The extracellular matrix Component Psl provides fast-acting antibiotic defense in Pseudomonas aeruginosa biofilms

Abstract

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive "non-producing" cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.

Figure 1. The exopolysaccharide Psl promotes P. aeruginosa biofilm tolerance to both cationic and anionic antibiotics.

Results of the MBC-B assay reveal that removal of Psl increases sensitivity to positively charged colistin (A) and tobramycin (B) and polymyxin B (C). In addition, removal of Psl sensitizes biofilms to negatively charged ciprofloxacin (D). (*) indicates statistical significance from WT PAO1 as determined by a student's t-test (P<0.05). Error bars represent SEM (n = 3).

Figure 2. Psl protection is independent of biofilm age up to 24 hours.

(A) Cell number count for WT PAO1 and ΔpslAB at 6, 12, 18, and 24 hours in biofilm development (B) The MBC-B of WT PAO1 and ΔpslAB at 6, 12, 18, and 24 hours suggest that sensitivity to antibiotics was the result of modulating the matrix and not a consequence of cell number in immature biofilms. Error bars represent SEM (n = 3).

Figure 3. Over-expression of psl increases biofilm tolerance to colistin.

Opposite to ΔpslAB, Psl over-producing strain P _BAD- psl shows increased tolerance to colistin. (A) Strains that naturally lack (PA14) and over-express Psl (CF127) have similar sensitivities as the synthetic ΔpslAB and P _BAD- psl, respectively. (B) Since Psl production was directly controllable in the over-producing strain by modulating the amount of the inducer (arabinose) in the culture medium, we could determine if tolerance correlated to the levels of Psl in the biofilm matrix. Biofilms that were formed in the presence of glucose tightly inhibited the expression of P _BAD- psl, which resulted in increased sensitivity to colistin similar to ΔpslAB. For the data presented, shades of blue represent strains that do not produce Psl while shades of red represent strains that over-produce Psl. (*) indicates a significantly lower bactericidal antibiotic concentration and (**) indicates a significantly higher bactericidal antibiotic concentration when compared to WT PAO1 as determined by a student's t-test (P<0.05). Error bars represent SEM (n = 3).

Figure 4. Polymyxin B interacts with the Psl extracellular matrix.

Images of WT PAO1, PA14, ΔpslAB, P_BAD- psl, and CF127 biofilms subjected to 5 µg/ml fluorescent polymyxin B after 2 hours of exposure. Polymyxin B accumulates in the extracellular material of P _BAD- psl and is less pronounced in WT PAO1 biofilms. Fluorescent Polymyxin B did not localize to the matrix in PA14 or ΔpslAB biofilms, but instead closely associated with the cell surface. For CF127, Polymyxin B was distributed around the periphery of microcolonies within the biofilm. Scale bars represent 10 µm.

Figure 5. Ionic strength of the challenge medium influences biofilm susceptibility to positively charged antibiotics.

Bacterial survival was assessed for WT PAO1, PA14, ΔpslAB, and P _BAD-psl after exposure to colistin (A), polymyxin B (B), tobramycin (C), and ciprofloxacin (D) in the presence or without NaCl. By increasing the ionic strength of the challenge medium with NaCl, electrostatic interactions are predicted to be reduced, leading to an increased efficacy of the positively charged antibiotics colistin, polymyxin B, and tobramycin, but not of ciprofloxacin. Error bars represent SD (n = 3).

Figure 6. Psl contributes to colistin tolerance for biofilms grown under flow.

Colistin kill kinetics for 18-hour old biofilms WT PAO1, Psl deficient, and over-producing Psl strains. The Psl deficient strain was substantially more susceptible to colistin compared to the wild type, whereas over-producing Psl was less sensitive. Error bars represent SD (n = 3).

Figure 7. Psl producing cells offer a protective advantage to Psl deficient cells.

Mixed P _BAD- psl and ΔpslAB (red cells) biofilm population before and after (A) treatment with colistin, cells in the biofilm were removed from the 96 well plate and imaged. (B) MBC-B results for mixed culture biofilms reveal that the presence of ΔpslAB sensitized the P _BAD- psl to lower concentrations of colistin. (C) A monoculture of ΔpslAB survived exposure to 4 µg/ml of colistin without the requirement of Psl in the matrix. ΔpslAB can survive increasing concentrations when part of a joint biofilm with P _BAD- psl, up to 32 µg/ml. Error bars represent SEM (n = 3). Scale bars represent 10 µm.

Figure 8. Psl provides a protective advantage for E. coli and S. aureus.

Both E. coli (A, E) and S. aureus (B, F) form mixed species biofilms with ΔpslAB and P _BAD- psl. E. coli did not require the presence of P. aeruginosa to form a biofilm at the liquid-air interface on polystyrene (E); however, S. aureus formed a biofilm at the liquid-air interface only in the presence of P. aeruginosa (F). Psl-mediated protection was extendable to E. coli biofilms co-cultured with P _BAD- psl. The addition of 32 µg/ml of colistin eradicated monospecies and ΔpslAB E. coli mixed species biofilms; however, E. coli cells co-cultured with P _BAD- psl were protected (C). Similar effects were observed for S. aureus, although a protective effect was also observed with ΔpslAB cells (D). E. coli cells were identified via expression of GFP while P. aeruginosa cells were non-fluorescent (E). S. aureus cells were identified with hexidium iodide (red) where as P. aeruginosa cells were stained with Syto 9 (green) (F). Scale bars represent 5 µm (E) and 10 µm (F).