Title: Characterization of Bacteriophages Virulent for Clostridium perfringens and Identification of Phage Lytic Enzymes as Alternatives to Antibiotics for Potential Control of the Bacterium Author
Submitted to: United States Animal Health Association Proceedings
Publication Type: Proceedings
Publication Acceptance Date: March 1, 2013
Publication Date: June 3, 2013
Citation: Seal, B.S. 2013. Characterization of Bacteriophages Virulent for Clostridium perfringens and Identification of Phage Lytic Enzymes as Alternatives to Antibiotics for Potential Control of the Bacterium. United States Animal Health Association Proceedings. 116:577-583. Interpretive Summary: Bacteriophages are viruses that infect and kill their bacterial host and bacteriophage therapy is the use of these viruses to treat pathogenic bacterial infections. There has been a renewed interest in the use of bacteriophages or their gene products as alternatives to the use of antibiotics due to concerns about bacteria that are resistant to antibiotic treatment or use of antibiotics in animal feeds as growth promoters. Clostridium perfringens is an anaerobic bacterium (unable to grow in oxygen) and is widely distributed in the environment. The bacterium frequently occurs in the intestines of humans and many domestic or feral animals, while spores of the organism persist in soil and areas subject to human or animal fecal pollution. Perfringens food poisoning is the third most common cause of bacterial food-borne disease in the United States. The bacterium also causes human diseases such as gas gangrene, enterotoxin diseases of lambs or calves and necrotic enteritis of chickens during poultry production. To combat these these diseases and investigate the possible use of bacteriophages to control Clostridium perfringens, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lyse and kill the bacterium. The bacterial viruses were members of bacteriophages with long or short, non-contractile tails and small double-stranded DNA genomes. Structural proteins of the purified phages were identified by biochemical methods and the genomes of the viruses were determined by nucleotide sequencing. All the bacteriophages identified had genes encoding potential bacterium-specific enzymatic activities capable of digesting their host Clostridium perfringens. Consequently, potential new alternatives to antibiotics were discovered that could eventually be used to control diseases caused by this bacterium without having deleterious effects on other beneficial bacteria.
Technical Abstract: Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal, and poultry diseases. There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial agents. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages in both the USA and Russian Federation that could lyse C. perfringens in spot assays. From the bacteriophage collections highly lytic viruses were isolated and the double-stranded deoxyribonucleic acid (DNA) genomes of the viruses were sequenced to completion. DNA sequencing of six bacteriophage genomes completed at PMSRU and four genomes in collaboration with Russian investigators resulted in identification of unique amidases as well as bacteriophage encoded proteins that potentially contain lysozyme and endopeptidase activities. Three recombinant bacteriophage lytic enzyme genes encoding putative amidases have been cloned, their proteins expressed as recombinants and isolated to homogeneity, then demonstrated to species-specifically lyse C. perfringens. No other bacteria beyond the genus were lysed by the recombinant bacteriophage proteins indicating that beneficial bacteria will not be killed by these novel antimicrobials. These bacteriophage lytic enzymes will have possibilities for use in agriculture and medical applications as potential replacements for current antibiotics that may have diminished activity.