#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Antibacterial efficacy of cold atmospheric plasma against Enterococcus faecalis planktonic cultures and biofilms in vitro


Autoři: Felix Theinkom aff001;  Larissa Singer aff001;  Fabian Cieplik aff002;  Sylvia Cantzler aff003;  Hannes Weilemann aff003;  Maximilian Cantzler aff003;  Karl-Anton Hiller aff002;  Tim Maisch aff001;  Julia L. Zimmermann aff004
Působiště autorů: Department of Dermatology, University Hospital Regensburg, Regensburg, Germany aff001;  Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Regensburg, Germany aff002;  terraplasma GmbH, Garching, Germany aff003;  terraplasma medical GmbH, Garching, Germany aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pone.0223925

Souhrn

Nosocomial infections have become a serious threat in our times and are getting more difficult to handle due to increasing development of resistances in bacteria. In this light, cold atmospheric plasma (CAP), which is known to effectively inactivate microorganisms, may be a promising alternative for application in the fields of dentistry and dermatology. CAPs are partly ionised gases, which operate at low temperature and are composed of electrons, ions, excited atoms and molecules, reactive oxygen and nitrogen species. In this study, the effect of CAP generated from ambient air was investigated against Enterococcus faecalis, grown on agar plates or as biofilms cultured for up to 72 h. CAP reduced the colony forming units (CFU) on agar plates by > 7 log10 steps. Treatment of 24 h old biofilms of E. faecalis resulted in CFU-reductions by ≥ 3 log10 steps after CAP treatment for 5 min and by ≥ 5 log10 steps after CAP treatment for 10 min. In biofilm experiments, chlorhexidine (CHX) and UVC radiation served as positive controls and were only slightly more effective than CAP. There was no damage of cytoplasmic membranes upon CAP treatment as shown by spectrometric measurements for release of nucleic acids. Thus, membrane damage seems not to be the primary mechanism of action for CAP towards E. faecalis. Overall, CAP showed pronounced antimicrobial efficacy against E. faecalis on agar plates as well as in biofilms similar to positive controls CHX or UVC.

Klíčová slova:

Nucleic acids – Antimicrobials – Bacterial biofilms – Biofilms – Biofilm culture – Enterococcus faecalis – Ultraviolet C – Plasmas


Zdroje

1. O'Neill Jea. Tackling drug-resistant infections globally: final report and recommendations. 2016.

2. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis. 2009;48(1):1–12. Epub 2008/11/28. doi: 10.1086/595011 19035777.

3. Guiton PS, Hung CS, Hancock LE, Caparon MG, Hultgren SJ. Enterococcal biofilm formation and virulence in an optimized murine model of foreign body-associated urinary tract infections. Infect Immun. 2010;78(10):4166–75. Epub 2010/08/11. doi: 10.1128/IAI.00711-10 20696830; PubMed Central PMCID: PMC2950371.

4. Kajihara T, Nakamura S, Iwanaga N, Oshima K, Hirano K, Miyazaki T, et al. Comparative efficacies of daptomycin, vancomycin, and linezolid in experimental enterococcal peritonitis. J Infect Chemother. 2017;23(7):498–501. Epub 2017/01/22. doi: 10.1016/j.jiac.2016.12.002 28108098.

5. Fernandes SC, Dhanashree B. Drug resistance & virulence determinants in clinical isolates of Enterococcus species. Indian J Med Res. 2013;137(5):981–5. Epub 2013/06/14. 23760387; PubMed Central PMCID: PMC3734693.

6. Duggan JM, Sedgley CM. Biofilm formation of oral and endodontic Enterococcus faecalis. J Endod. 2007;33(7):815–8. Epub 2007/09/07. doi: 10.1016/j.joen.2007.02.016 17804318.

7. Mohamed JA, Huang DB. Biofilm formation by enterococci. J Med Microbiol. 2007;56(Pt 12):1581–8. Epub 2007/11/24. doi: 10.1099/jmm.0.47331-0 18033823.

8. Ch'ng JH, Chong KKL, Lam LN, Wong JJ, Kline KA. Biofilm-associated infection by enterococci. Nat Rev Microbiol. 2018. Epub 2018/10/20. doi: 10.1038/s41579-018-0107-z 30337708.

9. Marsh PD, Bradshaw DJ. Dental plaque as a biofilm. J Ind Microbiol. 1995;15(3):169–75. Epub 1995/09/01. 10.1007/bf01569822 8519474.

10. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet. 2001;358(9276):135–8. Epub 2001/07/21. 10.1016/s0140-6736(01)05321-1 11463434.

11. Cieplik F, Deng D, Crielaard W, Buchalla W, Hellwig E, Al-Ahmad A, et al. Antimicrobial photodynamic therapy—what we know and what we don't. Crit Rev Microbiol. 2018:1–19. Epub 2018/05/12. 10.1080/1040841X.2018.1467876 29749263.

12. Bush K, Courvalin P, Dantas G, Davies J, Eisenstein B, Huovinen P, et al. Tackling antibiotic resistance. Nat Rev Microbiol. 2011;9(12):894–6. Epub 2011/11/04. 10.1038/nrmicro2693 22048738; PubMed Central PMCID: PMC4206945.

13. Šimončicová J, Kryštofová S., Medvecká V. et al. Technical applications of plasma treatments: current state and perspectives. Appl Microbiol Biotechnol. 2019. https://doi.org/10.1007/s00253-019-09877-x.

14. Matthes R, Assadian O, Kramer A. Repeated applications of cold atmospheric pressure plasma does not induce resistance in Staphylococcus aureus embedded in biofilms. GMS Hyg Infect Control. 2014;9(3):Doc17. Epub 2014/10/07. 10.3205/dgkh000237 25285261; PubMed Central PMCID: PMC4184041.

15. Maisch T, Shimizu T, Isbary G, Heinlin J, Karrer S, Klampfl TG, et al. Contact-free inactivation of Candida albicans biofilms by cold atmospheric air plasma. Appl Environ Microbiol. 2012;78(12):4242–7. Epub 2012/04/03. 10.1128/AEM.07235-11 22467505; PubMed Central PMCID: PMC3370520.

16. Duan Y, Huang C, Yu QS. Cold plasma brush generated at atmospheric pressure. Rev Sci Instrum. 2007;78(1):015104. Epub 2007/05/17. 10.1063/1.2409624 17503943.

17. Weiss M, Daeschlein G, Kramer A, Burchardt M, Brucker S, Wallwiener D, et al. Virucide properties of cold atmospheric plasma for future clinical applications. J Med Virol. 2017;89(6):952–9. Epub 2016/10/04. 10.1002/jmv.24701 27696466.

18. Heinlin J, Maisch T, Zimmermann JL, Shimizu T, Holzmann T, Simon M, et al. Contact-free inactivation of Trichophyton rubrum and Microsporum canis by cold atmospheric plasma treatment. Future Microbiol. 2013;8(9):1097–106. Epub 2013/09/12. 10.2217/fmb.13.86 24020738.

19. Maisch T, Shimizu T, Li YF, Heinlin J, Karrer S, Morfill G, et al. Decolonisation of MRSA, S. aureus and E. coli by cold-atmospheric plasma using a porcine skin model in vitro. PLoS One. 2012;7(4):e34610. Epub 2012/05/05. 10.1371/journal.pone.0034610 22558091; PubMed Central PMCID: PMC3338731.

20. Hüfner A, Steffen H., Holtfreter B., Schlüter R., Duske K., Matthes R., von Woedtke T., Weltmann K., Kocher T. and Jablonowski L. Effects of Non‐Thermal Atmospheric Pressure Plasma and Sodium Hypochlorite Solution on Enterococcus faecalis Biofilm: An Investigation in Extracted Teeth. Wiley Online Library. 2017. 10.1002/ppap.201600064

21. Brun P, Brun P, Vono M, Venier P, Tarricone E, Deligianni V, et al. Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma. PLoS One. 2012;7(3):e33245. Epub 2012/03/21. 10.1371/journal.pone.0033245 22432007; PubMed Central PMCID: PMC3303808.

22. Isbary G, Morfill G, Schmidt HU, Georgi M, Ramrath K, Heinlin J, et al. A first prospective randomized controlled trial to decrease bacterial load using cold atmospheric argon plasma on chronic wounds in patients. Br J Dermatol. 2010;163(1):78–82. Epub 2010/03/13. 10.1111/j.1365-2133.2010.09744.x 20222930.

23. Rehman MU, Jawaid P, Uchiyama H, Kondo T. Comparison of free radicals formation induced by cold atmospheric plasma, ultrasound, and ionizing radiation. Arch Biochem Biophys. 2016;605:19–25. Epub 2016/04/18. 10.1016/j.abb.2016.04.005 27085689.

24. Li Y, Sun K, Ye G, Liang Y, Pan H, Wang G, et al. Evaluation of Cold Plasma Treatment and Safety in Disinfecting 3-week Root Canal Enterococcus faecalis Biofilm In Vitro. J Endod. 2015;41(8):1325–30. Epub 2015/06/02. 10.1016/j.joen.2014.10.020 26027875.

25. Mai-Prochnow A, Clauson M, Hong J, Murphy AB. Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma. Sci Rep. 2016;6:38610. Epub 2016/12/10. 10.1038/srep38610 27934958; PubMed Central PMCID: PMC5146927.

26. Ziuzina D, Han L, Cullen PJ, Bourke P. Cold plasma inactivation of internalised bacteria and biofilms for Salmonella enterica serovar Typhimurium, Listeria monocytogenes and Escherichia coli. Int J Food Microbiol. 2015;210:53–61. Epub 2015/06/22. 10.1016/j.ijfoodmicro.2015.05.019 26093991.

27. Rupf S, Idlibi AN, Marrawi FA, Hannig M, Schubert A, von Mueller L, et al. Removing biofilms from microstructured titanium ex vivo: a novel approach using atmospheric plasma technology. PLoS One. 2011;6(10):e25893. Epub 2011/10/22. 10.1371/journal.pone.0025893 22016784; PubMed Central PMCID: PMC3189945.

28. Habib M, Hottel T., & Hong L. Antimicrobial effects of non-thermal atmospheric plasma as a novel root canal disinfectant. 2014. 10.1016/j.cpme.2014.07.002

29. Maisch T, Shimizu T, Mitra A, Heinlin J, Karrer S, Li YF, et al. Contact-free cold atmospheric plasma treatment of Deinococcus radiodurans. J Ind Microbiol Biotechnol. 2012;39(9):1367–75. Epub 2012/05/16. 10.1007/s10295-012-1137-6 22584820.

30. Battista JR, Earl AM, Park MJ. Why is Deinococcus radiodurans so resistant to ionizing radiation? Trends Microbiol. 1999;7(9):362–5. Epub 1999/09/02. 10.1016/s0966-842x(99)01566-8 10470044.

31. Almeida A, Shao Y. Genome watch: Keeping tally in the microbiome. Nat Rev Microbiol. 2018;16(3):124. Epub 2018/01/31. 10.1038/nrmicro.2018.13 29379216.

32. Shimizu T, Lachner V, Zimmermann JL. Surface Microdischarge Plasma for Disinfection. 2017;7(2):175–85. 10.1615/PlasmaMed.2017019455

33. Cieplik F, Pummer A, Regensburger J, Hiller KA, Spath A, Tabenski L, et al. The impact of absorbed photons on antimicrobial photodynamic efficacy. Front Microbiol. 2015;6:706. Epub 2015/08/04. 10.3389/fmicb.2015.00706 26236292; PubMed Central PMCID: PMC4502582.

34. Cieplik F, Spath A, Regensburger J, Gollmer A, Tabenski L, Hiller KA, et al. Photodynamic biofilm inactivation by SAPYR—an exclusive singlet oxygen photosensitizer. Free Radic Biol Med. 2013;65:477–87. Epub 2013/07/31. 10.1016/j.freeradbiomed.2013.07.031 23891675.

35. Pratten J, Wills K, Barnett P, Wilson M. In vitro studies of the effect of antiseptic-containing mouthwashes on the formation and viability of Streptococcus sanguis biofilms. J Appl Microbiol. 1998;84(6):1149–55. Epub 1998/08/26. 10.1046/j.1365-2672.1998.00462.x 9717301.

36. Miles AA, Misra SS, Irwin JO. The estimation of the bactericidal power of the blood. J Hyg (Lond). 1938;38(6):732–49. Epub 1938/11/01. 10.1017/s002217240001158x 20475467; PubMed Central PMCID: PMC2199673.

37. Cieplik F, Steinwachs VS, Muehler D, Hiller KA, Thurnheer T, Belibasakis GN, et al. Phenalen-1-one-Mediated Antimicrobial Photodynamic Therapy: Antimicrobial Efficacy in a Periodontal Biofilm Model and Flow Cytometric Evaluation of Cytoplasmic Membrane Damage. Front Microbiol. 2018;9:688. Epub 2018/04/24. 10.3389/fmicb.2018.00688 29681899; PubMed Central PMCID: PMC5897782.

38. Chen CZ, Cooper SL. Interactions between dendrimer biocides and bacterial membranes. Biomaterials. 2002;23(16):3359–68. Epub 2002/07/09. 10.1016/s0142-9612(02)00036-4 12099278.

39. Boyce JM, Pittet D. Guideline for Hand Hygiene in Health-Care Settings: recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Infect Control Hosp Epidemiol. 2002;23(12 Suppl):S3–40. Epub 2003/01/08. 10.1086/503164 12515399.

40. Kollmuss M, Kist S, Obermeier K, Pelka AK, Hickel R, Huth KC. Antimicrobial effect of gaseous and aqueous ozone on caries pathogen microorganisms grown in biofilms. Am J Dent. 2014;27(3):134–8. Epub 2014/09/12. 25208360.

41. Walters J. How antibiotics work: protein synthesis. Prof Nurse. 1993;8(12):788–91. Epub 1993/09/01. 8367507.

42. Kapoor G, Saigal S, Elongavan A. Action and resistance mechanisms of antibiotics: A guide for clinicians. J Anaesthesiol Clin Pharmacol. 2017;33(3):300–5. Epub 2017/11/08. 10.4103/joacp.JOACP_349_15 29109626; PubMed Central PMCID: PMC5672523.

43. Becker S, Zimmermann JL, Baumeister P, Brunner TF, Shimizu T, Li YF, et al. Effects of cold atmospheric plasma (CAP) on bacteria and mucosa of the upper aerodigestive tract. Auris Nasus Larynx. 2019;46(2):294–301. Epub 2018/08/14. 10.1016/j.anl.2018.07.008 30098846.

44. Lis KA, Boulaaba A, Binder S, Li Y, Kehrenberg C, Zimmermann JL, et al. Inactivation of Salmonella Typhimurium and Listeria monocytogenes on ham with nonthermal atmospheric pressure plasma. PLoS One. 2018;13(5):e0197773. Epub 2018/05/26. 10.1371/journal.pone.0197773 29795627; PubMed Central PMCID: PMC5967798 GmbH. The scientific cooperation with Drs Zimmermann and Li with TiHo-Hannover dates back to the time when they were both employed at the Max Planck Institute for extraterrestrial Physics. Since their appointments at terraplasma GmbH (a spin-out start-up from the Max Planck Society) they have been given freedom to carry on with the basic research that was started earlier and is continuing. Their contribution to the current paper has been scientific—involving study design, analysis and interpretation and preparation of the manuscript. Ms Binder is a Bio-Engineer and helped in preparing the specialized plasma equipment used in the research and optimizing it for bactericidal and virucidal effects, without which the success of the study would not have been possible. The specialized plasma source was designed and manufactured at terraplasma and sold at cost price to TiHo-Hannover. terraplasma GmbH made no financial contribution other than continuing salary payments for (JZ, SyB and YFL). There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

45. Wende K, Bekeschus S, Schmidt A, Jatsch L, Hasse S, Weltmann KD, et al. Risk assessment of a cold argon plasma jet in respect to its mutagenicity. Mutat Res Genet Toxicol Environ Mutagen. 2016;798–799:48–54. Epub 2016/03/21. 10.1016/j.mrgentox.2016.02.003 26994493.

46. Yang Y, Guo J, Zhou X, Liu Z, Wang C, Wang K, et al. A novel cold atmospheric pressure air plasma jet for peri-implantitis treatment: An in vitro study. Dent Mater J. 2018;37(1):157–66. Epub 2017/11/28. 10.4012/dmj.2017-030 29176301.

47. Weiss M, Barz J, Ackermann M, Utz R, Ghoul A, Weltmann KD, et al. Dose-Dependent Tissue-Level Characterization of a Medical Atmospheric Pressure Argon Plasma Jet. ACS Appl Mater Interfaces. 2019;11(22):19841–53. Epub 2019/05/10. 10.1021/acsami.9b04803 31071258.

48. Gan L, Zhang S, Poorun D, Liu D, Lu X, He M, et al. Medical applications of nonthermal atmospheric pressure plasma in dermatology. J Dtsch Dermatol Ges. 2018;16(1):7–13. Epub 2017/12/07. 10.1111/ddg.13373 29211323.

49. Joaquin JC, Kwan C, Abramzon N, Vandervoort K, Brelles-Marino G. Is gas-discharge plasma a new solution to the old problem of biofilm inactivation? Microbiology. 2009;155(Pt 3):724–32. Epub 2009/02/28. 10.1099/mic.0.021501-0 19246743.

50. Laroussi M. Nonthermal decontamination of biological media by atmospheric-pressure plasmas: review, analysis, and prospects. IEEE Transactions on Plasma Science. 2002;30. 10.1109/TPS.2002.804220

51. Dezest M, Bulteau AL, Quinton D, Chavatte L, Le Bechec M, Cambus JP, et al. Oxidative modification and electrochemical inactivation of Escherichia coli upon cold atmospheric pressure plasma exposure. PLoS One. 2017;12(3):e0173618. Epub 2017/03/31. 10.1371/journal.pone.0173618 28358809; PubMed Central PMCID: PMC5373509.

52. Ziuzina D, Boehm D, Patil S, Cullen PJ, Bourke P. Cold Plasma Inactivation of Bacterial Biofilms and Reduction of Quorum Sensing Regulated Virulence Factors. PLoS One. 2015;10(9):e0138209. Epub 2015/09/22. 10.1371/journal.pone.0138209 26390435; PubMed Central PMCID: PMC4577073.

53. Ureyen Kaya B, Kececi AD, Guldas HE, Cetin ES, Ozturk T, Oksuz L, et al. Efficacy of endodontic applications of ozone and low-temperature atmospheric pressure plasma on root canals infected with Enterococcus faecalis. Lett Appl Microbiol. 2014;58(1):8–15. Epub 2013/08/29. 10.1111/lam.12148 23980743.

54. Jiang C, Schaudinn C, Jaramillo DE, Webster P, Costerton JW. In Vitro Antimicrobial Effect of a Cold Plasma Jet against Enterococcus faecalis Biofilms. ISRN Dent. 2012;2012:295736. Epub 2012/03/31. 10.5402/2012/295736 22461988; PubMed Central PMCID: PMC3302053.

55. Modic M, McLeod NP, Sutton JM, Walsh JL. Cold atmospheric pressure plasma elimination of clinically important single- and mixed-species biofilms. Int J Antimicrob Agents. 2017;49(3):375–8. Epub 2017/02/06. 10.1016/j.ijantimicag.2016.11.022 28161488.

56. Lis KA, Kehrenberg C, Boulaaba A, von Kockritz-Blickwede M, Binder S, Li Y, et al. Inactivation of multidrug-resistant pathogens and Yersinia enterocolitica with cold atmospheric-pressure plasma on stainless-steel surfaces. Int J Antimicrob Agents. 2018;52(6):811–8. Epub 2018/09/04. 10.1016/j.ijantimicag.2018.08.023 30176354.

57. Flynn PB, Higginbotham S, Alshraiedeh NH, Gorman SP, Graham WG, Gilmore BF. Bactericidal efficacy of atmospheric pressure non-thermal plasma (APNTP) against the ESKAPE pathogens. Int J Antimicrob Agents. 2015;46(1):101–7. Epub 2015/05/13. 10.1016/j.ijantimicag.2015.02.026 25963338.

58. Ballout H, Hertel M, Doehring J, Kostka E, Hartwig S, Paris S, et al. Effects of plasma jet, dielectric barrier discharge, photodynamic therapy and sodium hypochlorite on infected curved root canals. J Biophotonics. 2018;11(3). Epub 2017/10/13. 10.1002/jbio.201700186 29024574.

59. Armand A, Khani M, Asnaashari M, AliAhmadi A, Shokri B. Comparison study of root canal disinfection by cold plasma jet and photodynamic therapy. Photodiagnosis Photodyn Ther. 2019. Epub 2019/04/27. 10.1016/j.pdpdt.2019.04.023 31026615.

60. Du TF, Tang XZ, Shi Q, Gan K, Zhu JF, Cao YG. [Killing activity of nonequilibrium plasma against young and old Enterococcus faecalis biofilms with long-term exposure in infected root canals in vitro]. Zhonghua Kou Qiang Yi Xue Za Zhi. 2018;53(10):681–7. Epub 2018/11/06. 10.3760/cma.j.issn.1002-0098.2018.10.007 30392225.

61. Boxhammer V, Li YF, Koritzer J, Shimizu T, Maisch T, Thomas HM, et al. Investigation of the mutagenic potential of cold atmospheric plasma at bactericidal dosages. Mutat Res. 2013;753(1):23–8. Epub 2013/02/19. 10.1016/j.mrgentox.2012.12.015 23416235.

62. Maisch T, Bosserhoff AK, Unger P, Heider J, Shimizu T, Zimmermann JL, et al. Investigation of toxicity and mutagenicity of cold atmospheric argon plasma. Environ Mol Mutagen. 2017;58(3):172–7. Epub 2017/04/04. 10.1002/em.22086 28370324.

63. Herbst SR, Hertel M, Ballout H, Pierdzioch P, Weltmann KD, Wirtz HC, et al. Bactericidal Efficacy of Cold Plasma at Different Depths of Infected Root Canals In Vitro. Open Dent J. 2015;9:486–91. Epub 2015/01/01. 10.2174/1874210601509010486 26962378; PubMed Central PMCID: PMC4768658.

64. Han L, Patil S, Boehm D, Milosavljevic V, Cullen PJ, Bourke P. Mechanisms of Inactivation by High-Voltage Atmospheric Cold Plasma Differ for Escherichia coli and Staphylococcus aureus. Appl Environ Microbiol. 2016;82(2):450–8. Epub 2015/11/01. 10.1128/AEM.02660-15 26519396; PubMed Central PMCID: PMC4711144.

65. Zahradka K, Slade D, Bailone A, Sommer S, Averbeck D, Petranovic M, et al. Reassembly of shattered chromosomes in Deinococcus radiodurans. Nature. 2006;443(7111):569–73. Epub 2006/09/29. 10.1038/nature05160 17006450.

66. Cox MM, Battista JR. Deinococcus radiodurans—the consummate survivor. Nat Rev Microbiol. 2005;3(11):882–92. Epub 2005/11/02. 10.1038/nrmicro1264 16261171.

67. Arjunan KP, Sharma VK, Ptasinska S. Effects of atmospheric pressure plasmas on isolated and cellular DNA-a review. Int J Mol Sci. 2015;16(2):2971–3016. Epub 2015/02/03. 10.3390/ijms16022971 25642755; PubMed Central PMCID: PMC4346876.


Článok vyšiel v časopise

PLOS One


2019 Číslo 11
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#