Detection and Quantification of Listeria monocytogenes in Ready-to-eat Vegetables, Frozen Vegetables and Sprouts Examined by Culture Methods and Real-time PCR

Monika Moravkova^1, , Veronika Verbikova¹, Veronika Michna¹, Vladimir Babak¹, Hana Cahlikova², Renata Karpiskova² and Petr Kralik¹

¹Department of Food and Feed Safety, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic

²Department of Bacteriology, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic

Cite this paper:
Monika Moravkova, Veronika Verbikova, Veronika Michna, Vladimir Babak, Hana Cahlikova, Renata Karpiskova and Petr Kralik. Detection and Quantification of Listeria monocytogenes in Ready-to-eat Vegetables, Frozen Vegetables and Sprouts Examined by Culture Methods and Real-time PCR. Journal of Food and Nutrition Research. 2017; 5(11):832-837. doi: 10.12691/jfnr-5-11-6

Abstract

In this study, a total of 175 samples of ready-to-eat vegetables, frozen vegetables and sprouted seeds originating in 10 states of the European Union and from 32 manufacturers were collected during a period of one year and examined for the presence of Listeria monocytogenes using standard culture methods and qPCR. In addition to these methods, isolation of Listeria monocytogenes was also carried out following a unified sample preparation for combined downstream use in culture and qPCR analysis. Standard culture and culture preceded by unified sample preparation, showed that L. monocytogenes was present in 6.9% and 11.4% of analyzed samples, respectively, in low numbers. Application of qPCR revealed only 2.3% of samples to be positive for L. monocytogenes in small quantities (less than 10 cells/gram). A statistically significant higher occurrence of L. monocytogenes was seen in frozen vegetables compared to ready to eat vegetables (p<0.01; Fisher’s exact post-hoc tests with Bonferroni’s correction) or sprouts (p<0.05; Fisher’s exact post-hoc tests with Bonferroni’s correction). Therefore, temperature abuse in food containing pieces of frozen vegetables without any processing such as cooking, may pose health risks, especially for sensitive individuals such as pregnant women, children, the elderly, and immunocompromised individuals.

Keywords:
Listeria monocytogenes Foodborne pathogens Molecular methods qPCR Ready-to-eat

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]	Callejon, R.M., Rodriguez-Naranjo, M.I., Ubeda, C., Hornedo-Ortega, R., Garcia-Parrilla, M.C. and Troncoso, A.M, “Reported foodborne outbreaks due to fresh produce in the United States and European Union: trends and causes” Foodborne Pathogens and Disease, 12 (1). 32-38. January 2015.
[2]	Chapela, M.J., Garrido-Maestu, A. and Cabado, A.G, “Detection of foodborne pathogens by qPCR: A practical approach for food industry applications.” Cogent Food and Agriculture, 1 (1). 1013771. March 2015.
[3]	Losio, M.N., Pavoni, E., Bilei, S., Bertasi, B., Bove, D., Capuano, F., Farneti, S., Blasi, G., Comin, D., Cardamone, C., Decastelli, L., Delibato, E., De Santis, P., Di Pasquale, S., Gattuso, A., Goffredo, E., Fadda, A., Pisanu, M. and De Medici, D, “Microbiological survey of raw and ready-to-eat leafy green vegetables marketed in Italy” International Journal of Food Microbiology, 210. 88-91. October 2015.
[4]	Berrada, H., Soriano, J.M., Pico, Y. and Manes, J, “Quantification of Listeria monocytogenes in salads by real time quantitative PCR” International Journal of Food Microbiology, 107 (2). 202-206. March 2006.
[5]	Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M.A., Roy, S.L., Jones, J.L. and Griffin, P.M, “Foodborne illness acquired in the United States-major pathogens” Emerging Infectious Diseases, 17 (1). 7-15. January 2011.
[6]	de Noordhout, C.M., Devleesschauwer, B., Angulo, F.J., Verbeke, G., Haagsma, J., Kirk, M., Havelaar, A. and Speybroeck, N, “The global burden of listeriosis: a systematic review and meta-analysis” Lancet Infectious Diseases, 14 (11). 1073-1082. November 2014.
[7]	European Food Safety Authority (EFSA). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015. EFSA Journal 14 (12):4634. 2016.
[8]	Hutchison, M.L., Walters, L.D., Avery, S.M., Synge, B.A. and Moore, A, “Levels of zoonotic agents in British livestock manures” Letters in Applied Microbiology, 39 (2). 207-214. July 2004.
[9]	Pappelbaum, K., Grif, K., Heller, I., Wuirzner, R., Hein, I., Ellerbroek, L. and Wagner, M, “Monitoring hygiene on- and at-line is critical for controlling Listeria monocytogenes during produce processing” Journal of Food Protection, 71 (4). 735-741. April 2008.
[10]	International Organization for Standardization. Microbiology of food and animal feeding stuffs - horizontal method for detection and enumeration of Listeria monocytogenes, Part 1 – Detection Method (EN ISO 11290-1:1996). International Organization for Standardization, Geneva, Switzerland. 1996.
[11]	International Organization for Standardization. Microbiology of food and animal feeding stuffs — horizontal method for detection and enumeration of Listeria monocytogenes, Part 2 – Detection Method (EN ISO 11290-2:1998). International Organization for Standardization, Geneva, Switzerland. 1998.
[12]	Vojkovska, H., Kubikova, I. and Kralik, P, “Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables” Letters in Applied Microbiology, 60 (3). 265-272. March 2015.
[13]	Rodriguez-Lazaro, D., Hernandez, M. and Pla, M, “Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay” FEMS Microbiology Letters, 233 (2). 257-267. April 2004.
[14]	Malorny, B., Lofstrom, C., Wagner, M., Kramer, N. and Hoorfar, J, “Enumeration of salmonella bacteria in food and feed samples by real-time PCR for quantitative microbial risk assessment” Applied and Environmental Microbiology, 74 (5). 1299-1304. March 2008.
[15]	Caponigro, V., Ventura, M., Chiancone, I., Amato, L., Parente, E. and Piro, F, “Variation of microbial load and visual quality of ready-to-eat salads by vegetable type, season, processor and retailer” Food Microbiology, 27 (8). 1071-1077. December 2010.
[16]	De Giusti, M., Aurigemma, C., Marinelli, L., Tufi, D., De Medici, D., Di Pasquale, S., De Vito, C. and Boccia, A, “The evaluation of the microbial safety of fresh ready-to-eat vegetables produced by different technologies in Italy” Journal of Applied Microbiology, 109 (3). 996-1006. March 2010.
[17]	Froder, H., Martins, C.G., De Souza, K.L., Landgraf, M., Franco, B.D. and Destro, M.T, “Minimally processed vegetable salads: microbial quality evaluation” Journal of Food Protection, 70 (5). 1277-1280. May 2007.
[18]	Cordano, A.M. and Jacquet, C, “Listeria monocytogenes isolated from vegetable salads sold at supermarkets in Santiago, Chile: prevalence and strain characterization” International Journal of Food Microbiology, 132 (2-3). 176-179. June 2009.