It is well established that in nature, bacteria are found primarily as residents of surface-associated communities called biofilms. These structures form in a sequential process initiated by attachment of cells to a surface, followed by the formation of matrix-enmeshed microcolonies, and culminating in dispersion of the bacteria from the mature biofilm. In the present study, we have demonstrated that, during growth,Pseudomonas aeruginosaproduces an organic compound we have identified ascis-2-decenoic acid, which is capable of inducing the dispersion of established biofilms and of inhibiting biofilm development. When added exogenously toP. aeruginosaPAO1 biofilms at a native concentration of 2.5 nM,cis-2-decenoic acid was shown to induce the dispersion of biofilm microcolonies. This molecule was also shown to induce dispersion of biofilms, formed byEscherichia coli,Klebsiella pneumoniae,Proteus mirabilis,Streptococcus pyogenes,Bacillus subtilis,Staphylococcus aureus, and the yeastCandida albicans. Active at nanomolar concentrations,cis-2-decenoic acid appears to be functionally and structurally related to the class of short-chain fatty acid signaling molecules such as diffusible signal factor, which act as cell-to-cell communication molecules in bacteria and fungi.

Persister cells, which are tolerant to antimicrobials, contribute to biofilm recalcitrance to therapeutic agents. In turn, the ability to kill persister cells is believed to significantly improve efforts in eradicating biofilm-related, chronic infections. While much research has focused on elucidating the mechanism(s) by which persister cells form, little is known about the mechanism or factors that enable persister cells to revert to an active and susceptible state. Here, we demonstrate that cis -2-decenoic acid ( cis -DA), a fatty acid signaling molecule, is able to change the status of Pseudomonas aeruginosa and Escherichia coli persister cells from a dormant to a metabolically active state without an increase in cell number. This cell awakening is supported by an increase of the persister cells' respiratory activity together with changes in protein abundance and increases of the transcript expression levels of several metabolic markers, including acpP , 16S rRNA, atpH , and ppx . Given that most antimicrobials target actively growing cells, we also explored the effect of cis -DA on enhancing antibiotic efficacy in killing persister cells due to their inability to keep a persister cell state. Compared to antimicrobial treatment alone, combinational treatments of persister cell subpopulations with antimicrobials and cis -DA resulted in a significantly greater decrease in cell viability. In addition, the presence of cis -DA led to a decrease in the number of persister cells isolated. We thus demonstrate the ability of a fatty acid signaling molecule to revert bacterial cells from a tolerant phenotype to a metabolically active, antimicrobial-sensitive state.

Biofilms are complex communities of microorganisms in organized structures attached to surfaces. Importantly, biofilms are a major cause of bacterial infections in humans, and remain one of the most significant challenges to modern medical practice. Unfortunately, conventional therapies have shown to be inadequate in the treatment of most chronic biofilm infections based on the extraordinary innate tolerance of biofilms to antibiotics. Antagonists of quorum sensing signaling molecules have been used as means to control biofilms. QS and other cell-cell communication molecules are able to revert biofilm tolerance, prevent biofilm formation and disrupt fully developed biofilms, albeit with restricted effectiveness. Recently however, it has been demonstrated that Pseudomonas aeruginosa produces a small messenger molecule cis-2-decenoic acid (cis-DA) that shows significant promise as an effective adjunctive to antimicrobial treatment of biofilms. This molecule is responsible for induction of the native biofilm dispersion response in a range of Gram-negative and Gram-positive bacteria and in yeast, and has been shown to reverse persistence, increase microbial metabolic activity and significantly enhance the cidal effects of conventional antimicrobial agents. In this manuscript, the use of cis-2-decenoic acid as a novel agent for biofilm control is discussed. Stimulating the biofilm dispersion response as a novel antimicrobial strategy holds significant promise for enhanced treatment of infections and in the prevention of biofilm formation.

A hallmark characteristic of biofilms is their extraordinary tolerance to antimicrobial agents. While multiple factors are thought to contribute to the high level of antimicrobial tolerance of biofilms, little is known about the timing of induction of biofilm tolerance. Here, we asked when over the course of their development do biofilms gain their tolerance to antimicrobial agents? We demonstrate that in Pseudomonas aeruginosa , biofilm tolerance is linked to biofilm development, with transition to the irreversible attachment stage regulated by the two-component hybrid SagS, marking the timing when biofilms switch to the high-level tolerance phenotype. Inactivation of sagS rendered biofilms but not planktonic cells more susceptible to tobramycin, norfloxacin, and hydrogen peroxide. Moreover, inactivation of sagS also eliminated the recalcitrance of biofilms to killing by bactericidal antimicrobial agents, a phenotype comparable to that observed upon inactivation of brlR , which encodes a MerR-like transcriptional regulator required for biofilm tolerance. Multicopy expression of brlR in a Δ sagS mutant restored biofilm resistance and recalcitrance to killing by bactericidal antibiotics to wild-type levels. In contrast, expression of sagS did not restore the susceptibility phenotype of Δ brlR mutant biofilms to wild-type levels, indicating that BrlR functions downstream of SagS. Inactivation of sagS correlated with reduced BrlR levels in biofilms, with the produced BrlR being impaired in binding to the previously described BrlR-activated promoters of the two multidrug efflux pump operons mexAB-oprM and mexEF-oprN . Our findings demonstrate that biofilm tolerance is linked to early biofilm development and SagS, with SagS contributing indirectly to BrlR activation.

In the present study, we report the identification of a putative enoyl-coenzyme A (CoA) hydratase/isomerase that is required for synthesis of the biofilm dispersion autoinducer cis -2-decenoic acid in the human pathogen Pseudomonas aeruginosa . The protein is encoded by PA14_54640 (PA0745), named dspI for d i sp ersion i nducer. The gene sequence for this protein shows significant homology to RpfF in Xanthomonas campestris . Inactivation of dspI was shown to abolish biofilm dispersion autoinduction in continuous cultures of P. aeruginosa and resulted in biofilms that were significantly greater in thickness and biomass than those of the parental wild-type strain. Dispersion was shown to be inducible in dspI mutants by the exogenous addition of synthetic cis -2-decenoic acid or by complementation of Δ dspI in trans under the control of an arabinose-inducible promoter. Mutation of dspI was also shown to abolish cis -2-decenoic acid production, as revealed by gas chromatography-mass spectrometry (GC-MS) analysis of cell-free spent culture medium. The transcript abundance of dspI correlated with cell density, as determined by quantitative reverse transcriptase (RT) PCR. This regulation is consistent with the characterization of cis -2-decenoic acid as a cell-to-cell communication molecule that regulates biofilm dispersion in a cell density-dependent manner.