Temperature-Dependent Phage Resistance of Listeria monocytogenes Epidemic Clone II

Listeria monocytogenes epidemic clone II (ECII) has been responsible for two multistate outbreaks in the United States in 1998-1999 and in 2002, in which contaminated ready-to-eat meat products (hot dogs and turkey deli meats, respectively) were implicated. However, ecological adaptations of ECII strains in the food-processing plant environment remain unidentified. In this study, we found that broad-host-range phages, including phages isolated from the processing plant environment, produced plaques on ECII strains grown at 37°C but not when the bacteria were grown at lower temperatures (30°C or below). ECII strains grown at lower temperatures were resistant to phage regardless of the temperature during infection and subsequent incubation. In contrast, the phage susceptibility of all other tested strains of serotype 4b (including epidemic clone I) and of strains of other serotypes and Listeria species was independent of the growth temperature of the bacteria. This temperature-dependent phage susceptibility of ECII bacteria was consistently observed with all surveyed ECII strains from outbreaks or from processing plants, regardless of the presence or absence of cadmium resistance plasmids. Phages adsorbed similarly on ECII bacteria grown at 25°C and at 37°C, suggesting that resistance of ECII strains grown at 25°C was not due to failure of the phage to adsorb. Even though the underlying mechanisms remain to be elucidated, temperature-dependent phage resistance may represent an important ecological adaptation of L. monocytogenes ECII in processed, cold-stored foods and in the processing plant environment, where relatively low temperatures prevail.Listeria monocytogenes is responsible for an estimated 2,500 cases of serious food-borne illness (listeriosis) and 500 deaths annually in the United States. It affects primarily pregnant women, newborns, the elderly, and adults with weakened immune systems. L. monocytogenes is frequently found in the environment and can grow at low temperatures, thus representing a serious hazard for cold-stored, ready-to-eat foods (18, 31).Two multistate outbreaks of listeriosis in the United States, in 1998-1999 and in 2002, respectively, were caused by contaminated ready-to-eat meats (hot dogs and turkey deli meats, respectively) contaminated by serotype 4b strains that represented a novel clonal group, designated epidemic clone II (ECII) (3, 4). ECII strains have distinct genotypes as determined by pulsed-field gel electrophoresis and various other subtyping tools, and harbor unique genetic markers (6, 8, 11, 19, 34). The genome sequencing of one of the isolates (L. monocytogenes H7858) from the 1998-1999 outbreak revealed the presence of a plasmid of ca. 80 kb (pLM80), which harbored genes mediating resistance to the heavy metal cadmium as well as genes conferring resistance to the quaternary ammonium disinfectant benzalkonium chloride (10, 29).Listeria phages (listeriaphage) have long been used for subtyping purposes (33), and extensive research has focused on the genomic characterization (2, 24, 26, 35), transducing potential (14), and biotechnological applications of selected phages (25). In addition, applications of listeriaphage as biocontrol agents in foods and the processing plant environment have been investigated (12, 15, 22). However, limited information exists on phages from processing plant environments and on the impact of environmental conditions on susceptibility of L. monocytogenes strains representing the major epidemic-associated clonal groups to such phages. We have found that strains harboring ECII-specific genetic markers can indeed be recovered from the environment of turkey-processing plants (9). Furthermore, environmental samples from such processing plants yielded phages with broad host range, which were able to infect L. monocytogenes strains of various serotypes, and different Listeria species (20). In this study, we describe the impact of growth temperature on susceptibility of L. monocytogenes ECII strains to phages, including phages isolated from turkey-processing plant environmental samples.