Extensive Reduction of Cell Viability and Enhanced Matrix Production in Pseudomonas aeruginosa PAO1 Flow Biofilms Treated with a d-Amino Acid Mixture

Abstract

Treatment of Pseudomonas aeruginosa PAO1 flow biofilms with a d-amino acid mixture caused significant reductions in cell biomass by 75% and cell viability by 71%. No biofilm disassembly occurred, and matrix production increased by 30%, thereby providing a thick protective cover for remaining viable or persister cells.

TEXT

The multifactorial tolerance of surface-attached complex microbial communities known as biofilms apparently renders conventional treatment strategies ineffective. Biofilms persist and are hard to eradicate because of mechanisms that involve restricted penetration of antimicrobials, differential physiological activity, presence of phenotypic variants and persisters, efflux systems, and enhanced repair systems (1, 2). Unless anti-biofilm strategies are able to block these tolerance mechanisms, biofilms will continue to exert detrimental effects, especially in the hospital and industrial settings.

Since d-amino acids are synthesized and released by many bacterial species, including the opportunistic human pathogen Pseudomonas aeruginosa, and have been shown to lack significant toxicity, the idea of using them to combat biofilm-associated infections is highly attractive (3). Recent findings reveal that d-amino acids regulate bacterial cell wall remodeling in stationary phase and cause biofilm dispersal in aging bacterial communities (3, 4). Incorporation of a d-amino acid mixture (d-leucine, d-methionine, d-tryptophan, and d-tyrosine) into the peptidoglycan has also been reported to induce biofilm disassembly in Bacillus subtilis (5). In standing cultures, exogenous d-amino acids prevent biofilm formation in some bacterial strains (5). To date, there are no reports on the susceptibility of flow chamber-grown biofilms to d-amino acids. Flow-chamber biofilm systems not only simulate more physiologically relevant biofilm environments but also allow nondestructive, real-time, high-resolution characterization and quantification of live biofilm structures and development. Hence, in the present study, we investigated whether a d-amino acid mixture could affect P. aeruginosa PAO1 biofilms grown under flow conditions using a multichannel microdevice flow system. With the use of confocal laser scanning microscopy (CLSM) and COMSTAT (6), we determined the effect of exogenous d-amino acid mixture on the structure, development, and viability of P. aeruginosa PAO1 flow biofilms.

Pseudomonas aeruginosa strain PAO1 with the green fluorescent protein (GFP)-expressing plasmid pMRP9-1 (7) was used to enable detection of cells by CLSM. Cultures for inoculation of the microdevice were prepared in Luria-Bertani (LB) medium supplemented with 300 μg ml⁻¹ carbenicillin for plasmid maintenance as described previously (8) but at 30°C. Biofilms were grown in the microdevice at 30°C supplied with 1/3-diluted LB medium at an 11-ml h⁻¹ flow rate. The multichannel microdevice flow system was assembled and prepared as described previously (8). In brief, this autoclavable, once-through, continuous-culture system consists of a stainless steel flow chamber with 9 parallel channels (channel dimensions, 1 by 1 by 30 mm) and a stainless steel bottom plate, covered with transparent silicon packing and a cover glass for CLSM imaging. To confirm viability, death, and distribution of cells, GFP-expressing P. aeruginosa PAO1 biofilms were stained with 30 μM propidium iodide (PI) (Molecular Probes, Invitrogen). For matrix visualization and quantification, biofilms were stained with 200 μl FilmTracer SYPRO Ruby (Molecular Probes, Invitrogen) per channel, incubated in the dark for 30 min at room temperature, and rinsed with filter-sterilized water. All images were acquired at 48 h and 64 h after inoculation in the microdevice using a Leica TCS SL upright microscope (Leica Microsystems, Germany) fitted with a 20×/0.7 objective and equipped with argon and GreNe (green helium neon) lasers. In treated biofilms, the 64-h time point corresponds to the end of a 16-h run of a d-amino acid mixture. Three-dimensional images and optical z sections at 2.5-μm intervals from the substratum to the top of biofilm were generated using Leica software. CLSM-captured image stacks were analyzed quantitatively using COMSTAT software written on the Matlab platform (6) based on total biomass, average thickness, and maximum thickness.

We first examined the effect of a d-amino acid mixture on P. aeruginosa PAO1 biofilm cells. The d-amino acid mixture (d-AA) described previously (5) was prepared at modified concentrations in 500 ml 1/3-diluted LB medium at final concentrations of 0.03 mM for d-leucine and d-methionine and 0.04 mM for d-tyrosine and d-tryptophan and supplied to 48-h-old biofilms in two test channels at an 11-ml h⁻¹ flow rate for 16 h, while control channels were continuously supplemented with 1/3-diluted LB medium. Fluorescence measurements of GFP expression showed that viable cells in untreated biofilms were able to grow in mass and thickness between 48 h and 64 h (Fig. 1A). In contrast, 16-h treatment with exogenous d-AA resulted in a reduction in live cell biomass by 75%, associated with a decrease in average thickness (78%) and maximum thickness (59%) of live cells (Fig. 1B).

Fig 1.

Change in live cell mass and thickness of P. aeruginosa PAO1 flow biofilms by the addition of d-AA. (A) Increasing biomass and average thickness of viable cells in untreated biofilms from 48 h (□) to 64 h (■) did not reach significant levels (P > 0.05, t test). (B) Biomass and average thickness of viable cells in biofilms show significant differences before (□) and after (■) 16-h treatment (64 h total) with d-AA (*, P < 0.05). Error bars indicate standard deviations of means from two independent experiments (2 image stacks/channel × 2 channels per experiment). Average thickness is the average depth of the biofilm, and maximum thickness locates the highest point (μm) above each (x,y) pixel in the bottom layer containing biomass.

The reduction in both biomass and thickness of viable cells led us to investigate whether this was due to cell death, cell detachment, or both. Viability detection in untreated and treated biofilms using GFP and PI, which fluoresced live cells green and stained dead cells red, respectively, revealed that d-AA killed 71% of the cells, since hardly any cell death in d-AA-free biofilms was observed (0.2%) (Fig. 2). The addition of d-AA also led to a 20% reduction in total biomass of live and dead cells after treatment (data not shown), which may indicate detachment of some dead cells from the biofilm or cell lysis. Our results confirm previous observations indicating the toxic activities of d-AA (9, 10), and a study to determine the mechanism of cell death is under way. Previous reports, however, elucidated the lethal or inhibitory effects of d-AA in a number of test bacterial species under static conditions, where the d-AA cause interference with the activity of peptidases and proteases, inhibition of cell wall synthesis, alterations in peptidoglycan metabolism when added at high concentrations (11), and incorporation into the peptidoglycan of bacterial cell walls, resulting in morphological and structural damage (9).

Fig 2.

Change in viability by the addition of d-AA. Representative CLSM images of untreated (top) and d-AA-treated (bottom) P. aeruginosa PAO1 biofilms are shown. (Left) Top-down view showing combined green (GFP) and red (propidium iodide) channels. (Middle and right) Three-dimensional reconstructions of viable, green fluorescent cells and dead, red fluorescent cells. Bar, 150 μm. Image size, 750 μm by 750 μm.

Under our conditions, the susceptibility of cells to d-AA varied within the depth of the biofilm. Cells in the upper regions (≥120 μm) and layers between 30 to 80 μm of treated biofilms were mostly killed and removed by d-AA (Fig. 3). Moreover, the greatest proportion of viable cells detected after treatment was at the bottom layers (0 to 20 μm). Similar studies have shown that conventional antimicrobial compounds specifically target metabolically active biofilm cells in the upper layers of P. aeruginosa flow chamber-grown biofilms and have been shown to be less effective for dormant cells in deeper layers due to low metabolic activity, nutrient or oxygen limitation, anaerobic conditions, and decreased penetration through the exopolysaccharide (EPS) (12–15). Interestingly, untreated and treated biofilms at depths below 60 μm showed contrasting distributions of both live and dead cells (Fig. 3). In untreated biofilms, live cells in the bottom layers were almost absent or covered a very low surface area, ranging from 0.1 to 15%. This is most likely due to low or undetectable expression of GFP in these oxygen-limited or anaerobic layers. In addition, dead cells were found only on or near the substratum, covering only up to 4% of the surface area (Fig. 3). In contrast, bottom layers of treated biofilms were covered with live and dead cells. In our opinion, cell removal and products of cell death in the upper and middle layers induced by d-AA influenced oxygen and nutrient concentrations and flow in the bottom layers and thereby caused alterations in the physiological state of some cells or cell differentiation from the dormant to the active state, which in turn made cells susceptible to d-AA or enhanced GFP expression in cells that survived the treatment.

Fig 3.

Live and dead cell distribution with depth in untreated and d-AA-treated P. aeruginosa PAO1 flow biofilms. In treated biofilms, the maximum surface coverage of dead cells was generally concentrated in regions between 30 to 80 μm while live cells were mostly localized in the bottom region (0 to 20 μm). Eight image stacks at 64 h were analyzed for each group (2 image stacks/channel × 2 channels × 2 experiments). Three-dimensional images and optical z-sections at 2.5 μm intervals from substratum to top of biofilm were analyzed quantitatively using COMSTAT.

While an exogenous d-amino acid mixture was toxic to biofilm cells, no biofilm disassembly occurred, and the biofilm matrix was not disrupted and instead showed increases in biomass of 30% and thickness of 2% after treatment (Fig. 4). Meanwhile, untreated biofilms showed a 22% decrease (P = 0.0103) in matrix biomass between 48 h and 64 h. Statistically significant differences were found between matrix biomasses of untreated and treated biofilms (P = 0.0092) based on measurements from 16 image stacks taken at 48 h or 64 h (4 image stacks/channel × 2 channels × 2 experiments). The greater biomass and average thickness of matrix in treated biofilms indicate that d-AA treatment enhances matrix formation. This can be partly explained by the effects of cell lysis induced by d-AA, which releases intracellular nutrients that are absorbed by the matrix (16, 17). This also demonstrates the ability of the matrix to provide a buffer against stress or changing environmental conditions.

Fig 4.

Effect of the d-AA mixture on the biofilm matrix. A P. aeruginosa PAO1 biofilm matrix was stained using FilmTracer SYPRO Ruby matrix stain and quantified with COMSTAT. The matrix of d-AA treated biofilms showed significantly greater biomass (A) and average thickness (B) than d-AA-free biofilms (control 64-h) (P < 0.05). In control, d-AA free biofilms, matrix biomass (A) and average thickness (B) decreased during the 16-h interval (from the 48-h to the 64-h time point).

Current information on the roles of d-amino acids in bacteria and their effects in biofilm formation are limited to biofilms grown in standing culture or assays (3–5, 10, 11, 18–20). Moreover, there is limited information describing the effects of d-AA on matrix structure and susceptibility. Our results indicate that under flow conditions, P. aeruginosa PAO1 biofilm cells are susceptible to d-AA. However, a fraction of cells are invulnerable and are able to survive the treatment and may well represent the existence of persister cells (14, 21, 22). Treated biofilms also do not demonstrate the observed phenomenon of active dispersal following cell death (19), since the matrix remains intact and undegraded and shows increased production rather than being dissolved. These results open new insights into the role of the matrix in biofilm tolerance, and further investigations on mechanisms by which d-AA cause cell death and increase matrix production will be necessary to develop the application of a d-amino acid mixture as an anti-biofilm agent.