Antimicrobial effect of low-temperature argon plasma on surgical infection in vitro

Introduction. Low-temperature argon plasma (LTAP) has been widely studied as an alternative approach to prevention of purulent infections in cases of reduced germicide effectiveness due to the developed pathogen resistance.

Objective. Survival assessment of opportunistic pathogens under the action of LTAP exposure in in vitro models.

Materials and methods. The study was carried out using ESKAPE clinical strains (a “superbug” group with a high epidemic potential for the formation of hospital strains) and reference strains from culture collections, as well as strain mixtures. A PLASMORAN plasma arc unit (Russia) was used as a LTAP source. One plasma generation mode, three distance variants (from the nozzle to the Petri dish culture plane — 10, 15, and 20 cm), four LTAP exposure options (15, 30, 45 s for bacteria and 30, 45, and 60 s for fungi) were used. Pathogens survival after LTAP exposure in vitro was assessed by bacterial growth inhibition.

Results. LTAP showed a significant antimicrobial action against clinical strains of ESKAPE Gram-negative bacteria K. pneumoniae, R. aeruginosa, A. baumanii, E. coli, Gram-positive bacteria MRSA and yeast-like fungi C. albicans under an exposure duration of 30–45 s for bacteria (the required dose of UV-A radiation is 37.8 J/m 2 UV-B 15.9 J/m² UV-C 34.2 J/m²) and a distance of 10–15 cm from the plasma generator nozzle. The antimicrobial effect is manifested in the absence of pathogen growth on culture media at the site of LTAP exposure at a certain exposure dose, duration, and distance. This effect ensures a decrease in the titer of viable microorganisms not only in monocultures, but also in bacterial associations, both in reference strains from culture collections and in ESKAPE clinical strains.

Conclusions. PLASMORAN-generated LTAP exhibits significant antibacterial and antifungal effects with respect to both reference strains from culture collections and ESKAPE clinical strains. The efficacy of LTAP action ensures a decrease in the titer of viable microorganisms from 10 8 –10 9  CFU to single CFU. The greatest effect of LTAP conditions (UV-A radiation dose of 37.8 J/m²; UV-B 15.9 J/m²; UV-C 34.2 J/m²) on bacterial cultures is observed under a distance of 10–15 cm and an exposure duration of 30–45 s. The optimal conditions for fungi (UV-A radiation dose of 168.6 J/m²; UV-B 68.4 J/m²; UV-C 159 J/m²) are a distance of 10 cm and an exposure duration of 60 s. Under standard dose, exposure duration, and distance, A. baumannii and E. faecium are more resistant to LTAP action than other studied bacteria. C. albicans is more resistant to LTAP action compared to bacteria, requiring a longer exposure and a shorter distance from the plasma generator nozzle to the treated surface. The results obtained require further study.

1. Brook I. Secondary bacterial infections complicating skin lesions. Journal of Medical Microbiology. 2002;51(10): 808–12. https://doi.org/10.1099/0022-1317-51-10-808

2. Mancuso G, Midiri A, Gerace E, Biondo C. Bacterial antibiotic resistance: the most critical pathogens. Pathogens. 2021;10(10):1310. https://doi.org/10.3390/pathogens10101310

3. Odonkor S, Addo K. Bacteria resistance to antibiotics: recent trends and challenges. International Journal of Biological & Medical Research. 2011;2(4):1204–10.

4. Brooks B, Brooks A. Therapeutic strategies to combat antibiotic resistance. Advanced Drug Delivery Reviews. 2014;78:14–27. https://doi.org/10.1016/j.addr.2014.10.027

5. Fair R, Tor Y. Antibiotics and bacterial resistance in the 21st century. Perspectives in Medicinal Chemistry. 2014;6:S14459. https://doi.org/10.4137/PMC.S14459

6. Strateva T, Yordanov D. Pseudomonas aeruginosa–a phenomenon of bacterial resistance. Journal of Medical Microbiology. 2009; 58(9):1133–48. https://doi.org/10.1099/jmm.0.009142-0

7. Vestby L, Gronseth T, Simm R, Nesse L. Bacterial biofilm and its role in the pathogenesis of disease. Antibiotics. 2020;9(2):59. https://doi.org/10.3390/antibiotics9020059

8. Prakash B, Veeregowda B, Krishnappa G. Biofilms: a survival strategy of bacteria. Current science. 2003;85:1299–307.

9. Bi Y, Xia G, Shi C, Wan J, Liu L, Chen Y, et al. Therapeutic strategies against bacterial biofilms. Fundamental Research. 2021;1(2):193–212. https://doi.org/10.1016/j.fmre.2021.02.003

10. Singh B, Ghosh S, Chauhan A. Development, dynamics and control of antimicrobial-resistant bacterial biofilms: a review. Environmental Chemistry Letters. 2021;19:1983–93. https://doi.org/10.1007/s10311-020-01169-5

11. Geisinger E, Isberg R. Interplay Between Antibiotic Resistance and Virulence During Disease Promoted by Multidrug-Resistant Bacteria. Journal of Infectious Diseases. 2017;215(1):9–17. https://doi.org/10.1093/infdis/jiw402

12. Pay GV, Rakitina DV, Sukhina MA, Yudin SM, Makarov VV, Maniya TR, et al. Study of the relationship between antibiotic resistance markers and virulence markers in NDM-positive Klebsiella pneumoniae strains circulating in various waters and human loci. Environmental Hygiene. 2021;100(12):1366–71 (In Russ.).

13. Venezia R, Orrico M, Houston E, Yin S, Naumova Y. Lethal activity of nonthermal plasma sterilization against microorganisms. Infection Control & Hospital Epidemiology. 2008;29(5):430–6. https://doi.org/10.1086/588003

14. Laroussi M. Cold plasma in medicine and healthcare: The new frontier in low temperature plasma applications. Frontiers in Physics. 2020;8:74. https://doi.org/10.3389/fphy.2020.00074

15. Karthik C, Sarngadharan S, Thomas V. Low-Temperature Plasma Techniques in Biomedical Applications and Therapeutics: An Overview. International Journal of Molecular Sciences. 2023;25(1):524. https://doi.org/10.3390/ijms25010524

16. Mohamed H, Nayak G, Rendine N, Wigdahl B, Krebs F, Bruggeman P, et al. Non-thermal plasma as a novel strategy for treating or preventing viral infection and associated disease. Frontiers in Physics. 2021;9:683118. https://doi.org/10.3389/fphy.2021.683118

17. Perucca M. Introduction to plasma and plasma technology. Plasma Technology for Hyperfunctional Surfaces: Food, Biomedical, and Textile Applications. Wiley-VCH Verlag GmbH & Co;2010:1–32. https://doi.org/10.1002/9783527630455.ch1

18. Hury S, Vidal D, Desor F, Pelletier J, Lagarde T. A parametric study of the destruction efficiency of Bacillus spores in low pressure oxygen-based plasmas. Letters in Applied Microbiology.1998;26(6):417–21. https://doi.org/10.1046/j.1472-765x.1998.00365.x

19. Lassen K, Nordby B, Grün R. The dependence of the sporicidal effects on the power and pressure of RF-generated plasma processes. Journal of Biomedical Materials Research Part B: Applied Biomaterials. 2005;74(1):553–9. https://doi.org/10.1002/jbm.b.30239

20. Shimizu T, Steffes B, Pompl R, Jamitzky F, Bunk W, Ramrath K, et al. Characterization of microwave plasma torch for decontamination. Plasma Processes and Polymers. 2008;5(6):577–82.

21. Yan D, Malyavko A, Wang Q, Ostrikov K, Sherman J, Keidar M. Multi-modal biological destruction by cold atmospheric plasma: capability and mechanism. Biomedicines. 2021;9(9):1259. https://doi.org/10.3390/biomedicines9091259

22. Lackmann J, Bandow J. Inactivation of microbes and macromolecules by atmospheric-pressure plasma jets. Applied Microbiology and Biotechnology.2014;98:6205–13. https://doi.org/10.1007/s00253-014-5781-9

23. Raji A, Vasu D, Pandiyaraj K, Ghobeira R, De Geyter N, Morent R, et al. Combinatorial effects of non-thermal plasma oxidation processes and photocatalytic activity on the inactivation of bacteria and degradation of toxic compounds in wastewater. RSC Advances. 2022;12(22):14246–59. https://doi.org/10.1039/d1ra09337a

24. Moreau M, Orange N, Feuilloley M. Non-thermal plasma technologies: new tools for bio-decontamination. Biotechnology Advances. 2008;26(6):610–7. https://doi.org/10.1016/j.biotechadv.2008.08.001

25. Sung S, Huh J, Yun M, Chang B, Jeong C, Jeon Y. Sterilization effect of atmospheric pressure non-thermal air plasma on dental instruments. The Journal of Advanced Prosthodontics. 2013;5(1):2–8. https://doi.org/10.4047/jap.2013.5.1.2

26. Busco G, Robert E, Chettouh-Hammas N, Pouvesle J, Grillon C. The emerging potential of cold atmospheric plasma in skin biology. Free Radical Biology and Medicine. 2020;161:290–304. https://doi.org/10.1016/j.freeradbiomed.2020.10.004

27. Lotfi M, Khani M, Shokri B. A review of cold atmospheric plasma applications in dermatology and aesthetics. Plasma Medicine. 2023;13:1. https://doi.org/10.1615/PlasmaMed.2023049359

28. Frolov SA, Kuzminov AM, Vyshegorodtsev DV, Korolik VYu, Tuktagulov NV, Zharkov EE, et al. Low-temperature argon plasma in the wounds treatment after hemorrhoidectomy. Koloproktologia. 2021;20(3):51–61 (In Russ.). https://doi.org/10.33878/2073-7556-2021-20-3-51-61

29. Shulutko AM, Osmanov ÉG, Novikova IV, Chochiya SL, Seredin VP, Macharadze AD. Plasma management in complex treatment of late inflammatory complications after injection contour plasty with polyacrylamide gel. Surgery. Journal named by Pirogov NI. 2017;9:59–63. https://doi.org/10.17116/hirurgia2017959-63

30. Pierdzioch P, Hartwig S, Herbst S, Raguse J, Dommisch H, Abu-Sirhan S, et al. Cold plasma: a novel approach to treat infected dentin–a combined ex vivo and in vitro study. Clinical Oral Investigations. 2016;20:2429–35. https://doi.org/10.1007/s00784-016-1723-5

31. Herbst S, Hertel M, Ballout H, Pierdzioch P, Weltmann K, Wirtz H, et al. Bactericidal efficacy of cold plasma at different depths of infected root canals in vitro. The Open Dentistry Journal. 2015;9:486. https://doi.org/10.2174/1874210601509010486

32. Scholtz V, Vaňková E, Kašparová P, Premanath R, Karunasagar I, Julák J. Non-thermal plasma treatment of ESKAPE pathogens: a review. Frontiers in Microbiology. 2021;12:737635. https://doi.org/10.3389/fmicb.2021.737635

33. Flynn P, Higginbotham S, Alshraiedeh N, Gorman S, Graham W, Gilmore B. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. International Journal of Antimicrobial Agents. 2015;46:101–7. https://doi.org/10.1016/j.ijantimicag.2015.02.026

34. Soušková H, Scholtz V, Julák J, Kommová L, Savická D, Pazlarová J. The survival of micromycetes and yeasts under the low-temperature plasma generated in electrical discharge. Folia Microbiologica. 2011;56:77–9. https://doi.org/10.1007/s12223-011-0005-5

35. Veerana M, Ketya W, Park G. Application of non-thermal plasma to fungal resources. Journal of Fungi. 2022,8(2):102. https://doi.org/10.3390/jof8020102

36. Tyczkowska-Sieroń E, Kałużewski T, Grabiec M, Kałużewski B, Tyczkowski J. Genotypic and phenotypic changes in Candida albicans as a result of cold plasma treatment. International Journal of Molecule Science. 2020;21:8100. https://doi.org/10.3390/ijms21218100