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. 2013 Aug;81(8):2705-13.
doi: 10.1128/IAI.00332-13. Epub 2013 May 20.

Clearance of Pseudomonas aeruginosa foreign-body biofilm infections through reduction of the cyclic Di-GMP level in the bacteria

Louise D Christensen  1 , Maria van Gennip, Morten T Rybtke, Hong Wu, Wen-Chi Chiang, Morten Alhede, Niels Høiby, Thomas E Nielsen, Michael Givskov, Tim Tolker-Nielsen
Affiliations

Clearance of Pseudomonas aeruginosa foreign-body biofilm infections through reduction of the cyclic Di-GMP level in the bacteria

Louise D Christensen et al. Infect Immun. 2013 Aug.

Abstract

Opportunistic pathogenic bacteria can engage in biofilm-based infections that evade immune responses and develop into chronic conditions. Because conventional antimicrobials cannot efficiently eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. It has recently been established that the secondary messenger cyclic diguanosine monophosphate (c-di-GMP) functions as a positive regulator of biofilm formation in several different bacteria. In the present study we investigated whether manipulation of the c-di-GMP level in bacteria potentially can be used for biofilm control in vivo. We constructed a Pseudomonas aeruginosa strain in which a reduction in the c-di-GMP level can be achieved via induction of the Escherichia coli YhjH c-di-GMP phosphodiesterase. Initial experiments showed that induction of yhjH expression led to dispersal of the majority of the bacteria in in vitro-grown P. aeruginosa biofilms. Subsequently, we demonstrated that P. aeruginosa biofilms growing on silicone implants, located in the peritoneal cavity of mice, dispersed after induction of the YhjH protein. Bacteria accumulated temporarily in the spleen after induction of biofilm dispersal, but the mice tolerated the dispersed bacteria well. The present work provides proof of the concept that modulation of the c-di-GMP level in bacteria is a viable strategy for biofilm control.

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Figures

Fig 1
Fig 1
Colony morphology of P. aeruginosa wspF/pPBAD-yhjH on agar plates without (A) or with (B) l-arabinose and of P. aeruginosa wspF/pJN105 on agar plates without (C) or with (D) l-arabinose.
Fig 2
Fig 2
Biofilm formation of PAO1/pPBAD-yhjH (left) and PAO1/pJN105 (right) in microtiter trays. l-Arabinose in different concentrations (0, 0.05, 0.1, or 0.2%) was added to the medium before inoculation, and biofilm growth was allowed for 24 h, after which the adherent biomass was quantified by crystal violet staining. The values are averages of three experiments with 12 replicates in each, and the bars indicate standard deviations (SD). There was a significant (P < 0.0001) decrease in the biomass of PAO1/pPBAD-yhjH when induced with l-arabinose compared to the same strain uninduced (evaluated by analysis of variance [ANOVA] test). No significant difference was observed when comparing PAO1/pJN105 induced with uninduced samples (evaluated by ANOVA test).
Fig 3
Fig 3
Quantification of P. aeruginosa bacteria dispersing from microtiter tray biofilms. Biofilms were allowed to develop for 24 h before inducing PBAD-driven yhjH expression with l-arabinose. Liquid culture samples were collected at intervals after yhjH induction and were plated to obtain CFU/ml. Circles represent PAO1/pPBAD-yhjH with l-arabinose (filled) or sodium chloride (open) in the medium. Squares represent PAO1/pJN105 with l-arabinose (filled) or sodium chloride (open) in the medium. There was a significant (P < 0.0001) increase in the CFU/ml in the liquid phase surrounding the biofilms of PAO1/pPBAD-yhjH induced with l-arabinose compared to the same strain uninduced or PAO1/pJN105 induced or uninduced, evaluated by ANOVA test. Error bars represent the SD.
Fig 4
Fig 4
Dispersal of flow-chamber-grown P. aeruginosa biofilms. Biofilms of GFP-labeled PAO1/pPBAD-yhjH (top) and PAO1/pJN105 (bottom) were allowed to develop in flow chambers for 24 h before PBAD-driven yhjH expression was induced. CLSM micrographs were acquired at times t = 0, 3, 10, and 15 h after yhjH induction. The images were acquired at the same location in the PAO1/pPBAD-yhjH biofilm (top) and PAO1/pJN105 biofilm (bottom). The microscope field corresponds to 220 by 220 μm.
Fig 5
Fig 5
Quantification of P. aeruginosa bacteria dispersing from flow-chamber biofilms. Biofilms were allowed to develop for 24 h before PBAD-driven yhjH expression was induced. Effluent was collected at intervals after yhjH induction, and the CFU/ml were determined. Circles represent PAO1/pPBAD-yhjH with l-arabinose (filled) or sodium chloride (open) in the medium. Squares represent PAO1/pJN105 with l-arabinose (filled) or sodium chloride (open) in the medium. There was a significant (P < 0.0001) increase in the CFU/ml of effluent for PAO1/pPBAD-yhjH induced with l-arabinose compared to the same strain uninduced or to PAO1/pJN105 induced or uninduced, evaluated by ANOVA. Error bars represent the SD.
Fig 6
Fig 6
Dispersal of P. aeruginosa biofilms in vivo. Mice had silicone implants, precolonized with either PAO1/pPBAD-yhjH (open circles) or PAO1/pJN105 (filled circles), inserted in the peritoneal cavity. At 24 and 26.5 h postinsertion, the mice received intraperitoneal injections of 2% arabinose. The mice were euthanized 2.5 h after the last injection, the implants were removed, and the CFU/implant were determined (filled and open triangles). The peritoneal cavity was then flushed with 0.9% NaCl, and the CFU/ml was determined (filled and open squares). Lastly, the spleens were removed, and the CFU/spleen was determined (filled and open diamantes). Lines represent medians. P values are shown in the figure.
Fig 7
Fig 7
Number of implant-associated P. aeruginosa evaluated over time. Mice had silicone implants, precolonized with either PAO1/pPBAD-yhjH or PAO1/pJN105, inserted in the peritoneal cavity. At 24 and 26.5 h postinsertion, the mice received intraperitoneal injections of 2% arabinose. The mice were euthanized over a time period of 15 h after the last arabinose injection. Implants were removed from the mice at 0, 1, 2.5, 5, 10, and 15 h after the last arabinose injection. The results shown in the figure were pooled from two experiments, and the CFU obtained at the different time points were normalized to the average initial CFU/implant of both experiments. The open squares represent PAO1/pPBAD-yhjH CFU/implant, and the closed circles represent PAO1/pJN105 CFU/implant. The number of mice assessed to each time point from each group is indicated in the figure. There was a significant difference between the recovered bacterial amounts from mice infected with PAO1/pPBAD-yhjH compared to mice infected with the vector control strain PAO1/pJN105 over time, P < 0.006, evaluated by ANOVA. Gray lines represent the medians.
Fig 8
Fig 8
Number of P. aeruginosa in the spleen followed over time. Mice had silicone implants, precolonized with either PAO1/pPBAD-yhjH or PAO1/pJN105, inserted in the peritoneal cavity. At 24 and 26.5 h postinsertion the mice received intraperitoneal injections of 2% arabinose. The mice were euthanized over a time period of 15 h after the last arabinose injection. Spleens were removed from the mice at 0, 1, 2.5, 5, 10, and 15 h after the last arabinose injection. The results shown in the figure were pooled from two experiments, and the CFU obtained at the different time points were normalized to the average initial CFU/implant of both experiments. The open squares represent PAO1/pPBAD-yhjH CFU/spleen, and the closed circles represent PAO1/pJN105 CFU/spleen. The number of mice assessed to each time point from each group is indicated in the figure. There was a significant difference between the recovered bacterial amounts from mice infected with PAO1/pPBAD-yhjH compared to mice infected with the vector control strain PAO1/pJN105 over time, P < 0.0004, evaluated by ANOVA. Gray lines represent the medians.
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