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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: Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl-L-alanine amidase as a potential antimicrobial to control the bacterium

Authors
item Tillman, Glenn -
item Simmons, Mustafa -
item Garrish, Johnna
item Seal, Bruce

Submitted to: Archives Of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 21, 2013
Publication Date: N/A

Interpretive Summary: Clostridium perfringens is a spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal and avian diseases as well as human foodborne illness. This bacterial pathogen can be present in the intestines of people or animals and is common in the environment. Concerns over antimicrobial resistance and use of antibiotic growth promoters in animal feeds may be justified with increasing incidences of antibiotic resistance among bacterial pathogens, including bacteria from healthy animals. Consequently, there is a need for developing new intervention methods including narrow-spectrum antimicrobials that selectively target pathogenic organisms while avoiding killing of beneficial bacteria. Bacteriophages are viruses that infect and kill their bacterial hosts by digesting the bacterial cell wall with various genetically encoded enzymes. Genomics approaches were utilized to identify a bacteriophage gene encoding a lytic protein in the C. perfringens genome. Subsequently, the putative amidase enzyme was cloned from the bacterium’s genomic DNA and expressed as a recombinant protein to specifically lyse C. perfringens isolates, but not other bacteria. Recombinant DNA enzymes such as phytases and carbohydrases are commercially produced and sold for feed additive purposes to improve monogastric food-animal production, such as for poultry and swine. Consequently, research was completed to further develop bacteriophage encoded lysin enzymes as feed-additive alternatives to antibiotics that specifically lyse and kill disease-causing bacteria such as Clostridium perfringens, but do not affect other potentially beneficial bacteria.

Technical Abstract: Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to identify putative prophage lysins or autolysins by BLAST analyses encoded by the genomes of C. perfringens isolates. A predicted N-acetylmuramoyl-L-alanine amidase or MurNAc-LAA (also known as peptidoglycan aminohydrolase, NAMLA amidase, NAMLAA, Amidase 3, and peptidoglycan amidase; EC 3.5.1.28) was identified that would hydrolyze the amide bond between N-acetylmuramoyl and L-amino acids in certain cell wall glycopeptides. The gene encoding this protein was subsequently cloned from genomic DNA of a C. perfringens isolate by polymerase chain reaction (PCR) and the gene product (PlyCpAmi) expressed to determine if it could be utilized as an antimicrobial to control the bacterium. By spot assay, lytic zones were observed for the purified amidase and the E. coli expression host cellular lysate containing the amidase gene. Turbidity reduction and plate counts of C. perfringens cultures were significantly reduced by the expressed protein and observed morphologies for cells treated with the amidase appeared vacuolated, non-intact and injured compared to the untreated cells. Among a variety of C. perfringens strains there was little gene sequence heterogeneity that varied from 1 to 21 nucleotide differences. The results demonstrate that it is possible to discover lytic proteins encoded in the genomes of bacteria that could be utilized to control bacterial pathogens.

Last Modified: 10/19/2014
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