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United States Department of Agriculture

Agricultural Research Service

Research Project: Pre-Harvest Interventions For Application During Poultry Production To Reduce Food-Borne Bacterial Pathogens

Location: Poultry Microbiological Safety Research

Title: Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Authors
item Volozhantsev, Nikolay -
item Oakley, Brian
item Morales, Cesar
item Verevkkin, Vladimir -
item Bannov, Vasily -
item Popova, Anactasiya -
item Zhilenkov, Eugeni -
item Svetoch, Edward -
item Garrish, Johnna
item Schegg, Kathleen -
item Woolsey, Rebekah -
item Quilici, David -
item Line, John
item Hiett, Kelli
item Siragusa, Gregory -
item Seal, Bruce

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 2, 2012
Publication Date: May 29, 2012
Citation: Volozhantsev, N.V., Oakley, B., Morales, C., Verevkkin, V.V., Bannov, V.A., Popova, A.V., Zhilenkov, E.L., Svetoch, E.A., Garrish, J.K., Schegg, K.M., Woolsey, R., Quilici, D.R., Line, J.E., Hiett, K.L., Siragusa, G.R., Seal, B.S. 2012. Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae. PLoS One. Volume/Page:7/e38283.

Interpretive Summary: Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium (unable to grow in the presence of oxygen) and is widely distributed in the environment. The bacterium frequently occurs in the intestines of humans and many domestic or feral animals. Spores of the organism persist in soil and areas subject to human or animal fecal pollution. Perfringens food poisoning is the term used to describe the common human food-borne illness and C. perfringens is the third most common cause of food poisoning 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. Phage therapy is the use of bacteriophages to treat pathogenic bacterial infections and 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. To address these concerns 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 the bacterium. Two bacteriophages designated phiCPV4 and phiZP2 were isolated in the Moscow Region of the Russian Federation and another closely related virus designated phiCP7R was isolated in the southeastern USA during international cooperative research projects. All three viruses were members of bacteriophages with 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 three viruses were very similar in genome content and three proteins were identified with potential enzymatic activities capable of digesting the host bacterium C. perfringens. Consequently, potential new alternatives to antibiotics were discovered that could eventually be used to control diseases caused by these 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 diseases. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lyse C. perfringens. Two bacteriophages designated phiCPV4 and phiZP2 were isolated in the Moscow Region of the Russian Federation and another closely related virus designated phiCP7R was isolated in the southeastern USA. All three viruses were members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of all three bacteriophages were approximately 18kbp encoding twenty-six to twenty-eight ORF’s with inverted terminal repeats and an average GC content of 34.6 percent. Structural proteins identified by mass spectrometry in the purified virion of phiCP7R included a pre-neck/appendage with putative lyase activity, major head, tail, connector or upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three clostridial podoviral genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. The predicted amino acid sequences of the phiCPV4 amidase were 98 percent similar to phiCP7R amidase sequences, while the proteins from these two phages were 90 percent similar to the predicted phiZP2 amidase protein. All three putative amidases contained a predicted bacterial SH3 domain at the C-terminal end of the protein presumably involved with binding of the protein to cell walls of C. perfringens. The predicted DNA polymerase type B protein sequences were most closely related phylogenetically to other members of the Podoviridae that included Bacillus phage phi29 representative of the Picovirinae. This relationship was further supported by whole-genome comparisons among picoviral bacteriophages.

Last Modified: 10/21/2014
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