Selection and Identification of a Listeria monocytogenes Target Strain for Pulsed Electric Field Process Optimization

The presence of Listeria monocytogenes in food constitutes a serious health hazard to consumers. This gram-positive pathogen has been detected in most raw foods, and its presence in processed products has caused numerous outbreaks of listeriosis, a disease with a high mortality rate (14, 35). Heat treatment is the most commonly used processing technique for inactivating L. monocytogenes in food. Alternative processing technologies such as pulsed electric field (PEF) are presently being developed to meet the increasing demand for safe food with fresh-like attributes (25). Gram-positive bacteria are more resistant to PEF processing than are gram-negative bacteria (e.g., reference 21). Reina et al. (34), for instance, reported a 4.2-log10 reduction in the count of L. monocytogenes Scott A when milk was treated for 600 μs at 30 kV/cm, whereas Grahl and Maerkl (18) found a similar degree of inactivation when Escherichia coli was treated in milk for only 200 μs at 22.4 kV/cm. Furthermore, the high PEF resistance of Listeria, compared to that of other gram-positive bacteria, was confirmed in various media (1, 12). Treatment with PEF for 360 μs at 20 kV/cm decreased L. monocytogenes and Staphylococcus aureus populations in phosphate buffer by 1 and 2 log10 CFU/ml, respectively (21). Therefore, destruction of L. monocytogenes is a critical measure of PEF efficacy in treatment of potentially contaminated food. Resistant microbial strains are problematic to the food industry but are also useful as “targets” for designing safe processes. Limited information is available in published literature on variations in resistance to PEF among strains of the same microorganism. Published research on the efficacy of PEF against L. monocytogenes is generally based on single-strain studies. L. monocytogenes strains vary only slightly in resistance to heat (13), but the variability of these strains to inactivation by PEF processing is not known. Thermal inactivation comprises multiple simultaneous mechanisms, whereas PEF largely relies upon one. Heat concurrently alters membrane permeability, denatures cell proteins, causes single-strand breaks in DNA, and degrades RNA (3, 17). Lethality of PEF is mainly caused by membrane disruption (e.g., references 24, 25, and 40). Therefore, variation among strains in susceptibility to processing is believed likelier with PEF than with heat. The variability of L. monocytogenes strains' inactivation by PEF processing, to our knowledge, has not been studied before and should be assessed to identify potential target strains. Genotyping of pathogens is widely used in epidemiological studies to facilitate tracking of virulent or processing-resistant strains in food-processing installations and to determine the contamination routes of these strains (9, 38). Genotyping is based on the detection of DNA polymorphism and is increasingly preferred to serotyping for its reproducibility and high discriminative power (2). Pulsed-field gel electrophoresis (PFGE) and ribotyping are generally the most sensitive genotyping methods (2, 20), but their cost, equipment unavailability, and poor adaptability to routine analysis are limiting the widespread use of these techniques (32). PFGE, however, is preferred to ribotyping in epidemiological studies of L. monocytogenes for its reproducibility and sensitivity (19, 20). Arbitrarily primed PCR (AP-PCR) is inexpensive to run, rapid, and easy to implement in most research laboratories (30). This study, therefore, compared AP-PCR and PFGE in identifying potential target strains for PEF processing. To help design or optimize safe PEF processes, the first objective of this study was to identify an L. monocytogenes target strain. Since cell resistance to PEF may vary with initial treatment temperature (1, 11), this factor was considered during selection of the target strain. Resistance of the target strain to PEF was verified in simple (0.1% NaCl, pH 7.0) and food type (50% acid whey, pH 4.2) media. The second objective was to determine if the target strain remained PEF resistant at different ages of the culture. Maintaining this resistance is a desirable property of a target strain because cells of the exponential phase may lack resistance against environmental and processing stress (27, 33). Finally, all L. monocytogenes strains used in the screening were typed by AP-PCR and PFGE. The genotyping was carried out to select a method sufficiently discriminatory to recognize the target strain and to correlate molecular types with PEF sensitivity.