جداسازی و شناسایی سویه‌های لاکتوکوکوس لاکتیس تولید کننده نایسین از پنیر لیقوان
محورهای موضوعی : علوم و صنایع غذایی
1 - 1. گروه بهداشت مواد غذایی و آبزیان، دانشکده دامپزشکی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران
2. مرکز تحقیقات بیوتکنولوژی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران
کلید واژه: باکتریوسین, نایسین, پنیر لیقوان, لاکتوکوکوس لاکتیس, نگهدارنده بیولوژیکی,
چکیده مقاله :
در این مطالعه سویه هایی از باکتری های اسید لاکتیک با قابلیت تولید باکتریوسین از نمونه های پنیر لیقوان جداسازی شدند. در ابتدا از 16 جدایه لاکتوکوکوس لاکتیس شناسایی شده، چهار جدایه از نظر فنوتیپی به‌عنوان تولید کننده باکتریوسین تعیین شدند. جدایه ها با روش لکه‌گذاری روی آگار بر رشد باکتری های گرم مثبت بیماریزای غذایی شامل استافیلوکوکوس اورئوس، باسیلوس سروئوس، کلستریدیوم پرفرینجنس و لیستریا مونوسیتوجنز اثر بازدارندگی نشان دادند. در جدایه های انتخاب شده واکنش زنجیره ای پلیمراز برای ژن کد کننده نایسین مثبت بود. سویه ها مواد ضدباکتریایی مقاوم به آنزیم آلفا آمیلاز اما حساس به تریپسین و پروتئیناز K تولید کردند. مقاومت حرارتی با تحمل 100 درجه سلسیوس به‌مدت 30 دقیقه مشخص شد. فعالیت ضدباکتریایی وابسته به pH بود و در pH 3 و 6 مشاهده شد اما در pH 9 تا حد زیادی کاهش یافت. پس از تیمار با SDS، Tween 20، Tween 80، Triton X-100، اوره و EDTA هیچ کاهشی در فعالیت ضدباکتریایی مشاهده نشد. طبق نتایج، این جدایه ها قابلیت استفاده در مواد غذایی به‌عنوان نگهدارنده بیولوژیکی را دارند.
چکیده انگلیسی:
In this study, strains of lactic acid bacteria capable of bacteriocin production were isolated from Lighvan cheese samples obtained from East Azerbaijan, Iran. Among 16 isolates initially identified as Lactococcus lactis, four isolates were phenotipically determined as bacteriocinogenic. The isolates showed inhibitory effect on the growth of Gram-positive food borne pathogens including Staphylococcus aureus, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes by the agar spot test. Selected isolates were PCR-positive for nisin-encoding gene. The strains produced antibacterial substances resistant to α-amylase but sensitive to trypsin and proteinase K. Thermo-stability was indicated after treatment at 100 °C for 30 minutes. The antibacterial activity was pH dependent and occurred in pH 3 and 6, but at pH 9 the antibacterial activity was mainly restricted. No loss in antibacterial activity was recorded after treatment with SDS, Tween20, Tween 80, Triton X-100, urea and EDTA. The results suggest the potential application of these isolates as biopreservatives in food products.
منابع و مأخذ:
· Alegría, A., Delgado, S., Roces, C., López, B. and Mayo, B. (2010). Bacteriocins produced by wild Lactococcus lactis strains isolated from traditional, starter-free cheeses made of raw milk. International Journal of Food Microbiology, 143(1), pp. 61–66.
· Ammor, S., Tauveron, G., Dufour, E. and Chevallier, I. (2006). Antibacterial activity of lactic acid bacteria against spoilage and pathogenic bacteria isolated from the same meat small-scale facility: 1-Screening and characterization of the antibacterial compounds. Food control, 17(6), pp. 454–461.
· Atrih, A., Rekhif, N., Milliere, J.B. and Lefebvre, G. (1993). Detection and characterization of a bacteriocin produced by Lactobacillus plantarum C19. Canadian Journal of Microbiology, 39(12), pp. 1173–1179.
· Bari, M.L., Ukuku, D.O., Kawasaki, T., Inatsu, Y., Isshiki, K. and Kawamoto, S. (2005). Combined efficacy of nisin and pediocin with sodium lactate, citric acid, phytic acid, and potassium sorbate and EDTA in reducing the Listeria monocytogenes population of inoculated fresh-cut produce. Journal of Food Protection, 68(7), pp.1381–1387.
· Bello, B. D., Rantsiou, K., Bellio, A., Zeppa, G., Ambrosoli, R., Civera, T., et al. (2010). Microbial ecology of artisanal products from North West of Italy and antimicrobial activity of the autochthonous populations. LWT e Food Science and Technology, 43, pp. 1151–1159.
· Bergogne-Bérézin, E. and Joly-Guillou, M.L. (1991). Hospital infection with Acinetobacter spp.: an increasing problem. Journal of Hospital Infection, 18, pp. 250–255.
· Bergogne-Bérézin, E. (1995). Outbreaks caused by bacteria with novel multiple resistance: towards zero therapeutical options? The increased significance of outbreaks of Acinetobacter spp.: the need for control and new agents. Journal of Hospital Infection, 30, pp. 441–452.
· Bromberg, R., Moreno, I., Delboni, R.R., Cintra, H.C. and Oliveira, P.T.V. (2005). Characteristics of the bacteriocin produced by Lactococcus lactis subsp. cremoris CTC 204 and the effect of this compound on the mesophilic bacteria associated with raw beef. World Journal of Microbiology and Biotechnology, 21(3), pp. 351–358.
· Brewer, R., Adams, M.R. and Park, S.F. (2002). Enhanced inactivation of Listeria monocytogenes by nisin in the presence of ethanol. Letters in applied microbiology, 34(1), pp.18–21.
· Corroler, D., Mangin, N., Desmasures, N., Gueguen, M. (1998). An ecological study of lactococci isolated from raw milk in the Camembert cheese registered designation of origin area. Applied and Environmental Microbiology, 64 (12), pp. 4729–4735.
· Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.H. and Stackebrandt, E. (2006). The Prokaryotes: Bacteria: Firmicutes, Cyanobacteria. 4: 212.
· Forde, A. and Fitzgerald, G.F., 1999. Bacteriophage defence systems in lactic acid bacteria. In: Lactic Acid Bacteria: Genetics, Metabolism and Applications (pp. 89–113). Springer Netherlands.
· Gálvez, A., Valdivia, E., Abriouel, H., Camafeita, E., Mendez, E., Martínez-Bueno, M. and Maqueda, M. (1998). Isolation and characterization of enterocin EJ97, a bacteriocin produced by Enterococcus faecalis EJ97. Archives of microbiology, 171(1), pp. 59–65.
· Gaya, P., Babin, M., Medina, M., Nunez, M. (1999). Diversity among lactococci isolated from ewe's raw milk cheese. Journal of AppliedMicrobiology, 87(6), pp. 849–855.
· Hanifian, S., and Khani, S. (2012). Fate of Yersinia enterocolitica during manufacture, ripening and storage of Lighvan cheese. International Journal of Food Microbiology, 156(2), pp. 141–146.
· Huot E., Maghrous J., Barena-Gonzalez, C. (1996). Bacteriocin J46, a new bacteriocin produced by Lactococcus lactis subsp. cremoris J46: isolation and characterization of the protein and its gene. Anaerobe, 2(3): pp. 137–145.
· Hyink, O., Balakrishnan, M., and Tagg, J.R. (2005). Streptococcus rattus strain BHT produces both a class I two-component lantibiotic and a class II bacteriocin. FEMS Microbiology Letters, 252(2), pp. 235–241.
· Ivanova, I., Kabadjova, P., Pantev, A., Danova, S. and Dousset, X. (2000). Detection, purification and partial characterization of a novel bacteriocin substance produced by Lactococcus lactis subsp. lactis B14 isolated from boza-Bulgarian traditional cereal beverage. Biocatalysis, 41(6), pp. 47–53.
· Kelly, W.J., Asmundson, R.V. and Huang, C.M., 1996. Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum. Journal of Applied Bacteriology, 81(6), pp. 657–662.
· Klaenhammer, T.R., 1988. Bacteriocins of lactic acid bacteria. Biochimie, 70(3), pp. 337–349.
· Kruger, M.F., de Souza Barbosa, M., Miranda, A., Landgraf, M., Destro, M.T., Todorov, S.D. and de Melo Franco, B.D.G. (2013). Isolation of bacteriocinogenic strain of Lactococcus lactis subsp. lactis from Rocket salad (Eruca sativa Mill.) and evidences of production of a variant of nisin with modification in the leader-peptide. Food control, 33(2), pp. 467–476.
· Lee, N.K. and Paik, H.D. (2001). Partial characterization of lacticin NK24, a newly identified bacteriocin of Lactococcus lactis NK24 isolated from Jeot-gal. Food Microbiology, 18(1), pp. 17–24.
· López-Pedemonte, T.J., Roig-Sagués, A.X., Trujillo, A.J., Capellas, M. and Guamis, B. (2003). Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme. Journal of dairy science, 86(10), pp. 3075–3081.
· Marshall, V.M. (1991). Inoculated ecosystems in milk environments. Journal ofApplied Bacteriology, 73, pp. 127–135.
· Mannu, L., Paba, A. (2002). Genetic diversity of lactococci and enterococci isolated from home-made Pecorino Sardo ewe's milk cheese. Journal ofApplied Microbiology, 92(1), pp. 55–62.
· Mead, P. S., Slutsker, L., Dietz, V., McCaige, L. F., Bresee, J. S., Shapiro, C., Griffin, P. M. and Tauxe, R. V. (1999). Food related illness and death in the United States. Emerging Infectious Diseases 5, pp. 607–625.
· Millette, M., Luquet, F.M. and Lacroix, M. (2007). In vitro growth control of selected pathogens by Lactobacillus acidophilus and Lactobacillus casei fermented milk. Letters in applied microbiology, 44(3), pp. 314–319.
· Mirdamadi, S. and Ghazvini, S.A., 2015. A comparative study between inhibitory effect of L. lactis and nisin on important pathogenic bacteria in Iranian UF Feta cheese. Biological Journal of Microorganism, 3(12).
· Noonpakdee, W., Santivarangkna, C., Jumriangrit, P., Sonomoto, K. and Panyim, S. (2003). Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham, a traditional Thai fermented sausage. International Journal of Food Microbiology, 81(2), pp. 137–145.
· Nwuche, C.O., 2013. Isolation of bacteriocin-producing lactic acid bacteria from'Ugba'and'Okpiye', two locally fermented nigerian food condiments. Brazilian Archives of Biology and Technology, 56(1), pp. 101–106.
· Perin, L.M., Moraes, P.M., Viçosa, G.N., Júnior, A.S. and Nero, L.A. (2012). Identification of bacteriocinogenic Lactococcus isolates from raw milk and cheese capable of producing nisin A and nisin Z. International Dairy Journal, 25(1), pp. 46–51.
· Rodríguez, E., González, B., Gaya, P., Nuñez, M. and Medina, M. (2000). Diversity of bacteriocins produced by lactic acid bacteria isolated from raw milk. International Dairy Journal, 10(1), pp. 07–15.
· Rodríguez, J. M., Cintas, L. M., Casaus, P., Horn, N., Doddl, H. M. and Hernandez, P. E. (1995). Isolation of nisin-producing Lactococcus lactis strains from dry fermented sausages. Journal of Applied Bacteriology, 78(2), pp. 109–115.
· Sánchez, M.M., Delgado, T., Alonso, L. and Mayo, B. (2000). Phenotypic and genetic characterization of a selected set of Lactococcus lactis strains isolated from a starter-free farmhouse cheese. Food microbiology, 17(4), pp. 449–460.
· Sobrino-López, Á. and Martín-Belloso, O. (2006). Enhancing inactivation of Staphylococcus aureus in skim milk by combining high-intensity pulsed electric fields and nisin. Journal of Food Protection, 69(2), pp. 345–353.
· Tafreshi, S. H., Mirdamadi, S., Norouzian, D., Khatami, S. and Sardari, S. (2010). Effect on non-nutritional factors on nisin production. Journal of Biotechnology, 9(9), pp. 1382–1391.
· Todorov, S., Onno, B., Sorokine, O., Chobert, J.M., Ivanova, I. and Dousset, X. (1999). Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST 31 isolated from sourdough. International Journal of Food Microbiology, 48(3), pp. 167–177.
· Todorov, S.D. and Dicks, L.M. (2005). Pediocin ST18, an anti-listerial bacteriocin produced by Pediococcus pentosaceus ST18 isolated from boza, a traditional cereal beverage from Bulgaria. Process Biochemistry, 40(1), pp. 365–370.
· Todorov, S.D. (2008). Bacteriocin production by Lactobacillus plantarum AMA-K isolated from Amasi, a Zimbabwean fermented milk product and study of the adsorption of bacteriocin AMA-K to Listeria sp. Brazilian Journal of microbiology, 39(1), pp. 178–187.
· Topisirovic, L., Kojic, M., Fira, D., Golic, N., Strahinic, I. and Lozo, J. (2006). Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation. International Journal of Food Microbiology, 112(3), pp. 230–235.
· Wirawan, R. E., Klesse, N. A., Jack, R. W. and Tagg, J. R. (2006). Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis. Applied and Environmental Microbiology, 72(2), pp. 1148–1156.
· Wise, K.A. and Tosolini, F.A. (1990). Epidemiological surveillance of Acinetobacter species. Journal of Hospital Infection, 16(4), pp. 319–329.
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· Alegría, A., Delgado, S., Roces, C., López, B. and Mayo, B. (2010). Bacteriocins produced by wild Lactococcus lactis strains isolated from traditional, starter-free cheeses made of raw milk. International Journal of Food Microbiology, 143(1), pp. 61–66.
· Ammor, S., Tauveron, G., Dufour, E. and Chevallier, I. (2006). Antibacterial activity of lactic acid bacteria against spoilage and pathogenic bacteria isolated from the same meat small-scale facility: 1-Screening and characterization of the antibacterial compounds. Food control, 17(6), pp. 454–461.
· Atrih, A., Rekhif, N., Milliere, J.B. and Lefebvre, G. (1993). Detection and characterization of a bacteriocin produced by Lactobacillus plantarum C19. Canadian Journal of Microbiology, 39(12), pp. 1173–1179.
· Bari, M.L., Ukuku, D.O., Kawasaki, T., Inatsu, Y., Isshiki, K. and Kawamoto, S. (2005). Combined efficacy of nisin and pediocin with sodium lactate, citric acid, phytic acid, and potassium sorbate and EDTA in reducing the Listeria monocytogenes population of inoculated fresh-cut produce. Journal of Food Protection, 68(7), pp.1381–1387.
· Bello, B. D., Rantsiou, K., Bellio, A., Zeppa, G., Ambrosoli, R., Civera, T., et al. (2010). Microbial ecology of artisanal products from North West of Italy and antimicrobial activity of the autochthonous populations. LWT e Food Science and Technology, 43, pp. 1151–1159.
· Bergogne-Bérézin, E. and Joly-Guillou, M.L. (1991). Hospital infection with Acinetobacter spp.: an increasing problem. Journal of Hospital Infection, 18, pp. 250–255.
· Bergogne-Bérézin, E. (1995). Outbreaks caused by bacteria with novel multiple resistance: towards zero therapeutical options? The increased significance of outbreaks of Acinetobacter spp.: the need for control and new agents. Journal of Hospital Infection, 30, pp. 441–452.
· Bromberg, R., Moreno, I., Delboni, R.R., Cintra, H.C. and Oliveira, P.T.V. (2005). Characteristics of the bacteriocin produced by Lactococcus lactis subsp. cremoris CTC 204 and the effect of this compound on the mesophilic bacteria associated with raw beef. World Journal of Microbiology and Biotechnology, 21(3), pp. 351–358.
· Brewer, R., Adams, M.R. and Park, S.F. (2002). Enhanced inactivation of Listeria monocytogenes by nisin in the presence of ethanol. Letters in applied microbiology, 34(1), pp.18–21.
· Corroler, D., Mangin, N., Desmasures, N., Gueguen, M. (1998). An ecological study of lactococci isolated from raw milk in the Camembert cheese registered designation of origin area. Applied and Environmental Microbiology, 64 (12), pp. 4729–4735.
· Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.H. and Stackebrandt, E. (2006). The Prokaryotes: Bacteria: Firmicutes, Cyanobacteria. 4: 212.
· Forde, A. and Fitzgerald, G.F., 1999. Bacteriophage defence systems in lactic acid bacteria. In: Lactic Acid Bacteria: Genetics, Metabolism and Applications (pp. 89–113). Springer Netherlands.
· Gálvez, A., Valdivia, E., Abriouel, H., Camafeita, E., Mendez, E., Martínez-Bueno, M. and Maqueda, M. (1998). Isolation and characterization of enterocin EJ97, a bacteriocin produced by Enterococcus faecalis EJ97. Archives of microbiology, 171(1), pp. 59–65.
· Gaya, P., Babin, M., Medina, M., Nunez, M. (1999). Diversity among lactococci isolated from ewe's raw milk cheese. Journal of AppliedMicrobiology, 87(6), pp. 849–855.
· Hanifian, S., and Khani, S. (2012). Fate of Yersinia enterocolitica during manufacture, ripening and storage of Lighvan cheese. International Journal of Food Microbiology, 156(2), pp. 141–146.
· Huot E., Maghrous J., Barena-Gonzalez, C. (1996). Bacteriocin J46, a new bacteriocin produced by Lactococcus lactis subsp. cremoris J46: isolation and characterization of the protein and its gene. Anaerobe, 2(3): pp. 137–145.
· Hyink, O., Balakrishnan, M., and Tagg, J.R. (2005). Streptococcus rattus strain BHT produces both a class I two-component lantibiotic and a class II bacteriocin. FEMS Microbiology Letters, 252(2), pp. 235–241.
· Ivanova, I., Kabadjova, P., Pantev, A., Danova, S. and Dousset, X. (2000). Detection, purification and partial characterization of a novel bacteriocin substance produced by Lactococcus lactis subsp. lactis B14 isolated from boza-Bulgarian traditional cereal beverage. Biocatalysis, 41(6), pp. 47–53.
· Kelly, W.J., Asmundson, R.V. and Huang, C.M., 1996. Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum. Journal of Applied Bacteriology, 81(6), pp. 657–662.
· Klaenhammer, T.R., 1988. Bacteriocins of lactic acid bacteria. Biochimie, 70(3), pp. 337–349.
· Kruger, M.F., de Souza Barbosa, M., Miranda, A., Landgraf, M., Destro, M.T., Todorov, S.D. and de Melo Franco, B.D.G. (2013). Isolation of bacteriocinogenic strain of Lactococcus lactis subsp. lactis from Rocket salad (Eruca sativa Mill.) and evidences of production of a variant of nisin with modification in the leader-peptide. Food control, 33(2), pp. 467–476.
· Lee, N.K. and Paik, H.D. (2001). Partial characterization of lacticin NK24, a newly identified bacteriocin of Lactococcus lactis NK24 isolated from Jeot-gal. Food Microbiology, 18(1), pp. 17–24.
· López-Pedemonte, T.J., Roig-Sagués, A.X., Trujillo, A.J., Capellas, M. and Guamis, B. (2003). Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme. Journal of dairy science, 86(10), pp. 3075–3081.
· Marshall, V.M. (1991). Inoculated ecosystems in milk environments. Journal ofApplied Bacteriology, 73, pp. 127–135.
· Mannu, L., Paba, A. (2002). Genetic diversity of lactococci and enterococci isolated from home-made Pecorino Sardo ewe's milk cheese. Journal ofApplied Microbiology, 92(1), pp. 55–62.
· Mead, P. S., Slutsker, L., Dietz, V., McCaige, L. F., Bresee, J. S., Shapiro, C., Griffin, P. M. and Tauxe, R. V. (1999). Food related illness and death in the United States. Emerging Infectious Diseases 5, pp. 607–625.
· Millette, M., Luquet, F.M. and Lacroix, M. (2007). In vitro growth control of selected pathogens by Lactobacillus acidophilus and Lactobacillus casei fermented milk. Letters in applied microbiology, 44(3), pp. 314–319.
· Mirdamadi, S. and Ghazvini, S.A., 2015. A comparative study between inhibitory effect of L. lactis and nisin on important pathogenic bacteria in Iranian UF Feta cheese. Biological Journal of Microorganism, 3(12).
· Noonpakdee, W., Santivarangkna, C., Jumriangrit, P., Sonomoto, K. and Panyim, S. (2003). Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham, a traditional Thai fermented sausage. International Journal of Food Microbiology, 81(2), pp. 137–145.
· Nwuche, C.O., 2013. Isolation of bacteriocin-producing lactic acid bacteria from'Ugba'and'Okpiye', two locally fermented nigerian food condiments. Brazilian Archives of Biology and Technology, 56(1), pp. 101–106.
· Perin, L.M., Moraes, P.M., Viçosa, G.N., Júnior, A.S. and Nero, L.A. (2012). Identification of bacteriocinogenic Lactococcus isolates from raw milk and cheese capable of producing nisin A and nisin Z. International Dairy Journal, 25(1), pp. 46–51.
· Rodríguez, E., González, B., Gaya, P., Nuñez, M. and Medina, M. (2000). Diversity of bacteriocins produced by lactic acid bacteria isolated from raw milk. International Dairy Journal, 10(1), pp. 07–15.
· Rodríguez, J. M., Cintas, L. M., Casaus, P., Horn, N., Doddl, H. M. and Hernandez, P. E. (1995). Isolation of nisin-producing Lactococcus lactis strains from dry fermented sausages. Journal of Applied Bacteriology, 78(2), pp. 109–115.
· Sánchez, M.M., Delgado, T., Alonso, L. and Mayo, B. (2000). Phenotypic and genetic characterization of a selected set of Lactococcus lactis strains isolated from a starter-free farmhouse cheese. Food microbiology, 17(4), pp. 449–460.
· Sobrino-López, Á. and Martín-Belloso, O. (2006). Enhancing inactivation of Staphylococcus aureus in skim milk by combining high-intensity pulsed electric fields and nisin. Journal of Food Protection, 69(2), pp. 345–353.
· Tafreshi, S. H., Mirdamadi, S., Norouzian, D., Khatami, S. and Sardari, S. (2010). Effect on non-nutritional factors on nisin production. Journal of Biotechnology, 9(9), pp. 1382–1391.
· Todorov, S., Onno, B., Sorokine, O., Chobert, J.M., Ivanova, I. and Dousset, X. (1999). Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST 31 isolated from sourdough. International Journal of Food Microbiology, 48(3), pp. 167–177.
· Todorov, S.D. and Dicks, L.M. (2005). Pediocin ST18, an anti-listerial bacteriocin produced by Pediococcus pentosaceus ST18 isolated from boza, a traditional cereal beverage from Bulgaria. Process Biochemistry, 40(1), pp. 365–370.
· Todorov, S.D. (2008). Bacteriocin production by Lactobacillus plantarum AMA-K isolated from Amasi, a Zimbabwean fermented milk product and study of the adsorption of bacteriocin AMA-K to Listeria sp. Brazilian Journal of microbiology, 39(1), pp. 178–187.
· Topisirovic, L., Kojic, M., Fira, D., Golic, N., Strahinic, I. and Lozo, J. (2006). Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation. International Journal of Food Microbiology, 112(3), pp. 230–235.
· Wirawan, R. E., Klesse, N. A., Jack, R. W. and Tagg, J. R. (2006). Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis. Applied and Environmental Microbiology, 72(2), pp. 1148–1156.
· Wise, K.A. and Tosolini, F.A. (1990). Epidemiological surveillance of Acinetobacter species. Journal of Hospital Infection, 16(4), pp. 319–329.
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