Of at least 400 Listeria phages, only a few have been characterized as having wide host range among listeriae and no phages have been isolated from the environment of food processing plants, where contamination of highly processed ready to eat foods is likely to occur. To understand the ecology of listeriaphage and Listeria monocytogenes in processing plant environments, we pursued the isolation of Listeria-specific phages from environmental samples from four turkey-processing plants in the United States. Nine of twelve isolated phages showed wide host range, including multiple serotypes of L. monocytogenes, as well as other Listeria spp. (L. innocua, L. welshimeri, L. seeligeri, L. ivanovii). The majority of L. monocytogenes of the serotype 4b complex (serotypes 4b, 4d, 4e) could be readily infected by these wide host range phages, but many isolates of other serotypes (1/2a or 3a, and 1/2b or 3b), which represented the majority of L. monocytogenes isolated from the environmental samples, were resistant to infection. L. monocytogenes epidemic clone II (ECII) has been responsible for two multistate outbreaks in the United States in 1998-99 and 2002, in which contaminated ready-to-eat meat products (hot dogs and turkey deli meats, respectively) were implicated. We found that broad host range phages from the processing plant environments were unable to produce plaques on ECII strain lawns when the bacteria were grown at temperatures lower than 37°C (4, 10, 25, 30°C), regardless of the temperature during infection and subsequent incubations. Plaques were readily produced when the bacteria were grown at 37°C, regardless of the temperature during infection or subsequent incubation. In contrast, phage susceptibility of all other tested strains of serotype 4b (including epidemic clone I) and other serotypes appeared to be independent of the growth temperature (25°Cor 37°C) of the bacteria. This temperature-dependent phage susceptibility of ECII strains was not related to presence of plasmid pLM80 or to failure of the phage to adsorb.
To identify genes responsible for this temperature-dependent resistance, a mariner-based transposon was used for isolation of a mutant (J46C), which harbored a single transposon insertion and was phage susceptible at both 25°C and 37°C. The transposon was localized in ORF 2753, a member of a gene cassette that was identified during the genome sequencing of the ECII strain L. monocytogenes H7858 and that appeared to be unique to this strain. The deduced polypeptides from ORF 2753 and the adjacent ORF 2754 exhibited similarity with several restriction endonucleases and methylases, respectively, suggesting that they may constitute a restriction-modification system. Indeed, phage 20422-1 propagated in the mutant J46C at 25°C was able to infect a wild type ECII strain at both 25°C and 37°C, suggesting that the phage DNA was modified and thus protected against the restriction activity of ECII bacteria. A deletion of the ORF 2753 was constructed in the parental strain (H7550-Cds). The mutant (J25FII-7) was identical to the transposon mutant J46C in being susceptible to phage following growth at either 25°C or 37°C. At refrigerator temperatures, ECII contamination could induce phage killing by the R/M system, and food would not be protected from further listerial contamination by the phage whereas consumers might think the foods would still be protected by the approved phage additive. ORF 2753 deletion mutants such as J25FII-7 could be suggested for the preparation of listeriaphage, since they would methylate the phage genome and those phages could kill Listeria strains regardless of temperature of growth.
Further studies are needed to elucidate the molecular mechanism by which ECII-specific genes confer temperature-dependent resistance to phage, and to assess other possible roles of these genes in the ecology of the bacteria and during infection.
|School:||North Carolina State University|
|School Location:||United States -- North Carolina|
|Source:||DAI-B 70/01, Dissertation Abstracts International|
|Keywords:||Epidemic clone, Listeria monocytogenes, Phage resistance|
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