Pseudomonas aeruginosa PA14 Enhances the Efficacy of Norfloxacin against Staphylococcus aureus Newman Biofilms

Abstract

The thick mucus within the airways of individuals with cystic fibrosis (CF) promotes frequent respiratory infections that are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent pathogens that cause CF pulmonary infections, and both are among the most common etiologic agents of chronic wound infections. Furthermore, the ability of P. aeruginosa and S. aureus to form biofilms promotes the establishment of chronic infections that are often difficult to eradicate using antimicrobial agents. In this study, we found that multiple LasR-regulated exoproducts of P. aeruginosa, including 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), siderophores, phenazines, and rhamnolipids, likely contribute to the ability of P. aeruginosa PA14 to shift S. aureus Newman norfloxacin susceptibility profiles. Here, we observe that exposure to P. aeruginosa exoproducts leads to an increase in intracellular norfloxacin accumulation by S. aureus We previously showed that P. aeruginosa supernatant dissipates the S. aureus membrane potential, and furthermore, depletion of the S. aureus proton motive force recapitulates the effect of the P. aeruginosa PA14 supernatant on shifting norfloxacin sensitivity profiles of biofilm-grown S. aureus Newman. From these results, we hypothesize that exposure to P. aeruginosa PA14 exoproducts leads to increased uptake of the drug and/or an impaired ability of S. aureus Newman to efflux norfloxacin. Surprisingly, the effect observed here of P. aeruginosa PA14 exoproducts on S. aureus Newman susceptibility to norfloxacin seemed to be specific to these strains and this antibiotic. Our results illustrate that microbially derived products can alter the ability of antimicrobial agents to kill bacterial biofilms.IMPORTANCEPseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from multiple infection sites, including the lungs of individuals with cystic fibrosis (CF) and nonhealing diabetic foot ulcers. Coinfection with P. aeruginosa and S. aureus has been shown to produce worse outcomes compared to infection with either organism alone. Furthermore, the ability of these pathogens to form biofilms enables them to cause persistent infection and withstand antimicrobial therapy. In this study, we found that P. aeruginosa-secreted products dramatically increase the ability of the antibiotic norfloxacin to kill S. aureus biofilms. Understanding how interspecies interactions alter the antibiotic susceptibility of bacterial biofilms may inform treatment decisions and inspire the development of new therapeutic strategies.

Keywords: Pseudomonas aeruginosa; Staphylococcus aureus; antibiotics; biofilm; fluoroquinolone.

FIG 1

P. aeruginosa PA14 increases the susceptibility of early and mature S. aureus Newman biofilms to norfloxacin. (A and B) Biofilm disruption assays on plastic were performed with S. aureus Newman, P. aeruginosa PA14 supernatant (Pa sup), and norfloxacin (Norflox) at 12.5 μg/ml (A) or 25 μg/ml (B). Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and then S. aureus biofilm CFU values were determined. (C) Biofilm disruption assays on plastic were performed with S. aureus Newman, P. aeruginosa PA14 supernatant (Pa sup), and norfloxacin (Norflox) at 25 μg/ml. Biofilms were grown for 24 h and exposed to the above treatments for 18 h, and then S. aureus biofilm CFU values were determined. Each column displays the average from at least six biological replicates, each with three technical replicates. Error bars indicate standard deviation (SD). ns, not significant; *, P < 0.05; ***, P < 0.001; values were determined by ordinary one-way analysis of variance (ANOVA) and Tukey’s multiple comparison posttest.

FIG 2

Testing the contribution of P. aeruginosa-secreted products to the ability of P. aeruginosa PA14 to shift S. aureus Newman biofilm sensitivity to norfloxacin. Biofilm disruption assays on plastic were performed with S. aureus Newman, wild-type P. aeruginosa PA14 and the specified deletion mutant supernatants (Pa sup), and norfloxacin (Norflox) at 25 μg/ml (A, B) or 12.5 μg/ml (C). Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU values were determined. Each column displays the average from at least seven biological replicates (A) or the average from two biological replicates (B, C), each with three technical replicates. Error bars indicate SD. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; values were determined by ordinary one-way ANOVA and Tukey’s multiple comparison posttest.

FIG 3

Addition of exogenous HQNO or antimycin A does not alter S. aureus Newman biofilm sensitivity to norfloxacin. Biofilm disruption assays on plastic were performed with S. aureus (Sa) Newman, HQNO at 100 μg/ml, antimycin A at 100 μg/ml, and norfloxacin (Norflox) at 12.5 μg/ml (A) or 25 μg/ml (B). Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU values were determined. Each column displays the average from at least two biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; values were determined by ordinary one-way ANOVA and Tukey’s multiple comparison posttest.

FIG 4

Testing the effect of changing membrane fluidity on the efficacy of norfloxacin against S. aureus Newman biofilms. (A and B) Biofilm disruption assays on plastic were performed with S. aureus Newman, norfloxacin (Norflox) at 25 μg/ml and benzyl alcohol (BnOH) at 50 mM (A) or dimethyl sulfoxide (DMSO) at 1% and 6% (B). Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU values were determined. Each column displays the average from at least three biological replicates, each with three technical replicates. (C) Biofilm disruption assays on plastic were performed with S. aureus Newman, norfloxacin (Norflox) at 12.5 μg/ml, and the specified fatty acids at 100 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU values were determined. Each column displays the average from two biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; *, P < 0.05; values were determined by ordinary one-way ANOVA and Tukey’s multiple comparison posttest.

FIG 5

Exposure to P. aeruginosa PA14 exoproducts increases S. aureus Newman intracellular norfloxacin levels. Antibiotic internalization assays were performed with S. aureus Newman, wild-type P. aeruginosa PA14 and the specified deletion mutant supernatants (Pa sup), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) at 5 μM, and norfloxacin (Norflox) at 10 μg/ml. Intracellular norfloxacin levels within planktonic S. aureus cells were quantified by measuring the intrinsic fluorescence at 440 nm following excitation at 281 nm, and a standard curve was used to relate fluorescence values to the amount of intracellular norfloxacin (ng) in each sample. Values are reported as the amount of intracellular norfloxacin per OD₆₀₀ unit. Each column displays the average from at least two biological replicates, each with three technical replicates. (B) Biofilm disruption assays on plastic were performed with S. aureus Newman, CCCP at 5 μM, and norfloxacin (Norflox) at 25 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU values were determined. Each column displays the average from three biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; values were determined by ordinary one-way ANOVA and Tukey’s multiple comparison posttest.

FIG 6

Exposure to P. aeruginosa PA14 exoproducts does not alter expression of a subset of S. aureus Newman antibiotic transporters. Quantitative reverse transcription-PCR (qRT-PCR) assays were performed to measure the expression of four S. aureus antibiotic transporters (mdeA, mepA, sdrM, and norA) in S. aureus Newman biofilm populations exposed to norfloxacin (Norflox) at 10 μg/ml, supernatants from wild-type P. aeruginosa PA14 supernatant (Pa WT sup) and the ΔlasR deletion mutant (Pa ΔlasR sup), or medium alone. Expression was normalized to S. aureus rpoB and is presented as relative to expression in medium alone. Each column displays the average from three biological replicates, each with three technical replicates. Error bars indicate SD. None of the above treatment conditions resulted in significant differences in expression for any of the genes tested relative to expression in medium alone by multiple Student t tests and the Holm-Šidák method for correcting multiple comparisons.

FIG 7

Testing the susceptibility of biofilm-grown S. aureus JE2 antibiotic transporter mutants to norfloxacin. Biofilm disruption assays on plastic were performed with the S. aureus JE2 parental strain or the specified transposon mutant (identified by Nebraska transposon mutant library number and described in Table 1) and norfloxacin (Norflox) at 12.5 μg/ml or 25 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU values were determined. Each column displays the average from two biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; values were determined by ordinary one-way ANOVA and Bonferroni multiple comparison posttest.

FIG 8

Testing additional strains of S. aureus and P. aeruginosa. (A) Biofilm disruption assays on plastic were performed with S. aureus JE2, P. aeruginosa PA14 supernatant (PA14 sup), and norfloxacin (Norflox) at 12.5 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU values were determined. Each column displays the average from two biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; values were determined by ordinary one-way ANOVA and Tukey’s multiple comparison posttest. (B) Biofilm disruption assays on plastic were performed with S. aureus Newman, supernatant (sup) from the specified strains of P. aeruginosa (PA14, PAO1, 1587, and 1595), and norfloxacin (Norflox) at 12.5 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU values were determined. Each column displays the average from two biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; ***, P < 0.001; values were determined by ordinary one-way ANOVA and Dunnett’s multiple comparison posttest.

FIG 9

P. aeruginosa PA14 supernatant does not alter the susceptibility of S. aureus Newman biofilms to other fluoroquinolones. Biofilm disruption assays on plastic were performed with S. aureus Newman, P. aeruginosa PA14 supernatant (Pa sup), and ciprofloxacin (Cipro) (A), levofloxacin (Levo) (B), or moxifloxacin (Moxi) (C), each at 10 μg/ml. Biofilms were grown for 6 h and exposed to the above treatments for 18 h, and S. aureus biofilm CFU values were determined. Each column displays the average from two biological replicates, each with three technical replicates. Error bars indicate SD. ns, not significant; *, P < 0.05; **, P < 0.01; values were determined by ordinary one-way ANOVA and Tukey’s multiple comparison posttest.