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. 2024 Oct 3;12(10):e0092224.
doi: 10.1128/spectrum.00922-24. Epub 2024 Aug 28.

The role of exopolysaccharides Psl and Pel in resistance of Pseudomonas aeruginosa to the oxidative stressors sodium hypochlorite and hydrogen peroxide

Waleska S da Cruz Nizer  1 , Kira N Allison  1 , Madison E Adams  1 , Mario A Vargas  2 , Duale Ahmed  1 , Carole Beaulieu  1 , Deepa Raju  2 , Edana Cassol  1 , P Lynne Howell  2   3 , Joerg Overhage  1
Affiliations

The role of exopolysaccharides Psl and Pel in resistance of Pseudomonas aeruginosa to the oxidative stressors sodium hypochlorite and hydrogen peroxide

Waleska S da Cruz Nizer et al. Microbiol Spectr. .

Abstract

Pseudomonas aeruginosa is well-known for its antimicrobial resistance and the ability to survive in harsh environmental conditions due to an abundance of resistance mechanisms, including the formation of biofilms and the production of exopolysaccharides. Exopolysaccharides are among the major components of the extracellular matrix in biofilms and aggregates of P. aeruginosa. Although their contribution to antibiotic resistance has been previously shown, their roles in resistance to oxidative stressors remain largely elusive. Here, we studied the function of the exopolysaccharides Psl and Pel in the resistance of P. aeruginosa to the commonly used disinfectants and strong oxidizing agents NaOCl and H2O2. We observed that the simultaneous inactivation of Psl and Pel in P. aeruginosa PAO1 mutant strain ∆pslA pelF resulted in a significant increase in susceptibility to both NaOCl and H2O2. Further analyses revealed that Pel is more important for oxidative stress resistance in P. aeruginosa and that the form of Pel (i.e., cell-associated or cell-free) did not affect NaOCl susceptibility. Additionally, we show that Psl/Pel-negative strains are protected against oxidative stress in co-culture biofilms with P. aeruginosa PAO1 WT. Taken together, our results demonstrate that the EPS matrix and, more specifically, Pel exhibit protective functions against oxidative stressors such as NaOCl and H2O2 in P. aeruginosa.

Importance: Biofilms are microbial communities of cells embedded in a self-produced polymeric matrix composed of polysaccharides, proteins, lipids, and extracellular DNA. Biofilm bacteria have been shown to possess unique characteristics, including increased stress resistance and higher antimicrobial tolerance, leading to failures in bacterial eradication during chronic infections or in technical settings, including drinking and wastewater industries. Previous studies have shown that in addition to conferring structure and stability to biofilms, the polysaccharides Psl and Pel are also involved in antibiotic resistance. This work provides evidence that these biofilm matrix components also contribute to the resistance of Pseudomonas aeruginosa to oxidative stressors including the widely used disinfectant NaOCl. Understanding the mechanisms by which bacteria escape antimicrobial agents, including strong oxidants, is urgently needed in the fight against antimicrobial resistance and will help in developing new strategies to eliminate resistant strains in any environmental, industrial, and clinical setting.

Keywords: Pseudomonas aeruginosa; biofilms; exopolysaccharides; hydrogen peroxide; oxidative stress; reactive chlorine species; sodium hypochlorite.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Susceptibility of PAO1 WT and PAO1 ∆pslA, ∆pelF, and ∆pslA pelF to NaOCl. PAO1 WT and mutant strains were grown in polystyrene 96- or 12-well microplates for 24 h at 37°C in BM2 biofilm medium under static conditions and treated with NaOCl. (A) Susceptibility was evaluated by the MBC-B assay. (B) Fluorescence microscopy of PAO1 WT and ∆pslA, ∆pelF, and ∆pslA pelF mutant biofilms treated with 4 µg/mL and 8 µg/mL NaOCl. Biofilms were grown in flat-bottom polystyrene 96-well microplates, treated with NaOCl at 4 and 8 µg/mL NaOCl 1 h at 37°C under static conditions, and stained with DNA-intercalating 1:1 Syto9 and PI dyes and visualized by fluorescence microscopy at 20× magnification. Untreated biofilms were used as controls. Pictures are representative of three independent experiments with three replicates. Scale bars represent 125 µm. (C) Time kill kinetics of PAO1 WT, ∆pslA, ∆pelF, and ∆pslA pelF biofilms treated with 8 µg/mL NaOCl for 5, 15, 30, and 60 min. (D) Susceptibility of PAO1 WT and ∆pslA, ∆pelF, and ∆pslA pelF mutant strains of biofilms using the agar colony biofilm model. P. aeruginosa PAO1 WT, ∆pslA, ∆pelF, and ∆pslA pelF were grown overnight in LB media, harvested by centrifugation, washed twice with PBS, and the OD600nm was adjusted to 0.05 (5 × 107 CFU/mL). Ten microliters of bacterial culture were inoculated in the center of the semi-permeable membranes, and the plates were incubated for 48 h at 37°C to allow biofilm establishment. Colony biofilms were treated with NaOCl at 32 µg/mL, which were incubated for 1 h at 37°C and plated following the drop plate method. Data represent the mean ± standard deviation of at least three independent experiments. Data was analyzed by one-way ANOVA. *P < 0.05; ***P < 0.001; ****P < 0.0001. Uc: untreated control.
Fig 2
Fig 2
Susceptibility of PAO1 WT and PAO1 ∆pslA, ∆pelF, and ∆pslA pelF biofilms to H2O2. PAO1 WT and mutant strains were grown in polystyrene 96- or 12-well microplates for 24 h at 37°C in BM2 biofilm medium under static conditions and treated with H2O2. (A) Susceptibility was evaluated by the MBC-B assay. (B) Microscopy pictures of PAO1 WT and ∆pslA, ∆pelF, and ∆pslA pelF mutant biofilms treated with 6,250 µg/mL and 50,000 µg/ml H2O2. Biofilms were grown for 24 h at 37°C in BM2 biofilm medium in flat-bottom polystyrene 96-well microplates. H2O2 was diluted in BM2, and biofilms were treated for 1 h at 37°C under static conditions. Biofilms were stained with DNA-intercalating 1:1 Syto9 and PI dyes and visualized by fluorescence microscopy at 20× magnification. Untreated biofilms were used as the positive control. Pictures are representative of three independent experiments with three replicates each. Scale bars represent 125 µm. Data represent the mean ± standard deviation of at least three independent experiments. Data was analyzed by one-way ANOVA. **P < 0.01. Uc: untreated control.
Fig 3
Fig 3
Viability of (A) ∆pslA pelF and (B) E. faecalis cells after the treatment with NaOCl and H2O2 as monoculture or mixed culture. (A) CFU numbers of ∆pslA pelF (pUCP20) cells after NaOCl and H2O2 treatment of mono- and co-culture biofilms with PAO1 WT (pJN105). Mono- and co-culture biofilms were grown for 24 h at 37°C in BM2 biofilm medium in flat-bottom polystyrene 12-well microplates. Biofilms were washed and treated with NaOCl or H2O2 diluted in BM2 at 8 µg/mL and 6.25 mg/mL, respectively, and biofilms were treated for 1 h at 37°C under static conditions. Then, sodium thiosulfate at 10 mM was added to NaOCl-treated biofilms, and CFU was determined by the drop method. For PAO1 WT (pJN105) was selected in LB agar plates supplemented with 30 µg/mL gentamicin, and ∆pslA pelF (pUCP20) cells were selected in LB agar plates supplemented with 300 µg/mL carbenicillin. (B) CFU numbers of E. faecalis cells after the treatment with NaOCl and H2O2 as monoculture or mixed culture with PAO1 WT or ∆pslA pelF. Mono- and co-culture biofilms were grown for 24 h at 37°C in DMEM in flat-bottom polystyrene 12-well microplates. Biofilms were washed and treated with NaOCl or H2O2 diluted in BM2 at 8 µg/mL and 6.25 mg/mL, respectively, and biofilms were treated for 1 h at 37°C under static conditions. Then, sodium thiosulfate at 10 mM was added to NaOCl-treated biofilms, and CFU was determined by the drop method. P. aeruginosa was selected using BM2 agar plates, and E. faecalis was selected using LB plates supplemented with 5 µg/mL gentamicin and 12 µg/mL Polymyxin B. Data represent the mean ± standard deviation of at least three independent experiments. Data was analyzed by one-way ANOVA or t-test for comparison between two groups. *P < 0.05; ***P < 0.001.
Fig 4
Fig 4
Susceptibility of (A) PAO1 WT and PAO1 mutants ∆pslA and ∆pelF and (B) PAO1 ΔwspF Δpsl PBADpel biofilms to NaOCl. PAO1 WT and mutant strains were grown in polystyrene 12-well microplates for 24 h at 37°C in BM2 biofilm medium under static conditions and treated with NaOCl at 8, 12, or 16 µg/mL. Then, 10 mM sodium thiosulfate was added to quench the toxic effect of the remaining NaOCl, and CFU was determined by the drop method. Data represent the mean ± standard deviation of at least three independent experiments. Data were analyzed by one-way ANOVA or t-test for comparison between two groups. *P < 0.05; ****P < 0.0001.
Fig 5
Fig 5
Susceptibility of PA14 WT and PA14 mutants ∆pelA, ∆pelF, and PA14 pelAE218A biofilms to NaOCl. PAO1 WT and mutant strains were grown in polystyrene 96- or 12-well microplates for 24 h at 25°C in BM2 biofilm medium under static conditions and treated with NaOCl at 8 µg/mL. Then, 10 mM sodium thiosulfate was added to quench the toxic effect of the remaining NaOCl, and CFU was determined by the drop method. Data represent the mean ± standard deviation of at least three independent experiments. Data were analyzed by one-way ANOVA. *P < 0.05.
Fig 6
Fig 6
NaOCl susceptibility of PAO1 WT biofilms treated with the glycoside hydrolases PslGh and PelAh. PAO1 WT biofilms were grown in polystyrene 12-well microplates for 24 h at 37°C in BM2 biofilm medium under static conditions and treated with 2 µM of PslGh, PelAh, or a combination of PslGh and PelAh for 1 h at 25°C. Then, NaOCl at 12 µg/mL was added for 1 h, followed by 10 mM sodium thiosulfate to quench the toxic effect of the remaining NaOCl, and CFU was determined by the drop method. Heat-inactive enzymes were used as a control. Data represent the mean ± standard deviation of at least three independent experiments. Data were analyzed by one-way ANOVA.
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