Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that can infect the lungs of cystic fibrosis (CF) patients and persist in the form of antibiotic-tolerant aggregates in the mucus. It has recently been suggested that such aggregates are formed due to restricted bacterial motility independent of the production of extracellular matrix components, and that they do not rely on an extracellular matrix for antimicrobial tolerance. However, we show here that biofilm matrix overexpression, as displayed by various clinical isolates, significantly protects P. aeruginosa aggregates against antimicrobial treatment. Alginate-overproducing mucA mutant bacteria growing in aggregates showed highly increased antibiotic tolerance compared to wild-type bacteria in aggregates. Deletion of algD in the mucA mutant strain abrogated alginate production and reversed the antibiotic tolerance displayed by the aggregates to a level similar to that observed for aggregates formed by the wild type. The P. aeruginosa ΔwspF and ΔyfiR mutant strains both overproduce Pel and Psl exopolysaccharide, and when these bacteria grew in aggregates, they showed highly increased antibiotic tolerance compared to wild-type bacteria growing in aggregates. However, the ΔwspF and ΔyfiR mutant strains, deficient in Pel/Psl production due to additional ΔpelA ΔpslBCD deletions, formed aggregates that displayed antibiotic tolerance levels close to those of wild-type aggregates. These results suggest that biofilm matrix components, such as alginate, Pel, and Psl, do play a role in the tolerance toward antimicrobials when bacteria grow as aggregates.

Keywords: aggregates; antimicrobial tolerance; biofilm; extracellular matrix.

FIG 1

Aggregate formation and survival of Pseudomonas aeruginosa bacterial aggregates with matrix component deletions (Δpel Δpsl Δalg). Single bacterial cells were encapsulated in 0.8% LB agar and allowed to grow for 24 h to form bacterial aggregates. (A) Micrographs of gel plugs of PAO1 and of ΔpelA ΔpslBCD ΔalgD mutant after overnight (o.n.) incubation in 0.8% LB agar. Each aggregate is approximately 300 to 500 μm. (B) Survival of PAO1 compared to ΔpelA ΔpslBCD ΔalgD mutant. No statistical differences in survival were observed between the two strains upon antimicrobial treatment. The gel plugs were incubated for 3 h in saline without antibiotic (purple bars) or with either 50 μg/ml tobramycin (tob; magenta bars) or 10 μg/ml ciprofloxacin (cip or cipro; white bars), and bacteria were released from the gel and enumerated by plating; n = 3 to 5.

FIG 2

Aggregate morphology and bacterial survival of alginate-overproducing Pseudomonas aeruginosa. (A) Micrographs of aggregates encased in LB agar for PAO1, mucA mutant, resulting in mucoid colonies, and mucA ΔalgD mutant nonmucoid colonies. (B) Survival of PAO1, mucA mutant, and mucA ΔalgD mutant aggregates upon antibiotic treatment with 50 μg/ml tobramycin (tobra) or 10 μg/ml ciprofloxacin (cipro) for 3 h, n > 4. For details, see Fig. 1 legend.

FIG 3

Aggregate morphology and bacterial survival of Pel/Psl-overproducing Pseudomonas aeruginosa. (A) Micrographs of aggregates of PAO1, ΔwspF mutant, and ΔwspF ΔpelA ΔpslBCD mutant. (B) Survival of PAO1, ΔwspF mutant, and ΔwspF ΔpelA ΔpslBCD mutant aggregates treated with 50 μg/ml tobramycin (tob) or 10 μg/ml ciprofloxacin (cip) for 3 h. For details, see the Fig. 1 legend.

FIG 4

Aggregate morphology and survival of Pseudomonas aeruginosa ΔyfiR knockout mutant strains overproducing Pel/Psl. (A) Micrographs of PAO1, ΔyfiR mutant, and ΔpelA ΔpslBCD ΔyfiR mutant aggregates. (B) Survival of ΔyfiR mutant compared to PAO1 after treatment with 50 μg/ml tobramycin (tob) or 10 μg/ml ciprofloxacin (cip or cipro) for 3 h. (C) The difference between the reductions in CFU after antimicrobial treatment is shown for the ΔyfiR mutant strain compared to PAO1; n = 8. (D) Survival of the ΔyfiR ΔpelA ΔpslBCD mutant strain compared to PAO1 after treatment with 50 μg/ml tobramycin or 10 μg/ml ciprofloxacin for 3 h; n = 3. For details, see the Fig. 1 legend.