Nucleic acid-based methods to identify, detect and type pathogenic bacteria occurring in milk and dairy products

Foodborne illnesses caused by pathogenic microorganisms, including bacteria, viruses and parasites are among the most serious public health concerns worldwide. A recent document of the Center for Disease Control and Prevention (CDC) estimates that each year 1 out of 6 American (or 48 million people) get sick, 128,000 are hospitalized and 3,000 die due to foodborne diseases, with Norovirus, nontyphoidal Salmonella, Clostridium perfringens, Campylobacter spp. and Staphylococcus (S.) aureus being the top five pathogens causing domestically acquired foodborne illnesses [1]. In the European Union, during 2009, 5,550 food-borne outbreaks occurred, mainly due to Salmonella, viruses and bacterial toxins, causing 48,964 human cases, 4,356 hospitalisations and 46 deaths [2]. Bacteria such as Salmonella spp., Campylobacter spp., Listeria (L.) monocytogenes, Escherichia (E.) coli O157:H7 and (S.) aureus have generally been identified as etiologic agents of most food-borne illnesses, with milk and its derivatives products among the most frequently involved food matrices. Moreover, although "less hazardous", these pathogens are a constant threat to the agro-food security, since they can be used to contaminate the environment, crops and animals, causing heavy damage to public health, agriculture and environment [3]. Traditionally, cultivation methods, ranging from plate counting to biochemical characterization, have been used to monitor pathogenic microorganisms in foods. However, these methodologies are labour-intensive and time-consuming, requiring from days to weeks to get results, with the consequence that products are often released for sale before the microbiological results become available. Moreover, these traditional methods as well as their advanced (such as cell wall composition analysis, whole-cell protein fingerprinting and fatty acid analysis) and automated (miniaturised kits or devices) applications often lead to uncertain identification or even misidentification, especially in cases of phenotypically closely related species. Failure to detect pathogens can have adverse health effects as well as substantial economic losses and fatalities. New approaches based on the application of molecular methods have being developed in the last years, bringing new insights in the detection of pathogenic bacteria in milk and milk-based products. In this chapter, we will endeavour to touch upon several nucleic acid based methods (such as PCR and its derivatives, real time PCR, REAPFGE, fAFLP, etc.) and their application in milk and dairy products.