Innovative non-thermal plasma disinfection process inside sealed bags: Assessment of bactericidal and sporicidal effectiveness in regard to current sterilization norms

Abstract

In this work, we developed a device capable to generate a non-thermal plasma discharge inside a sealed bag. The aim of this study was to assess the effectiveness of the oxygen, nitrogen and argon plasma sterilization on Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis spores according to the NF EN 556 Norm. Moreover the bag integrity which is a critical key to maintain the sterile state of items after the end of the process was verified by Fourier Transform Infrared (FTIR) and X-ray Photoelectron Spectrometry (XPS) analyses. After plasma treatments, the bacterial counting showed a 6 log reduction of P. aeruginosa and S. aureus in 45 min and 120 min respectively whatever the gas used and a 4 log reduction of B. subtilis spores in 120 min with only oxygen plasma. These results were confirmed by Scanning Electron Microscopy (SEM) observations showing altered bacteria or spores and numerous debris. Taking into account the studied microorganisms, the oxygen plasma treatment showed the highest efficiency. FTIR and XPS analyses showed that this treatment induced no significant modification of the bags. To conclude this non-thermal plasma sterilization technique could be an opportunity to sterilize heat and chemical-sensitive medical devices and to preserve their sterile state after the end of the process.

Fig 1. Plasma sterilization prototype device and creation of plasma inside sealed bags.

(A) The vacuum chamber was equipped with 2 pumps, a radio frequency polarization plate and two magnetic coils. (B) Creation of N₂- gas plasma inside a sealed bags.

Fig 2. Survival curves of P. aeruginosa exposed to N₂, O₂, Ar non-thermal plasma (25 W; 14 G and 0.5 sccm).

Each data represents the mean of data obtained from at least six independent experiments made in triplicate. Statistical analysis was performed by the Student's t test (**p<0.01).

Fig 3. Survival curves of S. aureus exposed to N₂, O₂, Ar non-thermal plasma (25 W; 14 G and 0.5 sccm).

Each data represents the mean of data obtained from at least six independent experiments made in triplicate. Statistical analysis was performed by the Student's t test (*p<0.05).

Fig 4. Survival curves of B. subtilis spores exposed to O₂ non-thermal plasma (100 W; 14 G and 1 sccm).

Each data represents the mean of data obtained from at least six independent experiments made in triplicate.

Fig 5. Scanning electron micrograph of P. aeruginosa before and after plasma treatments (25 W; 14 G and 0.5 sccm), (G x 7,500) (Jeol JSM 5400LV).

(A) Untreated. (B, E) After N₂ plasma treatment. (C, F) After Ar plasma treatment. (D, G) After O₂ plasma treatment.

Fig 6. Scanning electron micrograph of S. aureus before and after plasma treatments (25 W; 14 G and 0.5 sccm), (G x 7,500).

(A) Untreated. (B, E, H) After N₂ plasma treatment. (C, F, I). After Ar plasma treatment. (D, G, J). After O₂ plasma treatment.

Fig 7. Scanning electron micrograph of B. subtilis spores before and after O₂ gas plasma treatments (100 W; 14 G and 1 sccm), (G x 7,500 for the left column and G x 20,0000 for the right column).

(A-B) Untreated. (C-F) After O₂ plasma treatment.

Fig 8. ATR-FTIR spectra of the PE film for untreated and 120 min plasma treatment at 100 W of O₂, Ar and N₂.

Fig 9. XPS survey scans of the PE bag film surfaces for untreated and 120 min plasma treatment at 100 W with O₂, Ar and N₂.

Fig 10. Comparison of the deconvolution of the XPS core level C1s spectra for untreated and 120 min plasma treatment at 100 W with N₂ and O₂.

(A) Control. (B) Nitrogen.