeruginosa Biofilms

Michael M. Maiden, Alessandra M. Agostinho Hunt, Mitchell P. Zachos, Jacob A. Gibson, Martin E. Hurwitz, Martha H. Mulks, Christopher M. Waters
Michael M. Maiden
aDepartments of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
cThe BEACON Center for The Study of Evolution in Action, Michigan State University, East Lansing, Michigan, USA
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Alessandra M. Agostinho Hunt
aDepartments of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
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Mitchell P. Zachos
aDepartments of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
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Jacob A. Gibson
aDepartments of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
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Martin E. Hurwitz
bDepartment of Pediatrics and Human Development, Michigan State University, East Lansing, Michigan, USA
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Martha H. Mulks
aDepartments of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
cThe BEACON Center for The Study of Evolution in Action, Michigan State University, East Lansing, Michigan, USA
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Christopher M. Waters
aDepartments of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
cThe BEACON Center for The Study of Evolution in Action, Michigan State University, East Lansing, Michigan, USA
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DOI: 10.1128/AAC.00146-18
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  • FIG 1
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    FIG 1

    Molecular structure of triclosan and type II fatty acid synthesis. (A) Chemical structure of triclosan, 2,4,4′-trichloro-2′-hydroxydiphenyl ether. (B) Type II fatty acid synthesis (FASII) pathway. Triclosan is known to disrupt FASII by inhibiting FabI. However, FabV is more resistant to triclosan. ACP, acyl carrier protein; CoA, coenzyme A.

  • FIG 2
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    FIG 2

    Triclosan enhances aminoglycoside killing of 24-h-old biofilms. Twenty-four-hour-old biofilms grown on minimum biofilm eradication concentration (MBEC) plates were treated for 6 h with 100 μM triclosan, 500 μM tobramycin, 100 μM gentamicin, or streptomycin, alone and in combination, and the number of viable cells within the biofilms was quantified using the BacTiter-Glo assay. The assay was performed at least three times in triplicate. The results represent the means ± the standard error of the mean (SEM). A one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison post hoc test was used to determine statistical significance compared to each aminoglycoside alone (*, P < 0.05).

  • FIG 3
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    FIG 3

    Triclosan enhances tobramycin killing at multiple concentrations. Twenty-four-hour-old biofilms grown on MBEC plates were treated for 6 h with 2-fold dilutions of equal concentrations of triclosan, tobramycin, and triclosan combined with tobramycin, and the number of viable cells within the biofilms was quantified using the BacTiter-Glo assay. The assay was performed at least three times in triplicate. The results represent the means ± the SEM.

  • FIG 4
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    FIG 4

    Triclosan enhances low concentrations of tobramycin. Twenty-four-hour-old biofilms grown on MBEC plates were treated for 6 h with checkerboard dilutions of triclosan combined with tobramycin. Numbers of viable cells within the biofilms were quantified using the BacTiter-Glo assay. The assay was performed at least three times in triplicate, and the mean is shown.

  • FIG 5
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    FIG 5

    Triclosan enhances the onset and maximum efficacy of tobramycin. Twenty-four-hour-old biofilms grown on MBEC plates were treated with triclosan (100 μM) tobramycin (500 μM), or a combination of the two. At 0, 2, 4, 6, and 8 h, the numbers of viable cells within the biofilms were determined using the BacTiter-Glo assay. The assay was performed at least three times in triplicate. The results represent means ± the SEM.

  • FIG 6
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    FIG 6

    Tobramycin and triclosan are effective against P. aeruginosa CF isolates. Twenty-four-hour-old biofilms grown on MBEC plates were treated with triclosan (100 μM,) tobramycin (500 μM), or a combination of the two for 6 h. The numbers of viable cells within the biofilms were quantified using the BacTiter-Glo assay. The assay was performed at least three times in triplicate. The results represent means ± the SEM. A one-way ANOVA followed by Bonferroni's multiple comparison post hoc test was used to determine statistical significance compared to tobramycin alone (*, P < 0.05). NS, not significant.

  • FIG 7
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    FIG 7

    Aminoglycosides combined with triclosan do not increase biofilm dispersal. (A) Twenty-four-hour-old biofilms grown on MBEC plates were treated with triclosan (100 μM), tobramycin (500 μM), gentamicin (100 μM), or streptomycin (100 μM), alone and in combination. The effect on biofilm biomass was quantified by staining with crystal violet. The experiment was performed at least five times in triplicate. The results represent the means ± SEM. A one-way ANOVA followed by Dunnett's multiple-comparison post hoc test was used to determine statistical significance compared to no treatment (*, P < 0.05). NS, not significant. (B) Twenty-four-hour-old biofilms grown in flow cells were treated with triclosan (100 μM), tobramycin (524 μM), or the combination for 6 h. Live cells are stained green, and dead cells are stained red. Representative images are shown: no treatment (top left quadrant), tobramycin alone (top right quadrant), triclosan alone (lower left quadrant), and a combination (lower right quadrant). Insets are shown for the live channel (A2, B2, C2, and D2) and for the dead channel (A3, B3, C3, and D3) for each condition.

  • FIG 8
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    FIG 8

    FabI inhibition by triclosan is not responsible for the synergy. (A) Twenty-four-hour-old biofilms grown on MBEC plates by a FabI P. aeruginosa-deficient strain (Tn::fabI mutant) were treated for 6 h with triclosan (100 μM), tobramycin (500 μM), gentamicin (100 μM), or streptomycin (100 μM), alone and in combination. (B) Twenty-four-hour-old biofilms grown on MBEC plates by PAO1 were treated for 6 h with triclocarban (100 μM) or tobramycin (500 μM), alone or in combination. The number of viable cells within the biofilms were quantified using the BacTiter-Glo assay. The assay was performed at least three times in triplicate. The results represent means ± the SEM. A one-way ANOVA followed by Bonferroni's multiple-comparison post hoc test was used to determine statistical significance compared to tobramycin alone (*, P < 0.05). NS, not significant.

  • FIG 9
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    FIG 9

    Tobramycin combined with triclosan kills persister cells. Twenty-hour-old stationary-phase cells were treated with triclosan (100 μM) or tobramycin (50 μM), alone and in combination, for 6 h. At 0, 2, 4, 6, 8, and 24 h, aliquots were taken for enumeration (CFU per milliliter). The experiment was performed three times in triplicate. The results represent means ± the SEM.

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  • TABLE 1

    EC50 values for aminoglycoside combinationsa

    AntibioticAdjuvantEC50 (μM)95% confidence interval (μM)
    TobramycinTriclosan20.507.65–83.78
    GentamicinTriclosan23.0612.08–44.02
    StreptomycinTriclosan28.9624.45–34.31
    • ↵a EC50 values were calculated using Prism version 5. Log10 (inhibitor) versus response–variable slope (four parameters) analyses were performed. EC50 values for aminoglycoside alone were not constructed, because no curve was established due to their ineffectiveness against biofilms.

  • TABLE 2

    Bacterial strains used in this study

    StrainCharacteristicsaSource or referenceb
    PAO1Standard reference strain, isolated in 1954 (64)Martha Mulks
    Tn::fabIISlacZ/hah38
    AMT0023_30Early isolate, 6 mo65
    AMT0023_34Late isolate, 8 yr65
    CF_115_JP. aeruginosa clinical CF isolate, MichiganMartha Mulks
    CF_110_NP. aeruginosa clinical CF isolate, MichiganMartha Mulks
    CF_110_OP. aeruginosa clinical CF isolate, MichiganMartha Mulks
    CF_131_MP. aeruginosa clinical CF isolate, MichiganMartha Mulks
    CF_300_AP. aeruginosa clinical CF isolate, MichiganMartha Mulks
    AU1054BCC clinical CF isolate, USAJ. J. LiPuma
    PC184BCC clinical CF isolate, Cleveland OhioJ. J. LiPuma
    AU2289BCC clinical CF isolate, MichiganJ. J. LiPuma
    H12424Soil, onion field, New YorkJ. J. LiPuma
    J2315BCC clinical CF isolate, Edinburgh, UKJ. J. LiPuma
    USA_300_JE2MRSA, wound, California61
    COLMRSA, Colindale Hospital, England61
    Newman (25904)MSSA, wound, endocarditisNeal Hammer ATCC
    Wichita (29213)MSSA, better biofilm formerATCC
    • ↵a BCC, Burkholderia cenocepacia complex; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive Staphylococcus aureus; ATCC, American Tissue Type Collection.

    • ↵b J. J. LiPuma, U.S. Burkholderia cenocepacia Research Laboratory and Repository, UM, Ann Arbor, MI.

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      Supplemental Figures S1 to S8

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Triclosan Is an Aminoglycoside Adjuvant for Eradication of Pseudomonas aeruginosa Biofilms
Michael M. Maiden, Alessandra M. Agostinho Hunt, Mitchell P. Zachos, Jacob A. Gibson, Martin E. Hurwitz, Martha H. Mulks, Christopher M. Waters
Antimicrobial Agents and Chemotherapy May 2018, 62 (6) e00146-18; DOI: 10.1128/AAC.00146-18

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Triclosan Is an Aminoglycoside Adjuvant for Eradication of Pseudomonas aeruginosa Biofilms
Michael M. Maiden, Alessandra M. Agostinho Hunt, Mitchell P. Zachos, Jacob A. Gibson, Martin E. Hurwitz, Martha H. Mulks, Christopher M. Waters
Antimicrobial Agents and Chemotherapy May 2018, 62 (6) e00146-18; DOI: 10.1128/AAC.00146-18
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KEYWORDS

Pseudomonas aeruginosa
biofilm
persister
tobramycin
triclosan

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