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Isolation and characterization of five novel probiotic strains from Korean infant and children faeces


Autoři: Sun-Young Kook aff001;  Eui-Chun Chung aff001;  Yaelim Lee aff001;  Dong Wan Lee aff001;  Seokjin Kim aff001
Působiště autorů: R&D Institute, BioEleven Co., Seoul, Republic of Korea aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0223913

Souhrn

Probiotics are dietary supplements containing viable, non-pathogenic microorganisms that interact with the gastrointestinal microflora and directly with the immune system. The possible health effects of probiotics include modulating the immune system and exerting antibacterial, anticancer, and anti-mutagenic effects. The purpose of this study was to isolate, identify, and characterize novel strains of probiotics from the faeces of Korean infants. Various assays were conducted to determine the physiological features of candidate probiotic isolates, including Gram staining, 16S rRNA gene sequencing, tolerance assays to stimulated gastric juice and bile salts, adherence ability assays, antibiotic susceptibility testing, and assays of immunomodulatory effects. Based on these morphological and biochemical characteristics, five potential probiotic isolates (Enterococcus faecalis BioE EF71, Lactobacillus fermentum BioE LF11, Lactobacillus plantarum BioE LPL59, Lactobacillus paracasei BioE LP08, and Streptococcus thermophilus BioE ST107) were selected. E. faecalis BioE EF71 and L. plantarum BioE LPL59 showed high tolerance to stimulated gastric juice and bile salts, and S. thermophilus BioE ST107 as well as these two strains exhibited stronger adherence ability than reference strain Lactobacillus rhamnosus GG. All five strains inhibited secretion of lipopolysaccharide-induced pro-inflammatory cytokines IL-6 and TNF-α in RAW264.7 macrophages in vitro. L. fermentum BioE LF11, L. plantarum BioE LPL59, and S. thermophilus BioE ST107 enhanced the production of anti-inflammatory cytokine IL-10. Overall, our findings demonstrate that the five novel strains have potential as safe probiotics and encouraged varying degrees of immunomodulatory effects.

Klíčová slova:

Antibiotic resistance – Bile – Caco-2 cells – Cytokines – Enterococcus faecalis – Gastrointestinal tract – Lactobacillus – Probiotics


Zdroje

1. Guarner F, Schaafsma G. Probiotics. International journal of food microbiology. 1998;39(3):237–8. doi: 10.1016/s0168-1605(97)00136-0 9553803

2. Kleerebezem M, Hols P, Bernard E, Rolain T, Zhou M, Siezen RJ, et al. The extracellular biology of the lactobacilli. FEMS microbiology reviews. 2010;34(2):199–230. doi: 10.1111/j.1574-6976.2010.00208.x 20088967

3. Gill HS, Rutherfurd KJ, Prasad J, Gopal PK. Enhancement of natural and acquired immunity by Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019). British Journal of Nutrition. 2000;83(2):167–76. doi: 10.1017/s0007114500000210 10743496

4. Nikoskelainen S, Ouwehand AC, Bylund G, Salminen S, Lilius E-M. Immune enhancement in rainbow trout (Oncorhynchus mykiss) by potential probiotic bacteria (Lactobacillus rhamnosus). Fish & shellfish immunology. 2003;15(5):443–52.

5. Garde S, Gómez-Torres N, Delgado D, Gaya P, Ávila M. Influence of reuterin-producing Lactobacillus reuteri coupled with glycerol on biochemical, physical and sensory properties of semi-hard ewe milk cheese. Food Research International. 2016;90:177–85. doi: 10.1016/j.foodres.2016.10.046 29195870

6. Nomoto K. Prevention of infections by probiotics. Journal of bioscience and bioengineering. 2005;100(6):583–92. doi: 10.1263/jbb.100.583 16473765

7. Kleerebezem M, Vaughan EE. Probiotic and gut lactobacilli and bifidobacteria: molecular approaches to study diversity and activity. Annual review of microbiology. 2009;63:269–90. doi: 10.1146/annurev.micro.091208.073341 19575569

8. Minocha A. Probiotics for preventive health. Nutrition in Clinical Practice. 2009;24(2):227–41. doi: 10.1177/0884533608331177 19321897

9. Rijkers GT, Bengmark S, Enck P, Haller D, Herz U, Kalliomaki M, et al. Guidance for substantiating the evidence for beneficial effects of probiotics: current status and recommendations for future research. The Journal of nutrition. 2010;140(3):671S–6S. doi: 10.3945/jn.109.113779 20130080

10. Kalliomaki M, Antoine JM, Herz U, Rijkers GT, Wells JM, Mercenier A. Guidance for substantiating the evidence for beneficial effects of probiotics: prevention and management of allergic diseases by probiotics. J Nutr. 2010;140(3):713S–21S. Epub 2010/02/05. doi: 10.3945/jn.109.113761 20130079.

11. Habil N, Al-Murrani W, Beal J, Foey AD. Probiotic bacterial strains differentially modulate macrophage cytokine production in a strain-dependent and cell subset-specific manner. Benef Microbes. 2011;2(4):283–93. Epub 2011/12/08. doi: 10.3920/BM2011.0027 22146688.

12. Ouwehand AC, Salminen S, Isolauri E. Probiotics: an overview of beneficial effects. Antonie Van Leeuwenhoek. 2002;82(1–4):279–89. Epub 2002/10/09. 12369194.

13. Okada Y, Tsuzuki Y, Hokari R, Komoto S, Kurihara C, Kawaguchi A, et al. Anti-inflammatory effects of the genus Bifidobacterium on macrophages by modification of phospho-I kappaB and SOCS gene expression. Int J Exp Pathol. 2009;90(2):131–40. Epub 2009/04/02. doi: 10.1111/j.1365-2613.2008.00632.x 19335551; PubMed Central PMCID: PMC2676698.

14. Herias MV, Hessle C, Telemo E, Midtvedt T, Hanson LA, Wold AE. Immunomodulatory effects of Lactobacillus plantarum colonizing the intestine of gnotobiotic rats. Clin Exp Immunol. 1999;116(2):283–90. Epub 1999/05/26. doi: 10.1046/j.1365-2249.1999.00891.x 10337020; PubMed Central PMCID: PMC1905288.

15. Anderson RC, Ulluwishewa D, Young W, Ryan LJ, Henderson G, Meijerink M, et al. Human oral isolate Lactobacillus fermentum AGR1487 induces a pro-inflammatory response in germ-free rat colons. Scientific reports. 2016;6:20318. Epub 2016/02/05. doi: 10.1038/srep20318 26843130; PubMed Central PMCID: PMC4740858.

16. Shida K, Kiyoshima-Shibata J, Nagaoka M, Watanabe K, Nanno M. Induction of interleukin-12 by Lactobacillus strains having a rigid cell wall resistant to intracellular digestion. J Dairy Sci. 2006;89(9):3306–17. doi: 10.3168/jds.S0022-0302(06)72367-0 16899663

17. Weiss G, Schaible UE. Macrophage defense mechanisms against intracellular bacteria. Immunological reviews. 2015;264(1):182–203. Epub 2015/02/24. doi: 10.1111/imr.12266 25703560; PubMed Central PMCID: PMC4368383.

18. Mudter J, Neurath MF. Il-6 signaling in inflammatory bowel disease: pathophysiological role and clinical relevance. Inflammatory bowel diseases. 2007;13(8):1016–23. doi: 10.1002/ibd.20148 17476678

19. Rodríguez JM, Murphy K, Stanton C, Ross RP, Kober OI, Juge N, et al. The composition of the gut microbiota throughout life, with an emphasis on early life. Microbial ecology in health and disease. 2015;26(1):26050.

20. Kook S-Y, Kim Y, Kang B, Choe YH, Kim Y-H, Kim S. Characterization of the fecal microbiota differs between age groups in Koreans. Intestinal research. 2018;16(2):246. doi: 10.5217/ir.2018.16.2.246 29743837

21. Chung H, Kim Y, Chun S, Ji GE. Screening and selection of acid and bile resistant bifidobacteria. International journal of food microbiology. 1999;47(1–2):25–32. doi: 10.1016/s0168-1605(98)00180-9 10357270

22. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic acids research. 1997;25(24):4876–82. doi: 10.1093/nar/25.24.4876 9396791

23. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution. 2016;33(7):1870–4. doi: 10.1093/molbev/msw054 27004904

24. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution. 1987;4(4):406–25. doi: 10.1093/oxfordjournals.molbev.a040454 3447015

25. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39(4):783–91. doi: 10.1111/j.1558-5646.1985.tb00420.x 28561359

26. Charteris W, Kelly P, Morelli L, Collins J. Antibiotic susceptibility of potentially probiotic Bifidobacterium isolates from the human gastrointestinal tract. Letters in Applied Microbiology. 1998;26(5):333–7. doi: 10.1046/j.1472-765x.1998.00342.x 9674160

27. Korhonen JM, Sclivagnotis Y, von Wright A. Characterization of dominant cultivable lactobacilli and their antibiotic resistance profiles from faecal samples of weaning piglets. J Appl Microbiol. 2007;103(6):2496–503. Epub 2007/11/30. doi: 10.1111/j.1365-2672.2007.03483.x 18045434.

28. Chon H, Choi B, Lee E, Lee S, Jeong G. Immunomodulatory effects of specific bacterial components of Lactobacillus plantarum KFCC11389P on the murine macrophage cell line RAW 264.7. J Appl Microbiol. 2009;107(5):1588–97. Epub 2009/06/03. doi: 10.1111/j.1365-2672.2009.04343.x 19486216.

29. Penders J, Thijs C, Vink C, Stelma FF, Snijders B, Kummeling I, et al. Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics. 2006;118(2):511–21. doi: 10.1542/peds.2005-2824 16882802

30. Drisko JA, Giles CK, Bischoff BJ. Probiotics in health maintenance and disease prevention. Alternative Medicine Review. 2003;8(2):143–55. 12777160

31. Heilig HG, Zoetendal EG, Vaughan EE, Marteau P, Akkermans AD, de Vos WM. Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA. Appl Environ Microbiol. 2002;68(1):114–23. doi: 10.1128/AEM.68.1.114-123.2002 11772617

32. Rolain T, Bernard E, Courtin P, Bron PA, Kleerebezem M, Chapot-Chartier M-P, et al. Identification of key peptidoglycan hydrolases for morphogenesis, autolysis, and peptidoglycan composition of Lactobacillus plantarum WCFS1. Microbial cell factories. 2012;11(1):137.

33. van den Nieuwboer M, van Hemert S, Claassen E, de Vos WM. Lactobacillus plantarum WCFS 1 and its host interaction: a dozen years after the genome. Microbial biotechnology. 2016;9(4):452–65. doi: 10.1111/1751-7915.12368 27231133

34. Chen T, Yu WH, Izard J, Baranova OV, Lakshmanan A, Dewhirst FE. The Human Oral Microbiome Database: a web accessible resource for investigating oral microbe taxonomic and genomic information. Database (Oxford). 2010;2010:baq013. Epub 2010/07/14. doi: 10.1093/database/baq013 20624719; PubMed Central PMCID: PMC2911848.

35. Akinterinwa O, Khankal R, Cirino PC. Metabolic engineering for bioproduction of sugar alcohols. Curr Opin Biotechnol. 2008;19(5):461–7. Epub 2008/09/02. doi: 10.1016/j.copbio.2008.08.002 18760354.

36. Bezkorovainy A. Probiotics: determinants of survival and growth in the gut. Am J Clin Nutr. 2001;73(2 Suppl):399S–405S. Epub 2001/02/07. doi: 10.1093/ajcn/73.2.399s 11157348.

37. Charteris WP, Kelly PM, Morelli L, Collins JK. Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. J Appl Microbiol. 1998;84(5):759–68. Epub 1998/07/23. doi: 10.1046/j.1365-2672.1998.00407.x 9674129.

38. Succi M, Tremonte P, Reale A, Sorrentino E, Grazia L, Pacifico S, et al. Bile salt and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. FEMS microbiology letters. 2005;244(1):129–37. Epub 2005/02/25. doi: 10.1016/j.femsle.2005.01.037 15727832.

39. Dunne C, O'Mahony L, Murphy L, Thornton G, Morrissey D, O'Halloran S, et al. In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. Am J Clin Nutr. 2001;73(2 Suppl):386S–92S. Epub 2001/02/07. doi: 10.1093/ajcn/73.2.386s 11157346.

40. Mainville I, Arcand Y, Farnworth ER. A dynamic model that simulates the human upper gastrointestinal tract for the study of probiotics. Int J Food Microbiol. 2005;99(3):287–96. Epub 2005/04/06. doi: 10.1016/j.ijfoodmicro.2004.08.020 15808363.

41. Chou LS, Weimer B. Isolation and characterization of acid- and bile-tolerant isolates from strains of Lactobacillus acidophilus. J Dairy Sci. 1999;82(1):23–31. Epub 1999/02/18. doi: 10.3168/jds.S0022-0302(99)75204-5 10022003.

42. Rodriguez E, Arques JL, Rodriguez R, Nunez M, Medina M. Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits. Lett Appl Microbiol. 2003;37(3):259–63. Epub 2003/08/09. doi: 10.1046/j.1472-765x.2003.01390.x 12904230.

43. Burns P, Sanchez B, Vinderola G, Ruas-Madiedo P, Ruiz L, Margolles A, et al. Inside the adaptation process of Lactobacillus delbrueckii subsp. lactis to bile. Int J Food Microbiol. 2010;142(1–2):132–41. Epub 2010/07/14. doi: 10.1016/j.ijfoodmicro.2010.06.013 20621375.

44. Nagpal R, Kumar A, Kumar M, Behare PV, Jain S, Yadav H. Probiotics, their health benefits and applications for developing healthier foods: a review. FEMS microbiology letters. 2012;334(1):1–15. Epub 2012/05/10. doi: 10.1111/j.1574-6968.2012.02593.x 22568660.

45. Lee YK, Puong KY, Ouwehand AC, Salminen S. Displacement of bacterial pathogens from mucus and Caco-2 cell surface by lactobacilli. J Med Microbiol. 2003;52(Pt 10):925–30. Epub 2003/09/16. doi: 10.1099/jmm.0.05009-0 12972590.

46. Schiffrin EJ, Rochat F, Link-Amster H, Aeschlimann JM, Donnet-Hughes A. Immunomodulation of human blood cells following the ingestion of lactic acid bacteria. J Dairy Sci. 1995;78(3):491–7. Epub 1995/03/01. doi: 10.3168/jds.S0022-0302(95)76659-0 7782506.

47. Fogh J, Fogh JM, Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. Journal of the National Cancer Institute. 1977;59(1):221–6. Epub 1977/07/01. doi: 10.1093/jnci/59.1.221 327080.

48. Kozak K, Charbonneau D, Sanozky-Dawes R, Klaenhammer T. Characterization of bacterial isolates from the microbiota of mothers' breast milk and their infants. Gut microbes. 2015;6(6):341–51. Epub 2016/01/05. doi: 10.1080/19490976.2015.1103425 26727418; PubMed Central PMCID: PMC4826109.

49. Yu X, Avall-Jaaskelainen S, Koort J, Lindholm A, Rintahaka J, von Ossowski I, et al. A Comparative Characterization of Different Host-sourced Lactobacillus ruminis Strains and Their Adhesive, Inhibitory, and Immunomodulating Functions. Frontiers in microbiology. 2017;8:657. Epub 2017/04/30. doi: 10.3389/fmicb.2017.00657 28450859; PubMed Central PMCID: PMC5390032.

50. Kristo I, Pitiriga V, Poulou A, Zarkotou O, Kimouli M, Pournaras S, et al. Susceptibility patterns to extended-spectrum cephalosporins among Enterobacteriaceae harbouring extended-spectrum beta-lactamases using the updated Clinical and Laboratory Standards Institute interpretive criteria. Int J Antimicrob Agents. 2013;41(4):383–7. Epub 2013/02/05. doi: 10.1016/j.ijantimicag.2012.12.003 23375981.

51. Ammor MS, Florez AB, Mayo B. Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria. Food Microbiol. 2007;24(6):559–70. Epub 2007/04/10. doi: 10.1016/j.fm.2006.11.001 17418306.

52. Liu C, Zhang ZY, Dong K, Yuan JP, Guo XK. Antibiotic resistance of probiotic strains of lactic acid bacteria isolated from marketed foods and drugs. Biomedical and environmental sciences: BES. 2009;22(5):401–12. Epub 2010/02/19. doi: 10.1016/S0895-3988(10)60018-9 20163065.

53. Klein A, Friedrich U, Vogelsang H, Jahreis G. Lactobacillus acidophilus 74–2 and Bifidobacterium animalis subsp lactis DGCC 420 modulate unspecific cellular immune response in healthy adults. Eur J Clin Nutr. 2008;62(5):584–93. Epub 2007/04/19. doi: 10.1038/sj.ejcn.1602761 17440520.

54. Temmerman R, Pot B, Huys G, Swings J. Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Int J Food Microbiol. 2003;81(1):1–10. Epub 2002/11/09. doi: 10.1016/s0168-1605(02)00162-9 12423913.

55. Sanchez Valenzuela A, Lavilla Lerma L, Benomar N, Galvez A, Perez Pulido R, Abriouel H. Phenotypic and molecular antibiotic resistance profile of Enterococcus faecalis and Enterococcus faecium isolated from different traditional fermented foods. Foodborne Pathog Dis. 2013;10(2):143–9. Epub 2012/12/25. doi: 10.1089/fpd.2012.1279 23259502.

56. Donado-Godoy P, Castellanos R, Leon M, Arevalo A, Clavijo V, Bernal J, et al. The Establishment of the Colombian Integrated Program for Antimicrobial Resistance Surveillance (COIPARS): A Pilot Project on Poultry Farms, Slaughterhouses and Retail Market. Zoonoses Public Health. 2015;62 Suppl 1:58–69. Epub 2015/04/24. doi: 10.1111/zph.12192 25903494.

57. Seputiene V, Bogdaite A, Ruzauskas M, Suziedeliene E. Antibiotic resistance genes and virulence factors in Enterococcus faecium and Enterococcus faecalis from diseased farm animals: pigs, cattle and poultry. Pol J Vet Sci. 2012;15(3):431–8. Epub 2012/12/12. 23214361.

58. Plaza-Diaz J, Fernandez-Caballero JA, Chueca N, Garcia F, Gomez-Llorente C, Saez-Lara MJ, et al. Pyrosequencing analysis reveals changes in intestinal microbiota of healthy adults who received a daily dose of immunomodulatory probiotic strains. Nutrients. 2015;7(6):3999–4015. Epub 2015/05/29. doi: 10.3390/nu7063999 26016655; PubMed Central PMCID: PMC4488769.

59. Plaza-Diaz J, Gomez-Llorente C, Campana-Martin L, Matencio E, Ortuno I, Martinez-Silla R, et al. Safety and immunomodulatory effects of three probiotic strains isolated from the feces of breast-fed infants in healthy adults: SETOPROB study. PLoS One. 2013;8(10):e78111. Epub 2013/11/10. doi: 10.1371/journal.pone.0078111 24205115; PubMed Central PMCID: PMC3810271.

60. Makelainen H, Tahvonen R, Salminen S, Ouwehand AC. In vivo safety assessment of two Bifidobacterium longum strains. Microbiol Immunol. 2003;47(12):911–4. Epub 2003/12/26. doi: 10.1111/j.1348-0421.2003.tb03464.x 14695440.

61. Medzhitov R, Janeway CA Jr. Innate immunity: impact on the adaptive immune response. Curr Opin Immunol. 1997;9(1):4–9. Epub 1997/02/01. doi: 10.1016/s0952-7915(97)80152-5 9039775.

62. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity. 1999;11(4):443–51. Epub 1999/11/05. doi: 10.1016/s1074-7613(00)80119-3 10549626.

63. Diaz-Ropero MP, Martin R, Sierra S, Lara-Villoslada F, Rodriguez JM, Xaus J, et al. Two Lactobacillus strains, isolated from breast milk, differently modulate the immune response. J Appl Microbiol. 2007;102(2):337–43. Epub 2007/01/24. doi: 10.1111/j.1365-2672.2006.03102.x 17241338.

64. Wu Y, Zhu C, Chen Z, Chen Z, Zhang W, Ma X, et al. Protective effects of Lactobacillus plantarum on epithelial barrier disruption caused by enterotoxigenic Escherichia coli in intestinal porcine epithelial cells. Veterinary immunology and immunopathology. 2016;172:55–63. Epub 2016/04/02. doi: 10.1016/j.vetimm.2016.03.005 27032504.

65. Bermudez-Brito M, Munoz-Quezada S, Gomez-Llorente C, Matencio E, Bernal MJ, Romero F, et al. Human intestinal dendritic cells decrease cytokine release against Salmonella infection in the presence of Lactobacillus paracasei upon TLR activation. PLoS One. 2012;7(8):e43197. Epub 2012/08/21. doi: 10.1371/journal.pone.0043197 22905233; PubMed Central PMCID: PMC3419202.

66. Tremaroli V, Backhed F. Functional interactions between the gut microbiota and host metabolism. Nature. 2012;489(7415):242–9. Epub 2012/09/14. doi: 10.1038/nature11552 22972297.

67. Merly L, Smith SL. Murine RAW 264.7 cell line as an immune target: are we missing something? Immunopharmacology and immunotoxicology. 2017;39(2):55–8. doi: 10.1080/08923973.2017.1282511 28152640

68. J. F. PHYLIP (Phylogeny inference package), Version 3.5c. Distributed by the author. Seattle, USA: Department of Genome Sciences, University of Washington1993.

69. Fitch WM. Toward Defining the Course of Evolution: Minimum Change for a Specific Tree Topology. Systematic Biology. 1971;20(4):406–16. doi: 10.1093/sysbio/20.4.406

70. Felsenstein J. Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol. 1981;17(6):368–76. Epub 1981/01/01. doi: 10.1007/bf01734359 7288891.


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