Keywords
food safety
hygienic quality of milk
How to Cite
Abstract
Raw cow milk is considered one of the most important vehicles for pathogenic bacteria like Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. These three bacteria are responsible for foodborne diseases. Routine microbiological methods to detect these microorganisms in cow milk can be complicated and time consuming. The aim of this work was to evaluate a method to simultaneously detect Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in experimentally contaminated cow milk. The assessed method combined a standard microbiological culture step, using a pre-enrichment medium that favors the growth of the three focal microorganisms: SEL broth, followed by a single PCR assay. A total of 43 interference bacterial strains were used to evaluate the method’s specificity. The detection rate for the microbiological method with standard culture media was 10 UFC/mL, and that of the PCR detection, following pre-enrichment in SEL broth, was 10 UFC/mL for S. enterica and L. monocytogenes and between 1 and 5 UFC/mL for E. coli O157:H7. The PCR method showed specificity for the reference strains. Simultaneous detection by multiple PCR using SEL broth was successful for the detection of S. enterica, E. coli O157:H7, and L. monocytogenes in samples of experimentally contaminated cow milk, featuring both a high detection rate and a high specificity. This approach promises to be a feasible routine procedure when testing milk samples in industry and public health control setups.
Middleton J, Saeman A, Fox L, Lombard J, Hogan J, Smith K J. The National Mastitis Council: A Global Organization for Mastitis Control and Milk Quality, 50 Years and Beyond, Journal of Mammary Gland Biology and Neoplasia, 19(3-4): 241-251, 2014.
doi: 10.1007/s10911-014-9328-6
Claeys W, Cardoen S, Daube G, De Block J, Dewettinck K, Dierick K, De Zutter L, Huyghebaert A, Imberechts H, Thiange P, Vandenplas Y, Herman L. Raw or heated cow milk consumption: Review of risks and benefits, Food Control, 31: 251-262, 2013.
doi: 10.1016/j.foodcont.2012.09.035
Centers for Disease Control and Prevention (CDC). Incidence and trends of infection with pathogens transmitted commonly through food—Foodborne Diseases Active Surveillance Network, 10 U.S. sites, 2006-2013, Morbidity and Mortality Weekly Report, 63(15): 328-332, 2014. PMID: 23594684.
Centers for Disease Control and Prevention (CDC). Vital signs: Listeria illnesses, deaths, and outbreaks- United States, 2009- 2011. Morbidity and Mortality Weekly Report, 62(22):448-52, 2013.
PMID: 23739339.
Ministerio de Salud y Protección Social – Unidad de Evaluación de riesgos para la inocuidad de Alimentos (UERIA). Identificación de riesgos biológicos asociados al consumo de leche cruda bovina en Colombia, 111p. 2011.
Ministerio de Protección Social, FAO. Perfil sanitario nacional de leche cruda para consumo humano directo, 26 p. 2014.
Settanni L, Corsetti A. The use of multiplex PCR to detect and differentiate food- and beverage-associated microorganisms: A review, Journal of Microbiological Methods, 69 (1): 1-22, 2007.
doi: 10.1016/j.mimet.2006.12.008
Omiccioli E, Amagliani G, Brandi Giorgio, Magnani M. A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk, Food Microbiology, 26 (6): 615-622, 2009.
doi: 10.1016/j.fm.2009.04.008
Germini A, Masola A, Carnevali P, Marchelli R. Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR, Food Control, 20 (8): 733-738, 2009.
doi: 10.1016/j.foodcont.2008.09.010
Denis E, Bielińska K, Wieczorek K, Osek J. Multiplex realtime PCRs for detection of Salmonella, Listeria monocytogenes, and verotoxigenic Escherichia coli in carcasses of slaughtered animals, Journal of Veterinary Research, 16(60): 287-292, 2016.
doi: 10.1515/jvetres-2016-0044
Kawasaki S, Fratamico PM, Horikoshi N, Okada Y, Takeshita K, Sameshima T, Kawamoto S. Evaluation of a multiplex PCR system for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in foods and in food subject to freezing, Foodborne Pathogens and Disease, 6 (1): 81-89, 2009.
doi: 10.1089/fpd.2008.0153
Fratamico PM, Strobaugh TP. Simultaneous detection of Salmonella spp., and Escherichia coli O157:H7 by multiplex PCR, Journal of Industrial Microbiology and Biotechnology, 21 (3): 92-98, 1998.
doi: 10.1038/sj.jim.2900520
Aslam M, Hogan J, Smith K. Development of a PCR based assay to detect Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella in milk, Food Microbiology, 20 (3): 345- 350, 2003.
doi: 10.1016/S0740-0020(02)00121-1
Kawasaki S, Horikoshi, N, Okada, Y, Takeshita, K, Sameshima, T, Shinichi, K. Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples, Journal of Food Protection, 68 (3): 551-556, 2005.
doi: 10.4315/0362-028X-68.3.551
Kim J, Demeke T, Clear R, Patrick S. Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenes and Salmonella Typhimurium in artificially inoculated wheat grain, International Journal of Food Microbiology, 111 (1): 21-25, 2006.
doi: 10.1016/j.ijfoodmicro.2006.04.032
Park Y, Lee R, Kim Y. Detection of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes in kimchi by multiplex polymerase chain reaction (mPCR), Journal of Microbiology, 44 (1): 92-97, 2006.
Calderón A, Rodríguez V. Prevalencia de mastitis bovina y su etiología infecciosa en sistemas especializados en producción de leche en el altiplano cundiboyacense (Colombia), Revista Colombiana de Ciencias Pecuarias, 21: 582-589, 2008.
Kim H, Bhunia A. SEL, a selective enrichment broth for simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes, Applied and Environmental Microbiology, 74 (15): 4853-4866, 2008.
doi: 10.1128/AEM.02756-07
INVIMA. Manual of techniques to analyze microbiological quality control of food for human consumption, 111 p, 1998.
doi: 10.1089/fpd.2008.0153
Feng P, Weagant SD, Jinneman K. Diarrheagenic Escherichia coli, Bacteriological Analytical Manual Chapter 4A. Maryland, USA: US Food and drug administration, 2016.
Retrieved from: h t t p : / / w w w. f d a . g ov/Food/Fo o d S c i e n c e Re s e a r ch/ LaboratoryMethods/ucm070080.htm
Andrews W, Hammack T. Salmonella. Bacteriological Analytical Manual, Chapter 5 Maryland, USA: US Food and drug administration, 2016.
Retrieved from: h t t p : / / w w w. f d a . g ov/Food/Fo o d S c i e n c e Re s e a r ch/ LaboratoryMethods/ucm2006949.htm
Hitchins AD, Jinneman K. Detection and enumeration of Listeria monocytogenes in foods, Bacteriological Analytical Manual Chapter 10, Maryland, USA: US Food and drug administration, 2016.
Retrieved from: h t t p : / / w w w. f d a . g ov/Food/Fo o d S c i e n c e Re s e a r ch/ LaboratoryMethods/ucm071400.htm
Wilson K. Preparation of Genomic DNA from Bacteria, Current Protocols in Molecular Biology, 56(1): 2.4.1-25B.4.17, 2001.
doi: 10.1002/0471142727.mb0204s56
Asadzadeh N, Javanmard A, Nassiry M. Comparison of rapid DNA extraction techniques for conventional PCR-RFLP analysis from mammalian whole blood cells, Journal of Molecular Genetics, 2 (3-4): 32-35, 2010.
doi: 10.3923/jmolgene.2010.32.35
Poutou R. Burbano M, Sierra S. Torres K. Carrascal AK, Mercado M. Estandarización de la extracción de ADN y validación de la PCR múltiple para detectar Listeria monocytogenes en queso, leche, carne de res y pollo, Universitas Scientiarum, 10 (2): 61-78, 2005.
Ames G, Noel K, Taber H, Spudich E, Nikaido K, Afong J. Fine-structure map of the histidine transport genes in Salmonella typhimurium, Journal of Bacteriology, 129(3):1289-1297. 1977.
PMID: 321422.
Freschi CR, Silva Carvalho LF; Oliveira CJ. Comparison of DNA-extraction methods and selective enrichment broths on the detection of Salmonella Typhimuriumin swine feces by Polymerase Chain Reaction (PCR), Brazilian Journal of Microbiology, 36 (4): 363-367, 2005.
doi: 10.1590/S1517-83822005000400011
Burbano E, Sierra S, Torres K, Mercado M, Carrascal A, Poutou R. Rapid DNA extraction and PCR validation for direct detection of Listeria monocytogenes in raw milk, Revista MVZ Córdoba, 11(1): 715-724, 2006.
doi: 10.21897/rmvz.456
Vanegas MC, Vásquez E, Martínez AJ, Rueda AM. Detection of Listeria monocytogenes in raw whole milk for human consumption in Colombia by real-time PCR, Food Control, 20(4): 430-432, 2009.
doi: 10.1016/j.foodcont.2008.07.007
Pollard DR, Johnson WM, Lior H, Tyler D, Rozee KR. Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction, Journal of Clinical Microbiology, 28(3); 540-545, 1990.
PMCID: PMC26965.
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