Assessment of a multiplex detection method for Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in cow milk

Univ. Sci. 24 (1): 277-294, 2019. doi: 10.11144/Javeriana.SC24-1.aoam Bogotá original article Assessment of a multiplex detection method for Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in cow milk Rocio Patiño Burbano1, *, Ana Karina Carrascal2, Jorge Luis Parra Arango3, José Luis Rodríguez Bautista4 Edited by Juan Carlos Salcedo-Reyes () 1. Animal Health Laboratory. Tibaitatá Research Center, AGROSAVIA. Mosquera, Colombia 2. Food Microbiology Laboratory, Environmental and Industrial Biotechnology Research Group, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia 3. Universidad de los Llanos, Villavicencio, Colombia. 4. Universidade Federal Rural do Rio de Janeiro, Programa de Pós-Graduação Seropédica, Brazil * Received: 22-11-2017 Accepted: 14-11-2018 Published on line: 06-03-2019 Citation: Patiño Burbano R, Carrascal AK, Parra Arango JL, Rodríguez Bautista JL. Assessment of a multiplex detection method for Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in cow milk, Universitas Scientiarum, 24 (1): 277-294, 2019. doi: 10.11144/Javeriana.SC24-1.aoam Funding: Ministerio de Agricultura y Desarrollo Rural y la Corporación colombiana de investigación agropecuaria, agrosavia. Electronic supplementary material: N.A. 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. Keywords: Foodborne diseases; food safety; hygienic quality of milk. Introduction The main components of cow milk are water, proteins, fat, carbohydrates, and minerals. These components exhibit quantitative variation both within and between individuals and their proportions in milk chiefly depend on cow race, feed, age, lactation period, time of the year, and milking system [1]. Universitas Scientiarum, Journal of the Faculty of Sciences, Pontificia Universidad Javeriana, is licensed under the Creative Commons Attribution 4.0 International Public License 278 A detection method of bacterias in milk Along its production chain, cow milk quality and safety might be threatened by biological, physical, and chemical hazards. Inadequate milking and manipulation practices, lack of boiling, and insufficient cooling methods during raw milk harvesting may lead to microbial growth in a very short period, thus increasing the risk of food poisoning and foodborne infections in consumers [2]. The most important biological contaminants of raw cow milk include bacteria like Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes [3]. These bacteria have been associated to food poisoning cases in the United States [4]. In 2014, a total of 140 food poisoning outbreaks were associated to Salmonella, 23 to Shiga toxin-producing E. coli, and 9 to L. monocytogenes; 80 out of the 140 Salmonella outbreaks were originated by consumption of food of animal origin. Among these, 15 cases were associated to the intake of unpasteurized dairy products, 3 cases due to consumption of pasteurized milk products, and 1 case involved a dairy product lacking information on hygienic protocols [4]. In Colombia, various investigations have demonstrated the presence of pathogenic microorganisms such as Brucella spp., L. monocytogenes, and Staphylococcus aureus in cow milk, and public health surveillance bodies have reported outbreaks of E. coli and coagulase-positive Staphylococcus associated with milk consumption [5, 6]. Commonly employed microbiological identification methods for Salmonella enterica, E. coli O157:H7, and L. monocytogenes in milk and dairy products rely on growing samples in selective media [7, 8]. Depending on the culture medium used, presence/absence of colonies is evaluated. Then, biochemical phenotyping and serotyping assays are performed. These culture-based microbiological detection methods take several days to identifying a given pathogen. In industry and public health surveillance setups, efficient and accurate testing methodologies are needed to ensure the timely release of consumer-safe milk and dairy products into the market. Molecular techniques are fast and efficient to detect pathogenic bacteria in food, particularly in milk. By combining both methodologies, the milk industry can have reliable test results over a short time period [7-10]. A promising molecular method for food testing is the multiplex PCR (mPCR). This method uses a group of target-specific primers to amplify different DNA fragments in a single reaction. With this technique, multiple microbial species can be simultaneously detected since the pool of primers consists of sequences targeted to a given bacterial species [11, 12]. This approach has been successfully applied to detect S. enterica, E. coli O157, and L. monocytogenes in milk [13], meat [14], cereals [15], and kimchi [16], offering the possibility to implement it as a routine technique. Universitas Scientiarum Vol. 24 (1): 277-294 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarum 279 Patiño Burbano et al. 2019 In the present study we assessed the effectiveness of the mPCR technique in detecting S. enterica, verotoxin-producing E. coli (E. coli O157:H7), and L. monocytogenes in experimentally contaminated cow milk. In parallel we conducted culture-based conventional microbiological identification tests, evaluating the use of a culture medium that favors the growth of the three focal bacteria. Materials and methods Bacterial strains The bacterial strains used in this study were obtained from different reference collections. T (...truncated)