Cold Plasma Inactivation of Bacterial Biofilms and Reduction of Quorum Sensing Regulated Virulence Factors

Abstract

The main objectives of this work were to investigate the effect of atmospheric cold plasma (ACP) against a range of microbial biofilms commonly implicated in foodborne and healthcare associated human infections and against P. aeruginosa quorum sensing (QS)-regulated virulence factors, such as pyocyanin, elastase (Las B) and biofilm formation capacity post-ACP treatment. The effect of processing factors, namely treatment time and mode of plasma exposure on antimicrobial activity of ACP were also examined. Antibiofilm activity was assessed for E. coli, L. monocytogenes and S. aureus in terms of reduction of culturability and retention of metabolic activity using colony count and XTT assays, respectively. All samples were treated 'inpack' using sealed polypropylene containers with a high voltage dielectric barrier discharge ACP generated at 80 kV for 0, 60, 120 and 300 s and a post treatment storage time of 24 h. According to colony counts, ACP treatment for 60 s reduced populations of E. coli to undetectable levels, whereas 300 s was necessary to significantly reduce populations of L. monocytogenes and S. aureus biofilms. The results obtained from XTT assay indicated possible induction of viable but non culturable state of bacteria. With respect to P. aeruginosa QS-related virulence factors, the production of pyocyanin was significantly inhibited after short treatment times, but reduction of elastase was notable only after 300 s and no reduction in actual biofilm formation was achieved post-ACP treatment. Importantly, reduction of virulence factors was associated with reduction of the cytotoxic effects of the bacterial supernatant on CHO-K1 cells, regardless of mode and duration of treatment. The results of this study point to ACP technology as an effective strategy for inactivation of established biofilms and may play an important role in attenuation of virulence of pathogenic bacteria. Further investigation is warranted to propose direct evidence for the inhibition of QS and mechanisms by which this may occur.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Experimental set up.

Either a microtiter plate containing bacterial biofilms or a petri dish containing P. aeruginosa cells TSB suspension or cell-free supernatant were placed inside the plastic container between the electrodes for direct ACP treatment. For indirect ACP treatment, a separate container was used and samples were placed outside the plasma discharge.

Fig 2. Surviving populations of bacterial biofilms after ACP treatment assessed by colony count assay.

(A) E. coli, (B) L. monocytogenes and (C) S. aureus: (Δ) untreated 24 h control, (◊) after direct and (□) indirect ACP treatment. Vertical bars represent standard deviation. Limit of detection 1.0 log10 CFU/ml. For each microorganism, all experiments were conducted in triplicate using independently grown cultures and replicated at least twice.

Fig 3. Percentage surviving populations of bacterial biofilms after ACP treatment assessed by XTT assay.

(A) E. coli, (B) L. monocytogenes and (C) S. aureus: (Δ) untreated 24 h control, (◊) after direct and (□) indirect ACP treatment. Vertical bars represent standard deviation. For each microorganism, all experiments were conducted in triplicate using independently grown cultures and replicated at least twice.

Fig 4. Scanning electron microscopy images of E. coli 48 h biofilms.

(A) untreated 0 h control (magnification 25 000X) and (B) after 300 s of direct ACP treatment (magnification 10 000X). Black arrows indicate cell debris; white arrows indicate intact cells.

Fig 5. ACP inhibition effects on P. aeruginosa virulence factors.

(A) pyocyanin in the presence of cells, (B) pyocyanin in the cell free medium (experiments repeated at least three times), (C) elastase Las B (experiments repeated at least five times), (D) planktonic cell concentration (experiments repeated at least six times) and (E) biofilm formation (experiments were performed in duplicate and replicated at least twice): (Δ) untreated 24 h controls, (◊) after direct and (□) indirect ACP treatment. Vertical bars represent standard deviation.

Fig 6. The effect of either untreated (0 h, 24 h controls) or ACP treated P. aeruginosa (P.a) and ACP treated TSB medium on growth/adherence of CHO-K1 cells.

Different letters indicate a significant difference in % absorbance levels. Vertical bars represent standard deviation. Experiments were performed in triplicate and replicated at least three times.