PMC

P. aeruginosa supernatant increases S. aureus biofilm sensitivity to chloroxylenol. Biofilm disruption assays on plastic were performed with the specified S. aureus clinical isolate, P. aeruginosa PA14 supernatant (Pa sup), and chloroxylenol at 100 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. Each column displays the average from two biological replicates, each with three technical replicates. Error bars indicate standard deviation (SD). Sa, S. aureus; bd, below detection; ns, not significant; **, P < 0.01; ***, P < 0.001, by ordinary one-way ANOVA and Bonferroni’s multiple-comparison posttest.

Exogenous HQNO increases S. aureus membrane fluidity. (A to C) Laurdan generalized polarization (GP) was performed with S. aureus (Sa) Newman, benzyl alcohol (BnOH) (A and B), HQNO (B), and the DMSO control (solvent for HQNO) (B) at the indicated concentrations and antimycin A at 100 μg/ml along with the ethanol (EtOH) control (solvent for antimycin A) (C). S. aureus was exposed to the above treatments for 1 h, and GP values were determined. Each column displays the average from at least three biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; ***, P < 0.001, by ordinary one-way ANOVA and Tukey’s multiple-comparison posttest.

Shifting membrane fluidity alters S. aureus biofilm sensitivity to chloroxylenol. (A to C) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, chloroxylenol (Chlor) at 100 μg/ml, benzyl alcohol (BnOH) at 50 mM (A), 1-heptanol at 50 mM (B), and dimethyl sulfoxide (DMSO) at 1% and 6% (C). Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. Each column displays the average from at least three biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; **, P < 0.01; ***, P < 0.001, by ordinary one-way ANOVA and Tukey’s multiple-comparison posttest.

P. aeruginosa supernatant increases S. aureus biofilm sensitivity to other membrane-targeting compounds. (A to E) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman; supernatants from wild-type P. aeruginosa PA14 and the specified mutants (Pa sup); and either biphenyl at 200 μg/ml (A), gramicidin at 100 μg/ml (B), trifluoperazine at 100 μg/ml (C), amitriptyline at 100 μg/ml (D), or octenidine dihydrochloride (Oct) at 5 μg/ml (E). Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. Each column displays the average from at least three biological replicates, each with three technical replicates. Error bars indicate standard deviation (SD). ns, not significant; *, P < 0.05; ***, P < 0.001, by ordinary one-way ANOVA and Tukey’s multiple-comparison posttest.

P. aeruginosa supernatant enhances the ability of chloroxylenol to kill difficult-to-treat S. aureus biofilms. (A) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, P. aeruginosa PA14 supernatant (Pa sup), and chloroxylenol (Chlor) at 100 μg/ml under normoxic or anoxic conditions. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. (B) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Col parental strain or hemB mutant, supernatants from wild-type P. aeruginosa PA14 and the ΔpqsL ΔpvdA ΔpchE mutant (Pa ΔΔΔ sup), and chloroxylenol (Chlor) at 100 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. (C) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, supernatants from wild-type P. aeruginosa PA14 and the ΔpqsL ΔpvdA ΔpchE mutant (Pa ΔΔΔ sup), and chloroxylenol (Chlor) at 100 μg/ml. Biofilms were grown for 24 h and exposed to the above treatments for 24 additional hours, and S. aureus biofilm CFU were determined. Each column displays the average from three biological replicates, each with three technical replicates. Error bars indicate standard deviations. bd, below detection; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, by ordinary one-way ANOVA and Tukey’s multiple-comparison posttest.

The P. aeruginosa exoproducts HQNO and siderophores increase S. aureus biofilm and planktonic sensitivity to chloroxylenol. (A and B) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, supernatants from wild-type P. aeruginosa PA14 and the ΔpqsL ΔpvdA ΔpchE deletion mutant (Pa ΔΔΔ sup), and chloroxylenol (Chlor) at 100 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm (A) and planktonic (B) CFU were determined. Data in panels A and B were from the same experiments. (C) Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, chloroxylenol (Chlor) at 100 μg/ml, and the specified concentrations of HQNO (dissolved in DMSO). Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU were determined. Each column displays the average from at least three biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; ***, P < 0.001, by ordinary one-way ANOVA and Tukey’s multiple-comparison posttest.