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: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 19, 2012
Publication Date: February 1, 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. Poultry Science. 92(2):526-533. 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 resurgent 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 help combat 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. Bacteriophages were isolated in the Moscow Region of the Russian Federation and other related viruses were isolated in the southeastern USA during international cooperative research projects. The bacterial viruses were members of bacteriophages with long or short, non-contractile tails and small double-stranded DNA genomes. Structural proteins of the phages were identified by biochemical methods and the genomes of the viruses were determined by nucleotide sequencing. All the bacteriophages identified had 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: There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently utilized antibiotics. 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. Countries that have complied with the ban on antimicrobial growth promoters (AGP) in feeds have reported increased incidences of C. perfringens-associated necrotic enteritis in poultry. To address these issues, new antimicrobial agents, putative lysins encoded by the genomes of bacteriophages, are being identified in our laboratory. Poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that could lyse the bacterium C. perfringens and produce clear plaques in spot assays. Bacteriophages were isolated that had long non-contractile tails, members of the family Siphoviridae, and with short non-contractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded N-acetylmuramoyl-l-alanine amidases, lysozyme-endopeptidases and a zinc carboxypeptidase domain that has not been previously reported in viral genomes. Putative phage lysin genes (ply) were cloned and expressed in Escherichia coli. The recombinant lysins were amidases capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays, but did not lyse any clostridia beyond the species. Consequently, bacteriophage gene products rather than the viruses themselves may eventually be utilized to target specific bacterial pathogens, such as C. perfringens, to control animal and human diseases without having deleterious effects on beneficial bacteria.