System for Large-Scale Production of the Bacteriocin Jensiniin P., Potential Agent for the Treatment of Foodborne Pathogens and Acne

NON-TECHNICAL SUMMARY: The societal cost related to foodborne illness is very significant. There are costs related to individuals and households which might include medical, physician, and laboratory costs; hospitalization or nursing home, medications, ambulance or other travel costs; lost of income, child care costs and lost of leisure time. It also costs the industry which include the cost of plant closings and cleanup, recalls, and implement programs to monitor, control, and improve food safety. There are also regulatory and public health sector costs which might include disease surveillance costs and costs related to the development of integrated database. The costs of outbreak itself could include costs of investigating outbreak and testing to contain an outbreak. In addition, the prevalence of drug resistant bacteria has hampered the nation??s ability to effectively control important pathogens. The research seeks to develop effective antibiotic displacing agents for the treatment of pathogenic infections. Bacteriocins are antimicrobial peptides produced by some bacteria which inhibit the growth of closely related species or non-related species across genera. Several bacteriocins exhibit a broader activity toward a number of Gram-positive food spoilage and/or pathogenic bacteria, including Listeria. Bacteriocins have been used as natural food preservatives in the food and agriculture industries to improve the microbiological quality of foods. The utilization of bacteriocin-producing bacteria in livestock has been evaluated as a potential alternative to antibiotic. Microcin-producing E. coli have been used to reduce Salmonella carriage in poultry. Lantibiotic-producing lactic acid bacteria have been used to reduce E. coli O157:H7 in cattle. The over prescription of antibiotics and excessive use of antibiotics in animal feed have led to emergence of multiple-drug-resistant pathogens. Bacteriocins offer an alternative to antibiotics with higher specificities which is less likely to induce drug-resistant in not targeted pathogens. In additions, they can act as probiotics which improve the health and growth rate of livestock. Common acne is a chronic inflammation of the sebaceous follicles that affects 85-100% of those aged 12-24. Although not life-threatening, acne causes psychological and social distress and can cause profound scarring with adverse long term social consequences. Acne causes considerable morbidity through soreness, disfigurement, and social handicap due to inflammatory lesions. Scarring occurs to some degree in 95% of all patients irrespective of the severity of inflammatory acne. The scarring causes long-term morbidity that requires specific therapy. Some patients are severely affected and require more than just acne therapy. Jenseniin P has the potential to be an alternative antimicrobial treatment agent. Its use can be expected to have no adverse impact on antibiotic resistance. It is produced by a food grade organism, present in foods, and likely will cause little to no adverse effects on the skin or the digestive system.

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APPROACH: The bench-scale cultivation of the antibacterial bacteriocin producer, Propionibacterium jensenii (P. jensenii) B1264, will be optimized using bioreactor under controlled pH, temperature, oxygen level, and various media compositions. This bacteriocin, jenseniin P, will be isolated and purified from the supernatant and its bioactivity assayed using a susceptible strain of lactobacillus delbrueckii as indicators. The properties and characteristics as well as the antibacterial activities of this will be evaluated. The amino acid sequence of jenseniin P will be determined with assistance from the Clemson University Genome Institute. The bacteriocin will then be commercially synthesized in small quantities with its amino acids systematically substituted with alanine for further testing to enhance its antibacterial activity against important foodborne pathogens. This bacteriocin has previously been shown to be bacteriacidal against acne causing bacteria, e.g. Propionibacterium acnes. Hence, its use as anti-acne agents will also be evaluated. The mode of action for jenseniin P against these foodborne pathogens will be determined in order to explore the potential usages of this bacteriocin for controlling of foodborne pathogens to improve food safety during production, transportation, and storage. The potential mode of resistance to jenseniin P will also be evaluated to minimize the emergence of drug-resistant foodborne pathogens or other non-targeted pathogens. The large-scale production of jenseniin P will be achieved by cloning of the genes encoding jenseniin P into expression vectors. Two approaches will be utilized to localize the genes involved in the production of jenseniin P as well as genes involved in the immunity of P. jensenii to the bacteriocin it produces. One approach will be to use the determined amino acid sequence as a probe to identify its nucleic acid counterpart. The other approach will be to isolate mutants which have lost the producing trait and to analyze which gene segments in them have been lost or changed. In the subsequent project, the jenseniin P genes identified in this proposed project will be cloned into microorganisms on the GRAS list (Generally Recognized as Safe) or into crops with important agricultural/economical value, e.g. tobacco plant, for large-scale production of the bacteriocin. The characteristics of the recombinant bacteriocins will be evaluated and compared to that produced in P. jensenii B1264 strain. The important milestones in this proposed research are 1) the optimization of bench-top production of jenseniin P, 2) the purification and characterization of jenseniin P, 3) the protein sequence determination and analysis of jenseniin P, 4) the mode of action for jenseniin P to bacteria and the mode of resistance for bacteria to jenseniin P, and 5) the localization of genes encoding jenseniin P production and immunity. Data collected in research will be disseminated to scientific community and general public via publications and two undergraduate/graduate courses, Micro 413/613 (Industrial Microbiology) and BioSci 493 (Senior Seminar) offered in the Department of Biological Sciences, Clemson University.