Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Exploration of Virulence Markers and Genes of Listeria monocytogenes Isolated from Animal Products

축산물유래 Listeria monocytogenes의 virulence marker 및 gene 조사

  • Yi, Chul-Hyeon (National Veterinary Research and Quarantine Service) ;
  • Song, Hyeon-Ho (College of Veterinary Medicine, Cheju National University) ;
  • Kim, Mi-Ryung (College of Veterinary Medicine, Cheju National University) ;
  • Kang, Ho-Jo (College of Veterinary Medicine, Gyeongsang National University) ;
  • Son, Won-Geun (College of Veterinary Medicine, Cheju National University)
  • Published : 2008.09.30
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Abstract

To investigate the epidemiological characteristics of 68 Listeria monocytogenes isolates, including 11 reference strains and 57 isolates from imported US beef, domestic meats(beef, pork, chicken meat), raw milk, and milk plants. L. monocytogenes was to evaluate the production of virulence proteins, such as hemolysin(LLO) and lecithinase(LCP), the adsorption of Congo red(CRA), and to detect virulence genes using the polymerase chain reaction(PCR). In the study of virulence protein production, 68(100%), 62(91.2%), and 54(79.4%) of the 68 L. monocytogenes strains were positive for LLO production, the LCP test, and the CRA test, respectively, while strains of other species, such as L. innocua, L. gray, L. murrayi, and L. welshimeri, were not. There were no significant differences between L. monocytogenes serotypes and the ability to produce LLO or LCP. L. monocytogenesstrains had very high hemolytic titers(2 to 16 fold), while the other Listeria species, other than L. ivanovii and L. seeligeri, did not. The hemolysin activities of L. monocytogenes, L. ivanovii, and L. seeligeri usually exceeded 1.0 HU/mg, while those of other Listeria spp. were less than 0.04 HU/mg. In the PCR assay, all of the L. monocytogenes strains contained the hlyA, plcA, plcB, inlA, and inlB virulence genes and produced a product of the expected size. In the PCR of the actA gene, the expected 385-bp product was seen in 39(57.4%) L. monocytogenesstrains, while an unexpected 268-bp product was seen in 29(42.6%) strains. Most L. monocytogenes strains isolated from Hanwoo beef produced the 385-bp actA gene product, while strains of imported US beef usually produced the 268-bp actA gene product. By contrast, no virulence gene products were amplified in the other Listeria spp.

본 연구는 축산물 유래 L. monocytogenes에 대한 역학적 연구로서 분리균의 hemolysin(LLO) 및 lecithinase(LCP)생산성, Congo red dye(CRA)흡수성 및 hemolysin activity를 조사하는 한편 inlA, inlBV, actA, hlyA, plcA 및 plcB의 virulence gene을 PCR법으로 분석하였다. LLO, LCP 및 CRA의 양성률은 L. monocytogenes의 경우 68균주 중 각각 100%, 94.1% 및 77.9%이었고, L. ivanovii와 L. seeligeri를 제외한 다른 Listeria spp.(L. innocua, L. gray, L. murrayi, L. welchimeri)는 음성이었다. LLO와 LCP간에는 통계적인 유의성은 없었으나 CRA는 약간 낮게 나타났으며(p<0.05), serotype 1/2b 및 4b 간에도 유의성이 인정되지 않았다. 면양적혈구에 대한 용혈성(MHU)에서 L. monocytogenes의 경우 2배에서 16배까지 다양한 반응을 보였으나 L. ivanovii와 L. seeligeri를 제외한 다른 Listeria spp.는 음성이었다. hemolysin activity(HU)는 L. monocytogenes의 경우 대부분의 균주가 1.0 HU/mg 이상이었으나 다른 Listeria spp.는 대부분 0.04 HU/mg 이하였다. PCR 증폭하여 virulence gene을 분석한 결과 모든 L. monocytogenes는 각기 예상한 크기의 PCR 증폭산물이 검출되어 hlyA, plcA, plcB, inlA 및 inlB gene을 보유하고 있음이 확인되었으나 다른 Listeria spp.는 어떠한 증폭산물도 보이지 않았다. 또한 actA gene에 대한 증폭산물은 385bp와 268bp 크기의 2종류로 각각 57.4%와 42.6%의 분포를 나타내었다. actA gene의 size 분포에서 국내산 쇠고기, 닭고기, 유가공장에서는 큰 size가 많았는데 반하여 미국산 수입쇠고기에서는 작은 size가 많은 것으로 나타났다.

Keywords

References

  1. 강호조, 손원근, 강광식: 동물유래 생식품, 사료 및 분변중 Listeria monocytogenes의 분포와 분리균의 특성에 관한 연구 1. 원유, 우육, 계육 및 동물분변에서 Listeria monocytogenes의 분포, 한국수의공중보건학회지, 17, 131-137 (1991)
  2. Allerberger, F. and Fritschel, S.J.: Use of automated ribotyping of Austrian Listeria monocytogenes isolated to support epidemiological typing. J. Microbiol. Methods., 35, 237-244 (1999) https://doi.org/10.1016/S0167-7012(99)00025-1
  3. Alvarez-Domonguez, C., Vazquez-Boland, J.A., Carrasco-Marth, E., Lopez-Mato, P. and Leyva-Cobion, F.: Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the Listerial surface protein ActA is involved in heparan sulfate receptor recognition. Infect. Immun., 65, 78-88 (1997)
  4. Amoril, J.G. and Bhunia, A.K.: Immunological and cytopathogenic properties of Listeria monocytogenes isolated from naturally contaminated meats. J. Food Safety., 19, 195-207 (1999) https://doi.org/10.1111/j.1745-4565.1999.tb00245.x
  5. Asao, T., Kinoshita, Y., Kozaki, S., Uemura, T. and Sakaguchi, G.: Purification and some properties of Aeromonas hydrophilia hemolysin. Infect. Immun., 46, 122-127 (1984)
  6. Barclay, R., Threlfall, D.R. and Leighton, I.: Haemolysis and extracellular enzymes of Listeria monocytogenes and Listeria ivanovii, Med. Microbiol., 30, 111-118 (1989) https://doi.org/10.1099/00222615-30-2-111
  7. Bortolussi, R. and Schlech, W.F.: Listeriosis. In Infectious diseases of the fetus and newborn infant, 4th Ed. (Remington, J.S. and klein, O. eds.) Saunders, Philadelphia, pp. 1055-1073 (1995)
  8. Bubert, A., Hein, I., Rauch, M., Lehner, A., Yoon, B., Goebel, W. and Wagner, M.: Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR. Appl. Environ. Microbiol,. 65, 4688-4692 (1999)
  9. Ericsson. H., Stalhandske, P., Danielsson-Tham, M.L., Bannerman, E., Bille, J., Jacquet, C., Rocourt, J. and Tham, W.: Division of Listeria monocytogenes serovar 4b strains into two groups by PCR and restriction enzyme analysis. Appl. Environ. Microbiol., 61, 3872-3874 (1995)
  10. Farber, J.M. and Daley, E.: Presence and growth of Liateria monocytogenes in naturally-contaminated meats. International J. Food Microbiol., 22, 33-42 (1994) https://doi.org/10.1016/0168-1605(94)90005-1
  11. Farber, J.M. and Peterkin, P.I.: Listeria monocytogenes, a foodborne pathogen, Microbiol. Rev., 55, 752-811 (1991)
  12. Farber, J.M. and Speirs, J.I.: Potential use of continuous cell lines to distinguishbetween pathogenic and nonpathogenic Listeria spp. J. Clin. Microbiol., 25, 1463-1466 (1987)
  13. Fletcher, D. L., Baltey, J.S., and Cox, N.A.: Recovery and serotype distribution of Listeria monocytogenes from broiler chickens in the southeastern United States. J. Food Protect., 52, 148-150 (1989) https://doi.org/10.4315/0362-028X-52.3.148
  14. Francis, M. S. and C. J. Thomas.: The Listeria monocytogenes gene ctpA encodes a putative P-type ATPase involved in copper transport. Mol. Gen. Genet., 253, 484-491 (1997) https://doi.org/10.1007/s004380050347
  15. Geoffroy, C., Gaillard, J.L., Alouf, J.E. and Berche, P.: Purification, characterization, and toxicity of the sulfhydryl-activated hemolysin listeriolysin O from Listeria monocytogenes. Infect. immun., 55, 1641-1646 (1987)
  16. Geoffroy, C., Gaillard, J.L., Alouf, J.E., and Berche, P.: Production of thiol-dependent haemolysins by Listeria monocytogenes and related species. J. Gen. Microbiol., 135, 481-487(1989)
  17. Goldfine, H.: The functions of the phosphatidylinositol-specific phospholipase C (PI-PLC), and the brosd range phospholipase C (PC-PLC) in the pathogenesis of Listeria monocytogenes. In Proceedings of the XII International Symposium on problems of Listeriosis, Perth. Western Australia pp. 204-205 (1995)
  18. Heisick, J.E., Wagner, D.E., and Nierman, M. L.: Listeria spp. found on fresh market produce. Appl. Environ. Microbiol., 55, 1925-1927 (1989)
  19. Jacquet, C., Thierry, D., Veit, P., Guesdon, J.L. and Rocourt, J.: Evaluation of an rDNA Listeria probe for Listeria monocytogenes typing. APMIS., 107, 624-630 (1999) https://doi.org/10.1111/j.1699-0463.1999.tb01451.x
  20. Jaradat, Z.W., Schutze, G.E. and Bhunia, A.K.: Genetic homogeneity among Listeria monocytogenes strains from infected patients and meat products from two geographic locations determined by phenotyping, ribotyping and PCR analysis of virulence genes. Int. J. food Microbiol., 76, 1-10 (2002) https://doi.org/10.1016/S0168-1605(02)00050-8
  21. Liewen, M.B., and Plautz, M.W.: Occurrence of Listeria monocytogenes in raw milk in Nebraska. J. Food Prot., 51, 840-841 (1988) https://doi.org/10.4315/0362-028X-51.11.840
  22. Marshall, S., Clark, C.G., Wang, G., Mulvey, M., Kelly, M.T. and Johnson, W.M.: Comparison of molecular methods for typing Vibrio parahaemolyticus. J. Clin. Microbiol., 37, 2437-2478 (1999)
  23. McLauchlin, J. and Jones, D.: Erysipelothrix and Listeria. In Topley and Wilson's Microbiology and Microbial Infections, 9th Ed (Borriello, S.P. and Duerden. B.I. eds) Systematic Bacteriology, vol. 2. Update 1. CD Rom London, Amold. (1999)
  24. Newton, L., Hall, S.M., Pelerin, M. and McLauchlin, J.: Listeriosis survillance 1991. PHLS Commun. Dis. Rep., 2, 142-144 (1992)
  25. Norrung, B. and J. K. Andersen.: Variations in virulence between different electrophoretic types of Listeria monocytogenes. Lett. Appl. Microbiol., 30, 228-232 (2000) https://doi.org/10.1046/j.1472-765x.2000.00704.x
  26. Nunes, Z.G. and Hofer, E. Evaluation of phenotypic markers associated with pathogenicity in the genus Listeria. Rev. Inst. Med. Trop., 30, 293-299 (1994)
  27. Portnoy, D.A., Chakraborty, T., Goebel, W. and Cossart, P.: Molecular determinants of Listeria monocytogenes pathogenesis. Infect. Immun., 60, 1263-1267 (1992)
  28. Proctor, M.E., Brosch, R., Mellen, J.W., Garrett, L.A., Kasper, C.W. and Luchansky, J.B.: Use of pulsed-field gel electrophoresis to link sporadic cases of invasive listeriosis with recalled chocolate milk. Appl. Environ. Microbiol., 61, 3177-3179 (1995)
  29. Rasmussen, O.F., Beck, T., Olsen, J.E., Dons, L. and Rossen, L.: Listeria monocytogenes isolates can be classified into two major types according to the sequence of the listeriolysin gene. Infect. Immun., 59, 3945-3951 (1991)
  30. Sampathkumar, B., Xavier, I.J., Yu, L.S.L. and Khachatourians, G.G.: Production of listerilysin O by Listeria monocytogenes (Scott A) under heat-shock conditions. Int. J. Food Microbiol., 48, 131-137 (1999) https://doi.org/10.1016/S0168-1605(99)00037-9
  31. SAS.: The SAS system for window. Stastical Analysis System Institute Inc. Carg. NC, (1998)
  32. Schlech, W.F., Lavigne, P.M. and Bortolussi, R.A.: Epidemic listeriosis; evidence for transmission by food. N. Engl. J. Med., 308, 203-206 (1983) https://doi.org/10.1056/NEJM198301273080407
  33. Seeliger, H.P.R., Schrettenjbrunner, A., Pongratz, G. and Hof, H.: Special position of strongly hemolytic strains of genus Listeria. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Erste Abt. Orig. Reihe A Med. Mikrobiol. Parasitol., 252, 176-190 (1982)
  34. Seeliger, H.P.R. and Hohne, K.: Serotyping of Listeria monocytogenes and related species. Methoda Microbiol., 13, 31-49 (1979) https://doi.org/10.1016/S0580-9517(08)70372-6
  35. Silva, M.C.D., Destro, M.T., Hofer, E. and Tibana, A.: Characterization and evaluation of some virulence markers of Listeria monocytogenes strains isolated from Brazilian cheeses using molecular, biochemical and serotyping techniques. Int. J. Food Microbiol., 63, 275-280 (2001) https://doi.org/10.1016/S0168-1605(00)00426-8
  36. Smerdon, W.J., Jones, R., McLauchlin, J. and Reacher, M.: Surveillance of listeriosis in England and Wales 1995-1999. Commun. Dis. Public Health., 4, 188-193 (2001)
  37. Van Langendonck, N., Bottreau, E., Bailly, S., Tabouret, M., Marly, J., Pardon, P. and Velge, P.: Tissue culture assays using Caco-2 cell line differentiate virulent from non-virulent Listeria monocytogenes strains. J. Appl. Microbiol., 85, 337-346 (1998) https://doi.org/10.1046/j.1365-2672.1998.00515.x
  38. Vazquez-Boland, J.A., Dominguez, L., Rodriguez-Ferri, E.F., and Suarez, G.: Purification and characterization of two Listeria ivanovii cytolysin, a sphingmyelinas C and a thiolactivated toxin (ivanolysin O). Infect. Immun., 57, 3928-3935 (1989)
  39. Vazquez-Boland, J.A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W., Gonzalez-Zorn, B., Wehland, J. and Kreft, J.: Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev., 14, 584-640 (2001) https://doi.org/10.1128/CMR.14.3.584-640.2001
  40. Wiedmann, M., J.L., Bruce, C., Keating, A.E, Johnson, P.L., McDonough, and Batt, C.A.: Ribotypes and virulence gene polymorphism suggest three distinct Listeria monocytogenes lincages with differences in pathogenic potential. Infect. Immun., 65, 2707-2716 (1997)