Pseudomonas aeruginosa PAO1 pyocin production affects population dynamics within mixed-culture biofilms

Abstract

Transcriptomic and phenotypic studies showed that pyocins are produced in Pseudomonas aeruginosa PAO1 aerobic and anaerobic biofilms. Pyocin activity was found to be high in slow-growing anaerobic biofilms but transient in aerobic biofilms. Biofilm coculture of strain PAO1 and a pyocin-sensitive isolate showed that pyocin production had a significant impact on bacterial population dynamics, particularly under anaerobic conditions.

FIG. 1.

Biofilm growth of P. aeruginosa strain PAO1 under aerobic and anaerobic conditions. White columns, biofilms grown under aerobic conditions; shaded columns, biofilms grown under anaerobic conditions. The temperature for both conditions was 37°C. Results shown are averages from two independent experiments.

FIG. 2.

Determination of pyocin activity in biofilms of strain PAO1. ΔprtN, the prtN mutant. (A) Cell density of 2-day biofilms. White and shaded columns represent biofilms grown under aerobic and anaerobic conditions, respectively. (B) Results obtained using the pyocin overlay assay. Ten microliters of neat supernatant and 10-fold dilutions of supernatant were spotted onto TSA and covered with a soft agar overlay containing an indicator strain (DWW1). A, aerobic biofilm supernatant; An, anaerobic biofilm supernatant. Similar results were obtained when proteinase K (100 μg/ml), which is active against S-type pyocins (6), was incorporated into the overlay agar. Although this observation does not eliminate the possibility that S-type pyocins are active against strain DWW1, it is clear from this result that bactericidal activity can be attributed to R- or F-type pyocins. Results obtained using the planktonic growth inhibition assay, showing the effect of biofilm supernatant (aerobic [C] or anaerobic [D]) on growth of indicator strain DWW1. Filled diamonds, control (no added supernatant); open squares, 10-μl PAO1 biofilm supernatant added; open triangle, 10-μl ΔprtN mutant biofilm supernatant added.

FIG. 3.

Bactericidal activities found at different biofilm time points (8, 14, 24, and 48 h). (A) Results obtained using the pyocin overlay assay. Ten microliters of neat supernatant from aerobic or anaerobic PAO1 biofilms was spotted onto TSA and covered with a soft agar overlay containing indicator strain DWW1. Lane 1, aerobic biofilms grown on 20% LB agar; lane 2, aerobic biofilms grown on 20% LB agar containing 1% KNO₃; lane 3, anaerobic biofilms grown on 20% LB agar containing 1% KNO₃. (B, C, and D) Results obtained using the planktonic growth inhibition assay. The results are averages for two independent biofilms harvested at each time point. Filled diamonds represent the control (no added supernatant), and results are also shown for cultures with biofilm supernatant added (10 μl) 8 h (open squares), 14 h (open triangles), 24 h (filled squares), and 48 h (filled triangles). Panels B and C show data for supernatants from aerobic PAO1 biofilms grown on 20% LB agar and on 20% LB agar containing 1% KNO₃, respectively. Panel D shows data for supernatant from the anaerobic PAO1 biofilms used. An overnight culture of strain DWW1 was used as the indicator inoculum (for panels B and C, 10 μl of 100-fold diluted indicator culture was used, and for panel D, 10 μl of neat indicator culture was used).

FIG. 4.

Effect of pyocin production on bacterial populations within mixed-culture biofilms incubated under aerobic and anaerobic conditions. Biofilm populations incubated under aerobic conditions (A) and anaerobic conditions (B). Shaded bars, total population (viable counts using LB agar); open bars, population isolated after culture on LB agar containing rifampin. prtN, isogenic prtN mutant of strain PAO1; DWW1 (referred to as DWW1 Rif^r in the text) spontaneous rifampin-resistant mutant obtained from CF patient isolate DWW1.