The role of individual exopolysaccharides in antibiotic tolerance of Pseudomonas aeruginosa aggregates

Abstract

The bacterium Pseudomonas aeruginosa is involved in chronic infections of cystic fibrosis lungs and chronic wounds. In these infections the bacteria are present as aggregates suspended in host secretions. During the course of the infections there is a selection for mutants that overproduce exopolysaccharides, suggesting that the exopolysaccharides play a role in the persistence and antibiotic tolerance of the aggregated bacteria. Here, we investigated the role of individual P. aeruginosa exopolysaccharides in aggregate-associated antibiotic tolerance. We employed an aggregate-based antibiotic tolerance assay on a set of P. aeruginosa strains that were genetically engineered to over-produce a single, none, or all of the three exopolysaccharides Pel, Psl, and alginate. The antibiotic tolerance assays were conducted with the clinically relevant antibiotics tobramycin, ciprofloxacin and meropenem. Our study suggests that alginate plays a role in the tolerance of P. aeruginosa aggregates toward tobramycin and meropenem, but not ciprofloxacin. However, contrary to previous studies we did not observe a role for Psl or Pel in the tolerance of P. aeruginosa aggregates toward tobramycin, ciprofloxacin, and meropenem.

Keywords: Pseudomonas aeruginosa; aggregates; antibiotic tolerance; biofilm; extracellular matrix.

FIGURE 1

Colony morphology of the P. aeruginosa wild-type, Epol–, Pel+, Psl+, Alg+, Epol+, and ΔwspF strains. The respective P. aeruginosa strains were streaked (upper row) or spotted (lower row) on LB plates supplemented with Congo Red and Coomassie Blue, and images of representative colonies were acquired after 24 hours incubation at 37°C. Scale bars correspond to 0.4 mm (upper row) or 2 mm (lower row).

FIGURE 2

Schematic illustration of the aggregate antibiotic tolerance assay used in the current study.

FIGURE 3

Curves showing the growth of the P. aeruginosa wild-type, Epol–, Pel+, Psl+, Alg+, Epol+, and ΔwspF aggregates in our assay. Equal numbers of bacteria from overnight cultures of the P. aeruginosa wild-type, Epol+, Epol–, Pel+, Psl+, Alg+, and ΔwspF strains were added to liquid agar and cast in petri dishes. The bacteria subsequently formed aggregates in the agar gels during incubation at 37°C, and at time intervals defined agar gel volumes were acquired from the agar plates by the use of a punch biopsy knife, and the number of colony forming units (CFU) per agar plug was determined by disintegration of the agar plugs and plating of the bacteria on agar plates followed by incubation. Average and standard deviation of three replicates are shown. The dotted line indicates the time point for antibiotic treatment in the antibiotic tolerance assay.

FIGURE 4

Morphology of aggregates formed by the P. aeruginosa wild-type, Epol–, Pel+, Psl+, Alg+, Epol+, and ΔwspF strains. The respective P. aeruginosa strains were grown as agar-embedded aggregates for 20 hours, upon which they were stained with Syto9 and images of representative aggregates were acquired by CLSM. Scale bars correspond to 60 μm.

FIGURE 5

Tolerance to tobramycin of aggregates formed by the P. aeruginosa wild-type, Epol–, Pel+, Psl+, Alg+, Epol+, and ΔwspF strains. The respective P. aeruginosa strains were grown as agar-embedded aggregates for 16 h. Subsequently, agar plugs containing agar-embedded aggregates were obtained from the agar plates using a punch biopsy knife. The agar plugs were then treated for 3 h with either 15 μg/ml tobramycin or saline. Next, the antibiotic was removed by a washing procedure and the agar plugs were disintegrated, serially diluted and spotted on LB agar plates and incubated for enumeration of the surviving bacteria (A) and calculation of fold reduction mediated by the antibiotic treatment (B). Panel (A) shows averages and standard deviations of six replicates, and the significance (two-way ANOVA) of the difference between the CFU values of select groups are indicated by stars: *p < 0.05; ^**p < 0.01; ^****p < 0.0001. The significance (one-way ANOVA) of the difference between the log reduction values of the wild-type and the other strains are indicated by stars in panel (B): ^****p < 0.0001.

FIGURE 6

Tolerance to meropenem of aggregates formed by the P. aeruginosa wild-type, Epol–, Pel+, Psl+, Alg+, Epol+, and ΔwspF strains. The respective P. aeruginosa strains were grown as agar-embedded aggregates for 16 h. Subsequently, agar plugs containing agar-embedded aggregates were obtained from the agar plates using a punch biopsy knife. The agar plugs were then treated for 3 h with either 15 μg/ml meropenem or saline. Next, the antibiotic was removed by a washing procedure and the agar plugs were disintegrated, serially diluted and spotted on LB agar plates and incubated for enumeration of the surviving bacteria (A) and calculation of fold reduction mediated by the antibiotic treatment (B). Panel (A) shows averages and standard deviations of six replicates, and the significance (two-way ANOVA) of the difference between the CFU values of select groups are indicated by stars: *p < 0.05; ^**p < 0.01; ^****p < 0.0001. The significance (one-way ANOVA) of the difference between the log reduction values of the wild-type and the other strains are indicated by stars in panel (B): ^***p < 0.001; ^****p < 0.0001.

FIGURE 7

Tolerance to ciprofloxacin of aggregates formed by the P. aeruginosa wild-type, Epol–, Pel+, Psl+, Alg+, Epol+, and ΔwspF strains. The respective P. aeruginosa strains were grown as agar-embedded aggregates for 16 h. Subsequently, agar plugs containing agar-embedded aggregates were obtained from the agar plates using a punch biopsy knife. The agar plugs were then treated for 3 h with either 2 μg/ml ciprofloxacin or saline. Next, the antibiotic was removed by a washing procedure and the agar plugs were disintegrated, serially diluted and spotted on LB agar plates and incubated for enumeration of the surviving bacteria (A) and calculation of fold reduction mediated by the antibiotic treatment (B). Panel (A) shows averages and standard deviations of six replicates, and the significance (two-way ANOVA) of the difference between the CFU values of select groups are indicated by stars: *p < 0.05; ^****p < 0.0001. There was no significant difference between the log reduction values shown in panel (B).

FIGURE 8

Tolerance to tobramycin (A) and meropenem (B) of aggregates formed by the P. aeruginosa Epol–, mucAEpol–, and Alg+ strains. The respective P. aeruginosa strains were grown as agar-embedded aggregates for 16 h. Subsequently, agar plugs containing agar-embedded aggregates were obtained from the agar plates using a punch biopsy knife. The agar plugs were then treated for 3 h with either 15 μg/ml tobramycin (A) or 15 μg/ml meropenem (B), or saline. Next, the antibiotic was removed by a washing procedure and the agar plugs were disintegrated, serially diluted and spotted on LB agar plates and incubated for enumeration of the surviving bacteria and calculation of fold reduction mediated by the antibiotic treatment. The significance (one-way ANOVA) of the difference between the log reduction values of the Epol– strain and the other strains are indicated by stars: ^****p < 0.0001.