Associational Resistance to Predation by Protists in a Mixed Species Biofilm

Abstract

Mixed species biofilms exhibit increased tolerance to numerous stresses compared to single species biofilms. The aim of this study was to examine the effect of grazing by the heterotrophic protist, Tetrahymena pyriformis, on a mixed species biofilm consisting of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Protozoan grazing significantly reduced the single species K. pneumoniae biofilm, and the single species P. protegens biofilm was also sensitive to grazing. In contrast, P. aeruginosa biofilms were resistant to predation. This resistance protected the otherwise sensitive members of the mixed species biofilm consortium. Rhamnolipids produced by P. aeruginosa were shown to be the primary toxic factor for T. pyriformis. However, a rhamnolipid-deficient mutant of P. aeruginosa (P. aeruginosa ΔrhlAB) maintained grazing resistance in the biofilm, suggesting the presence of at least one additional protective mechanism. P. aeruginosa with a deleted gene encoding the type III secretion system also resisted grazing. A transposon library was generated in the ΔrhlAB mutant to identify the additional factor involved in community biofilm protection. Results indicated that the Pseudomonas Quinolone Signal (PQS), a quorum sensing signaling molecule, was likely responsible for this effect. We confirmed this observation by showing that double mutants of ΔrhlAB and genes in the PQS biosynthetic operon lost grazing protection. We also showed that PQS was directly toxic to T. pyriformis. This study demonstrates that residing in a mixed species biofilm can be an advantageous strategy for grazing sensitive bacterial species, as P. aeruginosa confers community protection from protozoan grazing through multiple mechanisms. IMPORTANCE Biofilms have been shown to protect bacterial cells from predation by protists. Biofilm studies have traditionally used single species systems, which have provided information on the mechanisms and regulation of biofilm formation and dispersal, and the effects of predation on these biofilms. However, biofilms in nature are comprised of multiple species. To better understand how multispecies biofilms are impacted by predation, a model mixed-species biofilm was here exposed to protozoan predation. We show that the grazing sensitive strains K. pneumonia and P. protogens gained associational resistance from the grazing resistant P. aeruginosa. Resistance was due to the secretion of rhamnolipids and quorum sensing molecule PQS. This work highlights the importance of using mixed species systems.

Keywords: Klebsiella pneumoniae; Pseudomonas aeruginosa; Pseudomonas protegens; multispecies biofilm; predation; protection; protozoa.

FIG 1

Confocal micrographs of ungrazed and grazed single and mixed species biofilms formed by P. aeruginosa (PAO1 WT, yellow), P. protegens (Pf-5, cyan) and K. pneumoniae (KP-1, red). Biofilms were pregrown for 48 h, at which point either nothing or T. pyriformis was added and the biofilms were incubated for a further 48 h (96 h total). Panels (a to h) show the difference in biomass between the ungrazed and grazed single and mixed species biofilms and (i to l) panels show the presence or absence of T. pyriformis after grazing. No T. pyriformis were observed in the mixed or single species P. aeruginosa biofilms. Scale bar: 20 μm.

FIG 2

Predation of single and mixed species biofilms by T. pyriformis. (a) Biovolume per unit base area (μm³ μm⁻²) of single and mixed species ungrazed (black bars) and grazed (gray bars) biofilms after 48 h. (b) Enumeration of T. pyriformis in the grazed biofilms and M9 media controls with 10⁸ cells mL⁻¹ of heat killed bacteria (HKB). (c) Enumeration of T. pyriformis exposed to cell-free supernatants from single and mixed species biofilms. Ungrazed (black bars) and grazed (gray bars) biofilms after 48 h. (n = 3). *, P < 0.05. The different letters are used to indicate significant differences between groups (P < 0.05).

FIG 3

T. pyriformis predation on single and mixed species biofilms containing the P. aeruginosa ΔrhlA mutant. (a) Biovolume per unit base area (μm³ μm⁻²) of ungrazed (black bars) and grazed (gray bars) biofilms after 48 h. (b) The number of T. pyriformis after coincubation with biofilms and M9 media controls with 10⁸ cells mL⁻¹ of heat killed bacteria (HKB). (c) Enumeration of T. pyriformis exposed to cell-free supernatants from single and mixed species biofilms. Ungrazed (black bars) and grazed (gray bars) biofilms after 48 h. The different letters are used to indicate significant differences between groups (P < 0.05).

FIG 4

T. pyriformis predation of single and mixed species biofilms formed by P. aeruginosa ΔpscJ and ΔrhlA ΔpscJ strains. (a) Biovolume per unit base area (μm³ μm⁻²) of ungrazed (black bars) and grazed (gray bars) biofilms after 48 h. (b) Enumeration of T. pyriformis in biofilms and M9 media controls with 10⁸ cells mL⁻¹ of heat killed bacteria (HKB). (c) Enumeration of T. pyriformis exposed to cell-free supernatants from single and mixed species biofilms. Ungrazed (black bars) and grazed (gray bars) biofilms after 48 h. (n =3). The different letters are used to indicate significant differences between groups (P < 0.05).

FIG 5

Rhamnolipid quantification from single and mixed species biofilms. Biofilms were pregrown for 48 h, at which time, either nothing was added (nongrazed controls, gray bars) or T. pyriformis was added and the biofilms were incubated for a further 48 h (96 h total). The concentration of rhamnolipids produced by the biofilms was subsequently quantified by orcinol assays (n = 3). No significant differences were found between the grazed and ungrazed biofilms.

FIG 6

(a) Tetrahymena pyriformis survival in the biofilms of P. aeruginosa ΔrhlA transposon after 24 h of incubation. P. aeruginosa ΔrhlA transposon mutants are named according to the transposon insertion followed by the sample number. (b) Protozoan survival after exogenous addition of PQS. T. Pyriformis were exposed to different PQS concentrations dissolved in methanol after incubating for 2 h in M9 medium with heat killed bacteria. The maximum concentration of methanol was 1% of the total M9 volume. M9 medium containing 1% methanol without any PQS was used as the negative control. (c) T. pyriformis enumeration after 2 h in the biofilms of P. aeruginosa ΔrhlA transposon mutant biofilms with 50 μM PQS. Heat-killed bacteria with methanol but no PQS was used as a control. ***, P < 0.001. Differences were tested between mixed and single species biofilms. The number of protozoa was determined using light microscopy Data points represent the mean ± standard deviation of the mean (SD) (n = 3).