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, 191 (5), 1393-403

A Fatty Acid Messenger Is Responsible for Inducing Dispersion in Microbial Biofilms

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A Fatty Acid Messenger Is Responsible for Inducing Dispersion in Microbial Biofilms

David G Davies et al. J Bacteriol.

Abstract

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 aeruginosa produces an organic compound we have identified as cis-2-decenoic acid, which is capable of inducing the dispersion of established biofilms and of inhibiting biofilm development. When added exogenously to P. aeruginosa PAO1 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 by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Streptococcus pyogenes, Bacillus subtilis, Staphylococcus aureus, and the yeast Candida 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.

Figures

FIG. 1.
FIG. 1.
Microcolonies of P. aeruginosa biofilms grown in continuous culture demonstrating native dispersion response. Transmitted light image (A) and fluorescent image (B) showing the size dependence of the dispersion response. Biofilm microcolonies growing in continuous culture with dimensions of greater than 40 μm in diameter by 10-μm thickness show dispersion (left 3). Microcolonies below this minimum dimension remain “solid” (right two photomicrographs). Fluorescence indicates presence of cells (lacZ reporter on chromosome). All images are the same relative size at ×500 magnification. Bars, 40 μm. Arrows indicate void areas within a microcolony.
FIG. 2.
FIG. 2.
Treatment of P. aeruginosa mature biofilms with spent medium, CSM, and cis-2-decenoic acid. (A) At 30 min, biofilms grown in silicone tubing were exposed to spent medium or fresh medium. Bacteria in effluent were collected continuously for 100 min, and the cell density determined by OD600. (B) Biofilm grown in continuous culture in silicone tubing for 4 days and switched either to fresh medium for 1 h or CSM for 1 h. Extruded contents of control tube shows intact biofilm. Extruded contents of CSM-treated biofilm show dispersion. (C) Photomicrographs show the addition of CSM to mature biofilm grown in continuous culture in a microscope-mounted flow cell. Microcolony disaggregation is shown to begin at 7 min. After 30 min of exposure, the microcolony had completely disaggregated. Dispersed cells were actively motile (not visible in static image), indicating a change in phenotype compared to cells in intact microcolony (prior to CSM addition). (D) Addition of 10 μM cis-2-decenoic acid (cis-DA) to mature biofilm grown in continuous culture in a microscope-mounted flow cell. Microcolony disaggregation is shown to begin at 11 min. Complete microcolony disaggregation is shown within 30 min of exposure. Control biofilms treated with carrier fluid were not affected by treatment up to 1 h (data not shown).
FIG. 3.
FIG. 3.
Biofilm development in the continuous presence of CSM diluted in modified EPRI to concentration of spent medium. The average thickness (A) and surface area (B) of biofilms grown in the presence of CSM is significantly less than for untreated biofilms. Gray bars indicate biofilms grown in the absence of dispersion inducer. Black bars indicate biofilms grown in the presence of CSM. Error bars represent the standard deviation of 20 randomly selected microcolonies.
FIG. 4.
FIG. 4.
Dispersion of different bacterial biofilms by P. aeruginosa CSM using microtiter plate dispersion bioassay. The y axis indicates the number of cells released into the bulk liquid of 16 replicate wells in three replicate experiments, after treatment for 1 h with CSM or carrier control (−), containing sterile medium. Hatched line indicates level of dispersion in carrier control samples. All differences between CSM samples and controls are statistically significant at the indicated P value as determined by using the Student t test.
FIG. 5.
FIG. 5.
Microtiter plate dispersion bioassay. (A) ODs of cells released from biofilm-containing microtiter plate wells. White bar, control sample treated with EPRI alone; gray bar, sample treated with CSM. Black bars represent biofilms treated with C18 reversed-phase HPLC fractions of CSM eluted in an acetonitrile gradient from 2 to 75%. The results are the average of 16 replicate wells; error bars represent one standard deviation. The results from Student t test show P < 0.001 for CSM and 22-min HPLC samples. (B) Microtiter plate biofilm dispersion bioassay comparing various concentrations of cis-2-decenoic acid to spent medium. ODs of cells released from biofilm-containing microtiter plate wells. Negative control wells contained P. aeruginosa treated with 10% ethanol in EPRI. The gray bar represents biofilms treated with spent medium. The black bars represent biofilms treated with increasing concentrations of cis-2-decenoic acid in 10% ethanol. The results are the average of 16 replicate wells; error bars represent one standard deviation. Student t test indicated P < 0.001 for all cis-2-decenoic acid samples compared to control. (C [inset]) Structure of cis-2-decenoic acid.
FIG. 6.
FIG. 6.
Spectral analysis of P. aeruginosa CSM and cis-2-decenoic acid. (A) Product ion mass peaks for the 171 m/z molecule detected in active HPLC CSM fraction and for synthetic cis-2-decenoic acid. The y axis indicates the intensity; The x axis indicates m/z in positive ion mode. CSM sample matches peaks from synthetic cis-2-decenoic acid. Note that in MS, the peak intensity is not a direct indication of concentration. (B) GC-MS spectrum of P. aeruginosa CSM and cis-2-decenoic acid. CSM sample peak at 15.9 min, indicates solvent carrier. The y axis indicates intensity; the x axis indicates time in minutes. (C) FT-IR spectrum of P. aeruginosa CSM and cis-2-decenoic acid. The y axis indicates absorbance; the x axis indicates reciprocal centimeters.
FIG. 7.
FIG. 7.
Dispersion of different bacterial biofilms by cis-2-decenoic acid using microtiter plate dispersion bioassay. The y axis indicates number of cells released into the bulk liquid of 16 replicate wells in three replicate experiments, following treatment for 1 h with 0.01 μM cis-2-decenoic acid (CDA), or carrier control (−), containing medium plus 10% ethanol. The hatched line indicates level of dispersion in carrier control samples. All differences between cis-2-decenoic acid-treated samples and controls are statistically significant at the indicated P value as determined by using a Student t test.

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