Isolation and Characterization of Bacteriophages of Listeria monocytogenes with Potential to be Used as Biocontrol Agents for Food Safety

Introduction: Since the foodborne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that bacteriophages infecting this bacterium are abundant on raw milk samples. Significant progress has been made toward their applications in food and has resulted in the U.S. Food and Drug Administration approving the use of a bacteriophage-based additive for the control of Listeria monocytogenes in foods.

Purpose: To better understand the diversity and host range of listeriaphages on raw milk samples and to develop a diverse phage collection for further studies. 

Methods: A total of 10 raw milk samples were collected from a dairy farm in Sao Paulo state between July and December 2013. Six bacteriophages of L. monocytogenes were isolated using an enrichment protocol and the double agar layer method. The host ranges of the six bacteriophages were determined by performing spot tests with nine L. monocytogenes strains, including serotypes 4b, 4a, 1/2a, 1/2b. 

Results: While none of raw milk samples tested positive for L. monocytogenes, 30% of samples were positive for listeriaphages, containing up to > 2.5x10² PFU/ml. Host range characterization of the six phage isolates obtained, with a reference set of nine L. monocytogenes strains representing the major serotypes and four lineages, revealed considerable host range diversity. Serotype 1/2a strains were highly susceptible to phages and were lysed by 60 to 85% of phages tested. Overall, 34% of phage isolates showed a narrow host range (lysing 1 to 4 strains), while 67% of phages represented broad host range (lysing > 7 strains).

Significance: The extensive host range of phages observed suggests 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 cheese and milk) and other phage-based tools.

Acknowledgment: FAPESP (Proc. 2012/20287-0).