Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

Abstract

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium ("listeriaphages") are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 ؋ 10 4 PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing >11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools. Listeria monocytogenes is a Gram-positive pathogenic bacterium that can cause a severe food-borne disease, listeriosis, in humans and farm ruminants. L. monocytogenes has been isolated from a variety of environmental sources, e.g., water, soil, silage, vegetation, and food processing plants (3,17,18,23,29,42). A number of studies have reported a high prevalence of L. monocytogenes in dairy farm environments (5,19,21,33,55). In addition, a previous study has found a considerably higher prevalence of L. monocytogenes in dairy farm environments than in urban and natural environments (45). Ruminants, including cattle, sheep, and goats, are not only often fecal shedders of L. monocytogenes but are also hosts in which L. monocytogenes can cause a severe disease (41). Silage (i.e., fermented plant material that is commonly used as feed for ruminants), if spoiled or improperly fermented, has often been found to contain L. monocytogenes (1,20), including at high numbers (Ͼ10 7 CFU/g silage) (61). Spoiled silage has also been reported to be the most important source of L. monocytogenes responsible for listeriosis cases and outbreaks in ruminants (5, 20). The high prevalence of L. monocytogenes on dairy farms and particularly in silage not only suggests that these environments may represent a major reservoir for L. monocytogenes (34) but also suggests that silage may be a superior source for listeriaphage isolation.Bacteriophages ...

Comparative Genomic and Morphological Analyses of Listeria Phages Isolated from Farm Environments

“…Phages were isolated by using three L. monocytogenes host strains: FSL J1-208 (lineage IV, serotype 4a), F2365 (lineage I, serotype 4b), and MACK (lineage II, serotype 1/2a). These 12 Listeria phages exhibited a wide diversity of host ranges, as determined by spot tests against 13 L. monocytogenes isolates representing the nine most common serotypes as well as the four phylogenetic lineages of L. monocytogenes (16). Two Listeria phages, LP-083-2 and LP-030-3, were later isolated as low-level contaminants from LP-083-1 and LP-030-2 cultures, respectively.…”

“…VD13, an E. faecalis phage isolated from human urogenital secretions and first described in 1975 by Ackermann et al (17), was obtained from the Felix d'Herelle Reference Center for Bacterial Viruses (Quebec City, Quebec, Canada). Genome sizes of the Listeria phages were previously estimated by pulsed-field gel electrophoresis (PFGE) (16).…”

“…Phage lysates were prepared from previously purified stocks, as described previously (16). Lysates were then concentrated by polyethylene glycol precipitation (16). DNA was extracted from the concentrated stocks according to a modified version of the lambda DNA extraction protocol described previously by Sambrook and Russell (19).…”

“…Phage lysates were prepared from previously purified stocks, as described previously (16). Lysates were then concentrated by polyethylene glycol precipitation (16).…”

“…Recently, the unique Listeria phage P70 was described by Schmuki et al (15). P70 has a medium-sized genome (ϳ67 kb), similar in size to several phages isolated by our group from dairy farms in the state of New York (16). P70 also possesses an unusual elongated capsid not yet seen in other Listeria phages.…”