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. 2013 Feb;92(2):526-33.
doi: 10.3382/ps.2012-02708.

Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

Bruce S Seal  1
Affiliations

Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

Bruce S Seal. Poult Sci. 2013 Feb.

Abstract

There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently used antibiotics. Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a significant role in human foodborne disease as well as non-foodborne human, animal, and avian diseases. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated diseases 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 C. perfringens and produce clear plaques in spot assays. Bacteriophages were isolated that had long noncontractile tails, members of the family Siphoviridae, and with short noncontractile 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 could eventually be used to target bacterial pathogens, such as C. perfringens via a species-specific strategy, to control animal and human diseases without having deleterious effects on beneficial probiotic bacteria.

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Figures

Figure 1
Figure 1
Representative clear plaques and electron micrographs for Russian and US bacteriophages. (A) All phages were plaque-purified at least 3×. (B) Many phages had long noncontractile tails representative of the Siphoviridae. (C) Several phages were representative of the Podoviridae with short noncontractile tails. Color version available in the online PDF.
Figure 2
Figure 2
Genomic maps for bacteriophages virulent for Clostridium perfringens. (A) Genomic maps for members of the Siphoviridae, bacteriophages with long noncontractile tails. (B) Genomic maps for members of the Podoviridae, bacteriophages with short noncontractile tails.
Figure 3
Figure 3
Bacteriophage host restriction for Clostridium perfringens. Infection of the parent Clostridium perfringens strain Cp13 only supports replication of its individual bacteriophage and not that of other C. perfringens bacteriophages demonstrating viral host restriction.
Figure 4
Figure 4
Bacteriophage proteins identified with potential to lyse Clostridium perfringens. Potential lytic proteins encoded by bacteriophages included N-acetylmuramoyl-l-alanine amidases with C-terminal cell wall binding domains, tail proteins with lysozyme and peptidase activities as well as a pectate lyase and a previously unknown viral Zn-endopeptidase.
Figure 5
Figure 5
Lysis of Clostridium perfringens by a recombinant bacteriophage enzyme. Cells treated without phage lysin (A) or with lysin (B); note that all C. perfringens cells were completely digested with the addition of a recombinant bacteriophage-encoded lysin.
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