Article Figures & Data
Figures
- 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
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
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
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
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
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
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
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
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.
Tables
- TABLE 1
EC50 values for aminoglycoside combinationsa
Antibiotic Adjuvant EC50 (μM) 95% confidence interval (μM) Tobramycin Triclosan 20.50 7.65–83.78 Gentamicin Triclosan 23.06 12.08–44.02 Streptomycin Triclosan 28.96 24.45–34.31 ↵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
Strain Characteristicsa Source or referenceb PAO1 Standard reference strain, isolated in 1954 (64) Martha Mulks Tn::fabI ISlacZ/hah 38 AMT0023_30 Early isolate, 6 mo 65 AMT0023_34 Late isolate, 8 yr 65 CF_115_J P. aeruginosa clinical CF isolate, Michigan Martha Mulks CF_110_N P. aeruginosa clinical CF isolate, Michigan Martha Mulks CF_110_O P. aeruginosa clinical CF isolate, Michigan Martha Mulks CF_131_M P. aeruginosa clinical CF isolate, Michigan Martha Mulks CF_300_A P. aeruginosa clinical CF isolate, Michigan Martha Mulks AU1054 BCC clinical CF isolate, USA J. J. LiPuma PC184 BCC clinical CF isolate, Cleveland Ohio J. J. LiPuma AU2289 BCC clinical CF isolate, Michigan J. J. LiPuma H12424 Soil, onion field, New York J. J. LiPuma J2315 BCC clinical CF isolate, Edinburgh, UK J. J. LiPuma USA_300_JE2 MRSA, wound, California 61 COL MRSA, Colindale Hospital, England 61 Newman (25904) MSSA, wound, endocarditis Neal Hammer ATCC Wichita (29213) MSSA, better biofilm former ATCC
Supplemental material
- Supplemental file 1 -
Supplemental Figures S1 to S8
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