Evaluation of antibacterial effect of Rosmarinus officinalis L. essential oil on three food-borne pathogens in vegetables by propidium monoazide quantitative PCR
Subject Areas : Food Science and Technologyمریم Azizkhani 1 , پاتریسیا Elizaquivel 2
1 - Assistant Professor, Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran
2 - Departamento de Microbiología y Ecología, Universitat de València, Burjassot, Valencia, Spain
Keywords: Vegetables, PMA-qPCR, Rosemary essential oil, Bio-preservative, Bactericidal,
Abstract :
Essential oils (EOs) have long been applied as flavoring agents in foods. Nowadays, due to the antimicrobial properties of EOs, they have been used as natural food preservatives. In this study, initial experiments were performed in order to elucidate the minimum bactericidal concentration of Rosmarinus officinalis L.EO on Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes. Thereafter, PMA-qPCR was applied in order to selectively quantify living cells within a bacterial population treated with rosemary EO. Inactivation was obtained at EO concentrations of 1%, 0.45%, 0.9% for L. monocytogenes, E. coli O157:H7 and S. enterica, respectively. L. monocytogenes were totally killed within 45 min while it took 90 min for E. coli O157:H7and S. enterica. It was concluded that rosemary EO has the potential to be used as a natural food additive or bio-preservative since it was able to irreversibly inactivate the three tested pathogens at lower concentrations and short exposition times in comparison with the other EOs. In addition, PMA-qPCR approach proved quantitatively precise and specific to selectively detect live pathogenic bacteria in vegetables following inactivation with rosemary EO.
References:
Adams, R.P. (2001). Identification of essential oils components by gas chromatography/ quadrupole mass spectroscopy. Illinois: Allured Publishing Corporation.
Celiktas, O.Y., Kocabas, E.E.H., Bedir, E., Vardar Sukan F., Ozek, T. and Baser, K.H.C. (2007). Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations. Food Chemistry, 100: 553–559.
Debersac, P., Haydel, J.M., Amiot, M.J., Goudonnet, H., Artue, Y., Suschetet, M., et al. (2001). Introduction of cytochrome P450 and/or dhietoxication enzymes by various extracts of rosemary description of specific patterns. Food and Chemical Technology, 39(9): 907-918.
Elizaquivel, P., Gabald¢n, J.A. and Aznar, R. (2011). Quantification of Salmonella spp.,Listeria monocytogenes and Escherichia coli O157:H7 in non-spiked food products and evaluation of real-time PCR as a disgnostic tool in routine food analysis. Food Control, 22: 158-164.
Elizaquivel, P., Sanchez, G. and Aznar, R. (2012a). Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR. Food Control, 25: 704-708.
Elizaquivel, P., Sanchez, G. and Aznar, R. (2012b). Application of propidium monoazide quantitative PCR for selective detection of live Escherichia coli O157:H7 in vegetables after inactivation by essential oils. International Journal of Food Microbiology, 159(2): 115-121.
Fittipaldi, M., Nocker, A. and Codony, F. (2012). Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification.Journal of Microbiological Methods, 91(2): 276-289.
Fu, Y.J., Zu, Y.G., Chen, L.Y., Shil, X.G., Wang, Z., Sunl, S., et al. (2007a). Antimicrobial activity of clove and rosemary essential oils alone and in combination. Phytotherapy Research, 21(10): 989–994.
Fu, Y.J., Zu, Y.G., Chen, L.Y., Efferth, T., Lianch, H., Liu, Z., et al. (2007b). Investigation of antibacterial activity of rosemary essential oil against Propionibacterium acnes with atomic force microscopy. Planta Medica, 73(12): 1275-1280.
Glaser, P., Frangeul, L., Buchrieser, C., Rusniok, C., Amend, A., Baquero, F., et al. (2001). Comparative genomics of Listeria species. Science, 294: 849-852.
Hayashi, T., Makino, K., Ohnishi, M., Kurokawa, K., Ishii, K., Yokohama, K., et al. (2001). Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with laboratory strain K-12. DNA Research, 8: 11-22.
Hoorfar, J., Ahrens, P. and Radström, P. (2000). Automated 5ïnuclease PCR assay for identification of Salmonella enterica. Journal of Clinical Microbiology, 38(9): 3429-3435.
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Jamshidi, R., Afzali, Z. and Afzali, D. (2009). Chemical composition of hydrodistillation essential oil of rosemary in different origins in Iran and comparison with other countries. American-Eurasian Journal of Agricultural and Environmental Science, 5(1): 78-81.
Jarrar, N., Abu-Hijleh, A. and Adwan, K. (2010). Antibacterial activity of Rosmarinus officinalis L. alone and in combination with cefuroxime against methicillin–resistant Staphylococcus aureus. Asian Pacific Journal of Tropical Medicine, 3(2): 121-123.
Jinneman, K.C., Yoshitomi, K.J. and Weagant, S.D. (2003). Multiplex real-time PCR method to identify shiga toxin genes stx1 and stx2 and Escherichia coli O157:H7/H e serotype. Applied and Environmental Microbiology, 69(10): 6327-6333.
Josefsen, M.H., Lofstrom, C., Hansen, T.B., Christensen, L.S., Olsen, J.E. and Hoorfar, J. (2010). Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment. Applied and Environmental Microbiology, 76(15): 5097-5104.
Klancnik, A., Piskernik, S., Jersek, B., and Mozina, S.S. (2010). Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts.Journal of Microbiological Methods,81(2): 121-126.
Kordali, S., Cakir, A., Mavi, A., Kilic, H. and Yildirim, A. (2005). Screening of chemical composition and antifungal and antioxidant activities of the essential oils from three Turkish Artemisia species. Journal of Agriculture and Food Chemistry, 53: 1408–1416.
Kramer, M., Obermajer, N., Bogovic, M.B., Rogelj, I. and Kmetec, V. (2009). Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry. Applied Microbiological Biotechnology, 84(6): 1137-1147.
Lopes-Lutz, D., Alviano, D.S., Alviano, C.S. and Kolodziejczyk, P.P. (2008). Screening of chemical composition, antimicrobial and antioxidant activities of Artemisia essential oils. Phytochemistry, 69: 1732–1738.
Lovdal, T., Hovda, M.B., Bjorkblom, B. and Moller, S.G. (2011). Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua. Journal of Microbiological Methods, 85(2): 164-169.
Mathlouthi, N., Bouzaienne, T., Oueslati, I., Recoquillay, F., Hamdi, M., Urdaci, M., et al. (2010). Use of rosemary, oregano, and a commercial blend of essential oils in broiler chickens: In vitro antimicrobial activities and effects on growth performance. Journal of Animal Science, 90(3): 813-823.
McClelland, M., Sanderson, K.E., Spieth, J., Clifton, S.W., Latreille, P. and Courtney, L. (2001). Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature, 413(6858): 852-856.
Moghtader, M. and Afzali, D. (2009). Study of the antibacterial properties of the essential oil of rosemary. American-Eurasian Journal of Agricultural and Environmental Science, 5(3): 393-397.
Nocker, A. and Camper, A.K. (2006). Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Applied and Environmental Microbiology, 72(3): 1997-2004.
Nogva, H.K., Dromtorp, S.M., Nissen, H. and Rudi, K. (2003). Ethidium monoazide for DNA-based differenciation of viable and dead bacteria by 5’-nuclease PCR.BioTechniques, 34: 804-813.
Ozcan, M.M.L. and Chalchat, J.C. (2008). Chemical composition and antifungal activity of rosemary (Rosmarinus officinalis L.) oil from Turkey. International Journal of Food Science and Nutrition, 59(7-8): 691-698.
Pan,Y. and Breidt, F., Jr. (2007). Enumeration of viable Listeria monocytogenes cells by real time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. Applied and Environmental Microbiology, 73(24): 8028-8031.
Randrianarivelo, R., Sarter, S., Odoux, E., Brat, P., Lebrun, M., Romestand, B., et al. (2009). Composition and antimicrobial activity of essential oils of Cinnamosma fragrans. Food Chemistry, 114: 680–684.
Rodríguez-Lázaro, D., Hernandez, M. and Pla, M. (2004). Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay. FEMS Microbiology Letters, 233: 257-267.
Rudi, K., Nogva, H.K., Moen, B., Nissen, H., Bredholt, S., Moretro, T., et al. (2002). Development and application of new nucleic acid-based technologies for microbial community analyses in foods. International Journal of Food Microbiology, 78: 171-180.
Ruiz, A., Williams, S.K., Djeri, N., Hinton, A. Jr. and Rodrick, G.E. (2009). Nisin, rosemary, and ethylene diaminetetraacetic acid affect the growth of Listeria monocytogenes on ready-to-eat turkey ham stored at four degrees Celsius for sixty-three days. Poultry Science, 88(8): 1765-1772.
Schnetzinger, F., Pan, Y. and Nocker, A. (2013). Use of propidium monoazide and increased amplicon length reduce false-positive signals in quantitative PCR for bioburden analysis. Applied Microbiology and Biotechnology, 97: 2153-2162.
Slimani, S., Robyns, A., Jarraud, S., Molmeret, M., Dusserre, E. and Mazure, C. (2012). Evaluation of propidium monoazide (PMA) treatment directly on membrane filter for the enumeration of viable but non cultivable Legionella by qPCR. Journal of Microbiological Methods, 88(2): 319-321.
Soejima, T., Schlitt-Dittrich, F. and Yoshida, S.I. (2011). Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of enterobacteriaceae. Analytical Biochemistry, 418: 37-43.
Sökmen, A., Gulluce, M., Askin H.A., Daferera, D., Tepe, B., Polissiou, M., et al. (2004). The in vitro antimicrobial and antioxidant activities of the essential oils and methanol extracts of endemic Thymus spathulifolius. Food Control, 15: 627–634.
Wang, S. and Levin, R.E. (2006). Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR. Journal of Microbiological Methods, 64(1): 1-8.
Wentao, X., Wei, Q., Kunlun, H., Feng, G., Jiajia, Y., Heng, Z. and YunBo, L. (2007). Antibacterial effect of grapefruit seed extract on food-borne pathogens and its application in the preservation of minimally processed vegetables. Postharvest Biology and Technology, 45(1): 126-133.
Zegura, B., Dobnikb, D., Niderlc, M.H. and Filipiča, M. (2011). Antioxidant and antigenotoxic effects of rosemary (Rosmarinus officinalis L.) extracts in Salmonella typhimurium TA98 and HepG2 cells. Environmental Toxicology and Pharmacology, 32(2): 296–305.
Zhu, R.G., Li, T.P., Jia, Y.F. and Song, L.F. (2012). Quantitative study of viable Vibrioparahemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR. Journal of Microbiological Methods, 90: 262-266.
Adams, R.P. (2001). Identification of essential oils components by gas chromatography/ quadrupole mass spectroscopy. Illinois: Allured Publishing Corporation.
Celiktas, O.Y., Kocabas, E.E.H., Bedir, E., Vardar Sukan F., Ozek, T. and Baser, K.H.C. (2007). Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations. Food Chemistry, 100: 553–559.
Debersac, P., Haydel, J.M., Amiot, M.J., Goudonnet, H., Artue, Y., Suschetet, M., et al. (2001). Introduction of cytochrome P450 and/or dhietoxication enzymes by various extracts of rosemary description of specific patterns. Food and Chemical Technology, 39(9): 907-918.
Elizaquivel, P., Gabald¢n, J.A. and Aznar, R. (2011). Quantification of Salmonella spp.,Listeria monocytogenes and Escherichia coli O157:H7 in non-spiked food products and evaluation of real-time PCR as a disgnostic tool in routine food analysis. Food Control, 22: 158-164.
Elizaquivel, P., Sanchez, G. and Aznar, R. (2012a). Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR. Food Control, 25: 704-708.
Elizaquivel, P., Sanchez, G. and Aznar, R. (2012b). Application of propidium monoazide quantitative PCR for selective detection of live Escherichia coli O157:H7 in vegetables after inactivation by essential oils. International Journal of Food Microbiology, 159(2): 115-121.
Fittipaldi, M., Nocker, A. and Codony, F. (2012). Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification.Journal of Microbiological Methods, 91(2): 276-289.
Fu, Y.J., Zu, Y.G., Chen, L.Y., Shil, X.G., Wang, Z., Sunl, S., et al. (2007a). Antimicrobial activity of clove and rosemary essential oils alone and in combination. Phytotherapy Research, 21(10): 989–994.
Fu, Y.J., Zu, Y.G., Chen, L.Y., Efferth, T., Lianch, H., Liu, Z., et al. (2007b). Investigation of antibacterial activity of rosemary essential oil against Propionibacterium acnes with atomic force microscopy. Planta Medica, 73(12): 1275-1280.
Glaser, P., Frangeul, L., Buchrieser, C., Rusniok, C., Amend, A., Baquero, F., et al. (2001). Comparative genomics of Listeria species. Science, 294: 849-852.
Hayashi, T., Makino, K., Ohnishi, M., Kurokawa, K., Ishii, K., Yokohama, K., et al. (2001). Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with laboratory strain K-12. DNA Research, 8: 11-22.
Hoorfar, J., Ahrens, P. and Radström, P. (2000). Automated 5ïnuclease PCR assay for identification of Salmonella enterica. Journal of Clinical Microbiology, 38(9): 3429-3435.
International Organization for Standardization (ISO), (1998). Microbiology of food and animal feeding stuffs -- Horizontal method for the detection and enumeration of Listeria monocytogenes -- Part 2: Enumeration method. The European Standard EN. ISO No. 11290: 1998.
International Organization for Standardization (ISO), (2001). Microbiology of food and animal feeding stuffs — Horizontal method for the detection of Escherichia coli 0157. The European Standard EN. ISO No. 16654: 2001.
International Organization for Standardization (ISO), (2002). Microbiology of food and animal feeding stuffs -- Horizontal method for the detection of Salmonella spp. The European Standard EN. ISO No. 6579: 2002.
Jamshidi, R., Afzali, Z. and Afzali, D. (2009). Chemical composition of hydrodistillation essential oil of rosemary in different origins in Iran and comparison with other countries. American-Eurasian Journal of Agricultural and Environmental Science, 5(1): 78-81.
Jarrar, N., Abu-Hijleh, A. and Adwan, K. (2010). Antibacterial activity of Rosmarinus officinalis L. alone and in combination with cefuroxime against methicillin–resistant Staphylococcus aureus. Asian Pacific Journal of Tropical Medicine, 3(2): 121-123.
Jinneman, K.C., Yoshitomi, K.J. and Weagant, S.D. (2003). Multiplex real-time PCR method to identify shiga toxin genes stx1 and stx2 and Escherichia coli O157:H7/H e serotype. Applied and Environmental Microbiology, 69(10): 6327-6333.
Josefsen, M.H., Lofstrom, C., Hansen, T.B., Christensen, L.S., Olsen, J.E. and Hoorfar, J. (2010). Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment. Applied and Environmental Microbiology, 76(15): 5097-5104.
Klancnik, A., Piskernik, S., Jersek, B., and Mozina, S.S. (2010). Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts.Journal of Microbiological Methods,81(2): 121-126.
Kordali, S., Cakir, A., Mavi, A., Kilic, H. and Yildirim, A. (2005). Screening of chemical composition and antifungal and antioxidant activities of the essential oils from three Turkish Artemisia species. Journal of Agriculture and Food Chemistry, 53: 1408–1416.
Kramer, M., Obermajer, N., Bogovic, M.B., Rogelj, I. and Kmetec, V. (2009). Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry. Applied Microbiological Biotechnology, 84(6): 1137-1147.
Lopes-Lutz, D., Alviano, D.S., Alviano, C.S. and Kolodziejczyk, P.P. (2008). Screening of chemical composition, antimicrobial and antioxidant activities of Artemisia essential oils. Phytochemistry, 69: 1732–1738.
Lovdal, T., Hovda, M.B., Bjorkblom, B. and Moller, S.G. (2011). Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua. Journal of Microbiological Methods, 85(2): 164-169.
Mathlouthi, N., Bouzaienne, T., Oueslati, I., Recoquillay, F., Hamdi, M., Urdaci, M., et al. (2010). Use of rosemary, oregano, and a commercial blend of essential oils in broiler chickens: In vitro antimicrobial activities and effects on growth performance. Journal of Animal Science, 90(3): 813-823.
McClelland, M., Sanderson, K.E., Spieth, J., Clifton, S.W., Latreille, P. and Courtney, L. (2001). Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature, 413(6858): 852-856.
Moghtader, M. and Afzali, D. (2009). Study of the antibacterial properties of the essential oil of rosemary. American-Eurasian Journal of Agricultural and Environmental Science, 5(3): 393-397.
Nocker, A. and Camper, A.K. (2006). Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Applied and Environmental Microbiology, 72(3): 1997-2004.
Nogva, H.K., Dromtorp, S.M., Nissen, H. and Rudi, K. (2003). Ethidium monoazide for DNA-based differenciation of viable and dead bacteria by 5’-nuclease PCR.BioTechniques, 34: 804-813.
Ozcan, M.M.L. and Chalchat, J.C. (2008). Chemical composition and antifungal activity of rosemary (Rosmarinus officinalis L.) oil from Turkey. International Journal of Food Science and Nutrition, 59(7-8): 691-698.
Pan,Y. and Breidt, F., Jr. (2007). Enumeration of viable Listeria monocytogenes cells by real time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. Applied and Environmental Microbiology, 73(24): 8028-8031.
Randrianarivelo, R., Sarter, S., Odoux, E., Brat, P., Lebrun, M., Romestand, B., et al. (2009). Composition and antimicrobial activity of essential oils of Cinnamosma fragrans. Food Chemistry, 114: 680–684.
Rodríguez-Lázaro, D., Hernandez, M. and Pla, M. (2004). Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay. FEMS Microbiology Letters, 233: 257-267.
Rudi, K., Nogva, H.K., Moen, B., Nissen, H., Bredholt, S., Moretro, T., et al. (2002). Development and application of new nucleic acid-based technologies for microbial community analyses in foods. International Journal of Food Microbiology, 78: 171-180.
Ruiz, A., Williams, S.K., Djeri, N., Hinton, A. Jr. and Rodrick, G.E. (2009). Nisin, rosemary, and ethylene diaminetetraacetic acid affect the growth of Listeria monocytogenes on ready-to-eat turkey ham stored at four degrees Celsius for sixty-three days. Poultry Science, 88(8): 1765-1772.
Schnetzinger, F., Pan, Y. and Nocker, A. (2013). Use of propidium monoazide and increased amplicon length reduce false-positive signals in quantitative PCR for bioburden analysis. Applied Microbiology and Biotechnology, 97: 2153-2162.
Slimani, S., Robyns, A., Jarraud, S., Molmeret, M., Dusserre, E. and Mazure, C. (2012). Evaluation of propidium monoazide (PMA) treatment directly on membrane filter for the enumeration of viable but non cultivable Legionella by qPCR. Journal of Microbiological Methods, 88(2): 319-321.
Soejima, T., Schlitt-Dittrich, F. and Yoshida, S.I. (2011). Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of enterobacteriaceae. Analytical Biochemistry, 418: 37-43.
Sökmen, A., Gulluce, M., Askin H.A., Daferera, D., Tepe, B., Polissiou, M., et al. (2004). The in vitro antimicrobial and antioxidant activities of the essential oils and methanol extracts of endemic Thymus spathulifolius. Food Control, 15: 627–634.
Wang, S. and Levin, R.E. (2006). Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR. Journal of Microbiological Methods, 64(1): 1-8.
Wentao, X., Wei, Q., Kunlun, H., Feng, G., Jiajia, Y., Heng, Z. and YunBo, L. (2007). Antibacterial effect of grapefruit seed extract on food-borne pathogens and its application in the preservation of minimally processed vegetables. Postharvest Biology and Technology, 45(1): 126-133.
Zegura, B., Dobnikb, D., Niderlc, M.H. and Filipiča, M. (2011). Antioxidant and antigenotoxic effects of rosemary (Rosmarinus officinalis L.) extracts in Salmonella typhimurium TA98 and HepG2 cells. Environmental Toxicology and Pharmacology, 32(2): 296–305.
Zhu, R.G., Li, T.P., Jia, Y.F. and Song, L.F. (2012). Quantitative study of viable Vibrioparahemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR. Journal of Microbiological Methods, 90: 262-266.
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